CN101781636A - Proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene, construction method and application thereof - Google Patents

Abstract

The invention relates to gene engineering and virology, in particular to a proliferous type recombination oncolytic adenovirus containing 11-type adenovirus cilia protein gene of a human B subset, a construction method and application thereof. The oncolytic adenovirus can be 11 type adenovirus or heterozygous adenovirus constructed by utilizing the coding sequence of 11-type adenovirus cilia protein to replace the coding sequence of non-11 type adenovirus cilia protein. The adenovirus has selective multiplication capacity by carrying out effective control or mutation on genes essential to proliferation. As a conditional proliferous type combination virus with wider spectrum of infection and improved infectious ability, the recombination oncolytic adenovirus of the invention can be used for gene treatment, in particular to tumor gene treatment.

Description

A kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, its construction process and uses thereof

Technical field

The present invention relates to genetic engineering and virusology, relate in particular to a kind of proliferous type recombination oncolytic adenovirus that contains human B subgroup 11 type adenovirus cilia protein genes, its construction process and uses thereof.This adenovirus can be 11 type adenovirus, also can be that aminoacid sequence with 11 type adenovirus cilium heads replaces the cilium head aminoacid sequence of non-11 type adenovirus and the heterozygosis adenovirus that constitutes.This adenovirus has the selective proliferative ability by effective control or the sudden change that its propagation must gene be carried out.As the recombinant virus of the condition proliferous type with wider pattern of infection and improvement infection ability, recombination oncolytic adenovirus of the present invention can be used for especially genetic treatment of tumor of gene therapy.

Background technology

Malignant tumour is a big class disease of serious threat human life health, and along with The development in society and economy, the primary hazard factor of tumour not only is not effectively controlled, and grows in intensity on the contrary, is that its M ﹠ M continues to rise.The conventional treatment of malignant tumour is difficult to receive that the therapeutic index of the curative effect of expection, particularly chemotherapeutics is low at present, and toxic side effect is big.In recent years, the complex therapy that comprises gene therapy became a part indispensable in the clinical tumor.The recombinant adenovirus p53 injection liquid " Ad-p53 " of China becomes gene therapy for cancer medicine (Wang X etc., the Mol Cancer Ther.2008Jun of first listing in the world in listing in 2004; 7:1598-1603).

Adenovirus is the most frequently used virus vector of therapy of tumor, and it is a kind of double-stranded DNA virus, and genome is about 36kb, and capsid (capsid) is 20 body structures of rule, the about 80-110nm of diameter.Capsid contains 240 six conjuncted (hexon), 12 5-linked bodies (penton) and 12 ciliums (fiber), in addition also has some other small protein, as VI, VIII, IX, III a and IV a2 etc.Six conjuncted be the major protein that forms 20 gores of viral capsid, 12 tops are 5 5-linked body subunits and 3 mixtures that cilia protein constitutes, 12 ciliums (Fiber) are that substrate is stretched out by the capsid surface with the 5-linked body protein, and the cilium top forms cephalomere district (knob).The cephalomere district of 5-linked body and cilium can combine with the virus receptor of cell surface, plays important effect (Gall JG etc., J Virol.1998 Dec in the virus infected cell process; 72:10260-10264; Sakurai F etc., Curr Gene Ther.2007Aug; 7:229-238).

There are 51 known serotypes in human adenovirus family, is divided into 6 subgenus (A-F).The people's that gene therapy is commonly used 2 types (Ad2) and 5 types (Ad5) adenovirus all belong to the C subgroup on serological classification, 95% homology is arranged on dna sequence dna.The process of adenovirus infection cell is that the specific receptors that the cephalomere district from the adenovirus cilium adheres to cell surface begins.The shared a kind of acceptor of C subgroup adenovirus major and CBV, therefore this acceptor be called as COxsackie/adenovirus receptor (coxsackie/adenovirus receptor, CAR).But, hematopoietic cell, embryonic stem cell, mescenchymal stem cell, some epidermic cell and tumour cell (as bladder cancer, cervical cancer, colorectal carcinoma, prostate cancer, lung cancer, mammary cancer, tumor of head and neck, melanoma, neurospongioma etc.) surface in human body, CAR is that low the expression even expressed.Since with the adenovirus be in the gene therapy of carrier the adenovirus of CAR mediation to enter target cell be rate-limiting step in the transgenosis, and transfection efficiency is relevant with cell surface CAR expression level.The CAR of low expression level restricts the major cause of adenovirus transduction efficiency beyond doubt, promptly shows resistance effect (Kim J etc., the J Virol.2002 of cell to C subgroup adenovirus carrier; 76:1892-1903; Cripe TP etc., Cancer Res.2001 Apr1; 61:2953-2960; Suzuki K etc., Clin Cancer Res.2001; 7:120-126; Wesseling JG etc., Gene Therapy.2001; 8:969-976; Bao Y etc., Prostate.2005 Sep 1; 64:401-407; Stoff-Khalili MA etc., Gene Ther.2005 Dec; 12:1696-1706).Be that the C subgroup adenovirus of acceptor also exists and can cause the very fast removing virus of the Ad5 type adenovirus neutralizing antibody that extensively exists in serious toxic side effect, the human body in vivo with CAR.These problems have limited the widespread use of Ad5 and Ad2.

B subgroup adenovirus comprises 3 types, 7 types, 11 types, 16 types, 35 types, 50 type adenovirus, and the cells infected of this group adenovirus serves as absorption acceptor (Fleischli C etc., J Gen Virol.2007 Nov with the CD46 molecule mainly not by the CAR acceptor; 88:2925-2934; Pache L etc., Virology.2008 Jun 5; 375:573-579; Pache L etc., J Virol.2008Aug; 82:7923-7931).CD46 is a kind of Complement Regulatory Protein of wide expression, and this albumen almost is expressed in except red corpuscle everyone peripheral blood cells, all kinds of stem cell and most of tumor cell surface.Therefore, utilize the adenovirus carrier of CD46 acceptor to be subjected to recently paying close attention to widely, people have proposed many strategies at the target sex modification problem of adenovirus carrier.Adenovirus Ad5/F35 carrier promptly is the hybrid vectors that a kind of shell is transformed, and its primary structure is on the basis of present Ad5 adenovirus carrier, and the cilium of its receptors bind position has been transformed into the cilium of 35 type adenovirus (Ad35).Therefore, the cell receptor of Ad5/F35 has become CD46 from the CAR of Ad5, the change of original infection absorption acceptor, improved its efficiency of infection to blood cell, stem cell, tumour cell, pattern of infection is wider than Ad5 commonly used now, and transduction efficiency is generally than Ad5 height (Stone D etc., Mol Ther.2007Dec; 15:2146-2153; Brouwer E etc., Cancer Gene Ther.2007Feb; 14:211-219).Experimental result confirms that the Ad5/F35 carrier obviously is better than 5 conventional type adenovirus carriers to the infection ability of the K562 cell in medullary system source, to the infection ability of bone-marrow-derived lymphocyte also a little more than the Ad5 carrier.The transformation of AD5/F35 has changed the infection characterization of carrier, but the toxic action of its pair cell not therefore increases, and the transformation carrier that confirms this mode has more wide application space in transduction such as stem cell, tumour cell and gene therapy research.

In addition, experiment confirm is arranged, may there be the special acceptor of another kind of cells infected in B subgroup adenovirus.Utilize Ad5/11-CMV-GFP and Ad5/35-CMV-GFP to infect the A549 and the Hela cell of CD46 positive expression respectively, the efficiency of infection of Ad5/11-CMV-GFP is all than Ad5/35-CMV-GFP height, and remove to infect the CHO-C2 cell of CD46 positive expression simultaneously with these two kinds of carriers, efficient (Stone D etc., J Virol.2005 about the same; 79:5090-5104).In addition, antiserum(antisera) experiment shows, the knob district of Ad11-Fiber can suppress combining of Ad35 and A549 cell fully, and the knob district of Ad35-Fiber can not suppress (MeiYF etc., the Virology.2002 of combining of Ad11 and A549 cell fully; 295:3043).Infect 11 kinds of esophageal cancer cells and 6 kinds of cancer cell of oral cavity with Ad5, Ad5/11 and Ad5/35 respectively, Ad5/11 and Ad5/35 are all high a lot of than Ad5 to the efficiency of infection of all tumour cells, and the efficiency of infection of Ad5/11 and Ad5/35 does not rely on expression level (the .Oncol Rep.2005 such as Ling Y of CD46; 14:831-835).Therefore infer that the acceptor that has other participates in regulating the infection of Ad11 and Ad35 pair cell.

Although with the adenovirus be carrier therapy of tumor experiment and clinical in obtained some curative effects, but, further improve adenovirus to the transduction efficiency of tumour cell, strengthen Antioncogene expression, make virus vector have more special target, thereby improving curative effect and security, is the emphasis direction of following therapy of tumor research.

People's 11 type adenovirus carriers are compared with traditional C group adenovirus carrier, and many advantages are arranged: the positive rate of neutralizing antibody is lower in the serum; And there is not cross-immune reaction between Ad5, the Ad35 yet; Cells infected does not rely on the CAR acceptor, and the many important cells that comprise hemopoietic stem cell, primary tumor cell, tumor stem cell are all had higher efficiency of infection; Hepatotoxicity is low, and immune side effect is little.Therefore, exploitation Ad11 carrier, most stem cells of efficient transfection and tumour cell, target is killed tumor stem cell, realizes thoroughly killing the purpose of tumour, and crucial meaning will be arranged.

The adenovirus carrier that adopts in the gene therapy scheme, most propagation defective adenovirals that do not have replication that adopt.Though the propagation defective adenoviral has shown certain security in vivo and in vitro, yet this type of adenovirus carrier of the great majority of using in clinical trial so far remains in that time of expression alien gene is short, immunogenicity is stronger, many shortcomings such as significant cytotoxicity are arranged during high titre, so clinical efficacy is not good.

Address the above problem, need a kind ofly can in tumour cell, have the proliferated specifically ability, can improve copy number of foreign gene and expression amount and can dissolve the condition replication competent adenovirus carrier of kill tumor cell, be i.e. the oncolytic adenovirus carrier.Both at home and abroad the oncolytic adenovirus development of projects is very fast, the H101 project of the three-dimensional company in domestic Shanghai go through for the end of the year 2005 listing (Yu W etc., Curr Cancer Drug Targets.2007 Mar; 7:141-148).More and more data proves that oncolytic adenovirus promises to be the anti-tumor medicine of a new generation.

Summary of the invention

Technical problem to be solved:

The adenovirus carrier that adopts in the gene therapy scheme, the human C subgroup 5 type adenovirus (Ad5) of most employings, the process of its cells infected depends on CAR.But, hematopoietic cell, embryonic stem cell, mescenchymal stem cell, some epidermic cell and tumor cell surface in human body, the low expression even of CAR expressed.Therefore, the CAR of low expression level becomes the major cause of restriction the type adenovirus transduction efficiency, promptly shows the resistance effect of cell to C subgroup adenovirus carrier.Be that the C subgroup adenovirus of acceptor also exists and can cause the very fast removing virus of the 5 type adenovirus neutralizing antibodies that extensively exist in serious toxic side effect, the human body in vivo with CAR.

The adenovirus carrier that adopts in the gene therapy scheme, most propagation defective adenovirals that do not have replication that adopt.Many shortcomings such as significant cytotoxicity are arranged during stronger, the high titre of short, immunogenicity of time of such adenovirus carrier ubiquity expression alien gene, influenced its clinical efficacy.

Therefore, further improve virus vector to the transduction efficiency of tumour cell, strengthen Antioncogene expression, make virus vector have more special target, thereby improve curative effect and security, be the emphasis direction of following therapy of tumor research.

The technical scheme that adopts:

The object of the present invention is to provide a kind of proliferous type recombination oncolytic adenovirus that can be used for multiple human body genetic treatment of tumor, this recombination oncolytic adenovirus can overcome current propagation defective adenoviral vector (as Ad-p53) transhipment and express the shortcoming of antioncogene inefficiency, no target, overcome with people Ad5 replication competent adenovirus carrier (as H101, ONYX-015) and only have single antitumor mechanism,, shortcoming such as security not good low the tumour cell infectivity, its antitumor curative effect is improved, and security is improved.

For achieving the above object, the invention provides a kind of proliferous type recombination oncolytic adenovirus of the B of containing subgroup 11 type adenovirus cilium head (knob) amino acid coding, it is characterized in that, the genome of this recombinant virus contains B subgroup 11 type adenovirus cilium head (knob) amino acid coding, described 11 type adenovirus cilia protein encoding sequences, both can be 11 type adenovirus, also can be that aminoacid sequence with human 11 type adenovirus cilium heads (knob) replaces the cilium head aminoacid sequence of non-11 type adenovirus and constitutes.

In an embodiment, the frame sequence of this proliferous type recombination oncolytic adenovirus is 5 type adenovirus (Ad5), and replacing the cilium head aminoacid sequence of 5 type adenovirus by the aminoacid sequence of 11 type adenovirus cilium heads, its cilia protein encoding sequence is shown in SEQ ID NO:1.Virus vector structure aspect, the improvement and the purposes of function that contains the proliferous type recombination oncolytic adenovirus of 11 type adenovirus cilia protein genes by two kinds of embodiment explanations, the one, adopt human telomerase reverse transcriptase (hTERT) promoter regulation E1a gene, hypoxia response elements (HRE) the regulation and control E1b district's gene (E1b-55kDa and E1b-19kDa gene) of choosing simultaneously, make up the proliferous type oncolytic adenovirus, the 2nd, E1b-55kDa is implemented sudden change, it is lost activity, thereby make the propagation of virus obtain tumour-specific.Aspect the external source gene Selection, the function and the purposes that contain the adenovirus mediated genetic expression of proliferous type recombination oncolytic of 11 type adenovirus cilia protein genes by two kinds of embodiment explanations, the one, carrier's chemokine CCL5 gene, the 2nd, carry green fluorescent protein reporter gene EGFP.

The proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes can be discerned CD46 membrane receptor and other special acceptors that may exist, thereby important cells such as the efficient infected person hemopoietic system cell of energy, mescenchymal stem cell, dendritic cell, tumor stem cell, primary tumor cell, and immunogenicity is lower, does not have cross-immune reaction with traditional C group adenovirus carrier; This adenoviral gene group makes it optionally in the tumour cell internal breeding, and does not breed substantially in normal cell by effective control of genetically deficient or generegulation; This adenovirus can be used as the carrier of transgenosis, inserts multiple antineoplaston gene in its genome simultaneously, and the oncotherapy gene is efficiently expressed with being replicated in the tumour cell of virus.By virus multiplication and antioncogene dual function, the specific killing tumour cell is used for the treatment of multiple human tumor.

Human adenovirus has A, B, C, D, 6 different subgenus of E, F, and they have nothing in common with each other to close preferendum, tumorigenicity and the history of disease of host cell.The present invention relates to adenovirus B subgroup 11 types.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, the cilia protein encoding sequence of adenoviral gene group is by people's 11 type adenovirus cilia protein genomic constitutions.Described 11 type adenovirus cilia protein genes both can be the cilia protein encoding sequences of complete wild-type 11 type adenovirus, also can be that the cilium head aminoacid sequence heterozygosis that aminoacid sequence by 11 type adenovirus cilium heads replaces non-11 type adenovirus forms.This virus can be discerned CD46 membrane receptor and other special acceptors that may exist, and be no longer dependent on the CAR acceptor, thereby make the pattern of infection of its infected person tumour cell wider, transduction efficiency is higher, has improved its efficiency of infection to important cells such as human hematopoietic system cell, mescenchymal stem cell, dendritic cell, tumor stem cell, primary tumor cells.In embodiments, 11 type cilia protein gene orders of described recombinant adenovirus are shown in SEQ ID NO:1, wherein: (1) 1-134 bit base: the tail albumen coded sequence of 5 type adenovirus ciliums; (2) 135-981 bit bases: the shaft of 11 type adenovirus ciliums, knob albumen coded sequence.

On the other hand, the invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, this adenoviral gene group is by the effective control of tumor-specific promoters to the propagation indispensable gene, make it optionally in the tumour cell internal breeding, and in normal cell, do not breed substantially, thereby make its target human tumor cells, guarantee adenovirus in tumour cell inner height propagation, the performance oncolysis.This tumor-specific promoters can be following any tumor-specific promoters or enhanser: (a) carcinomebryonic antigen promotor, enhanser and mutant sequence thereof; (b) afp promoter, enhanser and mutant sequence thereof; (c) receptor tyrosine kinase of Human epidermal growth factor receptor family (EGFRs) (comprising EGFR, Her-2, Her-3 and Her-4) promotor, enhanser and mutant sequence thereof; (d) breast cancer correlation antigen DF3/MUC1 promotor, enhanser and mutant sequence thereof; (e) promotor, enhanser and the mutant sequence thereof of vascular endothelial growth factor (VEGF) receptor KDR; (f) L-plastin promotor, enhanser and mutant sequence thereof; (g) member's of IAP family (IAP) promotor, enhanser and mutant sequence thereof; (h) promotor of prostaglandin(PG) specific antigens, enhanser and mutant sequence thereof; (i) the hypoxia response elements conserved sequence of HIF-1 (HIF-1) regulation and control; (j) transcription factor E2F promotor, enhanser and mutant sequence thereof; (k) human telomerase subunit promotor.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that at least one virus multiplication indispensable gene is controlled by a kind of tumor-specific promoters.This virus multiplication indispensable gene can be E1a.This oncolytic adenovirus can be expressed E1a duplicating in specifically inside tumor cell ground propagation, performance E1a antitumor action.E1a can be by activity, the lethal effect that improves immunocyte that suppresses Her-2/neu gene transcription, blocking-up NF-κ B, the number of ways such as expression that strengthen nm-23 nm23, inducing apoptosis of tumour cell, suppress tumor invasion and shift, improve the susceptibility of tumour cell chemotherapy, radiotherapy.This oncolytic adenovirus and chemotherapy and radiation share, and can strengthen the susceptibility of tumour cell to chemotherapy and radiation, further improve result of treatment.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that at least one virus multiplication indispensable gene is controlled by a kind of tumor-specific promoters.The virus multiplication indispensable gene of tumor-specific promoters control can also be E1b-55kDa, E1b-19kDa, E2 or E4, and this propagation indispensable gene can be a wild-type, also can be the genetic mutation of transforming through disappearance, sudden change etc.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, the forfeiture of its E1b-55kDa protein function, by the proteic gene order of coding E1b-55kDa is all lacked, point mutation, excalation, insertion sudden change or insert mode such as terminator codon.To be adenovirus duplicate essential and nonessential albumen when duplicating in tumour cell in normal cell propagation E1b-55kDa, the ability that the selectivity disappearance of E1b-55kDa encoding gene can make adenovirus keep propagation to duplicate in tumour cell, and in normal cell, lose replication.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, the forfeiture of its E1b-19kDa protein function by the proteic gene order of coding E1b-19kDa is all lacked, point mutation, excalation, insertion sudden change or insert mode such as terminator codon.Adenovirus E 1 b-19kDa gene and apoptosis suppressor Bc1-2 homology; the E1b-19kDa albumen of coding can suppress program or/and Bak starts the apoptosis in downstream in conjunction with Bax; E1b-19kDa can also destroy Fas mediated Apoptosis process, and the cell that protection is infected is avoided the alpha mediated lethal effect of TNF-.The E1b-19kDa gene inactivation, thereby should virus be improved in the specificity of tumour cell internal breeding, and the proliferation activity in normal cell is weakened.The forfeiture of E1b-19kDa protein function, not only can promote the recovery of apoptosis of tumor cells path, and help the quick removing of virus in normal cell and the snap-out release in tumour cell and send out, make the specificity of this oncolytic adenovirus treatment tumour better, usefulness is stronger.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, preset multiple clone site in its genome, can be used as the carrier of transgenosis.This site can be in E1a promotor upstream, also can be between E1a and E1b, or in the E3 district, various Antioncogene expression cassettes are easy to carry.Antioncogene is inserted this viral foreign gene multiple clone site, bring into play multiple antitumor usefulness.Along with the proliferated specifically of virus in tumour duplicates, the copy number and the expression amount of the Antioncogene that it carries are improved, thereby suppress growth of tumour cell or kill tumour cell.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, the selection of its Antioncogene can also be following any one: (a) Interferon, rabbit series immune-regulating factor gene; (b) interleukin-series immune-regulating factor gene; (c) colony-stimulating factor gene; (d) cancer suppressor gene; (e) proto-oncogene antisense sequences; (f) antibody gene of people's tumour Epidermal Growth Factor Receptor Family (EGFRs); (g) has the nucleotide sequence such as Endostatin, angiostatin and fragment thereof, plasminogen activating factors inhibitor, Canstatin, thrombospondin of angiogenesis inhibitor; (h) apoptosis inducing factor family gene; (i) vascular endothelial growth factor antibody gene; (j) medicine prerequisite transforming gene (claiming suicide gene again); (k) tumour necrosis factor gene.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, encoding sequence is subjected to the control of human cytomegalovirus (CMV) promotor in the Antioncogene expression cassette, this cis-acting elements inserts between the transcription initiation site and translation initiation site of Antioncogene sequence, guarantee that this gene protein efficiently expresses in the inside tumor of virus infection, the performance biologic activity.Antioncogene can also be controlled by following promotor: (a) carcinomebryonic antigen promotor, enhanser and mutant sequence thereof; (b) afp promoter, enhanser and mutant sequence thereof; (c) receptor tyrosine kinase of Human epidermal growth factor receptor family (EGFRs) (comprising EGFR, Her-2, Her-3 and Her-4) promotor, enhanser and mutant sequence thereof; (d) breast cancer correlation antigen DF3/MUC1 promotor, enhanser and mutant sequence thereof; (e) promotor, enhanser and the mutant sequence thereof of vascular endothelial growth factor (VEGF) receptor KDR; (f) L-plastin promotor, enhanser and mutant sequence thereof; (g) member's of IAP family (IAP) promotor, enhanser and mutant sequence thereof; (h) promotor of prostaglandin(PG) specific antigens, enhanser and mutant sequence thereof; (i) the hypoxia response elements conserved sequence of HIF-1 (HIF-1) regulation and control; (j) transcription factor E2F promotor, enhanser and mutant sequence thereof; (k) human telomerase subunit promotor.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that, the tailer sequence that adds of encoding sequence is SV40 Poly A in the Antioncogene expression cassette, and this cis-acting elements inserts after translation stop codon of gene nucleotide series.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that at least one virus multiplication indispensable gene is controlled by a kind of tumor-specific promoters.Can connect by polysome entry site (IRES) sequence between its a plurality of propagation indispensable genes, make it regulated and control by tumor-specific promoters, or regulate and control respectively by a plurality of tumor-specific promoters.Among the embodiment, control E1a and E1b gene respectively, human telomerase reverse transcriptase (hTERT) promotor control E1a gene, people's hypoxia response elements (HRE) regulation and control E1b district's gene (E1b-55kDa and E1b-19kDa gene) with double-promoter.

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that this virus mutant is operably connected by the cis-acting elements of human adenovirus carrier, tumor-specific promoters, human 11 type adenovirus cilia protein genes and Antioncogene expression cassette and forms.This proliferous type oncolytic adenovirus is produced by the reorganization of Cre-loxP recombinase diced system fixed point in 293 cells by skeleton plasmid that contains human 11 type adenovirus cilia protein genes and the adenovirus shuttle plasmid that carries Antioncogene.Its skeleton plasmid sequence that contains human 11 type adenovirus cilia protein genes is shown in SEQ ID NO:2, wherein: (1) 1-85 bit base: adenovirus ITR sequence; (2) 562-700 bit base: SV40 the AN sequence; (3) 1245-2294 bit bases: pSP13Cre sequence; (4) 2375-2716 bit base: HCMV the IE sequence; (5) 2962-3819 bit bases: amp encoding sequence; (6) 4901-4934 bit bases: LoxP sequence; (7) 31516-31649 bit bases: the tail encoding sequence of 5 type adenovirus cilia proteins; (8) 31649-32496 bit bases: shaft, the knob encoding sequence of 11 type adenovirus cilia protein genes; (8) 35554-35655 bit bases: adenovirus ITR sequence; (9) all the other: 5 type adenoviral gene group sequences, see the pBHGloxdeltaE1 of MicrobixBiosystems company, the 3Cre sequence.

The technique effect of realizing:

1. the invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, this adenovirus has the multiple clone site of foreign gene, can be used for making up the adenovirus product of multiple Antioncogene treatment, for genetic treatment of tumor is set up a kind of curative effect reliable technique platform.

2. oncolytic adenovirus provided by the invention has multiple antitumor mechanism and specificity advantage: 1) genome of this recombinant virus contains 11 type adenovirus cilia protein albumen coded sequences, make virus can discern the CD46 membrane receptor, thus important cells such as efficient infected person hemopoietic system cell, mescenchymal stem cell, dendritic cell, tumor stem cell, primary tumor cell; 2) with the necessary genetic expression of tumor-specific promoters regulating and controlling adenovirus propagation, make virus propagation and destruction tumour cell in the human tumor cell of most of types, its regulation and control have preciseness and security; 3) efficient and specific expressed E1a in tumour cell promotes virus multiplication, itself also has antitumor action simultaneously; 4) will the encode nucleotide sequence of Antioncogene inserts in this viral genome, and along with the proliferated specifically of virus in tumour duplicates, the copy number of the Antioncogene that it carries and expression amount increase, thereby suppress tumor growth.

3. oncolytic adenovirus provided by the invention has better broad spectrum and efficiency of infection than the domestic gene therapy product recombinant P 53 adenovirus injection liquid " Ad-p53 " that has gone on the market at present, and curative effect is improved; Have the security of better therapeutic and Geng Gao than the domestic replicative adenovirus product H101 that has gone on the market at present, be adapted to the treatment of human most type malignant tumours.

The accompanying drawing summary

Fig. 1 represents to contain the structure flow process of the adenovirus carrier pCLON9-F11 of 11 type adenovirus cilia protein gene coded sequences.

Fig. 2 represents the structure flow process of adenovirus packaging plasmid pPE3.

Fig. 3 represents that pPE3-F11 makes up flow process.

Fig. 4 represents the L02 normal liver cell, and MOI=10 infects Ad5-EGFP, and fluorocyte accounts for about 25%.

Fig. 5 represents the L02 normal liver cell, and MOI=10 infects SG511-EGFP, and fluorocyte accounts for about 85%.

Fig. 6 represents the SMMC-7721 liver cancer cell, and MOI=10 infects Ad5-EGFP, and fluorocyte accounts for about 35%.

Fig. 7 represents the SMMC-7721 liver cancer cell, and MOI=10 infects SG511-EGFP, and fluorocyte accounts for about 95%.

Fig. 8 CCL5 gene primer amplification 1-4 adenovirus clone that represents to choose all can amplify the 349bp band, and (P) is consistent with positive control, illustrates that the 1-4 adenovirus all contains the CCL5 gene.M1:100bp DNA Ladder wherein; N: negative control; P: positive control (pENTR12-CCL5); The 1-4:SG511-CCL5 virus clone

Fig. 9 represent SG511-CCL5 in liver cancer cell along with the time is how much multiples propagation, have good multiplication capacity, and the propagation multiple in normal cell strain BJ is very low.

Figure 10 represents that SG511-CCL5 treatment group compares with other control groups, demonstrate stronger inhibition tumor growth effect, gross tumor volume is significantly less than control group always, remarkable significant difference (p＜0.05) is arranged, illustrate that chimeric replication competent adenovirus SG511-CCL5 has very strong antitumor action to liver cancer animal model, can obviously suppress the growth of tumor development.

Detailed Description Of The Invention

Set forth embodiments of the present invention below by embodiment; described embodiment only is used for the exemplary the present invention of demonstrating; it does not limit protection scope of the present invention, and those skilled in the art are included among the present invention by the technical scheme of equal modification and improvement according to the present invention.

In an embodiment, first scheme is controlled 5 type adenovirus E 1 a with human telomerase reverse transcriptase (hTERT) promotor, and personnel selection hypoxia response elements (HRE) regulation and control E1b district's genes (E1b-55kDa and E1b-19kDa gene) are example; Alternative plan is implemented sudden change to E1b-55kDa, and it is lost activity, thereby makes the propagation of virus obtain tumour-specific, makes up the oncolytic adenovirus carrier that contains 11 type adenovirus cilia protein genes.In the concrete enforcement of embodiment, be that example is set up two kinds of strategies with human chemokine CCL5 gene and green fluorescent protein reporter gene (EGFP), make up the adenovirus carrier that carries goal gene.

Embodiment:

Embodiment 1: the structure that contains the adenovirus carrier pCLON9-F11 of 11 type adenovirus cilia protein gene coded sequences

The first step: synthesize and contain 11 type adenovirus cilia protein gene coded sequences shown in SEQ ID NO:3, name Ad5F11, wherein: (1) 236-369 bit base: 5 type adenovirus cilium tail albumen coded sequences; (2) 367-1216 bit bases: 11 type adenovirus cilium shaft, knob albumen coded sequence; (3) all the other bases: 5 type adenoviral gene group sequences, see the pBHGloxdeltaE1 of Microbix Biosystems company, the 3Cre sequence.

Second step: synthetic VT176 sequence (SEQ ID NO:4) and VT177 sequence (SEQ IDNO:5) make up the pCLON9 carrier.The Ad5F11 sequence is cut through the PacI+HindIII enzyme, reclaims the 2656bp fragment, and the carrier of cutting with the pCLON9/PacI+HindIII enzyme is connected, is built into pCLON9-F11, cuts and checks order and identify affirmation through enzyme.Construction step as shown in Figure 1.

Homologous recombination in the embodiment 2:BJ5183 cell

The first step: pCLON9-F11 cuts through the HindIII+AatII enzyme, reclaims the 5913bp fragment.

Second step: synthetic ADP sequence (SEQ ID NO:6) as shown in Figure 2, makes up adenovirus packaging plasmid pPE3.

The 3rd step: the pPE3 carrier is cut through the PacI enzyme, reclaims linearized vector.PCLON9-F11/HindIII+AatII 4 microlitres, pPE3/PacI 2 microlitres, common transformed competence colibacillus intestinal bacteria BJ5183 (Qbiogene company).Bed board is cultivated, and the picking clone carries out enzyme and cuts evaluation, and correct person's called after pPE3-F11 extracts purifying through the full-page proof plasmid, is stored in the profound hypothermia refrigerator.PPE3-F11 makes up flow process as shown in Figure 3.

Embodiment 3: the adenovirus that contains Ad11 adenovirus cilia protein gene is in external infectivity experiment to culturing cell

The first step: control 5 type adenovirus E 1 a with human telomerase reverse transcriptase (hTERT) promotor, insert site NotI+SwaI, making up adenovirus carrier pQW-hTERT-Ep-HRE (sees: patent ZL028138198, virus of a kind of proliferated specifically inside tumor cell that efficiently expresses antioncogene and uses thereof; Now be renamed as pSG502).HTERT promoter regulation E1a gene wherein, hypoxia response elements (HRE) the regulation and control E1b district's gene (E1b-55kDa and E1b-19kDa gene) of choosing simultaneously.Described hTERT promotor core sequence is shown in SEQ ID NO:7, and hypoxia response elements HRE sequence is shown in SEQ ID NO:8.

Second step: EGFP gene (SEQ ID NO:9) is inserted pSG502, be built into pSG502-EGFP, it is EcoRI+SalI that EGFP inserts the site.

Second step: contain the adenoviral plasmid pSG502-EGFP of EGFP gene and skeleton plasmid pPE3-F11 cotransfection HEK293 cell and (purchase company in Canadian Microbix Biosystems, this cell is transformed by the 5 type adenovirus DNAs of shearing, contain 5 type adenovirus E 1 districts, adenovirus DNA has high transfection efficiency to it, produce adenovirus by reorganization in the cell with infectivity), carry out reorganization in the cell.Transfection concrete grammar step is referring to the LipoFectamine2000 test kit process specifications of Invitrogen company.Recombinant products is identified correct person's called after SG511-EGFP through PCR.EGFP gene PCR primer is: upstream primer 5 '-GCTCTAGAGAATTCACCATGGTGAGCAAGGGC-3 ' (SEQ ID NO:10), downstream primer 5 '-CGGGATCCGTCGACTTATTACCTAGATCCGGTGG-3 ' (SEQ ID NO:11), amplified fragments 793bp.

The 3rd step: with the EcoRI+SalI site among the EGFP gene insertion adenoviral plasmid pDC315 (MicrobixBiosystems company), be built into pDC315-EGFP, carry out recombinating in the cell with skeleton plasmid pPE3, recombinant products is identified correct person's called after Ad5-EGFP through PCR, in contrast virus.This contrast virus is for containing the virus vector of 5 type adenovirus cilia protein genes.

The 4th step: normal cell strain and tumor cell line are respectively by 5 * 10 ⁵Cells/well is layered in 6 orifice plates, cultivates at 37 ℃ of incubator 5%CO2, changes serum-free liquid 1ml in second day, adds recombination oncolytic adenovirus SG511-EGFP and Ad5-EGFP respectively by MOI=10 then, and fluorescent microscope is observed down after 24 hours.

Observations: be the L02 normal liver cell among Fig. 4, MOI=10 infects Ad5-EGFP, and fluorocyte accounts for about 25%.Be the L02 normal liver cell among Fig. 5, MOI=10 infects SG511-EGFP, and fluorocyte accounts for about 85%.Be the SMMC-7721 liver cancer cell among Fig. 6, MOI=10 infects Ad5-EGFP, and fluorocyte accounts for about 35%.Be the SMMC-7721 liver cancer cell among Fig. 7, MOI=10 infects SG511-EGFP, and fluorocyte accounts for about 95%.Show that the SG511-EGFP infection ability obviously is better than Ad5-EGFP.

Embodiment 4: contain the reorganization of the oncolytic adenovirus of 11 type adenovirus cilia protein gene coded sequences, E1b-55kDa inactivation

Adenovirus carrier pXC-delE1b (the Qian Qijun etc. of the E1b-55kDa inactivation that successfully constructs in earlier stage with us, Chinese Medical Journal .2002,82:557-560) be the basis, with pPE3-F11 cotransfection HEK293 cell, reorganization contains the adenovirus SG211 of 11 type adenovirus cilia protein gene coded sequences, this virus E1b-55kDa inactivation, the ability of acquisition special propagation in tumour cell.

Embodiment 5: the construction of recombinant adenovirus containing of carrying Antioncogene

The invention provides a kind of recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, it is characterized in that,, make it optionally in the tumour cell internal breeding by effective control of genetically deficient or generegulation; Preset multiple clone site in its genome, various Antioncogene expression cassettes can be carried in this site.The oncolytic adenovirus carrier is controlled 5 type adenovirus E 1 a genes with human telomerase reverse transcriptase (hTERT) promotor, hypoxia response elements (HRE) the regulation and control E1b district genes (E1b-55kDa and E1b-19kDa gene) of choosing simultaneously are example, and Antioncogene is an example with human chemokine CCL5 gene.The adenovirus carrier that makes up non-propagation simultaneously is contrast virus.

The first step: control 5 type adenovirus E 1 a with human telomerase reverse transcriptase (hTERT) promotor, insert site NotI+SwaI, make up adenovirus carrier pSG502 and (see: patent ZL028138198).Wherein hTERT promotor core sequence is shown in SEQ ID NO:7.

Second step: the synthetic human chemokine CCL5 gene nucleotide series (SEQ IDNO:12) that contains, its 5 ' end and 3 ' end are introduced EcoRI and BamHI site respectively, after cutting, inserts the EcoRI+BamHI enzyme plasmid pENTR11 (Invitrogen LifeTechnology Inc.) and pDC315 multiple clone site district respectively, constitute pENTR12-CCL5 and pDC315-CCL5, recombinate with skeleton plasmid pPE3-F11 respectively, recombinant products is cut through enzyme and is identified correct person's called after pPE3-F11-CCL5 and Ad5F11-CCL5 again.

Embodiment 6: the interior reorganization of cell and the amplification purification that contain the oncolytic adenovirus of 11 type adenovirus cilia protein genes

The invention provides a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, tumour cell is had good infection ability and target.

The first step: the 5 type adenoviral plasmid pSG502 that will build, with the heterozygosis adenovirus carrier plasmid pPE3-F11-CCL5 that contains 11 type adenovirus cilia protein genes, by the common transfection HEK293 of LipoFectamine2000 cell.PPE3-F11-CCL5 contains 5 type adenovirus right arms, disappearance E1 district, and its recombinase system Cre/LoxP can guarantee the efficient reorganization of virus.9-14 behind the carrier cotransfection days, virus plaque appears successively, (concrete grammar is referring to GeneTransfer and Expression Protocols through three virus plaque purifying, Murray EJ chief editor, Humana Press, Clifton, New Jersey), reorganization obtains oncolytic adenovirus SG511-CCL5.

Second step: the evaluation of recombination oncolytic adenovirus, get rid of wild-type adenovirus with adenovirus E 1 a gene primer, identify with people CCL5 gene primer whether recombinant adenovirus contains the CCL5 sequence.Adenovirus E 1 a gene primer: upstream primer 5 '-cggaattcaccatgagacatattatc-3 ' (SEQ ID NO:13), downstream primer 5 '-gcgtcgacttatggcctggggcg-3 ' (SEQID NO:14), amplified fragments 1005bp.People CCL5 gene primer is: upstream primer 5 '-CGGAATTCACCATGAAGGTCTCCGCGGCAG-3 ' (SEQ ID NO:15), downstream primer 5 '-CGGGATCCCTATAACTGGAAGTCATCATCCATTG-3 ' (SEQ ID NO:16), amplified fragments 349bp.

As shown in Figure 8, with people CCL5 gene primer amplification 1-4 adenovirus clone, all can amplify the 349bp band, (P) is consistent with positive control, illustrates that the 1-4 adenovirus all contains the CCL5 gene.

The 3rd step: the recombination oncolytic adenovirus is amplification in a large number in the HEK293 cell, (concrete grammar is referring to GeneTransfer andExpression Protocols, and Murray EJ edits, Humana Press for the method purification of adenoviral of application caesium chloride density gradient centrifugation, Clifton, NewJersey).The titre that records recombination oncolytic adenovirus SG511-CCL5 with the TCID50 method reaches 2.79 * 10 ¹⁰Pf μ/ml.

Embodiment 7: the tumour cell internal breeding experiment in vitro culture of recombination oncolytic adenovirus SG511-CCL5

Normal cell strain and tumor cell line are respectively by 5 * 10 ⁵Cells/well is layered in 6 orifice plates, at 37 ℃ of incubator 5% CO ₂Cultivate, changed serum-free liquid 1ml in second day, press MOI=5 then and add recombination oncolytic adenovirus SG511-CCL5, cultivate after 90 minutes, (PBS) washes twice with phosphate buffered saline buffer, with viral flush away, cultivate with the nutrient solution that adds 5% foetal calf serum, collected at 0 hour, 48 hours, 96 hours respectively, freeze thawing three times detects virus titer (method is referring to the AdEasyTM operational manual of U.S. Qbiogene company) with the TCID50 method.With 0 hour virus titer was reference, calculate virus at 48 hours, 96 hours propagation multiple, found that SG511-CCL5 in liver cancer cell along with the time is multiples propagation how much, have good multiplication capacity, and the propagation multiple in normal cell strain BJ is very low, specifically as shown in Figure 9.

Embodiment 8: recombination oncolytic adenovirus SG511-CCL5 experimentation on animals

The first step: 4～6 age in week 50 of BALB/C nude mices, weight in average 22～27g is provided by Shanghai Chinese Academy of Sciences laboratory animal cultivation center, cleaning level Animal Lab. is raised.

Second step: extracorporeal culture human liver cancer cell SMMC-7721, the adherent growth cell in vegetative period of taking the logarithm, 0.25% trysinization, resuspended behind centrifugal, the collecting cell with PBS liquid, centrifugal 2 minutes of 3000g room temperature, abandon supernatant, use the resuspended back of PBS liquid centrifugal collecting cell again, adjust concentration of cell suspension to 5 * 10 ⁷Individual/ml.

The 3rd step: in the right ribbed back subcutaneous vaccination SMMC-7721 of portion of nude mice cell, 0.1ml/ only.After inoculating about three weeks, the subcutaneous grain of rice size that grows of visible inoculation position is hardened, and the transplanted tumor model builds up.Begin treatment when treating the growth in thickness of knurl body to 7-9mm.Select the SMMC-7721 transplanted tumor that 40 Subcutaneous tumors grow to 7-9mm and treat, nude mice is divided into 5 groups at random.Route of administration is a multi-point injection in the direct knurl.

The 4th step: observe animation and the periodic measurement tumor size of mouse every day, major diameter (A) and vertical diameter (B), the by formula (A * B of a tumour of periodic measurement ²Tumor size is calculated in)/2, observes the curative effect of various dose group.

Experiment be provided with band CCL5 gene non-proliferous type adenovirus Ad5F11-CCL5 and (its title is not adenovirus hominis 5/11 mosaic type SG511 with the replication competent adenovirus SG511 of CCL5 gene, preservation day is on January 9th, 2009, preserving number is CCTCC-V2000903, and the preservation place is Chinese typical culture collection center) for contrasting virus.Found that SG511-CCL5 treatment group compares with other control groups, demonstrate stronger inhibition tumor growth effect, gross tumor volume is significantly less than control group always, remarkable significant difference (p＜0.05) is arranged, illustrate that heterozygosis replication competent adenovirus SG511-CCL5 has very strong antitumor action to liver cancer animal model, can obviously suppress growth of tumor development (see figure 10).SG511-CCL5 treatment group is compared with other control groups, demonstrate stronger inhibition tumor growth effect, gross tumor volume is significantly less than control group always, remarkable significant difference (p＜0.05) is arranged, illustrate that chimeric replication competent adenovirus SG511-CCL5 has very strong antitumor action to liver cancer animal model, can obviously suppress the growth of tumor development.

Sequence table

＜110〉The 2nd Army Medical College east hospital of liver and gall surgical department

＜120〉a kind of proliferous type recombination oncolytic adenovirus that contains 11 type adenovirus cilia protein genes, its construction process and uses thereof

<130>IDC080XXX

<160>16

<170>PatentIn?version?3.2

<210>1

<211>981

<212>DNA

＜213〉11 cilium sequences

<400>1

atgaagcgcg?caagaccgtc?tgaagatacc?ttcaaccccg?tgtatccata?tgacacggaa 60

accggtcctc?caactgtgcc?ttttcttact?cctccctttg?tatcccccaa?tgggtttcaa 120

gagagtcccc?ctggagttct?tactttaaaa?tgtttaaccc?cactaacaac?cacaggcgga 180

tctctacagc?taaaagtggg?agggggactt?acagtggatg?acaccaacgg?ttttttgaaa 240

gaaaacataa?gtgccaccac?accactcgtt?aagactggtc?actctatagg?tttaccacta 300

ggagccggat?tgggaacgaa?tgaaaataaa?ctttgtatca?aattaggaca?aggacttaca 360

ttcaattcaa?acaacatttg?cattgatgac?aatattaaca?ccttatggac?aggagtcaac 420

cccaccgaag?ccaactgtca?aatcatgaac?tccagtgaat?ctaatgattg?caaattaatt 480

ctaacactag?ttaaaactgg?agcactagtc?actgcatttg?tttatgttat?aggagtatct 540

aacaatttta?atatgctaac?tacacacaga?aatataaatt?ttactgcaga?gctgtttttc 600

gattctactg?gtaatttact?aactagactc?tcatccctca?aaactccact?taatcataaa 660

tcaggacaaa?acatggctac?tggtgccatt?actaatgcta?aaggtttcat?gcccagcacg 720

actgcctatc?ctttcaatga?taattctaga?gaaaaagaaa?actacattta?cggaacttgt 780

tactacacag?ctagtgatcg?cactgctttt?cccattgaca?tatctgtcat?gcttaaccga 840

agagcaataa?atgacgagac?atcatattgt?attcgtataa?cttggtcctg?gaacacagga 900

gatgccccag?aggtgcaaac?ctctgctaca?accctagtca?cctccccatt?taccttttac 960

tacatcagag?aagacgactg?a 981

<210>2

<211>35655

<212>DNA

＜213〉Ad5/11 recombinant vectors sequence

<220>

<221>misc_feature

<222>(721)..(721)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(849)..(849)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(874)..(874)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(1118)..(1118)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(1197)..(1197)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(2254)..(2254)

＜223〉n is a, c, g, or t

<400>2

ttattttgga?ttgaagccaa?tatgataatg?agggggtgga?gtttgtgacg?tggcgcgggg 60

cgtgggaacg?gggcgggtga?cgtagtagtg?tggcggaagt?gtgatgttgc?aagtgtggcg 120

gaacacatgt?aagcgacgga?tgtggcaaaa?gtgacgtttt?tggtgtgcgc?cggatccaca 180

ggacgggtgt?ggtcgccatg?atcgcgtagt?cgatagtggc?tccaagtagc?gaagcgagca 240

ggactgggcg?gcggccaaag?cggtcggaca?gtgctccgag?aacgggtgcg?catagaaatt 300

gcatcaacgc?atatagcgct?agcagcacgc?catagtgact?ggcgatgctg?tcggaatgga 360

cgatatcccg?caagaggccc?ggcagtaccg?gcataaccaa?gcctatgcct?acagcatcca 420

gggtgacggt?gccgaggatg?acgatgagcg?cattgttaga?tttcatacac?ggtgcctgac 480

tgcgttagca?atttaactgt?gataaactac?cgcattaaag?cttatcgttc?gaaatttggg 540

ggatcttcga?tgctagacga?tccagacatg?ataagataca?ttgatgagtt?tggacaaacc 600

acaactagaa?tgcagtgaaa?aaaatgcttt?atttgtgaaa?tttgtgatgc?tattgcttta 660

tttgtaacca?ttataagctg?caataaacaa?gttgctcgaa?gtcgacgatc?cgaacaaacg 720

ncccaacacc?cgtgcgtttt?attctgtctt?tttattgccg?atcccctcag?aagaactcgt 780

caagaaggcg?atagaaggcg?atgcgctgcg?aatcgggagc?ggcgataccg?taaagcacga 840

ggaagcggnc?agcccattcg?ccgccatgtt?tttnagcaat?atcacgggta?gccaacgcta 900

tgtcctgata?gcggtccgcc?acacccagcc?tcgacaattc?caaccttacc?caagagttcg 960

ccaaactcag?acatcacttt?agcaaaaccg?cgccgtgctt?cttcctcggt?ggcattcatc 1020

acgaaatgtt?cagcactacg?catacttttg?gacaggaaac?gcaacggata?ttgagtcaat 1080

atcaggcatt?ctatcgctca?gctttacagt?gacaatgncg?gctggcgact?gaatattagt 1140

gcttacagac?agcactacat?attttccgtc?gatgttgaaa?tcctttctca?tatgtcncca 1200

taaatatcaa?ataattatag?caatcattta?cgcgttaatg?gctaatcgcc?atcttccagc 1260

aggcgcacca?ttgcccctgt?ttcactatcc?aggttacgga?tatagttcat?gacaatattt 1320

acattggtcc?agccaccagc?ttgcatgatc?tccggtattg?aaactccagc?gcgggccata 1380

tctcgcgcgg?ctccgacacg?ggcactgtgt?ccagaccagg?ccaggtatct?ctgaccagag 1440

tcatccttag?cgccgtaaat?caatcgatga?gttgcttcaa?aaatcccttc?cagggcgcga 1500

gttgatagct?ggctggtggc?agatggcgcg?gcaacaccat?tttttctgac?ccggcaaaac 1560

aggtagttat?tcggatcatc?agctacacca?gagacggaaa?tccatcgctc?gaccagttta 1620

gttaccccca?ggctaagtgc?cttctctaca?cctgcggtgc?taaccagcgt?tttcgttctg 1680

ccaatatgga?ttaacattct?cccaccgtca?gtacgtgaga?tatctttaac?cctgatcctg 1740

gcaatttcgg?ctatacgtaa?cagggtgtta?taagcaatcc?ccagaaatgc?cagattacgt 1800

atatcctggc?agcgatcgct?attttccatg?agtgaacgaa?cctggtcgaa?atcagtgcgt 1860

tcgaacgcta?gagcctgttt?tgcacgttca?ccggcatcaa?cgttttcttt?tcggatccgc 1920

cgcataacca?gtgaaacagc?attgctgtca?cttggtcgtg?gcagcccgga?ccgacgatga 1980

agcatgttta?gctggcccaa?atgttgctgg?atagttttta?ctgccagacc?gcgcgcctga 2040

agatatagaa?gataatcgcg?aacatcttca?ggttctgcgg?gaaaccattt?ccggttattc 2100

aacttgcacc?atgccgccca?cgaccggcaa?acggacagaa?gcattttcca?ggtatgctca 2160

gaaaacgcct?ggcgatccct?gaacatgtcc?atcaggttct?tgcgaacctc?atcactcgtt 2220

gcatcgaccg?gtaatgcagg?caaattttgg?tgtncggtca?gtaaattgga?caccttcctc 2280

ttcttcttgg?gcatggtcga?gtctagactc?gagggatcct?cgactcgaag?actgatcccc 2340

aggctggatc?ggtcccggtg?tcttctatgg?aggtcaaaac?agcgtggatg?gcgtctccag 2400

gcgatctgac?ggttcactaa?acgagctctg?cttatataga?cctcccaccg?tacacgccta 2460

ccgcccattt?gcgtcaatgg?ggcggagttg?ttacgacatt?ttggaaagtc?ccgttgattt 2520

tggtgccaaa?acaaactccc?attgacgtca?atggggtgga?gacttggaaa?tccccgtgag 2580

tcaaaccgct?atccacgccc?attgatgtac?tgccaaaacc?gcatcaccat?ggtaatagcg 2640

atgactaata?cgtagatgta?ctgccaagta?ggaaagtccc?ataaggtcat?gtactgggca 2700

taatgccagg?gaattagatc?cactagacga?tgataagctg?tcaaacatga?gaattcttga 2760

agacgaaagg?gcctcgtgat?acgcctattt?ttataggtta?atgtcatgat?aataatggtt 2820

tcttagacgt?caggtggcac?ttttcgggga?aatgtgcgcg?gaacccctat?ttgtttattt 2880

ttctaaatac?attcaaatat?gtatccgctc?atgagacaat?aaccctgata?aatgcttcaa 2940

taatattgaa?aaaggaagag?tatgagtatt?caacatttcc?gtgtcgccct?tattcccttt 3000

tttgcggcat?tttgccttcc?tgtttttgct?cacccagaaa?cgctggtgaa?agtaaaagat 3060

gctgaagatc?agttgggtgc?acgagtgggt?tacatcgaac?tggatctcaa?cagcggtaag 3120

atccttgaga?gttttcgccc?cgaagaacgt?tttccaatga?tgagcacttt?taaagttctg 3180

ctatgtggcg?cggtattatc?ccgtgttgac?gccgggcaag?agcaactcgg?tcgccgcata 3240

cactattctc?agaatgactt?ggttgagtac?tcaccagtca?cagaaaagca?tcttacggat 3300

ggcatgacag?taagagaatt?atgcagtgct?gccataacca?tgagtgataa?cactgcggcc 3360

aacttacttc?tgacaacgat?cggaggaccg?aaggagctaa?ccgctttttt?gcacaacatg 3420

ggggatcatg?taactcgcct?tgatcgttgg?gaaccggagc?tgaatgaagc?cataccaaac 3480

gacgagcgtg?acaccacgat?gcctgcagca?atggcaacaa?cgttgcgcaa?actattaact 3540

ggcgaactac?ttactctagc?ttcccggcaa?caattaatag?actggatgga?ggcggataaa 3600

gttgcaggac?cacttctgcg?ctcggccctt?ccggctggct?ggtttattgc?tgataaatct 3660

ggagccggtg?agcgtgggtc?tcgcggtatc?attgcagcac?tggggccaga?tggtaagccc 3720

tcccgtatcg?tagttatcta?cacgacgggg?agtcaggcaa?ctatggatga?acgaaataga 3780

cagatcgctg?agataggtgc?ctcactgatt?aagcattggt?aactgtcaga?ccaagtttac 3840

tcatatatac?tttagattga?tttaaaactt?catttttaat?ttaaaaggat?ctaggtgaag 3900

atcctttttg?ataatctcat?gaccaaaatc?ccttaacgtg?agttttcgtt?ccactgagcg 3960

tcagaccccg?tagaaaagat?caaaggatct?tcttgagatc?ctttttttct?gcgcgtaatc 4020

tgctgcttgc?aaacaaaaaa?accaccgcta?ccagcggtgg?tttgtttgcc?ggatcaagag 4080

ctaccaactc?tttttccgaa?ggtaactggc?ttcagcagag?cgcagatacc?aaatactgtc 4140

cttctagtgt?agccgtagtt?aggccaccac?ttcaagaact?ctgtagcacc?gcctacatac 4200

ctcgctctgc?taatcctgtt?accagtggct?gctgccagtg?gcgataagtc?gtgtcttacc 4260

gggttggact?caagacgata?gttaccggat?aaggcgcagc?ggtcgggctg?aacggggggt 4320

tcgtgcacac?agcccagctt?ggagcgaacg?acctacaccg?aactgagata?cctacagcgt 4380

gagcattgag?aaagcgccac?gcttcccgaa?gggagaaagg?cggacaggta?tccggtaagc 4440

ggcagggtcg?gaacaggaga?gcgcacgagg?gagcttccag?ggggaaacgc?ctggtatctt 4500

tatagtcctg?tcgggtttcg?ccacctctga?cttgagcgtc?gatttttgtg?atgctcgtca 4560

ggggggcgga?gcctatggaa?aaacgccagc?aacgcggcct?ttttacggtt?cctggccttt 4620

tgctggcctt?ttgctcacat?gttctttcct?gcgttatccc?ctgattctgt?ggataaccgt 4680

attaccgcct?ttgagtgagc?tgataccgct?cgccgcagcc?gaacgaccga?gcgcagcgag 4740

tcagtgagcg?aggaagcgga?agagcgcctg?atgcggtatt?ttctccttac?gcatctgtgc 4800

ggtatttcac?accgcatatg?gtgcactctc?agtacaatct?gctctgatgc?cgcatagtta 4860

agccagtaat?cgaattcaag?cttgtcgact?cgaagatcca?ataacttcgt?atagcataca 4920

ttatacgaag?ttataagtac?tgaattcgga?tctgggcgtg?gttaagggtg?ggaaagaata 4980

tataaggtgg?gggtcttatg?tagttttgta?tctgttttgc?agcagccgcc?gccgccatga 5040

gcaccaactc?gtttgatgga?agcattgtga?gctcatattt?gacaacgcgc?atgcccccat 5100

gggccggggt?gcgtcagaat?gtgatgggct?ccagcattga?tggtcgcccc?gtcctgcccg 5160

caaactctac?taccttgacc?tacgagaccg?tgtctggaac?gccgttggag?actgcagcct 5220

ccgccgccgc?ttcagccgct?gcagccaccg?cccgcgggat?tgtgactgac?tttgctttcc 5280

tgagcccgct?tgcaagcagt?gcagcttccc?gttcatccgc?ccgcgatgac?aagttgacgg 5340

ctcttttggc?acaattggat?tctttgaccc?gggaacttaa?tgtcgtttct?cagcagctgt 5400

tggatctgcg?ccagcaggtt?tctgccctga?aggcttcctc?ccctcccaat?gcggtttaaa 5460

acataaataa?aaaaccagac?tctgtttgga?tttggatcaa?gcaagtgtct?tgctgtcttt 5520

atttaggggt?tttgcgcgcg?cggtaggccc?gggaccagcg?gtctcggtcg?ttgagggtcc 5580

tgtgtatttt?ttccaggacg?tggtaaaggt?gactctggat?gttcagatac?atgggcataa 5640

gcccgtctct?ggggtggagg?tagcaccact?gcagagcttc?atgctgcggg?gtggtgttgt 5700

agatgatcca?gtcgtagcag?gagcgctggg?cgtggtgcct?aaaaatgtct?ttcagtagca 5760

agctgattgc?caggggcagg?cccttggtgt?aagtgtttac?aaagcggtta?agctgggatg 5820

ggtgcatacg?tggggatatg?agatgcatct?tggactgtat?ttttaggttg?gctatgttcc 5880

cagccatatc?cctccgggga?ttcatgttgt?gcagaaccac?cagcacagtg?tatccggtgc 5940

acttgggaaa?tttgtcatgt?agcttagaag?gaaatgcgtg?gaagaacttg?gagacgccct 6000

tgtgacctcc?aagattttcc?atgcattcgt?ccataatgat?ggcaatgggc?ccacgggcgg 6060

cggcctgggc?gaagatattt?ctgggatcac?taacgtcata?gttgtgttcc?aggatgagat 6120

cgtcataggc?catttttaca?aagcgcgggc?ggagggtgcc?agactgcggt?ataatggttc 6180

catccggccc?aggggcgtag?ttaccctcac?agatttgcat?ttcccacgct?ttgagttcag 6240

atggggggat?catgtctacc?tgcggggcga?tgaagaaaac?ggtttccggg?gtaggggaga 6300

tcagctggga?agaaagcagg?ttcctgagca?gctgcgactt?accgcagccg?gtgggcccgt 6360

aaatcacacc?tattaccggg?tgcaactggt?agttaagaga?gctgcagctg?ccgtcatccc 6420

tgagcagggg?ggccacttcg?ttaagcatgt?ccctgactcg?catgttttcc?ctgaccaaat 6480

ccgccagaag?gcgctcgccg?cccagcgata?gcagttcttg?caaggaagca?aagtttttca 6540

acggtttgag?accgtccgcc?gtaggcatgc?ttttgagcgt?ttgaccaagc?agttccaggc 6600

ggtcccacag?ctcggtcacc?tgctctacgg?catctcgatc?cagcatatct?cctcgtttcg 6660

cgggttgggg?cggctttcgc?tgtacggcag?tagtcggtgc?tcgtccagac?gggccagggt 6720

catgtctttc?cacgggcgca?gggtcctcgt?cagcgtagtc?tgggtcacgg?tgaaggggtg 6780

cgctccgggc?tgcgcgctgg?ccagggtgcg?cttgaggctg?gtcctgctgg?tgctgaagcg 6840

ctgccggtct?tcgccctgcg?cgtcggccag?gtagcatttg?accatggtgt?catagtccag 6900

cccctccgcg?gcgtggccct?tggcgcgcag?cttgcccttg?gaggaggcgc?cgcacgaggg 6960

gcagtgcaga?cttttgaggg?cgtagagctt?gggcgcgaga?aataccgatt?ccggggagta 7020

ggcatccgcg?ccgcaggccc?cgcagacggt?ctcgcattcc?acgagccagg?tgagctctgg 7080

ccgttcgggg?tcaaaaacca?ggtttccccc?atgctttttg?atgcgtttct?tacctctggt 7140

ttccatgagc?cggtgtccac?gctcggtgac?gaaaaggctg?tccgtgtccc?cgtatacaga 7200

cttgagaggc?ctgtcctcga?gcggtgttcc?gcggtcctcc?tcgtatagaa?actcggacca 7260

ctctgagaca?aaggctcgcg?tccaggccag?cacgaaggag?gctaagtggg?aggggtagcg 7320

gtcgttgtcc?actagggggt?ccactcgctc?cagggtgtga?agacacatgt?cgccctcttc 7380

ggcatcaagg?aaggtgattg?gtttgtaggt?gtaggccacg?tgaccgggtg?ttcctgaagg 7440

ggggctataa?aagggggtgg?gggcgcgttc?gtcctcactc?tcttccgcat?cgctgtctgc 7500

gagggccagc?tgttggggtg?agtactccct?ctgaaaagcg?ggcatgactt?ctgcgctaag 7560

attgtcagtt?tccaaaaacg?aggaggattt?gatattcacc?tggcccgcgg?tgatgccttt 7620

gagggtggcc?gcatccatct?ggtcagaaaa?gacaatcttt?ttgttgtcaa?gcttggtggc 7680

aaacgacccg?tagagggcgt?tggacagcaa?cttggcgatg?gagcgcaggg?tttggttttt 7740

gtcgcgatcg?gcgcgctcct?tggccgcgat?gtttagctgc?acgtattcgc?gcgcaacgca 7800

ccgccattcg?ggaaagacgg?tggtgcgctc?gtcgggcacc?aggtgcacgc?gccaaccgcg 7860

gttgtgcagg?gtgacaaggt?caacgctggt?ggctacctct?ccgcgtaggc?gctcgttggt 7920

ccagcagagg?cggccgccct?tgcgcgagca?gaatggcggt?agggggtcta?gctgcgtctc 7980

gtccgggggg?tctgcgtcca?cggtaaagac?cccgggcagc?aggcgcgcgt?cgaagtagtc 8040

tatcttgcat?ccttgcaagt?ctagcgcctg?ctgccatgcg?cgggcggcaa?gcgcgcgctc 8100

gtatgggttg?agtgggggac?cccatggcat?ggggtgggtg?agcgcggagg?cgtacatgcc 8160

gcaaatgtcg?taaacgtaga?ggggctctct?gagtattcca?agatatgtag?ggtagcatct 8220

tccaccgcgg?atgctggcgc?gcacgtaatc?gtatagttcg?tgcgagggag?cgaggaggtc 8280

gggaccgagg?ttgctacggg?cgggctgctc?tgctcggaag?actatctgcc?tgaagatggc 8340

atgtgagttg?gatgatatgg?ttggacgctg?gaagacgttg?aagctggcgt?ctgtgagacc 8400

taccgcgtca?cgcacgaagg?aggcgtagga?gtcgcgcagc?ttgttgacca?gctcggcggt 8460

gacctgcacg?tctagggcgc?agtagtccag?ggtttccttg?atgatgtcat?acttatcctg 8520

tccctttttt?ttccacagct?cgcggttgag?gacaaactct?tcgcggtctt?tccagtactc 8580

ttggatcgga?aacccgtcgg?cctccgaacg?gtaagagcct?agcatgtaga?actggttgac 8640

ggcctggtag?gcgcagcatc?ccttttctac?gggtagcgcg?tatgcctgcg?cggccttccg 8700

gagcgaggtg?tgggtgagcg?caaaggtgtc?cctgaccatg?actttgaggt?actggtattt 8760

gaagtcagtg?tcgtcgcatc?cgccctgctc?ccagagcaaa?aagtccgtgc?gctttttgga 8820

acgcggattt?ggcagggcga?aggtgacatc?gttgaagagt?atctttcccg?cgcgaggcat 8880

aaagttgcgt?gtgatgcgga?agggtcccgg?cacctcggaa?cggttgttaa?ttacctgggc 8940

ggcgagcacg?atctcgtcaa?agccgttgat?gttgtggccc?acaatgtaaa?gttccaagaa 9000

gcgcgggatg?cccttgatgg?aaggcaattt?tttaagttcc?tcgtaggtga?gctcttcagg 9060

ggagctgagc?ccgtgctctg?aaagggccca?gtctgcaaga?tgagggttgg?aagcgacgaa 9120

tgagctccac?aggtcacggg?ccattagcat?ttgcaggtgg?tcgcgaaagg?tcctaaactg 9180

gcgacctatg?gccatttttt?ctggggtgat?gcagtagaag?gtaagcgggt?cttgttccca 9240

gcggtcccat?ccaaggttcg?cggctaggtc?tcgcgcggca?gtcactagag?gctcatctcc 9300

gccgaacttc?atgaccagca?tgaagggcac?gagctgcttc?ccaaaggccc?ccatccaagt 9360

ataggtctct?acatcgtagg?tgacaaagag?acgctcggtg?cgaggatgcg?agccgatcgg 9420

gaagaactgg?atctcccgcc?accaattgga?ggagtggcta?ttgatgtggt?gaaagtagaa 9480

gtccctgcga?cgggccgaac?actcgtgctg?gcttttgtaa?aaacgtgcgc?agtactggca 9540

gcggtgcacg?ggctgtacat?cctgcacgag?gttgacctga?cgaccgcgca?caaggaagca 9600

gagtgggaat?ttgagcccct?cgcctggcgg?gtttggctgg?tggtcttcta?cttcggctgc 9660

ttgtccttga?ccgtctggct?gctcgagggg?agttacggtg?gatcggacca?ccacgccgcg 9720

cgagcccaaa?gtccagatgt?ccgcgcgcgg?cggtcggagc?ttgatgacaa?catcgcgcag 9780

atgggagctg?tccatggtct?ggagctcccg?cggcgtcagg?tcaggcggga?gctcctgcag 9840

gtttacctcg?catagacggg?tcagggcgcg?ggctagatcc?aggtgatacc?taatttccag 9900

gggctggttg?gtggcggcgt?cgatggcttg?caagaggccg?catccccgcg?gcgcgactac 9960

ggtaccgcgc?ggcgggcggt?gggccgcggg?ggtgtccttg?gatgatgcat?ctaaaagcgg 10020

tgacgcgggc?gagcccccgg?aggtaggggg?ggctccggac?ccgccgggag?agggggcagg 10080

ggcacgtcgg?cgccgcgcgc?gggcaggagc?tggtgctgcg?cgcgtaggtt?gctggcgaac 10140

gcgacgacgc?ggcggttgat?ctcctgaatc?tggcgcctct?gcgtgaagac?gacgggcccg 10200

gtgagcttga?gcctgaaaga?gagttcgaca?gaatcaattt?cggtgtcgtt?gacggcggcc 10260

tggcgcaaaa?tctcctgcac?gtctcctgag?ttgtcttgat?aggcgatctc?ggccatgaac 10320

tgctcgatct?cttcctcctg?gagatctccg?cgtccggctc?gctccacggt?ggcggcgagg 10380

tcgttggaaa?tgcgggccat?gagctgcgag?aaggcgttga?ggcctccctc?gttccagacg 10440

cggctgtaga?ccacgccccc?ttcggcatcg?cgggcgcgca?tgaccacctg?cgcgagattg 10500

agctccacgt?gccgggcgaa?gacggcgtag?tttcgcaggc?gctgaaagag?gtagttgagg 10560

gtggtggcgg?tgtgttctgc?cacgaagaag?tacataaccc?agcgtcgcaa?cgtggattcg 10620

ttgatatccc?ccaaggcctc?aaggcgctcc?atggcctcgt?agaagtccac?ggcgaagttg 10680

aaaaactggg?agttgcgcgc?cgacacggtt?aactcctcct?ccagaagacg?gatgagctcg 10740

gcgacagtgt?cgcgcacctc?gcgctcaaag?gctacagggg?cctcttcttc?ttcttcaatc 10800

tcctcttcca?taagggcctc?cccttcttct?tcttctggcg?gcggtggggg?aggggggaca 10860

cggcggcgac?gacggcgcac?cgggaggcgg?tcgacaaagc?gctcgatcat?ctccccgcgg 10920

cgacggcgca?tggtctcggt?gacggcgcgg?ccgttctcgc?gggggcgcag?ttggaagacg 10980

ccgcccgtca?tgtcccggtt?atgggttggc?ggggggctgc?catgcggcag?ggatacggcg 11040

ctaacgatgc?atctcaacaa?ttgttgtgta?ggtactccgc?cgccgaggga?cctgagcgag 11100

tccgcatcga?ccggatcgga?aaacctctcg?agaaaggcgt?ctaaccagtc?acagtcgcaa 11160

ggtaggctga?gcaccgtggc?gggcggcagc?gggcggcggt?cggggttgtt?tctggcggag 11220

gtgctgctga?tgatgtaatt?aaagtaggcg?gtcttgagac?ggcggatggt?cgacagaagc 11280

accatgtcct?tgggtccggc?ctgctgaatg?cgcaggcggt?cggccatgcc?ccaggcttcg 11340

ttttgacatc?ggcgcaggtc?tttgtagtag?tcttgcatga?gcctttctac?cggcacttct 11400

tcttctcctt?cctcttgtcc?tgcatctctt?gcatctatcg?ctgcggcggc?ggcggagttt 11460

ggccgtaggt?ggcgccctct?tcctcccatg?cgtgtgaccc?cgaagcccct?catcggctga 11520

agcagggcta?ggtcggcgac?aacgcgctcg?gctaatatgg?cctgctgcac?ctgcgtgagg 11580

gtagactgga?agtcatccat?gtccacaaag?cggtggtatg?cgcccgtgtt?gatggtgtaa 11640

gtgcagttgg?ccataacgga?ccagttaacg?gtctggtgac?ccggctgcga?gagctcggtg 11700

tacctgagac?gcgagtaagc?cctcgagtca?aatacgtagt?cgttgcaagt?ccgcaccagg 11760

tactggtatc?ccaccaaaaa?gtgcggcggc?ggctggcggt?agaggggcca?gcgtagggtg 11820

gccggggctc?cgggggcgag?atcttccaac?ataaggcgat?gatatccgta?gatgtacctg 11880

gacatccagg?tgatgccggc?ggcggtggtg?gaggcgcgcg?gaaagtcgcg?gacgcggttc 11940

cagatgttgc?gcagcggcaa?aaagtgctcc?atggtcggga?cgctctggcc?ggtcaggcgc 12000

gcgcaatcgt?tgacgctcta?ccgtgcaaaa?ggagagcctg?taagcgggca?ctcttccgtg 12060

gtctggtgga?taaattcgca?agggtatcat?ggcggacgac?cggggttcga?gccccgtatc 12120

cggccgtccg?ccgtgatcca?tgcggttacc?gcccgcgtgt?cgaacccagg?tgtgcgacgt 12180

cagacaacgg?gggagtgctc?cttttggctt?ccttccaggc?gcggcggctg?ctgcgctagc 12240

ttttttggcc?actggccgcg?cgcagcgtaa?gcggttaggc?tggaaagcga?aagcattaag 12300

tggctcgctc?cctgtagccg?gagggttatt?ttccaagggt?tgagtcgcgg?gacccccggt 12360

tcgagtctcg?gaccggccgg?actgcggcga?acgggggttt?gcctccccgt?catgcaagac 12420

cccgcttgca?aattcctccg?gaaacaggga?cgagcccctt?ttttgctttt?cccagatgca 12480

tccggtgctg?cggcagatgc?gcccccctcc?tcagcagcgg?caagagcaag?agcagcggca 12540

gacatgcagg?gcaccctccc?ctcctcctac?cgcgtcagga?ggggcgacat?ccgcggttga 12600

cgcggcagca?gatggtgatt?acgaaccccc?gcggcgccgg?gcccggcact?acctggactt 12660

ggaggagggc?gagggcctgg?cgcggctagg?agcgccctct?cctgagcggt?acccaagggt 12720

gcagctgaag?cgtgatacgc?gtgaggcgta?cgtgccgcgg?cagaacctgt?ttcgcgaccg 12780

cgagggagag?gagcccgagg?agatgcggga?tcgaaagttc?cacgcagggc?gcgagctgcg 12840

gcatggcctg?aatcgcgagc?ggttgctgcg?cgaggaggac?tttgagcccg?acgcgcgaac 12900

cgggattagt?cccgcgcgcg?cacacgtggc?ggccgccgac?ctggtaaccg?catacgagca 12960

gacggtgaac?caggagatta?actttcaaaa?aagctttaac?aaccacgtgc?gtacgcttgt 13020

ggcgcgcgag?gaggtggcta?taggactgat?gcatctgtgg?gactttgtaa?gcgcgctgga 13080

gcaaaaccca?aatagcaagc?cgctcatggc?gcagctgttc?cttatagtgc?agcacagcag 13140

ggacaacgag?gcattcaggg?atgcgctgct?aaacatagta?gagcccgagg?gccgctggct 13200

gctcgatttg?ataaacatcc?tgcagagcat?agtggtgcag?gagcgcagct?tgagcctggc 13260

tgacaaggtg?gccgccatca?actattccat?gcttagcctg?ggcaagtttt?acgcccgcaa 13320

gatataccat?accccttacg?ttcccataga?caaggaggta?aagatcgagg?ggttctacat 13380

gcgcatggcg?ctgaaggtgc?ttaccttgag?cgacgacctg?ggcgtttatc?gcaacgagcg 13440

catccacaag?gccgtgagcg?tgagccggcg?gcgcgagctc?agcgaccgcg?agctgatgca 13500

cagcctgcaa?agggccctgg?ctggcacggg?cagcggcgat?agagaggccg?agtcctactt 13560

tgacgcgggc?gctgacctgc?gctgggcccc?aagccgacgc?gccctggagg?cagctggggc 13620

cggacctggg?ctggcggtgg?cacccgcgcg?cgctggcaac?gtcggcggcg?tggaggaata 13680

tgacgaggac?gatgagtacg?agccagagga?cggcgagtac?taagcggtga?tgtttctgat 13740

cagatgatgc?aagacgcaac?ggacccggcg?gtgcgggcgg?cgctgcagag?ccagccgtcc 13800

ggccttaact?ccacggacga?ctggcgccag?gtcatggacc?gcatcatgtc?gctgactgcg 13860

cgcaatcctg?acgcgttccg?gcagcagccg?caggccaacc?ggctctccgc?aattctggaa 13920

gcggtggtcc?cggcgcgcgc?aaaccccacg?cacgagaagg?tgctggcgat?cgtaaacgcg 13980

ctggccgaaa?acagggccat?ccggcccgac?gaggccggcc?tggtctacga?cgcgctgctt 14040

cagcgcgtgg?ctcgttacaa?cagcggcaac?gtgcagacca?acctggaccg?gctggtgggg 14100

gatgtgcgcg?aggccgtggc?gcagcgtgag?cgcgcgcagc?agcagggcaa?cctgggctcc 14160

atggttgcac?taaacgcctt?cctgagtaca?cagcccgcca?acgtgccgcg?gggacaggag 14220

gactacacca?actttgtgag?cgcactgcgg?ctaatggtga?ctgagacacc?gcaaagtgag 14280

gtgtaccagt?ctgggccaga?ctattttttc?cagaccagta?gacaaggcct?gcagaccgta 14340

aacctgagcc?aggctttcaa?aaacttgcag?gggctgtggg?gggtgcgggc?tcccacaggc 14400

gaccgcgcga?ccgtgtctag?cttgctgacg?cccaactcgc?gcctgttgct?gctgctaata 14460

gcgcccttca?cggacagtgg?cagcgtgtcc?cgggacacat?acctaggtca?cttgctgaca 14520

ctgtaccgcg?aggccatagg?tcaggcgcat?gtggacgagc?atactttcca?ggagattaca 14580

agtgtcagcc?gcgcgctggg?gcaggaggac?acgggcagcc?tggaggcaac?cctaaactac 14640

ctgctgacca?accggcggca?gaagatcccc?tcgttgcaca?gtttaaacag?cgaggaggag 14700

cgcattttgc?gctacgtgca?gcagagcgtg?agccttaacc?tgatgcgcga?cggggtaacg 14760

cccagcgtgg?cgctggacat?gaccgcgcgc?aacatggaac?cgggcatgta?tgcctcaaac 14820

cggccgttta?tcaaccgcct?aatggactac?ttgcatcgcg?cggccgccgt?gaaccccgag 14880

tatttcacca?atgccatctt?gaacccgcac?tggctaccgc?cccctggttt?ctacaccggg 14940

ggattcgagg?tgcccgaggg?taacgatgga?ttcctctggg?acgacataga?cgacagcgtg 15000

ttttccccgc?aaccgcagac?cctgctagag?ttgcaacagc?gcgagcaggc?agaggcggcg 15060

ctgcgaaagg?aaagcttccg?caggccaagc?agcttgtccg?atctaggcgc?tgcggccccg 15120

cggtcagatg?ctagtagccc?atttccaagc?ttgatagggt?ctcttaccag?cactcgcacc 15180

acccgcccgc?gcctgctggg?cgaggaggag?tacctaaaca?actcgctgct?gcagccgcag 15240

cgcgaaaaaa?acctgcctcc?ggcatttccc?aacaacggga?tagagagcct?agtggacaag 15300

atgagtagat?ggaagacgta?cgcgcaggag?cacagggacg?tgccaggccc?gcgcccgccc 15360

acccgtcgtc?aaaggcacga?ccgtcagcgg?ggtctggtgt?gggaggacga?tgactcggca 15420

gacgacagca?gcgtcctgga?tttgggaggg?agtggcaacc?cgtttgcgca?ccttcgcccc 15480

aggctgggga?gaatgtttta?aaaaaaaaaa?agcatgatgc?aaaataaaaa?actcaccaag 15540

gccatggcac?cgagcgttgg?ttttcttgta?ttccccttag?tatgcggcgc?gcggcgatgt 15600

atgaggaagg?tcctcctccc?tcctacgaga?gtgtggtgag?cgcggcgcca?gtggcggcgg 15660

cgctgggttc?tcccttcgat?gctcccctgg?acccgccgtt?tgtgcctccg?cggtacctgc 15720

ggcctaccgg?ggggagaaac?agcatccgtt?actctgagtt?ggcaccccta?ttcgacacca 15780

cccgtgtgta?cctggtggac?aacaagtcaa?cggatgtggc?atccctgaac?taccagaacg 15840

accacagcaa?ctttctgacc?acggtcattc?aaaacaatga?ctacagcccg?ggggaggcaa 15900

gcacacagac?catcaatctt?gacgaccggt?cgcactgggg?cggcgacctg?aaaaccatcc 15960

tgcataccaa?catgccaaat?gtgaacgagt?tcatgtttac?caataagttt?aaggcgcggg 16020

tgatggtgtc?gcgcttgcct?actaaggaca?atcaggtgga?gctgaaatac?gagtgggtgg 16080

agttcacgct?gcccgagggc?aactactccg?agaccatgac?catagacctt?atgaacaacg 16140

cgatcgtgga?gcactacttg?aaagtgggca?gacagaacgg?ggttctggaa?agcgacatcg 16200

gggtaaagtt?tgacacccgc?aacttcagac?tggggtttga?ccccgtcact?ggtcttgtca 16260

tgcctggggt?atatacaaac?gaagccttcc?atccagacat?cattttgctg?ccaggatgcg 16320

gggtggactt?cacccacagc?cgcctgagca?acttgttggg?catccgcaag?cggcaaccct 16380

tccaggaggg?ctttaggatc?acctacgatg?atctggaggg?tggtaacatt?cccgcactgt 16440

tggatgtgga?cgcctaccag?gcgagcttga?aagatgacac?cgaacagggc?gggggtggcg 16500

caggcggcag?caacagcagt?ggcagcggcg?cggaagagaa?ctccaacgcg?gcagccgcgg 16560

caatgcagcc?ggtggaggac?atgaacgatc?atgccattcg?cggcgacacc?tttgccacac 16620

gggctgagga?gaagcgcgct?gaggccgaag?cagcggccga?agctgccgcc?cccgctgcgc 16680

aacccgaggt?cgagaagcct?cagaagaaac?cggtgatcaa?acccctgaca?gaggacagca 16740

agaaacgcag?ttacaaccta?ataagcaatg?acagcacctt?cacccagtac?cgcagctggt 16800

accttgcata?caactacggc?gaccctcaga?ccggaatccg?ctcatggacc?ctgctttgca 16860

ctcctgacgt?aacctgcggc?tcggagcagg?tctactggtc?gttgccagac?atgatgcaag 16920

accccgtgac?cttccgctcc?acgcgccaga?tcagcaactt?tccggtggtg?ggcgccgagc 16980

tgttgcccgt?gcactccaag?agcttctaca?acgaccaggc?cgtctactcc?caactcatcc 17040

gccagtttac?ctctctgacc?cacgtgttca?atcgctttcc?cgagaaccag?attttggcgc 17100

gcccgccagc?ccccaccatc?accaccgtca?gtgaaaacgt?tcctgctctc?acagatcacg 17160

ggacgctacc?gctgcgcaac?agcatcggag?gagtccagcg?agtgaccatt?actgacgcca 17220

gacgccgcac?ctgcccctac?gtttacaagg?ccctgggcat?agtctcgccg?cgcgtcctat 17280

cgagccgcac?tttttgagca?agcatgtcca?tccttatatc?gcccagcaat?aacacaggct 17340

ggggcctgcg?cttcccaagc?aagatgtttg?gcggggccaa?gaagcgctcc?gaccaacacc 17400

cagtgcgcgt?gcgcgggcac?taccgcgcgc?cctggggcgc?gcacaaacgc?ggccgcactg 17460

ggcgcaccac?cgtcgatgac?gccatcgacg?cggtggtgga?ggaggcgcgc?aactacacgc 17520

ccacgccgcc?accagtgtcc?acagtggacg?cggccattca?gaccgtggtg?cgcggagccc 17580

ggcgctatgc?taaaatgaag?agacggcgga?ggcgcgtagc?acgtcgccac?cgccgccgac 17640

ccggcactgc?cgcccaacgc?gcggcggcgg?ccctgcttaa?ccgcgcacgt?cgcaccggcc 17700

gacgggcggc?catgcgggcc?gctcgaaggc?tggccgcggg?tattgtcact?gtgcccccca 17760

ggtccaggcg?acgagcggcc?gccgcagcag?ccgcggccat?tagtgctatg?actcagggtc 17820

gcaggggcaa?cgtgtattgg?gtgcgcgact?cggttagcgg?cctgcgcgtg?cccgtgcgca 17880

cccgcccccc?gcgcaactag?attgcaagaa?aaaactactt?agactcgtac?tgttgtatgt 17940

atccagcggc?ggcggcgcgc?aacgaagcta?tgtccaagcg?caaaatcaaa?gaagagatgc 18000

tccaggtcat?cgcgccggag?atctatggcc?ccccgaagaa?ggaagagcag?gattacaagc 18060

cccgaaagct?aaagcgggtc?aaaaagaaaa?agaaagatga?tgatgatgaa?cttgacgacg 18120

aggtggaact?gctgcacgct?accgcgccca?ggcgacgggt?acagtggaaa?ggtcgacgcg 18180

taaaacgtgt?tttgcgaccc?ggcaccaccg?tagtctttac?gcccggtgag?cgctccaccc 18240

gcacctacaa?gcgcgtgtat?gatgaggtgt?acggcgacga?ggacctgctt?gagcaggcca 18300

acgagcgcct?cggggagttt?gcctacggaa?agcggcataa?ggacatgctg?gcgttgccgc 18360

tggacgaggg?caacccaaca?cctagcctaa?agcccgtaac?actgcagcag?gtgctgcccg 18420

cgcttgcacc?gtccgaagaa?aagcgcggcc?taaagcgcga?gtctggtgac?ttggcaccca 18480

ccgtgcagct?gatggtaccc?aagcgccagc?gactggaaga?tgtcttggaa?aaaatgaccg 18540

tggaacctgg?gctggagccc?gaggtccgcg?tgcggccaat?caagcaggtg?gcgccgggac 18600

tgggcgtgca?gaccgtggac?gttcagatac?ccactaccag?tagcaccagt?attgccaccg 18660

ccacagaggg?catggagaca?caaacgtccc?cggttgcctc?agcggtggcg?gatgccgcgg 18720

tgcaggcggt?cgctgcggcc?gcgtccaaga?cctctacgga?ggtgcaaacg?gacccgtgga 18780

tgtttcgcgt?ttcagccccc?cggcgcccgc?gcggttcgag?gaagtacggc?gccgccagcg 18840

cgctactgcc?cgaatatgcc?ctacatcctt?ccattgcgcc?tacccccggc?tatcgtggct 18900

acacctaccg?ccccagaaga?cgagcaacta?cccgacgccg?aaccaccact?ggaacccgcc 18960

gccgccgtcg?ccgtcgccag?cccgtgctgg?ccccgatttc?cgtgcgcagg?gtggctcgcg 19020

aaggaggcag?gaccctggtg?ctgccaacag?cgcgctacca?ccccagcatc?gtttaaaagc 19080

cggtctttgt?ggttcttgca?gatatggccc?tcacctgccg?cctccgtttc?ccggtgccgg 19140

gattccgagg?aagaatgcac?cgtaggaggg?gcatggccgg?ccacggcctg?acgggcggca 19200

tgcgtcgtgc?gcaccaccgg?cggcggcgcg?cgtcgcaccg?tcgcatgcgc?ggcggtatcc 19260

tgcccctcct?tattccactg?atcgccgcgg?cgattggcgc?cgtgcccgga?attgcatccg 19320

tggccttgca?ggcgcagaga?cactgattaa?aaacaagttg?catgtggaaa?aatcaaaata 19380

aaaagtctgg?actctcacgc?tcgcttggtc?ctgtaactat?tttgtagaat?ggaagacatc 19440

aactttgcgt?ctctggcccc?gcgacacggc?tcgcgcccgt?tcatgggaaa?ctggcaagat 19500

atcggcacca?gcaatatgag?cggtggcgcc?ttcagctggg?gctcgctgtg?gagcggcatt 19560

aaaaatttcg?gttccaccgt?taagaactat?ggcagcaagg?cctggaacag?cagcacaggc 19620

cagatgctga?gggataagtt?gaaagagcaa?aatttccaac?aaaaggtggt?agatggcctg 19680

gcctctggca?ttagcggggt?ggtggacctg?gccaaccagg?cagtgcaaaa?taagattaac 19740

agtaagcttg?atccccgccc?tcccgtagag?gagcctccac?cggccgtgga?gacagtgtct 19800

ccagaggggc?gtggcgaaaa?gcgtccgcgc?cccgacaggg?aagaaactct?ggtgacgcaa 19860

atagacgagc?ctccctcgtacgaggaggca?ctaaagcaag?gcctgcccac?cacccgtccc 19920

atcgcgccca?tggctaccgg?agtgctgggc?cagcacacac?ccgtaacgct?ggacctgcct 19980

ccccccgccg?acacccagca?gaaacctgtg?ctgccaggcc?cgaccgccgt?tgttgtaacc 20040

cgtcctagcc?gcgcgtccct?gcgccgcgcc?gccagcggtc?cgcgatcgtt?gcggcccgta 20100

gccagtggca?actggcaaag?cacactgaac?agcatcgtgg?gtctgggggt?gcaatccctg 20160

aagcgccgac?gatgcttctg?aatagctaac?gtgtcgtatg?tgtgtcatgt?atgcgtccat 20220

gtcgccgcca?gaggagctgc?tgagccgccg?cgcgcccgct?ttccaagatg?gctacccctt 20280

cgatgatgcc?gcagtggtct?tacatgcaca?tctcgggcca?ggacgcctcg?gagtacctga 20340

gccccgggct?ggtgcagttt?gcccgcgcca?ccgagacgta?cttcagcctg?aataacaagt 20400

ttagaaaccc?cacggtggcg?cctacgcacg?acgtgaccac?agaccggtcc?cagcgtttga 20460

cgctgcggtt?catccctgtg?gaccgtgagg?atactgcgta?ctcgtacaag?gcgcggttca 20520

ccctagctgt?gggtgataac?cgtgtgctgg?acatggcttc?cacgtacttt?gacatccgcg 20580

gcgtgctgga?caggggccct?acttttaagc?cctactctgg?cactgcctac?aacgccctgg 20640

ctcccaaggg?tgccccaaat?ccttgcgaat?gggatgaagc?tgctactgct?cttgaaataa 20700

acctagaaga?agaggacgat?gacaacgaag?acgaagtaga?cgagcaagct?gagcagcaaa 20760

aaactcacgt?atttgggcag?gcgccttatt?ctggtataaa?tattacaaag?gagggtattc 20820

aaataggtgt?cgaaggtcaa?acacctaaat?atgccgataa?aacatttcaa?cctgaacctc 20880

aaataggaga?atctcagtgg?tacgaaactg?aaattaatca?tgcagctggg?agagtcctta 20940

aaaagactac?cccaatgaaa?ccatgttacg?gttcatatgc?aaaacccaca?aatgaaaatg 21000

gagggcaagg?cattcttgta?aagcaacaaa?atggaaagct?agaaagtcaa?gtggaaatgc 21060

aatttttctc?aactactgag?gcgaccgcag?gcaatggtga?taacttgact?cctaaagtgg 21120

tattgtacag?tgaagatgta?gatatagaaa?ccccagacac?tcatatttct?tacatgccca 21180

ctattaagga?aggtaactca?cgagaactaa?tgggccaaca?atctatgccc?aacaggccta 21240

attacattgc?ttttagggac?aattttattg?gtctaatgta?ttacaacagc?acgggtaata 21300

tgggtgttct?ggcgggccaa?gcatcgcagt?tgaatgctgt?tgtagatttg?caagacagaa 21360

acacagagct?ttcataccag?cttttgcttg?attccattgg?tgatagaacc?aggtactttt 21420

ctatgtggaa?tcaggctgtt?gacagctatg?atccagatgt?tagaattatt?gaaaatcatg 21480

gaactgaaga?tgaacttcca?aattactgct?ttccactggg?aggtgtgatt?aatacagaga 21540

ctcttaccaa?ggtaaaacct?aaaacaggtc?aggaaaatgg?atgggaaaaa?gatgctacag 21600

aattttcaga?taaaaatgaa?ataagagttg?gaaataattt?tgccatggaa?atcaatctaa 21660

atgccaacct?gtggagaaat?ttcctgtact?ccaacatagc?gctgtatttg?cccgacaagc 21720

taaagtacag?tccttccaac?gtaaaaattt?ctgataaccc?aaacacctac?gactacatga 21780

acaagcgagt?ggtggctccc?gggttagtgg?actgctacat?taaccttgga?gcacgctggt 21840

cccttgacta?tatggacaac?gtcaacccat?ttaaccacca?ccgcaatgct?ggcctgcgct 21900

accgctcaat?gttgctgggc?aatggtcgct?atgtgccctt?ccacatccag?gtgcctcaga 21960

agttctttgc?cattaaaaac?ctccttctcc?tgccgggctc?atacacctac?gagtggaact 22020

tcaggaagga?tgttaacatg?gttctgcaga?gctccctagg?aaatgaccta?agggttgacg 22080

gagccagcat?taagtttgat?agcatttgcc?tttacgccac?cttcttcccc?atggcccaca 22140

acaccgcctc?cacgcttgag?gccatgctta?gaaacgacac?caacgaccag?tcctttaacg 22200

actatctctc?cgccgccaac?atgctctacc?ctatacccgc?caacgctacc?aacgtgccca 22260

tatccatccc?ctcccgcaac?tgggcggctt?tccgcggctg?ggccttcacg?cgccttaaga 22320

ctaaggaaac?cccatcactg?ggctcgggct?acgaccctta?ttacacctac?tctggctcta 22380

taccctacct?agatggaacc?ttttacctca?accacacctt?taagaaggtg?gccattacct 22440

ttgactcttc?tgtcagctgg?cctggcaatg?accgcctgct?tacccccaac?gagtttgaaa 22500

ttaagcgctc?agttgacggg?gagggttaca?acgttgccca?gtgtaacatg?accaaagact 22560

ggttcctggt?acaaatgcta?gctaactaca?acattggcta?ccagggcttc?tatatcccag 22620

agagctacaa?ggaccgcatg?tactccttct?ttagaaactt?ccagcccatg?agccgtcagg 22680

tggtggatga?tactaaatac?aaggactacc?aacaggtggg?catcctacac?caacacaaca 22740

actctggatt?tgttggctac?cttgccccca?ccatgcgcga?aggacaggcc?taccctgcta 22800

acttccccta?tccgcttata?ggcaagaccg?cagttgacag?cattacccag?aaaaagtttc 22860

tttgcgatcg?caccctttgg?cgcatcccat?tctccagtaa?ctttatgtcc?atgggcgcac 22920

tcacagacct?gggccaaaac?cttctctacg?ccaactccgc?ccacgcgcta?gacatgactt 22980

ttgaggtgga?tcccatggac?gagcccaccc?ttctttatgt?tttgtttgaa?gtctttgacg 23040

tggtccgtgt?gcaccggccg?caccgcggcg?tcatcgaaac?cgtgtacctg?cgcacgccct 23100

tctcggccgg?caacgccaca?acataaagaa?gcaagcaaca?tcaacaacag?ctgccgccat 23160

gggctccagt?gagcaggaac?tgaaagccat?tgtcaaagat?cttggttgtg?ggccatattt 23220

tttgggcacc?tatgacaagc?gctttccagg?ctttgtttct?ccacacaagc?tcgcctgcgc 23280

catagtcaat?acggccggtc?gcgagactgg?gggcgtacac?tggatggcct?ttgcctggaa 23340

cccgcactca?aaaacatgct?acctctttga?gccctttggc?ttttctgacc?agcgactcaa 23400

gcaggtttac?cagtttgagt?acgagtcact?cctgcgccgt?agcgccattg?cttcttcccc 23460

cgaccgctgt?ataacgctgg?aaaagtccac?ccaaagcgta?caggggccca?actcggccgc 23520

ctgtggacta?ttctgctgca?tgtttctcca?cgcctttgcc?aactggcccc?aaactcccat 23580

ggatcacaac?cccaccatga?accttattac?cggggtaccc?aactccatgc?tcaacagtcc 23640

ccaggtacag?cccaccctgc?gtcgcaacca?ggaacagctc?tacagcttcc?tggagcgcca 23700

ctcgccctac?ttccgcagcc?acagtgcgca?gattaggagc?gccacttctt?tttgtcactt 23760

gaaaaacatg?taaaaataat?gtactagaga?cactttcaat?aaaggcaaat?gcttttattt 23820

gtacactctc?gggtgattat?ttacccccac?ccttgccgtc?tgcgccgttt?aaaaatcaaa 23880

ggggttctgc?cgcgcatcgc?tatgcgccac?tggcagggac?acgttgcgat?actggtgttt 23940

agtgctccac?ttaaactcag?gcacaaccat?ccgcggcagc?tcggtgaagt?tttcactcca 24000

caggctgcgc?accatcacca?acgcgtttag?caggtcgggc?gccgatatct?tgaagtcgca 24060

gttggggcct?ccgccctgcg?cgcgcgagtt?gcgatacaca?gggttgcagc?actggaacac 24120

tatcagcgcc?gggtggtgca?cgctggccag?cacgctcttg?tcggagatca?gatccgcgtc 24180

caggtcctcc?gcgttgctca?gggcgaacgg?agtcaacttt?ggtagctgcc?ttcccaaaaa 24240

gggcgcgtgc?ccaggctttg?agttgcactc?gcaccgtagt?ggcatcaaaa?ggtgaccgtg 24300

cccggtctgg?gcgttaggat?acagcgcctg?cataaaagcc?ttgatctgct?taaaagccac 24360

ctgagccttt?gcgccttcag?agaagaacat?gccgcaagac?ttgccggaaa?actgattggc 24420

cggacaggcc?gcgtcgtgca?cgcagcacct?tgcgtcggtg?ttggagatct?gcaccacatt 24480

tcggccccac?cggttcttca?cgatcttggc?cttgctagac?tgctccttca?gcgcgcgctg 24540

cccgttttcg?ctcgtcacat?ccatttcaat?cacgtgctcc?ttatttatca?taatgcttcc 24600

gtgtagacac?ttaagctcgc?cttcgatctc?agcgcagcgg?tgcagccaca?acgcgcagcc 24660

cgtgggctcg?tgatgcttgt?aggtcacctc?tgcaaacgac?tgcaggtacg?cctgcaggaa 24720

tcgccccatc?atcgtcacaa?aggtcttgtt?gctggtgaag?gtcagctgca?acccgcggtg 24780

ctcctcgttc?agccaggtct?tgcatacggc?cgccagagct?tccacttggt?caggcagtag 24840

tttgaagttc?gcctttagat?cgttatccac?gtggtacttg?tccatcagcg?cgcgcgcagc 24900

ctccatgccc?ttctcccacg?cagacacgat?cggcacactc?agcgggttca?tcaccgtaat 24960

ttcactttcc?gcttcgctgg?gctcttcctc?ttcctcttgc?gtccgcatac?cacgcgccac 25020

tgggtcgtct?tcattcagcc?gccgcactgt?gcgcttacct?cctttgccat?gcttgattag 25080

caccggtggg?ttgctgaaac?ccaccatttg?tagcgccaca?tcttctcttt?cttcctcgct 25140

gtccacgatt?acctctggtg?atggcgggcg?ctcgggcttg?ggagaagggc?gcttcttttt 25200

cttcttgggc?gcaatggcca?aatccgccgc?cgaggtcgat?ggccgcgggc?tgggtgtgcg 25260

cggcaccagc?gcgtcttgtg?atgagtcttc?ctcgtcctcg?gactcgatac?gccgcctcat 25320

ccgctttttt?gggggcgccc?ggggaggcgg?cggcgacggg?gacggggacg?acacgtcctc 25380

catggttggg?ggacgtcgcg?ccgcaccgcg?tccgcgctcg?ggggtggttt?cgcgctgctc 25440

ctcttcccga?ctggccattt?ccttctccta?taggcagaaa?aagatcatgg?agtcagtcga 25500

gaagaaggac?agcctaaccg?ccccctctga?gttcgccacc?accgcctcca?ccgatgccgc 25560

caacgcgcct?accaccttcc?ccgtcgaggc?acccccgctt?gaggaggagg?aagtgattat 25620

cgagcaggac?ccaggttttg?taagcgaaga?cgacgaggac?cgctcagtac?caacagagga 25680

taaaaagcaa?gaccaggaca?acgcagaggc?aaacgaggaa?caagtcgggc?ggggggacga 25740

aaggcatggc?gactacctag?atgtgggaga?cgacgtgctg?ttgaagcatc?tgcagcgcca 25800

gtgcgccatt?atctgcgacg?cgttgcaaga?gcgcagcgat?gtgcccctcg?ccatagcgga 25860

tgtcagcctt?gcctacgaac?gccacctatt?ctcaccgcgc?gtacccccca?aacgccaaga 25920

aaacggcaca?tgcgagccca?acccgcgcct?caacttctac?cccgtatttg?ccgtgccaga 25980

ggtgcttgcc?acctatcaca?tctttttcca?aaactgcaag?atacccctat?cctgccgtgc 26040

caaccgcagc?cgagcggaca?agcagctggc?cttgcggcag?ggcgctgtca?tacctgatat 26100

cgcctcgctc?aacgaagtgc?caaaaatctt?tgagggtctt?ggacgcgacg?agaagcgcgc 26160

ggcaaacgct?ctgcaacagg?aaaacagcga?aaatgaaagt?cactctggag?tgttggtgga 26220

actcgagggt?gacaacgcgc?gcctagccgt?actaaaacgc?agcatcgagg?tcacccactt 26280

tgcctacccg?gcacttaacc?taccccccaa?ggtcatgagc?acagtcatga?gtgagctgat 26340

cgtgcgccgt?gcgcagcccc?tggagaggga?tgcaaatttg?caagaacaaa?cagaggaggg 26400

cctacccgca?gttggcgacg?agcagctagc?gcgctggctt?caaacgcgcg?agcctgccga 26460

cttggaggag?cgacgcaaac?taatgatggc?cgcagtgctc?gttaccgtgg?agcttgagtg 26520

catgcagcgg?ttctttgctg?acccggagat?gcagcgcaag?ctagaggaaa?cattgcacta 26580

cacctttcga?cagggctacg?tacgccaggc?ctgcaagatc?tccaacgtgg?agctctgcaa 26640

cctggtctcc?taccttggaa?ttttgcacga?aaaccgcctt?gggcaaaacg?tgcttcattc 26700

cacgctcaag?ggcgaggcgc?gccgcgacta?cgtccgcgac?tgcgtttact?tatttctatg 26760

ctacacctgg?cagacggcca?tgggcgtttg?gcagcagtgc?ttggaggagt?gcaacctcaa 26820

ggagctgcag?aaactgctaa?agcaaaactt?gaaggaccta?tggacggcct?tcaacgagcg 26880

ctccgtggcc?gcgcacctgg?cggacatcat?tttccccgaa?cgcctgctta?aaaccctgca 26940

acagggtctg?ccagacttca?ccagtcaaag?catgttgcag?aactttagga?actttatcct 27000

agagcgctca?ggaatcttgc?ccgccacctg?ctgtgcactt?cctagcgact?ttgtgcccat 27060

taagtaccgc?gaatgccctc?cgccgctttg?gggccactgc?taccttctgc?agctagccaa 27120

ctaccttgcc?taccactctg?acataatgga?agacgtgagc?ggtgacggtc?tactggagtg 27180

tcactgtcgc?tgcaacctat?gcaccccgca?ccgctccctg?gtttgcaatt?cgcagctgct 27240

taacgaaagt?caaattatcg?gtacctttga?gctgcagggt?ccctcgcctg?acgaaaagtc 27300

cgcggctccg?gggttgaaac?tcactccggg?gctgtggacg?tcggcttacc?ttcgcaaatt 27360

tgtacctgag?gactaccacg?cccacgagat?taggttctac?gaagaccaat?cccgcccgcc 27420

aaatgcggag?cttaccgcct?gcgtcattac?ccagggccac?attcttggcc?aattgcaagc 27480

catcaacaaa?gcccgccaag?agtttctgct?acgaaaggga?cggggggttt?acttggaccc 27540

ccagtccggc?gaggagctca?acccaatccc?cccgccgccg?cagccctatc?agcagcagcc 27600

gcgggccctt?gcttcccagg?atggcaccca?aaaagaagct?gcagctgccg?ccgccaccca 27660

cggacgagga?ggaatactgg?gacagtcagg?cagaggaggt?tttggacgag?gaggaggagg 27720

acatgatgga?agactgggag?agcctagacg?aggaagcttc?cgaggtcgaa?gaggtgtcag 27780

acgaaacacc?gtcaccctcg?gtcgcattcc?cctcgccggc?gccccagaaa?tcggcaaccg 27840

gttccagcat?ggctacaacc?tccgctcctc?aggcgccgcc?ggcactgccc?gttcgccgac 27900

ccaaccgtag?atgggacacc?actggaacca?gggccggtaa?gtccaagcag?ccgccgccgt 27960

tagcccaaga?gcaacaacag?cgccaaggct?accgctcatg?gcgcgggcac?aagaacgcca 28020

tagttgcttg?cttgcaagac?tgtgggggca?acatctcctt?cgcccgccgc?tttcttctct 28080

accatcacgg?cgtggccttc?ccccgtaaca?tcctgcatta?ctaccgtcat?ctctacagcc 28140

catactgcac?cggcggcagc?ggcagcggca?gcaacagcag?cggccacaca?gaagcaaagg 28200

cgaccggata?gcaagactct?gacaaagccc?aagaaatcca?cagcggcggc?agcagcagga 28260

ggaggagcgc?tgcgtctggc?gcccaacgaa?cccgtatcga?cccgcgagct?tagaaacagg 28320

atttttccca?ctctgtatgc?tatatttcaa?cagagcaggg?gccaagaaca?agagctgaaa 28380

ataaaaaaca?ggtctctgcg?atccctcacc?cgcagctgcc?tgtatcacaa?aagcgaagat 28440

cagcttcggc?gcacgctgga?agacgcggag?gctctcttca?gtaaatactg?cgcgctgact 28500

cttaaggact?agtttcgcgc?cctttctcaa?atttaagcgc?gaaaactacg?tcatctccag 28560

cggccacacc?cggcgccagc?acctgtcgtc?agcgccatta?tgagcaagga?aattcccacg 28620

ccctacatgt?ggagttacca?gccacaaatg?ggacttgcgg?ctggagctgc?ccaagactac 28680

tcaacccgaa?taaactacat?gagcgcggga?ccccacatga?tatcccgggt?caacggaatc 28740

cgcgcccacc?gaaaccgaat?tctcttggaa?caggcggcta?ttaccaccac?acctcgtaat 28800

aaccttaatc?cccgtagttg?gcccgctgcc?ctggtgtacc?aggaaagtcc?cgctcccacc 28860

actgtggtac?ttcccagaga?cgcccaggcc?gaagttcaga?tgactaactc?aggggcgcag 28920

cttgcgggcg?gctttcgtca?cagggtgcgg?tcgcccgggc?agggtataac?tcacctgaca 28980

atcagagggc?gaggtattca?gctcaacgac?gagtcggtga?gctcctcgct?tggtctccgt 29040

ccggacggga?catttcagat?cggcggcgcc?ggccgtcctt?cattcacgcc?tcgtcaggca 29100

atcctaactc?tgcagacctc?gtcctctgag?ccgcgctctg?gaggcattgg?aactctgcaa 29160

tttattgagg?agtttgtgcc?atcggtctac?tttaacccct?tctcgggacc?tcccggccac 29220

tatccggatc?aatttattcc?taactttgac?gcggtaaagg?actcggcgga?cggctacgac 29280

tgaatgttaa?gtggagaggc?agagcaactg?cgcctgaaac?acctggtcca?ctgtcgccgc 29340

cacaagtgct?ttgcccgcga?ctccggtgag?ttttgctact?ttgaattgcc?cgaggatcat 29400

atcgagggcc?cggcgcacgg?cgtccggctt?accgcccagg?gagagcttgc?ccgtagcctg 29460

attcgggagt?ttacccagcg?ccccctgcta?gttgagcggg?acaggggacc?ctgtgttctc 29520

actgtgattt?gcaactgtcc?taaccttgga?ttacatcaag?atctttgttg?ccatctctgt 29580

gctgagtata?ataaatacag?aaattaaaat?atactggggc?tcctatcgcc?atcctgtaaa 29640

cgccaccgtc?ttcacccgcc?caagcaaacc?aaggcgaacc?ttacctggta?cttttaacat 29700

ctctccctct?gtgatttaca?acagtttcaa?cccagacgga?gtgagtctac?gagagaacct 29760

ctccgagctc?agctactcca?tcagaaaaaa?caccaccctc?cttacctgcc?gggaacgtac 29820

gagtgcgtca?ccggccgctg?caccacacct?accgcctgac?cgtaaaccag?actttttccg 29880

gacagacctc?aataactctg?tttaccagaa?caggaggtga?gcttagaaaa?cccttagggt 29940

attaggccaa?aggcgcagct?actgtggggt?ttatgaacaa?ttcaagcaac?tctacgggct 30000

attctaattc?aggtttctct?agaatcgggg?ttggggttat?tctctgtctt?gtgattctct 30060

ttattcttat?actaacgctt?ctctgcctaa?ggctcgccgc?ctgctgtgtg?cacatttgca 30120

tttattgtca?gctttttaaa?cgctggggtc?gccacccaag?atgattaggt?acataatcct 30180

aggtttactc?acccttgcgt?cagcccacgg?taccacccaa?aaggtggatt?ttaaggagcc 30240

agcctgtaat?gttacattcg?cagctgaagc?taatgagtgc?accactctta?taaaatgcac 30300

cacagaacat?gaaaagctgc?ttattcgcca?caaaaacaaa?attggcaagt?atgctgttta 30360

tgctatttgg?cagccaggtg?acactacaga?gtataatgtt?acagttttcc?agggtaaaag 30420

tcataaaact?tttatgtata?cttttccatt?ttatgaaatg?tgcgacatta?ccatgtacat 30480

gagcaaacag?tataagttgt?ggcccccaca?aaattgtgtg?gaaaacactg?gcactttctg 30540

ctgcactgct?atgctaatta?cagtgctcgc?tttggtctgt?accctactct?atattaaata 30600

caaaagcaga?cgcagcttta?ttgaggaaaa?gaaaatgcct?taatttacta?agttacaaag 30660

ctaatgtcac?cactaactgc?tttactcgct?gcttgcaaaa?caaattcaaa?aagttagcat 30720

tataattaga?ataggattta?aaccccccgg?tcatttcctg?ctcaatacca?ttcccctgaa 30780

caattgactc?tatgtgggat?atgctccagc?gctacaacct?tgaagtcagg?cttcctggat 30840

gtcagcatct?gactttggcc?agcacctgtc?ccgcggattt?gttccagtcc?aactacagcg 30900

acccacccta?acagagatga?ccaacacaac?caacgcggcc?gccgctaccg?gacttacatc 30960

taccacaaat?acaccccaag?tttctgcctt?tgtcaataac?tgggataact?tgggcatgtg 31020

gtggttctcc?atagcgctta?tgtttgtatg?ccttattatt?atgtggctca?tctgctgcct 31080

aaagcgcaaa?cgcgcccgac?cacccatcta?tagtcccatc?attgtgctac?acccaaacaa 31140

tgatggaatc?catagattgg?acggactgaa?acacatgttc?ttttctctta?cagtatgatt 31200

aaatgagaca?tgattcctcg?agtttttata?ttactgaccc?ttgttgcgct?tttttgtgcg 31260

tgctccacat?tggctaagta?atagttaatt?aagatcttat?tccctttaac?taataaaaaa 31320

aaataataaa?gcatcactta?cttaaaatca?gttagcaaat?ttctgtccag?tttattcagc 31380

agcacctcct?tgccctcctc?ccagctctgg?tattgcagct?tcctcctggc?tgcaaacttt 31440

ctccacaatc?taaatggaat?gtcagtttcc?tcctgttcct?gtccatccgc?acccactatc 31500

ttcatgttgt?tgcagatgaa?gcgcgcaaga?ccgtctgaag?ataccttcaa?ccccgtgtat 31560

ccatatgaca?cggaaaccgg?tcctccaact?gtgccttttc?ttactcctcc?ctttgtatcc 31620

cccaatgggt?ttcaagagag?tccccctgga?gttcttactt?taaaatgttt?aaccccacta 31680

acaaccacag?gcggatctct?acagctaaaa?gtgggagggg?gacttacagt?ggatgacacc 31740

aacggttttt?tgaaagaaaa?cataagtgcc?accacaccac?tcgttaagac?tggtcactct 31800

ataggtttac?cactaggagc?cggattggga?acgaatgaaa?ataaactttg?tatcaaatta 31860

ggacaaggac?ttacattcaa?ttcaaacaac?atttgcattg?atgacaatat?taacacctta 31920

tggacaggag?tcaaccccac?cgaagccaac?tgtcaaatca?tgaactccag?tgaatctaat 31980

gattgcaaat?taattctaac?actagttaaa?actggagcac?tagtcactgc?atttgtttat 32040

gttataggag?tatctaacaa?ttttaatatg?ctaactacac?acagaaatat?aaattttact 32100

gcagagctgt?ttttcgattc?tactggtaat?ttactaacta?gactctcatc?cctcaaaact 32160

ccacttaatc?ataaatcagg?acaaaacatg?gctactggtg?ccattactaa?tgctaaaggt 32220

ttcatgccca?gcacgactgc?ctatcctttc?aatgataatt?ctagagaaaa?agaaaactac 32280

atttacggaa?cttgttacta?cacagctagt?gatcgcactg?cttttcccat?tgacatatct 32340

gtcatgctta?accgaagagc?aataaatgac?gagacatcat?attgtattcg?tataacttgg 32400

tcctggaaca?caggagatgc?cccagaggtg?caaacctctg?ctacaaccct?agtcacctcc 32460

ccatttacct?tttactacat?cagagaagac?gactgagccc?aagaataaag?aatcgtttgt 32520

gttatgtttc?aacgtgttta?tttttcaatt?gcagaaaatt?tcaagtcatt?tttcattcag 32580

tagtatagcc?ccaccaccac?atagcttata?cagatcaccg?taccttaatc?aaactcacag 32640

aaccctagta?ttcaacctgc?cacctccctc?ccaacacaca?gagtacacag?tcctttctcc 32700

ccggctggcc?ttaaaaagca?tcatatcatg?ggtaacagac?atattcttag?gtgttatatt 32760

ccacacggtt?tcctgtcgag?ccaaacgctc?atcagtgata?ttaataaact?ccccgggcag 32820

ctcacttaag?ttcatgtcgc?tgtccagctg?ctgagccaca?ggctgctgtc?caacttgcgg 32880

ttgcttaacg?ggcggcgaag?gagaagtcca?cgcctacatg?ggggtagagt?cataatcgtg 32940

catcaggata?gggcggtggt?gctgcagcag?cgcgcgaata?aactgctgcc?gccgccgctc 33000

cgtcctgcag?gaatacaaca?tggcagtggt?ctcctcagcg?atgattcgca?ccgcccgcag 33060

cataaggcgc?cttgtcctcc?gggcacagca?gcgcaccctg?atctcactta?aatcagcaca 33120

gtaactgcag?cacagcacca?caatattgtt?caaaatccca?cagtgcaagg?cgctgtatcc 33180

aaagctcatg?gcggggacca?cagaacccac?gtggccatca?taccacaagc?gcaggtagat 33240

taagtggcga?cccctcataa?acacgctgga?cataaacatt?acctcttttg?gcatgttgta 33300

attcaccacc?tcccggtacc?atataaacct?ctgattaaac?atggcgccat?ccaccaccat 33360

cctaaaccag?ctggccaaaa?cctgcccgcc?ggctatacac?tgcagggaac?cgggactgga 33420

acaatgacag?tggagagccc?aggactcgta?accatggatc?atcatgctcg?tcatgatatc 33480

aatgttggca?caacacaggc?acacgtgcat?acacttcctc?aggattacaa?gctcctcccg 33540

cgttagaacc?atatcccagg?gaacaaccca?ttcctgaatc?agcgtaaatc?ccacactgca 33600

gggaagacct?cgcacgtaac?tcacgttgtg?cattgtcaaa?gtgttacatt?cgggcagcag 33660

cggatgatcc?tccagtatgg?tagcgcgggt?ttctgtctca?aaaggaggta?gacgatccct 33720

actgtacgga?gtgcgccgag?acaaccgaga?tcgtgttggt?cgtagtgtca?tgccaaatgg 33780

aacgccggac?gtagtcatat?ttcctgaagc?aaaaccaggt?gcgggcgtga?caaacagatc 33840

tgcgtctccg?gtctcgccgc?ttagatcgct?ctgtgtagta?gttgtagtat?atccactctc 33900

tcaaagcatc?caggcgcccc?ctggcttcgg?gttctatgta?aactccttca?tgcgccgctg 33960

ccctgataac?atccaccacc?gcagaataag?ccacacccag?ccaacctaca?cattcgttct 34020

gcgagtcaca?cacgggagga?gcgggaagag?ctggaagaac?catgtttttt?tttttattcc 34080

aaaagattat?ccaaaacctc?aaaatgaaga?tctattaagt?gaacgcgctc?ccctccggtg 34140

gcgtggtcaa?actctacagc?caaagaacag?ataatggcat?ttgtaagatg?ttgcacaatg 34200

gcttccaaaa?ggcaaacggc?cctcacgtcc?aagtggacgt?aaaggctaaa?cccttcaggg 34260

tgaatctcct?ctataaacat?tccagcacct?tcaaccatgc?ccaaataatt?ctcatctcgc 34320

caccttctca?atatatctct?aagcaaatcc?cgaatattaa?gtccggccat?tgtaaaaatc 34380

tgctccagag?cgccctccac?cttcagcctc?aagcagcgaa?tcatgattgc?aaaaattcag 34440

gttcctcaca?gacctgtata?agattcaaaa?gcggaacatt?aacaaaaata?ccgcgatccc 34500

gtaggtccct?tcgcagggcc?agctgaacat?aatcgtgcag?gtctgcacgg?accagcgcgg 34560

ccacttcccc?gccaggaacc?ttgacaaaag?aacccacact?gattatgaca?cgcatactcg 34620

gagctatgct?aaccagcgta?gccccgatgt?aagctttgtt?gcatgggcgg?cgatataaaa 34680

tgcaaggtgc?tgctcaaaaa?atcaggcaaa?gcctcgcgca?aaaaagaaag?cacatcgtag 34740

tcatgctcat?gcagataaag?gcaggtaagc?tccggaacca?ccacagaaaa?agacaccatt 34800

tttctctcaa?acatgtctgc?gggtttctgc?ataaacacaa?aataaaataa?caaaaaaaca 34860

tttaaacatt?agaagcctgt?cttacaacag?gaaaaacaac?ccttataagc?ataagacgga 34920

ctacggccat?gccggcgtga?ccgtaaaaaa?actggtcacc?gtgattaaaa?agcaccaccg 34980

acagctcctc?ggtcatgtcc?ggagtcataa?tgtaagactc?ggtaaacaca?tcaggttgat 35040

tcatcggtca?gtgctaaaaa?gcgaccgaaa?tagcccgggg?gaatacatac?ccgcaggcgt 35100

agagacaaca?ttacagcccc?cataggaggt?ataacaaaat?taataggaga?gaaaaacaca 35160

taaacacctg?aaaaaccctc?ctgcctaggc?aaaatagcac?cctcccgctc?cagaacaaca 35220

tacagcgctt?cacagcggca?gcctaacagt?cagccttacc?agtaaaaaag?aaaacctatt 35280

aaaaaaacac?cactcgacac?ggcaccagct?caatcagtca?cagtgtaaaa?aagggccaag 35340

tgcagagcga?gtatatatag?gactaaaaaa?tgacgtaacg?gttaaagtcc?acaaaaaaca 35400

cccagaaaac?cgcacgcgaa?cctacgccca?gaaacgaaag?ccaaaaaacc?cacaacttcc 35460

tcaaatcgtc?acttccgttt?tcccacgtta?cgtaacttcc?cattttaaga?aaactacaat 35520

tcccaacaca?tacaagttac?tccgccctaa?aacctacgtc?acccgccccg?ttcccacgcc 35580

ccgcgccacg?tcacaaactc?caccccctca?ttatcatatt?ggcttcaatc?caaaataagg 35640

tatattattg?atgat 35655

<210>3

<211>2673

<212>DNA

＜213〉contain the chimeric sequences of 5 type adenovirus tail sequences and 11 type adenovirus shaft, knob sequence

<400>3

gccgttaatt?aagatcttat?tccctttaac?taataaaaaa?aaataataaa?gcatcactta 60

cttaaaatca?gttagcaaat?ttctgtccag?tttattcagc?agcacctcct?tgccctcctc 120

ccagctctgg?tattgcagct?tcctcctggc?tgcaaacttt?ctccacaatc?taaatggaat 180

gtcagtttcc?tcctgttcct?gtccatccgc?acccactatc?ttcatgttgt?tgcagatgaa 240

gcgcgcaaga?ccgtctgaag?ataccttcaa?ccccgtgtat?ccatatgaca?cggaaaccgg 300

tcctccaact?gtgccttttc?ttactcctcc?ctttgtatcc?cccaatgggt?ttcaagagag 360

tccccctgga?gttcttactt?taaaatgttt?aaccccacta?acaaccacag?gcggatctct 420

acagctaaaa?gtgggagggg?gacttacagt?ggatgacacc?aacggttttt?tgaaagaaaa 480

cataagtgcc?accacaccac?tcgttaagac?tggtcactct?ataggtttac?cactaggagc 540

cggattggga?acgaatgaaa?ataaactttg?tatcaaatta?ggacaaggac?ttacattcaa 600

ttcaaacaac?atttgcattg?atgacaatat?taacacctta?tggacaggag?tcaaccccac 660

cgaagccaac?tgtcaaatca?tgaactccag?tgaatctaat?gattgcaaat?taattctaac 720

actagttaaa?actggagcac?tagtcactgc?atttgtttat?gttataggag?tatctaacaa 780

ttttaatatg?ctaactacac?acagaaatat?aaattttact?gcagagctgt?ttttcgattc 840

tactggtaat?ttactaacta?gactctcatc?cctcaaaact?ccacttaatc?ataaatcagg 900

acaaaacatg?gctactggtg?ccattactaa?tgctaaaggt?ttcatgccca?gcacgactgc 960

ctatcctttc?aatgataatt?ctagagaaaa?agaaaactac?atttacggaa?cttgttacta 1020

cacagctagt?gatcgcactg?cttttcccat?tgacatatct?gtcatgctta?accgaagagc 1080

aataaatgac?gagacatcat?attgtattcg?tataacttgg?tcctggaaca?caggagatgc 1140

cccagaggtg?caaacctctg?ctacaaccct?agtcacctcc?ccatttacct?tttactacat 1200

cagagaagac?gactgagccc?aagaataaag?aatcgtttgt?gttatgtttc?aacgtgttta 1260

tttttcaatt?gcagaaaatt?tcaagtcatt?tttcattcag?tagtatagcc?ccaccaccac 1320

atagcttata?cagatcaccg?taccttaatc?aaactcacag?aaccctagta?ttcaacctgc 1380

cacctccctc?ccaacacaca?gagtacacag?tcctttctcc?ccggctggcc?ttaaaaagca 1440

tcatatcatg?ggtaacagac?atattcttag?gtgttatatt?ccacacggtt?tcctgtcgag 1500

ccaaacgctc?atcagtgata?ttaataaact?ccccgggcag?ctcacttaag?ttcatgtcgc 1560

tgtccagctg?ctgagccaca?ggctgctgtc?caacttgcgg?ttgcttaacg?ggcggcgaag 1620

gagaagtcca?cgcctacatg?ggggtagagt?cataatcgtg?catcaggata?gggcggtggt 1680

gctgcagcag?cgcgcgaata?aactgctgcc?gccgccgctc?cgtcctgcag?gaatacaaca 1740

tggcagtggt?ctcctcagcg?atgattcgca?ccgcccgcag?cataaggcgc?cttgtcctcc 1800

gggcacagca?gcgcaccctg?atctcactta?aatcagcaca?gtaactgcag?cacagcacca 1860

caatattgtt?caaaatccca?cagtgcaagg?cgctgtatcc?aaagctcatg?gcggggacca 1920

cagaacccac?gtggccatca?taccacaagc?gcaggtagat?taagtggcga?cccctcataa 1980

acacgctgga?cataaacatt?acctcttttg?gcatgttgta?attcaccacc?tcccggtacc 2040

atataaacct?ctgattaaac?atggcgccat?ccaccaccat?cctaaaccag?ctggccaaaa 2100

cctgcccgcc?ggctatacac?tgcagggaac?cgggactgga?acaatgacag?tggagagccc 2160

aggactcgta?accatggatc?atcatgctcg?tcatgatatc?aatgttggca?caacacaggc 2220

acacgtgcat?acacttcctc?aggattacaa?gctcctcccg?cgttagaacc?atatcccagg 2280

gaacaaccca?ttcctgaatc?agcgtaaatc?ccacactgca?gggaagacct?cgcacgtaac 2340

tcacgttgtg?cattgtcaaa?gtgttacatt?cgggcagcag?cggatgatcc?tccagtatgg 2400

tagcgcgggt?ttctgtctca?aaaggaggta?gacgatccct?actgtacgga?gtgcgccgag 2460

acaaccgaga?tcgtgttggt?cgtagtgtca?tgccaaatgg?aacgccggac?gtagtcatat 2520

ttcctgaagc?aaaaccaggt?gcgggcgtga?caaacagatc?tgcgtctccg?gtctcgccgc 2580

ttagatcgct?ctgtgtagta?gttgtagtat?atccactctc?tcaaagcatc?caggcgcccc 2640

ctggcttcgg?gttctatgta?aactaagctt?cgc 2673

<210>4

<211>51

<212>DNA

＜213〉primer sequence

<400>4

aattgaccgg?tctcgagact?agtggatccg?cggccgcatc?tagattaatt?a 51

<210>5

<211>51

<212>DNA

＜213〉primer sequence

<400>5

agcttaatta?atctagatgc?ggccgcggat?ccactagtct?cgagaccggt?c 51

<210>6

<211>1067

<212>DNA

＜213〉5 type adenovirus ADP gene orders

<400>6

tgacacatgc?agctcccgga?gacggtcaca?gcttgtctgt?aagcggatgc?cgggagcaga 60

caagcccgtc?agggcgcgtc?agcgggtgtt?ggcgggtgtc?ggggctggct?taactatgcg 120

gcatcagagc?agattgtact?gagagtgcac?catatgcggt?gtgaaatacc?gcacagatgc 180

gtaaggagaa?aataccgcat?caggcgccat?tcgccattca?ggctgcgcaa?ctgttgggaa 240

gggcgatcgg?tgcgggcctc?ttcgctatta?cgccagctgg?cgaaaggggg?atgtgctgca 300

aggcgattaa?gttgggtaac?gccagggttt?tcccagtcac?gacgttgtaa?aacgacggcc 360

agtgaattga?ccggtctcga?gactagtgga?tccgcggccg?ccgctaccgg?acttacatct 420

accacaaata?caccccaagt?ttctgccttt?gtcaataact?gggataactt?gggcatgtgg 480

tggttctcca?tagcgcttat?gtttgtatgc?cttattatta?tgtggctcat?ctgctgccta 540

aagcgcaaac?gcgcccgacc?acccatctat?agtcccatca?ttgtgctaca?cccaaacaat 600

gatggaatcc?atagattgga?cggactgaaa?cacatgttct?tttctcttac?agtatgatta 660

aatgagacat?gattcctcga?gtttttatat?tactgaccct?tgttgcgctt?tttttgtgcg 720

tgctccacat?tggctaagta?atagttaatt?aagcttggcg?taatcatggt?catagctgtt 780

tcctgtgtga?aattgttatc?cgctcacaat?tccacacaac?atacgagccg?gaagcataaa 840

gtgtaaagcc?tggggtgcct?aatgagtgag?ctaactcaca?ttaattgcgt?tgcgctcact 900

gcccgctttc?cagtcgggaa?acctgtcgtg?ccagctgcat?taatgaatcg?gccaacgcgc 960

ggggaagagg?cggtttgcgt?attgggcgct?cttccgcttc?ctcgctcact?gactcgctgc 1020

gctcggtcgt?tcggctgcgg?cgagcggtat?cagctcactc?aaaggcg 1067

<210>7

<211>271

<212>DNA

＜213〉hTERT promotor core sequence

<400>7

agatctcaca?gacgcccagg?accgcgcttc?ccacgtggcg?gagggactgg?ggacccgggc 60

acccgtcctg?ccccttcacc?ttccagctcc?gcctcctccg?cgcggacccc?gccccgtccc 120

gacccctccc?gggtccccgg?cccagccccc?tccgggccct?cccagcccct?ccccttcctt 180

tccgcggccc?cgccctctcc?tcgcggcgcg?agtttcaggc?agcgctgcgt?cctgctgcgc 240

acgtgggaag?ccctggcccc?ggccactcga?g 271

<210>8

<211>247

<212>DNA

＜213〉HRE sequence

<400>8

actagtccac?agtgcatacg?tgggctccaa?caggtcctct?tccacagtgc?atacgtgggc 60

tccaacaggt?cctcttccac?agtgcatacg?tgggctccaa?caggtcctct?tccacagtgc 120

atacgtgggc?tccaacaggt?cctcttccac?agtgcatacg?tgggctccaa?caggtcctct 180

tggtaggcgt?gtacggtggg?aggtctatat?aagcagagct?cgtttagtga?accgtcagat 240

cactagt 247

<210>9

<211>747

<212>DNA

＜213〉EGFP reporter gene sequence

<400>9

atggtgagca?agggcgagga?gctgttcacc?ggggtggtgc?ccatcctggt?cgagctggac 60

ggcgacgtaa?acggccacaa?gttcagcgtg?tccggcgagg?gcgagggcga?tgccacctac 120

ggcaagctga?ccctgaagtt?catctgcacc?accggcaagc?tgcccgtgcc?ctggcccacc 180

ctcgtgacca?ccctgaccta?cggcgtgcag?tgcttcagcc?gctaccccga?ccacatgaag 240

cagcacgact?tcttcaagtc?cgccatgccc?gaaggctacg?tccaggagcg?caccatcttc 300

ttcaaggacg?acggcaacta?caagacccgc?gccgaggtga?agttcgaggg?cgacaccctg 360

gtgaaccgca?tcgagctgaa?gggcatcgac?ttcaaggagg?acggcaacat?cctggggcac 420

aagctggagt?acaactacaa?cagccacaac?gtctatatca?tggccgacaa?gcagaagaac 480

ggcatcaagg?tgaacttcaa?gatccgccac?aacatcgagg?acggcagcgt?gcagctcgcc 540

gaccactacc?agcagaacac?ccccatcggc?gacggccccg?tgctgctgcc?cgacaaccac 600

tacctgagca?cccagtccgc?cctgagcaaa?gaccccaacg?agaagcgcga?tcacatggtc 660

ctgctggagt?tcgtgaccgc?cgccgggatc?actctcggca?tggacgagct?gtacaagtcc 720

ggactcagat?ccaccggatc?taggtaa 747

<210>10

<211>32

<212>DNA

＜213〉primer sequence

<400>10

gctctagaga?attcaccatg?gtgagcaagg?gc 32

<210>11

<211>34

<212>DNA

＜213〉primer sequence

<400>11

cgggatccgt?cgacttatta?cctagatccg?gtgg 34

<210>12

<211>345

<212>DNA

＜213〉human chemokine CCL5 sequence

<400>12

gaattcacca?tgaaggtctc?cgcggcagcc?ctcgctgtca?tcctcattgc?tactgccctc 60

tgcgctcctg?catctgcctc?cccatattcc?tcggacacca?caccctgctg?ctttgcctac 120

attgcccgcc?cactgccccg?tgcccacatc?aaggagtatt?tctacaccag?tggcaagtgc 180

tccaacccag?cagtcgtctt?tgtcacccga?aagaaccgcc?aagtgtgtgc?caacccagag 240

aagaaatggg?ttcgggagta?catcaactct?ttggagatga?gcttagactt?ggagatgtta 300

gctccctata?tcccaatgga?tgatgacttc?cagttatagg?gatcc 345

<210>13

<211>26

<212>DNA

＜213〉primer sequence

<400>13

cggaattcac?catgagacat?attatc 26

<210>14

<211>23

<212>DNA

＜213〉primer sequence

<400>14

gcgtcgactt?atggcctggg?gcg 23

<210>15

<211>30

<212>DNA

＜213〉primer sequence

<400>15

cggaattcac?catgaaggtc?tccgcggcag 30

<210>16

<211>34

<212>DNA

＜213〉primer sequence

<400>16

cgggatccct?ataactggaa?gtcatcatcc?attg 34

Claims (12)

1. a proliferous type recombination oncolytic adenovirus is characterized in that this recombinant adenovirus contains the amino acid coding of B subgroup 11 type adenovirus cilium heads; And at least one virus multiplication indispensable gene of this viral genome is suddenlyd change or is lacked and lose function, or described virus operationally inserts cis-acting elements between the transcription initiation site of propagation indispensable gene sequence and translation initiation site ATG, and at least one virus multiplication indispensable gene transcribes in the described element controlling gene group.

2. the proliferous type recombination oncolytic adenovirus of claim 1, the aminoacid sequence that it is characterized in that this recombinant adenovirus cilium head is the aminoacid sequence of human B subgroup 11 type adenovirus cilium heads.

3. proliferous type recombination oncolytic adenovirus according to claim 1, it is characterized in that described virus between the transcription initiation site of propagation indispensable gene sequence and translation initiation site ATG operationally insertion be the cis-acting elements of tumor-specific promoters.

4. proliferous type recombination oncolytic adenovirus according to claim 3 is characterized in that described tumor-specific promoters is selected from:

(a) carcinomebryonic antigen promotor, enhanser and mutant sequence thereof;

(b) afp promoter, enhanser and mutant sequence thereof;

(c) receptor tyrosine kinase promotor, enhanser and the mutant sequence thereof of Human epidermal growth factor receptor family;

(d) breast cancer correlation antigen DF3/MUC1 promotor, enhanser and mutant sequence thereof;

(e) promotor of vascular endothelial growth factor receptor KDR, enhanser and mutant sequence thereof;

(f) L-plastin promotor, enhanser and mutant sequence thereof;

(g) IAP family member's promotor, enhanser and mutant sequence thereof;

(h) promotor of prostaglandin(PG) specific antigens, enhanser and mutant sequence thereof;

(i) the hypoxia response elements conserved sequence of HIF-1 regulation and control;

(j) transcription factor E2F promotor, enhanser and mutant sequence thereof; With

(k) human telomerase subunit promotor, enhanser and mutant sequence thereof

In at least a.

5. proliferous type recombination oncolytic adenovirus according to claim 1 is characterized in that described virus multiplication indispensable gene is the gene of coding E1a, E1b-55kDa, E1b-19kDa, E2 or E4 wild-type or the process sudden change.

6. proliferous type recombination oncolytic adenovirus according to claim 1 is characterized in that described virus multiplication indispensable gene is through point mutation, inserts sudden change, inserts terminator codon, lacks and the E1b-55kDa or the E1b-19kDa gene of inactivation wholly or in part.

7. proliferous type recombination oncolytic adenovirus according to claim 1 is characterized in that its exogenous gene expression box that also has been operably connected.

8. proliferous type recombination oncolytic adenovirus according to claim 7 is characterized in that described foreign gene is selected from:

(a) gene of interferons immune-regulating factor;

(b) gene of interleukin-para-immunity regulatory factor;

(c) gene of colony-stimulating factor;

(d) cancer suppressor gene;

(e) antisense sequences of proto-oncogene;

(f) antibody gene of people's tumour Epidermal Growth Factor Receptor Family;

(g) nucleotide sequence of Endostatin, angiostatin and the fragment thereof of coding anti-angiogenesis, plasminogen activating factors inhibitor, Canstatin, thrombospondin;

(h) gene of apoptosis inducing factor family;

(i) gene of vascular endothelial growth factor antibody;

(j) suicide gene; With

(k) gene of tumour necrosis factor

In at least a.

9. proliferous type recombination oncolytic adenovirus according to claim 1 is characterized in that described virus vector is to be selected from least a in 2 types of C subgroup or the 5 type adenovirus.

10. the proliferous type recombination oncolytic adenovirus of claim 1, its preservation day is on January 9th, 2009, and deposit number is CCTCC-V200903, and the preservation place is Chinese typical culture collection center.

11. make up the method for the described proliferous type recombination oncolytic of claim 2 adenovirus, it is characterized in that replacing with the aminoacid sequence of human B subgroup 11 type adenovirus cilium heads the cilium head aminoacid sequence of non-11 type adenovirus by gene engineering method; And at least one the propagation indispensable gene that makes described virus is suddenlyd change or is lacked and lose function, or making described virus between transcription initiation site of breeding the indispensable gene sequence and translation initiation site ATG, operationally insert cis-acting elements, at least one virus multiplication indispensable gene transcribes in the described element controlling gene group.

12. the described proliferous type recombination oncolytic of claim 1 adenovirus carrier, it is as the carrier of gene and be used to prepare gene therapy medicament.

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