CN104651378B - A kind of hog cholera vaccine infectious CDNA and its construction method and application - Google Patents
A kind of hog cholera vaccine infectious CDNA and its construction method and application Download PDFInfo
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- CN104651378B CN104651378B CN201510052888.8A CN201510052888A CN104651378B CN 104651378 B CN104651378 B CN 104651378B CN 201510052888 A CN201510052888 A CN 201510052888A CN 104651378 B CN104651378 B CN 104651378B
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Abstract
The invention belongs to veterinary biologicses technical field.It is specifically related to a kind of to include C plants of infectious CDNA clones of hog cholera vaccine and the 5 ' terminal fusion pig rna plymerase i promoter sequences in the genome cDNA, insert the plasmid pASPIT of mouse rna plymerase i terminator sequence in 3 ' endsICS structure.The plasmid transfection can directly produce hog cholera vaccine C strain virus to pig source cell PK15.Convert plasmid pASPITICS Escherichia coli DH10B/pASPITICS,Escherichia coli DH10B/pASPITICS, send China typical culture collection center preservation, and preserving number is CCTCC NO：M2015062.This hog cholera vaccine C plants of infectious CDNA clones prepare high-titer hog cholera vaccine available for genetic modification or build the bivalent vaccine based on hog cholera vaccine carrier.
Description
Technical field
The invention belongs to veterinary biologicses technical field, relate to the use of reverse genetic manipulation and obtain a kind of weak poison of swine fever
C plants of infectious CDNA clones of vaccine, while being related to the infectious CDNA clones is building high-titer restructuring live vaccines of hog cholera and pig
Application in pest vaccine carrier.
Background technology
Swine fever is a kind of extremely strong viral disease of infectiousness of pig, and early stage is referred to as hog cholera (Hog Cholera, HC),
Various age pigs can be infected, popular throughout the year, morbidity and mortality are very high, endanger huge.The pathogen of swine fever is pig
Pestivirus (Classical swine fever virus, CSFV), is formulated in International Office of Epizootics (OIE)《International animal is defended
Think of a way allusion quotation》In, swine fever is put into one of infectious disease of 16 kinds of statutory reports of A classes.Swine fever takes place frequently in recent years for China, to pig industry
Serious financial consequences are caused, development and the export trade of pig industry is hampered.Strengthen effective prevention and control to swine fever, be to promote to support
The inevitable requirement that pig industry develops in a healthy way, improves culture benefit and allow its people to be able to eat quality-assured meat；Develop efficient swine fever control technology
And vaccine product, lifting swine fever prevention and control level is the key subjects of scientific and technical research.
CSFV genomes are the single-stranded positive RNA for being about 12.3kb, by 5' end non-coding regions (5'Untranstaled
Region, 5'UTR), open reading frame (ORF) one big and 3' end non-coding regions (3'UTR) constitute.ORF codings one
The individual polyprotein being made up of 3898 amino acid residues, polyprotein is compiled in translation process or after translation by virus itself
The protease of code and the protease of host cell are processed into 12 kinds of mature proteins, including 4 kinds of structural proteins C, Erns、E1、E2
With 8 kinds of non-structural protein Npro, P7, NS2, NS3, NS4A, S4B, NS5A and NS5B.Wherein, Erns, E1 and E2 insertion lipids it is double
In tunic, in virion surface formation cynapse, the absorption and invasion of mediate retroviral are CSFV virulence correlation factor and protection
Property antigen protein.E2 is often present in virion with homodimer or ElE2 heterodimers and host is thin
Hog cholera vaccine inoculation is prevention and the most effectual way for controlling swine fever.Early in nineteen fifty-one, China researcher screening
Go out the excellent CSFV crossdrifts strain (Yin Zhen, 1997) of immunogenicity, the strain as Chinese CSFV standards velogen strain continue to use to
It is modern.CSFV crossdrift strains are used at that time, and inactivation is made swine fever crystal violet glycerine inactivated vaccine, China is controlled rapidly after use
Swine fever epidemic situation.Between 1954-1956, the researcher of the unit such as China Veterinary Drugs Supervisory Inst. by CSFV velogen strains (including
The strong malicious Strain Shimen of standard) rabbit is adapted to, the continuous passage in rabbit body selects one plant to the basic avirulence of pig, and keep CSFV
The lapinized virus strain of immunogenicity, i.e. China's swine fever rabbitization attenuated vaccine strain (hog cholera lapinized vaccine,
HCLV), abbreviation hog cholera vaccine C plants.The successful development and application of hog cholera lapinised virus vaccine, prevention and control to China's swine fever
System has played decisive role.However, hog cholera lapinised virus vaccine widely using over 60 years, does not control the generation of swine fever.
Although dosage of inoculation and the immune time increase of vaccine, swine fever are not eradicated not only, in recent years, China's swine fever occurs
Many new features, show as being changed into from being very popular of taking place frequently and irregular distribute prevalence；Clinical symptoms are changed into non-from typical case
Classical swine fever, virus virulence reduction, the course of disease is changed into based on chronic by acute, subacute, persistent infection, placenta sense occurs
Congenital infection of dye, sow breeding difficulty and newborn piglet etc..Hog cholera vaccine strain viral titer in cell is not high, system
Standby high-titer vaccine difficulty be cause vaccine Vaccine effectiveness low and swine fever prevention and control difficulty one of major reason.The hair of swine fever
Raw, propagation and prevalence are the bottlenecks that serious restriction China pig industry develops in a healthy way, increases farmers' income and develop international trade.
The need for meeting China's prevention, control and final elimination swine fever, new generation high-titer hog cholera vaccine system
Product, improve hog cholera vaccine immune protection effectiveness, imperative.In addition, it is also reduction epidemic disease to improve hog cholera vaccine cell growth titre
Seedling manufacturing enterprise production cost, the basic assurance for improving vaccine quality and benefit.
On the other hand, CSFV infection causes the immunosupress of pig body, and infection CSFV pig body is easier occur annulus disease
The mixed infection of malicious, pig breeding and breathing syndrome virus, japanese encephalitis virus etc., causes the pig death rate higher, harm is more
It is huge.Based on live vaccines of hog cholera vector expression, other cause of disease protective antigens albumen build bivalent vaccine, can be while prevention and control swine fever
And related swine disease, with important application value.
The content of the invention
The primary and foremost purpose of the present invention is to provide a kind of full-length infectious CDNA of hog cholera vaccine, the full-length infectious CDNA
Contain the malicious full-length cDNA of hog cholera vaccine kind, in the terminal fusion pig rna plymerase i promoters of cDNA 5 ', inserted in cDNA 3 '
Enter mouse rna plymerase i terminator；
Further, the present invention provides the full-length infectious CDNA of C plants of hog cholera vaccine, and the full-length infectious CDNA is included
C plants of full-length cDNAs of hog cholera vaccine, in the terminal fusion pig rna plymerase i promoters of cDNA 5 ' (GenBank
No.L31782.1), its sequence is SEQ ID NO：1；Mouse RNA polymerase I terminators are inserted in cDNA 3 ', its sequence is SEQ
ID NO：2；
To achieve the above object, the present invention provides a kind of full-length infectious CDNA clone for building above-mentioned hog cholera vaccine C plants
Method, including：
1) using C plants of RNA of hog cholera vaccine as template, using according to C plants of full-length genome sequence (Genbank of hog cholera vaccine
No.AF091507) 6 pairs of specific PCR amplimers (SEQ ID NO of design synthesis：3-14), using RT-PCR, pig is obtained
C plants of the pestilence seedling cDNA fragments of full-length genome 6 (CSf1-6)；
2) strategy for connecting the PCR fragment expanded using digestion with restriction enzyme, T4 ligases, is cloned into matter
In grain carrier；
3) structure of hog cholera vaccine C plants of infectious CDNA clones plasmids：
(1) 5 ' are built and holds half long genome large fragment：Fragment CSf1, CSf2 and CSf3 are consecutively connected to low-copy
Plasmid pACNR1180 (is given) by N.Ruggli and J.D.Tratschin doctors, builds pA-CSf123；
(2) 3 ' are built and holds half long genome large fragment：Fragment CSf4, CSf5 and CSf6 are consecutively connected to low-copy
Plasmid pACNR1180, builds pA-CSf456；
(3) the recombinant plasmid pSPI-CSf123 of the end of hog cholera vaccine 5 ' of fusion pig rna plymerase i promoter is built；
A) prepared by pigs rna plymerase i promoter (pSPolI) fragment：According to pig rna plymerase i promoter sequence
(GenBank No.L31782.1) design promoter fragment pcr amplification primer thing (SEQ ID NO：15-16), with pig source PK-15
The DNA of cell is template, expands pig rna plymerase i promoter, and using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing is true
Card, -70 DEG C save backup；
B) the end 784bp fragment amplifications of hog cholera vaccines C strains 5 '：Hog cholera vaccine cDNA sequence is built, a pair of PCR of design draw
Thing (SEQ ID NO：17-18), the end 784bp fragments of amplification gene group 5 ', using pA-CSf123 recombinant plasmids as template, are used
DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup；
C) .pACNR1180 vector backbone segments are expanded：According to pACNR1180 carrier sequences (SEQ ID NO：19) design
Amplimer (SEQ ID NO：20-21), using pA-CSf123 recombinant plasmids as template, pACNR1180 carrier segments PCR is obtained
Product, using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup；
D) merges the pACNR1180 vector backbone segments of the end of hog cholera vaccine C strains 5 ' and pig rna plymerase i promoter
Amplification：Carried with the end 784bp fragments of hog cholera vaccine C strains 5 ', pig rna plymerase i promoter and pACNR1180 of above-mentioned preparation
Body fragment mixture is template, with SEQ ID NO：20 be sense primer, SEQ ID NO：18 be anti-sense primer, and PCR amplifications are obtained
The pACNR1180 vector backbone segment PCR primers A-SPI- of the end of hog cholera vaccine 5 ' and pig rna plymerase i promoter must be merged
CS-5 ', using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup；
E) merges the end of the hog cholera vaccine 5 ' recombinant plasmid pSPI-CSf123 structures of the promoter of pig RNA polymerase I：Point
Yong not the double digested plasmid pA-CSf123 and A-SPI-CS-5 ' DNA fragmentations of restriction enzyme PmlI and MluI, DNA times
Receive kits and prepare △ CSf123 and A-SPI-CS-5 ' fragments, two kinds of fragments press 1:2 mixing, the connection of T4DNA ligases,
Conversion, selects positive colony, and sequencing confirmation obtains pSPI-CSf123；
(4) terminator (T of fusion mouse RNA polymerase I is builtI) the end of hog cholera vaccine 3 ' recombinant plasmid pA-CSf456-
TI:
According to mouse rna plymerase i terminator sequence (SEQ ID NO：2) with the end sequence of hog cholera vaccine C strains 3 ', design
Long aligning primer (the SEQ ID NO of synthesis fusion mouse terminator sequence：9th, 22), using recombinant plasmid pA-CSf456 as template,
The end of the hog cholera vaccine 3 ' PCR primer CSf456-T of the terminator of fusion mouse RNA polymerase I is obtained by PCRIFragment；
The double digested plasmid pA-CSf456 and CSf456-T of restriction enzyme SphI and MluI is used respectivelyIFragment,
DNA QIAquick Gel Extraction Kit purifying prepares the linearisation pA-CSf456 and CSf456-T of double digestion cohesive endIFragment, two kinds of fragments
By 1:3-1:10 molar ratios are mixed, the connection of T4DNA ligases, conversion, select positive colony, and sequencing confirmation obtains pA-
CSf456-TI；
(5) pig rna plymerase i promoter (pSPolI) and mouse rna plymerase i terminator (T are based onI) hog cholera vaccine
Infectious CDNA clones pASPITI- CS is built：
By the end of the hog cholera vaccine 5 ' large fragment plasmid pSPI-CSf123 and 3 ' ends large fragment plasmid pA- of above-mentioned structure
CSf456-TIIt is double digested with BamHI and MluI respectively, reclaim linearisation pSPI-CSf123 and CSf456-TIFragment, two
Person presses 4:1 molar ratio is mixed, the connection of T4DNA ligases, conversion, selects positive colony, and sequencing confirmation obtains hog cholera vaccine
Infectious CDNA clones plasmid pASPITI- CS, transformed competence colibacillus E. coli DH10B, coating contain ampicillin
The agar plate culture of resistance, selects positive recombinant Escherichia coli DH10B/pASPITI- CS, Escherichia coli
DH10B/pASPITI- CS, was delivered in Chinese Wuhan Wuhan Universitys China typical culture collection on January 23rd, 2015
Heart preservation, deposit number is CCTCC NO：M2015062.
Another object of the present invention is to be used to the full-length infectious CDNA of C plants constructed of hog cholera vaccine save pig
Fever live vaccine kind poison.I.e.：Using liposome Lipofectamine2000, by hog cholera vaccine infectious CDNA plasmid pASPITI-
CS is transfected to pig source PK15 or SK6 cells, and transcription produces viral RNA in PK15/SK6 cells, then carries out virus protein
Translation, processing, virion assembling obtain genetic engineering live vaccines of hog cholera kind poison；
Another object of the present invention is by the full-length infectious CDNA clones plasmid of C plants constructed of hog cholera vaccine
pASPITI- CS is as carrier, for expressing foreign protein；
Further, the present invention also provides the side that construction expression foreign protein recombinates hog cholera vaccine infectious CDNA clones
Method, it comprises the following steps：1) C plants of full-length infectious CDNA clones of hog cholera vaccine are built, recombinant plasmid pASPIT is obtainedI-
CS；2) PCR amplifications obtain external source antigen-4 fusion protein gene, its 5 ' end fusion CSFV 7 amino acid coding of C protein, its
3 ' end fusion foot and mouth disease virus 2A albumen coded sequences；3) using multi-step over-lap PCR amplification foreign protein genes fusion piece
Section, hog cholera vaccine genome N is inserted into by the foreign gene of acquisitionproBetween C protein code area, obtain expressing foreign protein
Restructuring hog cholera vaccine be fitted together to infectious CDNA clones；
Described foreign protein is luciferase, and luciferase (Luc) gene is merged with foot and mouth disease virus 2A GFPs
For Luc2A, in order to Luc from hog cholera vaccine polyprotein by correct processing, release.
It is another object of the present invention to provide application of the vaccine in prevention swine fever.
Plasmid pASPITI- CS can be widely used for hog cholera vaccine modification transformation, expression exogenous antigen albumen, to prepare efficiently
Valency live vaccines of hog cholera or restructuring bivalent and multivalence swine fever carrier live.
A kind of New Kind of Vaccine for Classical Swine Fever infectious CDNA clones of the present invention have the following advantages that：Classical swine fever vaccine infection
, it is necessary to by cDNA clone is linearized, using T7DNA polymerase in-vitro transcription viral genes during cDNA clone generation virus
Group vRNA, virus, complex steps, usual inefficiency are obtained by vRNA with liposome transfection to the rescue of pig source cell；The present invention
Infectious CDNA clones include in pig source cell the pig rna plymerase i promoter of directly synthesis virus genome RNA
With mouse rna plymerase i terminator.By the way that infectious CDNA clones are transfected into pig source cell, hog cholera vaccine can be directly produced
Virus, and efficiency improves tens of to hundreds times；Therefore, with it is simple, quickly and efficiently the features such as, it is adaptable to build, screen height
Potency live vaccines of hog cholera kind poison；Foreign gene is inserted into the N of hog cholera vaccine genome encoding by the present inventionproBetween C protein
Expression, there is the 2A GFPs of Self cleavage function in the terminal fusion foot and mouth disease virus of foreign gene 3 ', can make foreign protein from
Correct cutting and effectively release in the polyprotein of hog cholera vaccine encoding viral, available for the expression of other swine disease pathogen antigens,
It is suitable for building the bivalent based on live vaccines of hog cholera carrier or polyvaccine.
Brief description of the drawings
Fig. 1 builds schematic diagram for a kind of restructuring hog cholera vaccine infectious CDNA clones；
Fig. 2 is each cDNA clone fragment electrophoretic analysis chart in infectious CDNA clones building process；
Fig. 3 is cDNA grams of the hog cholera vaccine constituted based on pig rna plymerase i promoter and mouse rna plymerase i terminator
Grand recombinant plasmid (A) and operation principle schematic diagram (B)；
Fig. 4 is to produce hog cholera vaccine Viral diagnosis figure based on new infectious CDNA clones transfectional cell：
A. hog cholera vaccine is viral in CSFV NS3 protein antibodies immunofluorescence dyeing detection PK15 cells；
B. specific primer RT-PCR detects hog cholera vaccine viral nucleic acid in PK15 cells；
Fig. 5 is to produce expression foreign protein luciferase Recombinant Swine pest vaccine virus based on new infectious CDNA clones
Detection figure：
A. CSFV NS3 protein antibodies immunofluorescence dyeing detects PK15 cell Recombinant Swine pest vaccine viruses；
B. uciferase activity in Recombinant Swine pest vaccine virus PK15 cell pyrolysis liquids is infected.
Fig. 6 is Temperature changing after restructuring vaccinated pig and the strong malicious lethal challenges of control experiment pig CSFV；
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The reality provided
It is only the explanation to the inventive method to apply example, without limiting remaining content that the present invention is disclosed in any way.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as《Molecular cloning：It is real
Test room guide》(New York:Cold Spring Harborlaboratory Press, 2001) described in condition carry out.
【Embodiment 1】A kind of new infectious CDNA clones construction method for preparing hog cholera vaccine kind poison
1. the design synthesis of primer
According to C plants of full-length genome sequences of hog cholera vaccine (Genbank No.AF091507), design 6 pairs of specificity of synthesis
Pcr amplification primer thing, the external synthesis for C plants of the hog cholera vaccine cDNA fragments of full-length genome 6.Each primer gives birth to work by Shanghai
Company synthesizes.Hog cholera vaccine C pnca gene groups cDNA is using the external reverse transcription synthesis of 9 nucleotides random primer pd (N) 9, pd (N) 9
It is Biocolor Products (Britain).
It is as follows from each cDNA fragment lengths in the end of genome 5 ' to 3 ', 6 pairs of specific primer sequences and numbering successively：
1) (the CSf1 of fragments 1：1-2431nt) pcr amplification primer thing
Sense primer P1F (SEQ ID NO.3):
5′-GCGTCGACTCTTAATACGACTCACTATAGTATACGAGGTTAGTTCATTC-3′
Anti-sense primer P1R (SEQ ID NO.4):
5′-CCAGTTACTAGTAACAGCCATACCACACCTTGC-3′
(amplified production is 2459bp, contains SalI sites at its 5 ' end, and SpeI sites are contained at 3 ' ends)
2) (the CSf2 of fragments 2：2414-4457nt) pcr amplification primer thing
Sense primer P2F (SEQ ID NO.5)
5′-GCTGTTACTAGTAACTGGGGCACAAGGCCGG-3′
Anti-sense primer P2R (SEQ ID NO.6):
5′-CATGGTACCTGTCATTGAACTTGTC-3′
(amplified production is 2043bp, contains SpeI sites at its 5 ' end, and KpnI sites are contained at 3 ' ends)
3) (the CSf3 of fragments 3：4441-6458nt) pcr amplification primer thing
Sense primer P3F (SEQ ID NO.7)
5′-CAATGACAGGTACCATGTTGCCATTG-3′
Anti-sense primer P3R (SEQ ID NO.8):
5′-CACCCTCAGGTTAGATGGATCC-3′
(amplified production is 2217bp, contains KpnI sites at its 5 ' end, and BamHI sites are contained at 3 ' ends)
4) (the CSf4 of fragments 4：6434-8681nt) pcr amplification primer thing
Sense primer P4F (SEQ ID NO.9)
5′-AGAGGATCCATCTAACCTGAGGG-3′
Anti-sense primer P4R (SEQ ID NO.10):
5′-ATTTCTGCAGAGAAATGAACCTTCC-3′
(amplified production is 2247bp, contains BamHI sites at its 5 ' end, and PstI sites are contained at 3 ' ends)
5) (the CSf5 of fragments 5：8652-11169nt) pcr amplification primer thing
Sense primer P5F (SEQ ID NO.11)
5′-GAGGAGGAAGGTTCATTTCTCTGCAG-3′
Anti-sense primer P 5R (SEQ ID NO.12):
5′-CATTTAGCATGCTGTTGCCCG-3′
(amplified production is 2517bp, contains PstI sites at its 5 ' end, and SphI sites are contained at 3 ' ends)
6) (the CSf6 of fragments 6：11149-12310nt) pcr amplification primer thing
Sense primer P6F (SEQ ID NO.13)
5′-CGGGCAACAGCATGCTAAATG-3′
Anti-sense primer P6R (SEQ ID NO.14):
5′-TACGCGTGAGCCCGGGCCGTTAG-3′
(amplified production is 1174bp, contains SphI sites at its 5 ' end, and MluI sites are contained at 3 ' ends)
2. hog cholera vaccine geneome RNA is extracted and cDNA synthesis
Hog cholera vaccine geneome RNA with total RNA extraction reagent box (two step method total serum IgE extraction agent box,
Invitrogen), the step of being introduced by kit operational manual extracts total serum IgE (including hog cholera vaccine geneome RNA).Tool
Body：
Hog cholera vaccine C strain virus is infected after PK15 cells, infection 72h, discards culture medium, 1ml is added into cellReagent, mixing is stored at room temperature 5min afterwards for several times；The chloroform of 1/5 volume is added, lid is tightened acute manually
Strong mixing 15s, room temperature places 2-3min；12000g centrifuges 15min in 4 DEG C, draws upper strata aqueous phase and is managed to another EP, is added
Isometric isopropanol, 10min is stored at room temperature after fully mixing；12000g centrifuges 10min in 4 DEG C；Supernatant is removed, 1ml is added
75% ethanol (ice precooling) washs RNA precipitate；Rotating speed 7500g centrifuges 5min in 4 DEG C；Supernatant is blotted, RNA precipitate is air-dried.
The RNA of extraction is dissolved in 15.5 μ l without in RNase distilled waters (Ambion companies), 2 μ l reverse transcriptions pd (N9) are added
Random primer (100ng/ μ l), 70 DEG C, 5min puts rapidly ice bath 5min, adds reverse transcription mixed liquor：5 × reverse transcriptase is buffered
Liquid (RT Buffer) 5 μ l, dNTPs (10 μM) 1 μ l, RNasin 0.5 μ l (40U/ μ l), the μ l of M-MLV 1 (200U/ μ l).In PCR
Following react is carried out on thermal cycler：37 DEG C of 60min, 95 DEG C of denaturation 10min, put ice bath, obtain the single-stranded cDNA of hog cholera vaccine ,-
70 DEG C freeze it is standby.3.PCR expands hog cholera vaccine cDNA fragments
Hog cholera vaccine cDNA using above-mentioned preparation is distinguished as template with above-mentioned 6 pairs of specific primers a pair of PCR therein
Corresponding genetic fragment is expanded, amplification reaction system and specific reaction condition are as follows：
Said components are mixed with pipettor, following react is carried out on PCR thermal cyclers：94 DEG C of 60 Sec of denaturation, then
94 DEG C of 30Sec, 55 DEG C of 30Sec, 72 DEG C of 90Sec carry out 35 circulations, obtain the PCR primer of fragment 1 (CSf1).Identical anti-
Answer and P2F/R is separately added under system and amplification condition to P6F/R primer pairs, fragment 2 is obtained successively to fragment 6 (CSf2-CSf6)
PCR primer.
PCR reagent used includes mononucleotide mixture dNTPs, 5 × PCR buffer solutions and pfu archaeal dna polymerases, is
Sheng Gong bioengineering Co., Ltd product, similarly hereinafter.
Agarose gel electrophoresis detection PCR primer after PCR amplifications.As shown in Fig. 2 swimming lane 1 is standard molecular weight, swimming lane
2-7 is followed successively by amplified production CSf1, CSf2, CSf3, CSf4, CSf5 and CSf6.
4. recovery product
By step 3 expand the various PCR primers of acquisition using Ago-Gel DNA QIAquick Gel Extraction Kits (D2500-02,
Omega Products) recovery purifying PCR primer；
5.PCR product clonings are to pMD18-T carriers
1) .PCR amplified productions end adds " A " to react
Reaction system and condition are as follows：
2) .PCR amplified productions are connected to pMD18-T carriers：Using pMD18-T kits (Takara Products), press
Kit specification is operated.The μ l of pMD18-T vector plasmid DNAs 0.5 are taken, end adds the μ l of pcr amplification product 2.5 of " A ", added
The μ l of coupled reaction mixture Solution I 3, add no RNase double distilled waters to the μ l of cumulative volume 10, mix, 4 DEG C connected
Night or 16 DEG C of connection 60min.
3) fragments are converted and positive clone identification with plasmid vector attachment：Connection product is added to 100 μ l DH5 α
In competent cell, ice bath 30min；42 DEG C of accurate response 90Sec；1~2min of ice bath；Add 800 μ l LB culture mediums, 37 DEG C
Shaken cultivation 45min；The overnight incubation on the Agar Plating containing X-Gal, IPTG, Amp.Selected from flat board white
Color bacterium colony, is expanded with the specific primer PCR of respective segments, agarose cohesion electrophoresis, it was observed that amplified production is to be connected with mesh
Fragment positive colony.Positive bacteria is expanded in LB fluid nutrient mediums, plasmid (plasmid is extracted by kit specification operation
Extracts kit D6943-02, Omega Products), follow-up cDNA clone is used for after being confirmed through sequencing analysis and is built.
6. the structure of C plants of infectious CDNA clones plasmids of hog cholera vaccine
1) builds 5 ' and holds half long genome large fragment：Fragment CSf1, CSf2 and CSf3 are consecutively connected to low-copy
Plasmid pACNR1180 builds pA-CSf123.
Restriction enzyme SalI and SpeI double digested plasmid pMD18T-CSf1 and plasmid pMD18T- are used respectively
CSf2, DNA QIAquick Gel Extraction Kit (are operated) purifying to prepare CSf1 fragments and the pMD18T-CSf2 linearized by kit specification,
Two kinds of fragments press 4:Positive colony is selected in 1 mixing, the connection of T4DNA ligases, conversion, and digestion identification obtains pMD18T-
CSf12。
Carried respectively with the double digested pMD18T-CSf12 and pACNR1180 plasmids of restriction enzyme SpeI and KpnI
Body, DNA QIAquick Gel Extraction Kits (by kit specification operation) purifying prepares CSf12 fragments and linearisation pACNR1180 plasmids,
Both press 4:Positive colony is selected in 1 mixing, the connection of T4DNA ligases, conversion, and digestion identification obtains recombinant plasmid pA-
CSf12。
The double digested plasmid pMD8T-CSf3 of restriction enzyme KpnI and BamHI and pA-CSf12, DNA are used respectively
QIAquick Gel Extraction Kit (by kit specification operation) purifying prepares CSf3 fragments and linearisation pA-CSf12, and both press 4:1 mixes
Close, the connection of T4DNA ligases, conversion, select positive colony, sequencing confirmation obtains recombinant plasmid pA-CSf123, i.e., 5 ' ends half
Long genome large fragment.
2) builds 3 ' and holds half long genome large fragment：That is fragment CSf4, CSf5 and CSf6's is sequentially connected.
The double digested plasmid pMD18T-CSf4 and pFastBacHTA of restriction enzyme BamHI and PstI is used respectively
(Invitrogen Products), are separately recovered linearized vector pFastBacHTA and CSf4 fragment, by 4:1 molar ratio is mixed
Close, the connection of T4DNA ligases, conversion, select positive colony, digestion identification obtains plasmid pFastBacHTA-CSf4.
With the double digested plasmid pMD18T-CSf5 and pFastBacHTA-CSf4 of restriction enzyme PstI and SphI
The pFastBacHTA-CSf4 plasmids of plasmid, recovery purifying CSf5 fragments and linearisation, both press 4:1 mixing, T4DNA connections
Enzyme connection, conversion, select positive colony, and digestion identification obtains recombinant plasmid pFastBacHTA-CSf45.
Restriction enzyme SphI and MluI double digested pMD18T-CSf6 and pACNR1180 is used respectively, is reclaimed
CSf6 fragments and linearization plasmid pACNR1180, both press 4:1 mixing, the connection of T4DNA ligases, conversion select positive gram
Grand, digestion identification obtains recombinant plasmid pA-CSf6.
Respectively with the double digested plasmid pFastBacHTA-CSf45 of restriction enzyme BamHI and SphI and
PACNR1180-CSf6, reclaims CSf45 fragments and linearization plasmid pACNR1180-CSf6, and both press 4:1 mixing, T4DNA connects
Enzyme connection, conversion are connect, positive colony is selected, sequencing confirmation obtains recombinant plasmid pA-CSf456, i.e., 3 ' half long genomes of end are big
Fragment (Fig. 1).
3) builds the recombinant plasmid pSPI-CSf123 of the end of hog cholera vaccine 5 ' of fusion pig rna plymerase i promoter
A) prepared by pigs rna plymerase i promoter (pSPolI) fragment：According to pig rna plymerase i promoter sequence
(GenBank No.L31782.1) designs promoter fragment pcr amplification primer thing：
Sense primer SPI-F (SEQ ID NO.15):
5′-CCGGATCCGGAGTGTTTCCCTGTCG-3′
Anti-sense primer SPI-R (SEQ ID NO.16):
5′-AGGGTACCTCTAGACTCGAGCGTCTCCATCTACCTGGTGACAGAAAAGGCG-3′
Fragment PCR is expanded：Using pig source PK-15 cells as material, base is extracted using QIAamp DNAmini kit kits
Because of a group DNA.Operating procedure is operated by kit specification.Using genomic DNA as template, SPI-F/SPI-R is used for primer,Expanded by following reaction system and condition
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 30Sec carry out 33 circulations, obtain pSPolI fragment PCR products.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
B) the end 784bp fragment amplifications of hog cholera vaccines C strains 5 '：
Build hog cholera vaccine cDNA sequence, the end 784bp fragments of design one couple of PCR primers amplification gene group 5 '；
Sense primer CS-F (SEQ ID NO.17):
5′-CAGGTAGATGTATACGAGGTTAGTTCATTCTCG-3′
Anti-sense primer CS-R (SEQ ID NO.18):
5′-CCATCGATGCACACATAAG-3′；
Fragment PCR is expanded：Using pA-CSf123 recombinant plasmids as template, CS-F/CS-R is used for primer,By following reaction System and condition amplification：
Said components are mixed, following react is carried out on PCR thermal cyclers：94 DEG C of denaturation 60Sec, then 94 DEG C
30Sec, 55 DEG C of 30Sec, 72 DEG C of 90Sec carry out 30 circulations, obtain the end of genome 5 ' 784bp fragment PCR products.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
C) .pACNR1180 vector backbone segments are expanded：
Amplimer is designed according to pACNR1180 carrier sequences (SEQ ID NO X3)
Sense primer ACNRF (SEQ ID NO.20):5′-TGGACTAAGGTAATTTCTAACGGC-3′
Anti-sense primer ACNRR (SEQ ID NO.21):5′-ACTCCGGAGTCGACGTCAGGTGGC-3′
Fragment PCR is expanded：Using pA-CSf123 recombinant plasmids as template, ACNRF/ACNRR is used for primer,By following anti- System and condition is answered to expand：
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 90Sec carry out 33 circulations, obtain pACNR1180 carrier segments PCR primers.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
D) merges the end of hog cholera vaccine 5 ' and the pACNR1180 vector backbone segments of pig rna plymerase i promoter expand
Increase：
Carried with the end of the hog cholera vaccine 5 ' 784bp fragments, pig rna plymerase i promoter and pACNR1180 of above-mentioned preparation
Body fragment mixture is template, by sense primer of ACNRF, CS-R be anti-sense primer, PCR amplifications obtain fusion hog cholera vaccine
5 ' ends and the pACNR1180 vector backbone segments of pig rna plymerase i promoter.
Expanded by following reaction system and condition
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 120Sec carry out 33 circulations, obtain the fusion end of hog cholera vaccine 5 ' and pig rna plymerase i
The pACNR1180 vector backbone segment PCR primers pASPI-CS-5 ' of promoter.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
E) merges the end of the hog cholera vaccine 5 ' recombinant plasmid pASPI-CSf123 structures of the promoter of pig RNA polymerase I：
The double digested plasmid pA-CSf123 and pASPI-CS-5 ' DNA of restriction enzyme PmlI and MluI are used respectively
Fragment, DNA QIAquick Gel Extraction Kits purifying prepares △ CSf123 and pASPI-CS-5 ' fragments, and two kinds of fragments press 1:2 mixing, T4DNA
Ligase connection, conversion, select positive colony, and sequencing confirmation obtains pASPI-CSf123.
4) builds the end of the hog cholera vaccine 3 ' recombinant plasmid pA-CSf456-T of the terminator of fusion mouse RNA polymerase II
According to mouse rna plymerase i terminator sequence (SEQ ID NO.2) and the end sequence of hog cholera vaccine C strains 3 ', design
The long aligning primer of synthesis fusion mouse terminator sequence, the end of hog cholera vaccine cDNA sequence 3 ' is fused to by PCR by mouse terminator
End：
Sense primer：Sense primer P4F (SEQ ID NO.9)
5′-AGAGGATCCATCTAACCTGAGGG-3′
Anti-sense primer CS-RTI(SEQ ID NO.22):
5′-CGACGCGTCGGAGTACTGGTCGACCTCCGAAGTTGGGGGGGAGGGCCGTTAGAAATTAC-3′
(underscore part is mouse terminator sequence)
Using recombinant plasmid pA-CSf456 as template, upstream and downstream primer is respectively P4F and CS-RTI,By following reaction system Enter performing PCR amplification with condition：
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 60Sec carry out 33 circulations, obtain the hog cholera vaccine 3 ' of the terminator of fusion mouse RNA polymerase I
End PCR primer CSf456-TIFragment.
The double digested plasmid pA-CSf456 and CSf456-T of restriction enzyme SphI and MluI is used respectivelyIFragment,
The purifying of DNA QIAquick Gel Extraction Kits prepares linearisation pA-CSf456 and CSf456-T with double digestion cohesive endIFragment, two kinds
Fragment presses 1:3-1:10 molar ratios are mixed, the connection of T4DNA ligases, conversion, select positive colony, and sequencing confirmation obtains pA-
CSf456-TI。
5) is based on pig rna plymerase i promoter (pSPolI) and mouse rna plymerase i terminator (TI) hog cholera vaccine
Infectious CDNA clones pASPITI- CS is built：
By the end of the hog cholera vaccine 5 ' large fragment plasmid pSPI-CSf123 and 3 ' ends large fragment plasmid pA- of above-mentioned structure
CSf456-TIIt is double digested with BamHI and MluI respectively, reclaim linearisation pSPI-CSf123 and CSf456-TIFragment piece
Section, both press 4:1 molar ratio is mixed, the connection of T4DNA ligases, conversion, selects positive colony, and sequencing confirmation obtains swine fever
Vaccine infection cDNA clone plasmid pASPITI- CS (Fig. 3 A)；
By above-mentioned hog cholera vaccine cDNA clone plasmid pASPITI- CS transformed competence colibacillus E. coli DH10B,
The agar plate culture containing amicillin resistance is coated with, selects and is cultivated in positive recombinant inoculation LB, extract DNA, will
Escherichia coli DH10B/pASPITI-CS, Escherichia containing hog cholera vaccine cDNA clone plasmid pASPITI-CS
coli DH10B/pASPITI- CS, Chinese Wuhan Wuhan Universitys Chinese Typical Representative culture guarantor is delivered on January 23rd, 2015
Tibetan center preservation, deposit number is CCTCC NO：M2015062.
6) prepared by hog cholera vaccine kind poison of the based on new strategy hog cholera vaccine infectious CDNA clones
When preparing hog cholera vaccine kind poison, plasmid containing infectious CDNA clones is taken to preserve bacterium E.coli DH10B/ first
pASPITIOn-CS streak inoculation LB flat boards, 37 DEG C of overnight incubations, next day is chosen in single bacterium colony inoculation LB liquid medium, and 200
Rev/min shaking, 37 DEG C of overnight incubations, collect bacterium solution, by kit specification operation extract plasmid (plasmid extraction kit
D6943-02, Omega Products), agarose cohesion electrophoresis detection, -70 DEG C save backup.
Take growth 6 porocyte culture plates in, density be 80% individual layer pig source PK15 or SK6 cells, using liposome
Lipofectamine2000, transfection cDNA clone plasmid pASPITI- CS (presses liposome into PK15/SK6 cells
Lipofectamine2000 transfection reagent boxes specification is operated), plasmid-transfected cells are Opti-MEM with nutrient solution
(Invitrogen Products).Transfect after 6h, use the DMEM containing 2% hyclone instead, continue to cultivate after 72h, frozen-thawed cell
Culture, 10000 revs/min are centrifuged 10 minutes, remove precipitation, collect supernatant, and detection hog cholera vaccine kind poison is produced.
Hog cholera vaccine virus produces detection：Take transfection pASPITIAfter-CS plasmids cultivate 72h pig source SK6 with rabbit-anti-
NS3 polyclonal antibodies are primary antibody, and secondary antibody is fluorescein (Alexa Flour488) mark goat anti-rabbit igg antibody (Invitrogen
Products) carry out indirect IF staining detection hog cholera vaccine kind poison.As a result as shown in Figure 4 A, (green is glimmering for stained positive
Light) cell for produce hog cholera vaccine virocyte.Sense is harvested after collecting cell cracking supernatant inoculation PK15 cells, culture 72h
Cell is contaminated, total serum IgE is extracted and carries out RT-PCR detection viral nucleic acids.As a result as shown in Figure 4 B, it is inoculated with prepared hog cholera vaccine kind poison
PK15 cells in detection with the presence of hog cholera vaccine virus genome RNA.
Based on new strategy hog cholera vaccine infectious CDNA clones pASPITI- CS is transfected to PK15/SK6 cellular rescue swine fevers
Vaccine kind poison operation principle be：pASPITIIt is poly- in pig RNA that-CS plasmids include hog cholera vaccine C pnca genes group cDNA, the cDNA
Under synthase I promoters and the control of mouse rna plymerase i terminator, transcription produces hog cholera vaccine disease in pig source cell PK15/SK6
Virus gene group vRNA, translates by template of vRNA, processes generation virus protein, while the vRNA, which replicate, produces filial generation disease
Virus gene group vRNA, then carries out virion assembling, produces hog cholera vaccine progeny virus (as shown in Figure 3 B).
Amplicon virus is passed in PK15/SK6 cell lines, genetic engineering hog cholera vaccine kind poison, packing, -70 DEG C of jellies are obtained
Deposit standby.
【Embodiment 2】Express the structure of foreign protein luciferase Recombinant Swine pest vaccine virus
Encoded according to C plants of full-length genome sequences of hog cholera vaccine (Genbank No.AF091507), sea cucumber luciferase
Sequence (GenBank No.AF025846.2), foot and mouth disease virus (FMDV) 2A coded sequences (GenBank No.M95781.1),
The various RT-PCR of design synthesis or pcr amplification primer thing.Each primer is synthesized by Shanghai Sheng Gong companies.
1. build the recombinant plasmid pASPI-CSf123/RLuc of the end of fusion luciferase gene hog cholera vaccine 5 '
A) the nearly 5 ' terminal restriction restriction endonuclease PmlI site fragments of hog cholera vaccines genome (are located at genome 290-
900nt) prepare：Primer, the nearly 5 ' terminal restriction restriction endonuclease of PCR amplification gene groups are designed according to hog cholera vaccine genome sequence
PmlI site fragments：
Sense primer CS-fPmF (SEQ ID NO.23)：
5′-CACCACGTGATGGGAGTACG-3′
Anti-sense primer CS-fPmR (SEQ ID NO.24):
5′-TCCCACTCGCGCCATC-3′；
Using pASPI-CSf123 as template, using CS-fPmF/CS-fPmR primers,Expanded by following reaction system and condition
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 30Sec carry out 33 circulations, obtain CS-fPm fragment PCR products.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
B) merges the luciferase encoding gene fragment preparation of foot and mouth disease virus (FMDV) 2A albumen coded sequences：According to
Luciferase gene sequence (GenBank No.AF025846.2) and (FMDV) 2A albumen coded sequences (GenBank
No.M95781.1 primer, PCR amplification fusion RLuc2A fragments, 7 amino of sense primer C protein containing hog cholera vaccine N-terminal) are designed
Coding sequences, anti-sense primer coded sequence containing 2A：
Sense primer Luc2AF (SEQ ID NO.25):
5′-CACCACGTGATGGGAGTACG-3′
Anti-sense primer Luc2AR (SEQ ID NO.26):
5′-CCACTTGCGCCATCATCGGAGGGCCCTGGGTTGGACTCGACGTCTCCGGCCAACTTGAGAAGGTCA AAGTTTTGTTCATTTTTGAG-3′
Underscore part is 2A genes.
With pRL-TK plasmids (Promega Products) for template, using Luc2AF/Luc2AFR primers,By following reaction System and condition amplificationLuc2A fusion sequences：
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 30Sec carry out 33 circulations, obtain RLuc2A fragment PCR products.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
C) the nearly 5 ' terminal restriction restriction endonuclease SpeI site fragments of hog cholera vaccines genome (are located at genome 878-
2442nt) prepare：Primer, the nearly 5 ' terminal restriction restriction endonuclease of PCR amplification gene groups are designed according to hog cholera vaccine genome sequence
SpeI site fragments：
Sense primer CS-fSpF (SEQ ID NO.27):
5′-CACCACGTGATGGGAGTACG-3′
Anti-sense primer CS-fSpR (SEQ ID NO.28):
5′-TCCCACTCGCGCCATC-3′；
Using pASPI-CSf123 plasmids as template, using CS-fSpF/CS-fSpR primers,By following reaction system and condition Amplification
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 45Sec carry out 33 circulations, obtain CS-fSp fragment PCR products.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
D) merges the end large fragment CSf123/Luc of luciferase gene hog cholera vaccine 5 ' and prepared
With the nearly 5 ' terminal restriction restriction endonuclease PmlI site fragments CS-fPm of the hog cholera vaccine genome of above-mentioned preparation,
RLuc2A fragments and the nearly 5 ' terminal restriction restriction endonuclease SpeI site fragments CS-fSp mixtures of genome are template, with CS-
FPmF is that sense primer, CS-fSpR are anti-sense primer, PCR amplifications：
Expanded by following reaction system and condition
Said components are mixed, following react is carried out on PCR thermal cyclers：98 DEG C of denaturation 30Sec, then 98 DEG C
10Sec, 55 DEG C of 30Sec, 72 DEG C of 90Sec carry out 33 circulations, obtain RLuc2A code areas and are fused to the end of hog cholera vaccine 5 ' with frame
Terminal sequence, PCR primer CS-fPm-RLuc2A-CS-fSp fragments.
Using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup.
E) merges the recombinant plasmid pASPI-CSf123/Luc structures of the end of luciferase gene hog cholera vaccine 5 '
The double digested plasmid pSPI-CSf123 and CS-fPm- of restriction enzyme PmlI and SpeI is used respectively
RLuc2A-CS-fSp DNA fragmentations, DNA QIAquick Gel Extraction Kit purifying prepares linearized vector and CS-fPm-RLuc2A-CS-fSp
Fragment, two kinds of fragments press 1:3-1:10 molar ratios are mixed, the connection of T4DNA ligases, conversion, select positive colony, and sequencing is true
Card, obtains pASPI-CSf123/RLuc recombinant plasmids.
2. merge luciferase gene hog cholera vaccine recombinant plasmid pASPITI- CS/RLuc is built
By the end of the hog cholera vaccine 5 ' large fragment plasmid pASPI-CSf123/RLuc and 3 ' ends large fragment of above-mentioned structure
Plasmid pA-CSf456-TIIt is double digested with BamHI and MluI respectively, reclaim linearisation pSPI- CSf123/Luc and
CSf456-TISegments-segment, both press 4:1 molar ratio is mixed, the connection of T4DNA ligases, conversion, is selected positive colony, is surveyed
Sequence is confirmed, and obtains hog cholera vaccine infectious CDNA clones plasmid pASPITI-CS/RLuc；
3. it is prepared by expressing luciferase Recombinant Swine pestilence seed poison
By above-mentioned hog cholera vaccine cDNA clone plasmid pASPITI- CS/Luc transformed competence colibacillus E. colis
DH10B, is coated with the agar plate culture containing amicillin resistance, selects and is cultivated in positive recombinant inoculation LB, extracts plasmid
DNA。
The pig source PK15 or SK6 cells of 80% individual layer of growth are taken, using liposome Lipofectamine2000, transfection
CDNA clone plasmid pASPITI- CS/Luc (presses liposome Lipofectamine2000 transfection reagents into PK15/SK6 cells
Box specification is operated), transfectional cell is Opti-MEM (Invitrogen Products) with nutrient solution.Transfect after 6h, use instead and contain
The DMEM of 2% hyclone, continues to cultivate after 72h, and frozen-thawed cell culture collects viral supernatant liquid.Swine fever virus resistant NS3
Antibody mediated immunity fluoroscopic examination is shown, transfects pASPITIThe PK15 cellular rescues of-CS/Luc DNAs go out hog cholera vaccine virus
(Fig. 5 A).
Amplicon virus is passed in PK15/SK6 cell lines, the Recombinant Swine pestilence seed poison of expressing luciferase is obtained, point
Dress, -70 DEG C freeze it is standby.
4. the chimeric hog cholera vaccine expressing protein uciferase activity of restructuring is determined
A) takes stand density 85-95% individual layer PK15 cells, by the Recombinant Swine pestilence seed toxogen of expressing luciferase
Liquid press cell after serial doubling dilution inoculation PK15 cells, virus inoculation add the DMEM nutrient solutions of 2% hyclone in 37 DEG C,
5%CO2CMC model 24h, harvesting blots nutrient solution, PBS washings cell is once.
B) uciferase activities are determined, and the operation of kit (Promega) specification is determined by uciferase activity.Specifically
Ground：4ml 5 × sea cucumber luciferase lysis buffer is taken, in the aseptic deionized water for adding 16ml, is mixed, preparation 1 × thin
Cellular lysate liquid, 1 × cell pyrolysis liquid is added in each hole of 24 orifice plates, 50 μ l/ holes, lysis at room temperature 15~20min of cell, phase
Between shake 24 orifice plates, so that all cells are cleaved；5 μ l cell pyrolysis liquids and 5 μ l luciferase substrates solution are taken to add respectively
Into 1.5ml EP pipes mix, immediately by mixed solution be put into luciferase detector (_20/
In 20Luminometer), fluorescent value is read, time delay is set to 10s.Experimental result is shown, is compared with negative control cell,
Infection restructuring CSFV PK15 cells express high levels uciferase activity (Fig. 5 B).
5. the chimeric hog cholera vaccine virus titer of restructuring is determined
Take be grown in 12 orifice plates, density 85-95% individual layer PK15 cells, by Recombinant Swine pestilence seed toxogen liquid dilute
Cell adds the DMEM nutrient solutions of 2% hyclone in 37 DEG C, 5%CO after MOI=0.01 inoculating cells, virus inoculation2Condition is trained
Support, respectively at the 6th after infection virus, 12,24,48,72h, take out infection virocyte (include supernatant), freeze thawing twice, from
The heart collects supernatant, uses anti-NS3 polyclonal antibodies for primary antibody, and indirect IF staining determines virus TCID50.As a result show
Show, titre of the chimeric hog cholera vaccine virus of restructuring on PK15 cells is about 104TCID50/ml。
【Embodiment 3】Recombinate the anti-CSFV of hog cholera vaccine induction pig body immune efficacy detection
1. pig body immunity inoculation
Choose and be not inoculated with swine fever, be good for through Immunofluorescence test CSFV is negative, ELISA detection CSFV serum antibodies are negative
2 monthly age of health piglet, is randomly divided into two groups, for vaccination experiments group and non-vaccine inoculation control group.
Specifically, the newborn 6 week old sodium selenites for not being inoculated with hog cholera vaccine are taken, vena cava anterior blood sampling is divided into two parts, one
Part separation serum, CSFV antibody is detected for ELISA；Another whole blood is detected for CSFV.Specific detection method：Take
Pig whole blood freezing-thawing and cracking, centrifuges supernatant, carries out 10 times with serum-free DMEM and be serially diluted, and is inoculated with PK15 cells, culture
Indirect IF staining, inverted fluorescence microscope observation are carried out after 72h by primary antibody of NS3 antibody.It is all immunofluorescence dye occur
Cytochrome is determined as the positive.Choose CSFV and negative antibody piglet 6 is randomly divided into two groups, subfield is raised, to 8 week old
Blood sampling detects serum antibody again, and negative antibody sodium selenite is used to test.
Vaccination experiments group：Every pig is inoculated with 1 × 10 through intramuscular injection path4TCID50Recombinant vaccine C strain virus.It is non-
Vaccine inoculation negative control group：Using through the isometric PBS of intramuscular injection.
After vaccine inoculation, measurement of bldy temperature and clinical observation are carried out to experimental group and each head pig of control group daily.After immune
The 7th, 14 and 21 days, gather pig vena cava anterior blood, separate serum, determine CSFV neutralizing antibodies.
2. pig body challenge test
Pig body immunity inoculation is after 23 days, with 5 × 105TCID50CSFV crossdrift velogen strain virus liquids, using collunarium approach
Malicious infection is attacked, each head temperature of pig body change, record pig morbidity clinical symptoms and death toll are measured after poison infection daily in attacking；Respectively
In attacking the 4th after poison, vena cava anterior blood sampling in 8,14 and 21 days, separate serum, detect change of serum C SFV neutralize antibody titers.
Neutralizing antibody is detected
(1) presses 1 × 105Cell/ml concentration inoculation PK15 cell suspensions (100 μ l/ in the well culture plate of flat-bottomed microtiter 96
Hole)；5%CO237 DEG C of cultures extremely form 70%~80% cell monolayer；
(2) Swine serum to be detected is inactivated 30min by 56 DEG C, is made 2 times with serum-free EMEM nutrient solutions and is serially diluted；Will
The serum to be checked of dilution is with containing 100TCID50/ 50 μ l CSFV virus liquids are mixed in equal volume, put 37 DEG C of incubation 1-2h；
(3) washs PK15 cell monolayers with serum-free DMEM (SH30002.01, HyClone Products), adds blood
Clearly/virus mixed liquor (100 μ l/ holes), 37 DEG C of incubation 1h, sucks serum/virus mixed liquor, adds containing 2% calf serum
DMEM nutrient solutions, 5%CO2, 37 DEG C continue to cultivate 48-72h；
(4) takes out Tissue Culture Plate, and the IIF as described in implementing 2 steps 3 detects serum neutralization titer
(every part of blood serum sample is repeated 2 times experiment).Specifically, it is inoculation serum/about 90% individual layer PK15 of virus mixed liquor inoculation is thin
After born of the same parents, culture cell 48-72h, culture medium is discarded, cell is washed with PBS 3 times, the acetonformaldehyde (1 of precooling:1) -20 DEG C of fixations
1h, successively using rabbit-anti CSFV NS3 as primary antibody (1:300 dilution), goat anti-rabbit igg-FITC be secondary antibody (1:400 dilutions) it is immune glimmering
Light is dyed, and result is observed under Olympus inverted fluorescence microscopes.
(5) neutralize antibody titers judge：Can neutralize the infective serum highest extension rates of CSFV, (unstressed configuration is dyed
Reaction) it is used as the neutralization titer of serum.
As a result as shown in table 1, CSFV velogen strains start on the 3rd day after attacking poison infection, vaccinated pig CSFV neutralizing antibodies effect
Valency is persistently raised, and neutralizing antibody is can't detect in non-vaccine inoculation negative control group Swine serum.
Table 1 recombinates serum neutralize antibody titers after hog cholera vaccine Pigs Inoculated and the strong malicious lethal challenges of control pig body CSFV
3) immunoprotection of the strong malicious lethal challenges of the anti-CSFV of vaccine inoculations induction pig body
Observation result is shown (Fig. 6 and table 2) after experiment pig is infected with CSFV strong virus attacks, non-vaccine inoculation negative control
Group experiment pig is attacked after poison, and mean body temperature started to increase to over 40 DEG C in the 3rd day, the 4th day all 3 temperature of pig body more than 41 DEG C, and
Continue to that all pigs are dead.There is 1 temperature of pig body slightly to raise (40.7) in vaccine immunization pig of the present invention, continue 5
It and then recovery are normal, and body temperature is normal always for remaining 2 pig, all vaccinated pigs survivals.
Table 2 recombinates Temperature changing and survival condition after hog cholera vaccine Pigs Inoculated and the strong malicious lethal infections of control pig body CSFV
SEQUENCE LISTING
<110>Wuhan University
<120>A kind of hog cholera vaccine infectious CDNA and its construction method and application
<130>
<160> 28
<170> PatentIn version 3.3
<210> 1
<211> 388
<212> DNA
<213>Pig rna plymerase i promoter sequence
<400> 1
cggagtgttt ccctgtcggt cggtcgatcg gtcgggaggt ggggaccggc ctgagctgga 60
tggtgtgtcc tggattttgg gggagccaag tccccgtctg gagctccgga cagaccgata 120
cctgcccgcg tgggcaagcc gggaagggct tcccggctgg ccggccggct ccacctcctt 180
catgtccctg tcccttccct gcggtcacgc tccccgggtc gaccagatgg ctctgagagc 240
gctgggtctg gcgactctag ggcagggctg ggggacaagt gtccggatgg gggttccggg 300
gataccccca cgtcctgtgg gtgggccccg ctgctgggca tggacatttt tcgcggccga 360
aatacgcctt ttctgtcacc aggtagat 388
<210> 2
<211> 34
<212> DNA
<213>Mouse terminator sequence
<400> 2
tcccccccaa cttcggaggt cgaccagtac tccg 34
<210> 3
<211> 49
<212> DNA
<213> Artificial
<223> P1F
<400> 3
gcgtcgactc ttaatacgac tcactatagt atacgaggtt agttcattc 49
<210> 4
<211> 33
<212> DNA
<213> Artificial
<223> P1R
<400> 4
ccagttacta gtaacagcca taccacacct tgc 33
<210> 5
<211> 31
<212> DNA
<213> Artificial
<223> P2F
<400> 5
gctgttacta gtaactgggg cacaaggccg g 31
<210> 6
<211> 25
<212> DNA
<213> Artificial
<223> P2R
<400> 6
catggtacct gtcattgaac ttgtc 25
<210> 7
<211> 26
<212> DNA
<213> Artificial
<223> P3F
<400> 7
caatgacagg taccatgttg ccattg 26
<210> 8
<211> 22
<212> DNA
<213> Artificial
<223> P3R
<400> 8
caccctcagg ttagatggat cc 22
<210> 9
<211> 23
<212> DNA
<213> Artificial
<223> P4F
<400> 9
agaggatcca tctaacctga ggg 23
<210> 10
<211> 25
<212> DNA
<213> Artificial
<223> P4R
<400> 10
atttctgcag agaaatgaac cttcc 25
<210> 11
<211> 26
<212> DNA
<213> Artificial
<223> P5F
<400> 11
gaggaggaag gttcatttct ctgcag 26
<210> 12
<211> 21
<212> DNA
<213> Artificial
<223> P5R
<400> 12
catttagcat gctgttgccc g 21
<210> 13
<211> 21
<212> DNA
<213> Artificial
<223> P6F
<400> 13
cgggcaacag catgctaaat g 21
<210> 14
<211> 23
<212> DNA
<213> Artificial
<223> P6R
<400> 14
tacgcgtgag cccgggccgt tag 23
<210> 15
<211> 25
<212> DNA
<213> Artificial
<223> SPI-F
<400> 15
ccggatccgg agtgtttccc tgtcg 25
<210> 16
<211> 51
<212> DNA
<213> Artificial
<223> SPI-R
<400> 16
agggtacctc tagactcgag cgtctccatc tacctggtga cagaaaaggc g 51
<210> 17
<211> 33
<212> DNA
<213> Artificial
<223> CS-F
<400> 17
caggtagatg tatacgaggt tagttcattc tcg 33
<210> 18
<211> 19
<212> DNA
<213> Artificial
<223> CS-R
<400> 18
ccatcgatgc acacataag 19
<210> 19
<211> 585
<212> DNA
<213>PACNR1180 is used for cloning region sequence
<400> 19
aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 60
ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 120
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacc tgaggtaatt 180
ataacccggg ccctatatat ggatccaatt gcaatgatca tcatgacaga tctgcgcgcg 240
atcgatatca gcgctttaaa tttgcgcatg ctagctatag ttctagaggt accggttgtt 300
aacgttagcc ggctacgtat actccggaat attaataggc ctaggatgca tatggcggcc 360
gcctgcagct ggcgccatcg atacgcgtac gtcgcgaccg cggacatgta cagagctcga 420
gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc 480
agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt 540
ttgagacaca acgtggcttt gttgaataaa tcgaactttt gctga 585
<210> 20
<211> 24
<212> DNA
<213> Artificial
<223> ACNRF
<400> 20
tggactaagg taatttctaa cggc 24
<210> 21
<211> 24
<212> DNA
<213> Artificial
<223> ACNRR
<400> 21
actccggagt cgacgtcagg tggc 24
<210> 22
<211> 59
<212> DNA
<213> Artificial
<223> CS-RTI
<400> 22
cgacgcgtcg gagtactggt cgacctccga agttgggggg gagggccgtt agaaattac 59
<210> 23
<211> 20
<212> DNA
<213> Artificial
<223> CS-fPmF
<400> 23
caccacgtga tgggagtacg 20
<210> 24
<211> 16
<212> DNA
<213> Artificial
<223> CS-fPmR
<400> 24
tcccactcgc gccatc 16
<210> 25
<211> 20
<212> DNA
<213> Artificial
<223> Luc2AF
<400> 25
caccacgtga tgggagtacg 20
<210> 26
<211> 86
<212> DNA
<213> Artificial
<223> Luc2AR
<400> 26
ccacttgcgc catcatcgga gggccctggg ttggactcga cgtctccggc caacttgaga 60
aggtcaaagt tttgttcatt tttgag 86
<210> 27
<211> 20
<212> DNA
<213> Artificial
<223> CS-fSpF
<400> 27
caccacgtga tgggagtacg 20
<210> 28
<211> 16
<212> DNA
<213> Artificial
<223> CS-fSpR
<400> 28
tcccactcgc gccatc 16
Claims (9)
1. a kind of plasmid pASPIT of the full-length infectious CDNA comprising hog cholera vaccine kind poisonI- CS, it is characterised in that：Described is complete
Long infectious CDNA includes C plants of full-length cDNAs of hog cholera vaccine, in the terminal fusion pig rna plymerase i promoters of cDNA 5 ', sequence
It is classified as SEQ ID NO：Shown in 1, in the ends of cDNA 3 ' insertion mouse rna plymerase i terminator, sequence is SEQ ID NO：Shown in 2；
Convert plasmid pASPITI- CS Escherichia coli DH10B/pASPITI-CS(E.coli DH10B/pASPITI- CS), in being deposited in
State's Type Tissue Collection, preserving number is CCTCC NO：M 2015062.
2. a kind of plasmid pASPIT built described in claim 1I- CS method, it is characterised in that the plasmid is by walking as follows
Suddenly prepare：
Step 1) preparation of hog cholera vaccine C pnca gene group cDNA fragments：
Using C plants of RNA of hog cholera vaccine as template, set according to C plants of full-length genome sequence Genbank No.AF091507 of hog cholera vaccine
Meter 6 pairs of specific PCR amplimers of synthesis, its sequence such as SEQ ID NO：Shown in 3-14, using RT-PCR, hog cholera vaccine is obtained
6 cDNA fragments CSf1-6 of C plants of full-length genomes；
Step 2) the long genome large fragment structure in the end of hog cholera vaccine C strains 5 ' half：
Fragment CSf1, CSf2 and CSf3 are consecutively connected to low-copy plasmid pACNR1180, pA-CSf123 is built；
Step 3) the long genome large fragment structure in the end of hog cholera vaccine C strains 3 ' half：
Fragment CSf4, CSf5 and CSf6 are consecutively connected to low-copy plasmid pACNR1180, pA-CSf456 is built；
Step 4) build fusion pig rna plymerase i promoter the end of hog cholera vaccine 5 ' recombinant plasmid pSPI-CSf123：
(1) prepared by pig rna plymerase i promoter pSPolI fragments：According to pig rna plymerase i promoter sequence GenBank
No.L31782.1 designs promoter fragment pcr amplification primer thing, its sequence such as SEQ ID NO：Shown in 15-16, with pig source PK-15
The DNA of cell is template, expands pig rna plymerase i promoter, and using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing is true
Card, -70 DEG C save backup；
(2) the end 784bp fragment amplifications of hog cholera vaccine C strains 5 '：Hog cholera vaccine cDNA sequence is built, one couple of PCR primers is designed, its
Sequence such as SEQ ID NO：Shown in 17-18, the end 784bp fragments of amplification gene group 5 ', using pA-CSf123 recombinant plasmids as mould
Plate, using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup；
(3) pACNR1180 vector backbone segments are expanded：According to pACNR1180 carrier sequence SEQ ID NO：19 design amplifications are drawn
Thing, its sequence such as SEQ ID NO：Shown in 20-21, using pA-CSf123 recombinant plasmids as template, pACNR1180 carrier bones are obtained
Frame fragment PCR products, using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup；
(4) the pACNR1180 vector backbone segments amplification of the end of fusion hog cholera vaccine C strains 5 ' and pig rna plymerase i promoter：
With the end 784bp fragments of hog cholera vaccine C strains 5 ', pig rna plymerase i promoter and the pACNR1180 carrier frameworks of above-mentioned preparation
DNA fragmentation mixture is template, with SEQ ID NO：20 be sense primer, SEQ ID NO：18 be anti-sense primer, and PCR amplifications are obtained
The pACNR1180 vector backbone segment PCR primers A-SPI- of the end of hog cholera vaccine 5 ' and pig rna plymerase i promoter must be merged
CS-5 ', using DNA QIAquick Gel Extraction Kit purified pcr products, sequencing confirmation, -70 DEG C save backup；
(5) end of the hog cholera vaccine 5 ' recombinant plasmid pSPI-CSf123 of the promoter of fusion pig RNA polymerase I is built：Respectively with limit
The double digested plasmid pA-CSf123 and A-SPI-CS-5 ' DNA fragmentations of property restriction endonuclease PmlI and MluI processed, DNA QIAquick Gel Extraction Kits
Purifying prepares △ CSf123 and A-SPI-CS-5 ' fragments, and two kinds of fragments press 1:2 mixing, the connection of T4DNA ligases, conversion, are selected
Positive colony, sequencing confirmation obtains pSPI-CSf123；
Step 5) build the terminator T of fusion mouse RNA polymerase IIThe end of hog cholera vaccine 3 ' recombinant plasmid pA-CSf456-TI：
According to mouse rna plymerase i terminator sequence SEQ ID NO：The end sequence of 2 and hog cholera vaccine C strains 3 ', design synthesis fusion
The long aligning primer of mouse terminator sequence, its sequence such as SEQ ID NO：9th, 23, using recombinant plasmid pA-CSf456 as template, lead to
Cross the end of the hog cholera vaccine 3 ' PCR primer CSf456-T that PCR obtains the terminator of fusion mouse RNA polymerase IIFragment；
The double digested plasmid pA-CSf456 and CSf456-T of restriction enzyme SphI and MluI is used respectivelyIFragment, DNA is reclaimed
Kits, prepare the linearisation pA-CSf456 and CSf456-T of double digestion cohesive endIFragment, two kinds of fragments press 1:3-
1:10 molar ratios are mixed, the connection of T4 DNA ligases, conversion, select positive colony, and sequencing confirmation obtains plasmid pA-
CSf456-TI；
Step 6) it is based on pig rna plymerase i promoter pSPolI and mouse rna plymerase i terminator TIHog cholera vaccine it is infectious
CDNA clone pASPITI- CS is built：
By the end of the hog cholera vaccine 5 ' large fragment plasmid pSPI-CSf123 and 3 ' ends large fragment plasmid pA- of above-mentioned structure
CSf456-TIIt is double digested with BamHI and MluI respectively, reclaim linearisation pSPI-CSf123 and A-CSf456-TIFragment, two
Person presses 4:1 molar ratio is mixed, the connection of T4 DNA ligases, conversion, selects positive colony, and sequencing confirmation obtains hog cholera vaccine
Infectious CDNA clones plasmid pASPITI-CS。
3. the plasmid pASPIT of claim 1IApplications of-the CS in saving hog cholera vaccine C plants, including use liposome
Lipofectamine2000, transfection cDNA clone plasmid pASPITI- CS is thin to the sensitive PK15 cells of CSFV or SK6
Born of the same parents, rescue obtains C plants of hog cholera vaccine kind poison and plants poison.
4. the plasmid pASPIT of claim 1IC plants of hog cholera vaccine the answering in the vaccine for preparing prevention swine fever that-CS rescues are obtained
With.
5. the plasmid pASPIT of claim 1I- CS is used for hog cholera vaccine and modifies transformation, to prepare high-titer hog cholera vaccine.
6. the plasmid pASPIT of claim 1I- CS is used to express exogenous antigen albumen, to prepare bivalent or polyvalent recombinant swine fever epidemic disease
Seedling.
7. the plasmid pASPIT of claim 1IApplications of-the CS as carrier in expression foreign protein, it is characterised in that including such as
Lower step：
1) external source antigen-4 fusion protein gene is obtained using the amplification of multi-step over-lap PCR, its 5 ' end fusion CSFV 7 ammonia of C protein
Base coding sequences, its 3 ' end fusion foot and mouth disease virus 2A albumen coded sequence；
2) the external source antigen-4 fusion protein gene of acquisition is inserted into hog cholera vaccine genome NproBetween C protein code area, table is obtained
Restructuring hog cholera vaccine up to foreign protein is fitted together to infectious CDNA clones.
8. the application described in claim 7, it is characterised in that：Described foreign protein be using luciferase as reporter gene, it is glimmering
Light element enzyme Luc genes are fused to Luc2A with foot and mouth disease virus 2A GFPs.
9. the application described in claim 7, it is characterised in that：Described foreign protein is other swine disease pathogen protective antigens
Albumen.
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| EP0351901A1 (en) * | 1988-06-23 | 1990-01-24 | Centraal Diergeneeskundig Instituut | Swine kidney cell culture, and its use in vaccine production |
| CN101914566A (en) * | 2010-06-08 | 2010-12-15 | 浙江大学 | Construction and application of E2 gene-based insertable swine fever virus cDNA vector |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0351901A1 (en) * | 1988-06-23 | 1990-01-24 | Centraal Diergeneeskundig Instituut | Swine kidney cell culture, and its use in vaccine production |
| CN101914566A (en) * | 2010-06-08 | 2010-12-15 | 浙江大学 | Construction and application of E2 gene-based insertable swine fever virus cDNA vector |
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| 利用体内转录系统对O型FMDV China99/S株的拯救;常艳燕;《甘肃农业大学硕士学位论文》;20081201;摘要 * |
| 动物RNA病毒反向遗传系统的研究和建立;郑海学;《中国农业科学院博士学位论文》;20070814;第1.2.3节，第1.2.3.2节，第11.1.6.1节，第11.2.1节 * |
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