CN104862336B - A kind of expression hepatitis B receptor --- construction method of the mouse model of people NTCP - Google Patents

Abstract

The invention discloses a kind of expression hepatitis B receptor --- construction methods of the mouse model of people NTCP.The present invention takes the transgenosis mode of hydrodynamic coefficients to be conducted into Mice Body by the micro-loop carrier of building carrier NTCP gene, makes one NTCP gene and expresses in mouse liver.The defect that this model will overcome existing experiment mice to be unable to natural infection HBV, building up one kind has hepatitis B functional receptor, can support HBV In vivo infection and the mouse model with normal immunological function.So as to really, comprehensively reflect clinical hepatitis B generation, development and chronicity process, for the pathogenic mechanism for further studying HBV, mechanism of action of the pathological change and body immune system of body in viral infection resisting and immune pathogenic course provides ideal animal model after HBV infection.

Description

A kind of expression hepatitis B receptor --- construction method of the mouse model of people NTCP

Technical field

The invention belongs to animal model preparation technical fields, and in particular to a kind of expression hepatitis B receptor --- people NTCP Mouse model construction method.

Background technique

Hepatitis type B virus (hepatitis B virus, HBV) infection is the weight that human health is influenced in global range Big problem.The chronic progressive liver inflammation lesion as caused by HBV infection, usually development is cirrhosis, liver cancer and eventually leads to It is dead.According to World Health Organization, global about 2,000,000,000 people had infected HBV, were at present HBV chronic infection there are about 3.5 hundred million people Person, the risk that these people progress to cirrhosis, hepatic failure or liver cancer are very high.30% cirrhosis and more than 50% in global range Liver cancer be as caused by HBV infection, every year because caused by HBV infection death toll be about 1,000,000-200 ten thousand.HBV infection Caused liver diseases are still a great public health problem urgently to be resolved.All the time, China is all HBV infection District occurred frequently, hepatitis B disease incidence is integrally in rising trend, and based on chronic HBV infection.Data shows: China is existing slow Property HBV infection person is about 93,000,000, and wherein chronic hepatitis B patient about 20,000,000, accounts for the 1/5 of Patients with Chronic HBV Infection By force.Guangdong is always the hotspot of virus hepatitis, and Monitoring on epidemic situation of infectious diseases information is shown: Guangdong Province's hepatitis B surface antigen is taken Band rate is 11. 10%, belongs to the high Endemic Area of hepatitis B, especially based on chronic adult's hepatitis B infection.It can be seen that China The prevention and treatment task of hepatitis B especially chronic hepatitis B is still very arduous.

Patients with Chronic HBV Infection and chronic hepatitis B patient are the main infections of HBV due to carrying a large amount of HBV Source.China all puts into a large amount of funds and manpower physical resources for the treatment of such crowd every year and prevents HBV in crowd Be propagated further.However, still lacking the means of effective treatment chronic HBV infection at present.One important reason is HBV The mechanism of chronic infection fails clear so far.And lack suitable animal model for a long time, seriously hinder the research of HBV into Exhibition.

Mankind HBV belongs to Hepadnaviridae, there is stronger species specificity, therefore setting up can be widely applied dynamic Object model not a duck soup.Lower infection people of HBV natural conditions, non-human primates (such as chimpanzee) and tree shrew.Chimp model Because its volume it is larger, it is at high cost and by ethics constraint etc. limit its application；And tree shrew model is then due to its infection effect Rate is low, is only capable of leading to of short duration low-grade infection, virus titer is very low, and is difficult to promote and apply.Researcher also once establishes HBV The small animal model of relevant other hepadnavirus carries out correlative study, such as duck hepatitis-B model, woodchuck hepatitis mould Type, ground squirrel hepatitis model etc., but these models have in pharmacokinetics and immunology genetic background with the mankind it is very big not Together, therefore all it can not become the ideal model of people HBV correlative study.

In recent years, since heredity and immune background understand, are easy to the advantages that raising, mouse is gradually by the pass of researcher Note becomes the optimal selection of building HBV animal model.Researcher also establishes people-mouse liver chimera HBV mouse model, base Because of HBV transfected mouse model and HBV transgene mouse model etc..But due to constantly multiple in vivo after HBV infection human body System, infection cycle liver cell, and thus cause a series of immune responses of body, or escape the monitoring of immune system and clear Remove, thus in vivo long-term existence and lead to the chronicity of hepatitis B, therefore to HBV pathogenic mechanism have one it is clear bright True understanding, and then effective treatment means are searched out, there is an urgent need to one kind intact immune system by researcher, and can People HBV is simulated in the animal model of the course of infection of human liver.Since mouse is not naturally by HBV infection, and built at present Vertical hepatitis B mouse model fails that mouse liver cell is overcome to infect this problem of HBV, thus is also unable to satisfy HBV research to dynamic The requirement of object model.Such as, people-mouse liver chimera HBV mouse model is able to achieve the infection duplication process of HBV, but this model is Mouse based on immune deficiency, therefore the immune response of Study Mouse can not be used for, and manufacture difficulty it is big, it is at high cost, at Power is low, it is difficult to be widely popularized utilization.Gene HBV transfected mouse model, in preparation process, HBV enters in Mice Body and can cause The immune response of host generates the cellular immunity and humoral immunity for being directed to HBV, however due to carrier and the antigen of HBV itself Property and HBV are unable to infecting mouse liver cell, cause HBV of short duration can only secrete, are not suitable for chronic HBV infection and chronic The research of type hepatitis.Although secretion HBV virus and antigen that current existing HBV transgenic mice can be stable, without each It is transfected or is transplanted, but since model is not by by the approach foundation of HBV infection, can not be used to carry out HBV entrance Infected body and HBV are in the intracorporal distribution research of machine;On the other hand, existing HBV transgenic mice is viral to HBV at present Antigen is in immune tolerance state, and body cannot generate the immune response to HBV, does not also generate hepatitis B lesion.Moreover, turning The acquisition of DNA murine needs expensive microinjection equipment and complicated operation, very time and effort consuming.The above reason results in The missing that can be used for the animal model of chronic HBV infection and the nonimmune tolerance of chronic hepatitis B, objectively limits to chronic Pathogenesis of hepatitis B, immunopathogenesis, therapeutic agent and the correlative study of immunization therapy.

How to establish it is a kind of it is cheap, easy to operate, have intact immune system, HBV can be simulated human liver's The small animal model of course of infection becomes critical issue urgently to be resolved needed for HBV research to meet.

The portal that HBV enters liver cell is viral receptor.Studies have shown that HBV has stringent host susceptibility and thermophilic Hepatocellular.The specific peptide fragment of antigen that infection early stage is present in HBV outer membrane is identified and is combined with specific receptors on liver cell, is mediated Cell entry cell starts the replicative cycle of HBV.Scholars in succession report can active hepatitis type B virus receptor, Such as polyerized human serum albumin, interior connection element II, Apolipoprotein H, IL-6, Glyceraldehyde 3-phosphate dehydrogenase, neutral metalloprotease Enzyme, human liver cell adds element etc., but does not determine the receptor to play a decisive role in HBV infection yet until the end of the year 2012.

Recently, the newest research achievement of China scientist confirm sodium ion taurocholate cotransport polypeptide (Na+/ Taurocholate Cotransporting Polypeptide, NTCP) be hepatitis type B virus critical functionality receptor. NTCP wide expression is most important sodium ion dependence taurocholate absorption system in liver cell in liver cell basolateral membrane, It is most important bile acid adsorbent system in liver, plays an important role in intestines liver circulation.The region Pre-S1 of HBV is special Identify NTCP, the infected liver cell in conjunction with it.Studies have shown that the NTCP by inhibiting the endogenous generation of liver cell, makes NTCP's Expression reduces, and reduces the content of the NTCP in liver cell, as a result considerably reduces sense of the HBV to people and tree shrew liver cell Dye.If NTCP is reduced in the HepaRG cell of enabled infection HBV, these cell infections HBV can be reduced.Usually it cannot infect Huh7 the and HepG2 cultured cell in vitro system of hepatitis B generates corresponding protein after importing NTCP gene, these cells are just It can be infected by HBV.It studies while showing: although NTCP has in multiple species such as mouse, rat, tree shrew, chimpanzee, people Expression, and have higher homology, but the difference of a few amino acid in particular combination domain leads to different plant species NTCP is identified to HBV and the greatest differences of binding ability, this is also narrow one of the reason of HBV host range.Machin NTCP generally can not in conjunction with hepatitis B, as long as but experiment show to be mutated the minimum region of its NTCP the preceding paragraph, be allowed to by people The corresponding amino acid substitution of NTCP can become effective HBV receptor, mediate infection of the HBV to machin liver cell.For This, people NTCP is considered as the hepatitis type B virus receptor of the most potential quality found so far, causes the very big of scholars Interest and concern, and be greatly prized by, this research achievement also brings preciousness simultaneously to establish ideal HBV infection model Opportunity.

But have not yet to see the mouse that people NTCP is transferred to building expression people NTCP in Mice Body by report so far in the prior art The method of model, reason may be due to: 1.NTCP is just proved to be hepatitis B functional receptor recently, and there has been no correlation models The report that mouse builds up；2. the importing form and introduction method of foreign gene directly influence external source base in animal model preparation Because of the presence and expression in animal.It is intended in different animal model preparation process by groping.It is existing for this problem Technology does not have effective solution scheme also.

Summary of the invention

The technical problem to be solved by the present invention is to lack the mouse model for expressing people NTCP in the prior art to overcome, especially It is a lack of the defect for continuing, stablizing the mouse model of expression people NTCP, provides a kind of expression hepatitis B receptor --- people The construction method of the mouse model of NTCP.

The purpose of the present invention is achieved by the following technical programs:

A kind of construction method of mouse model that expressing people NTCP, includes the following steps:

S1. people's NTCP gene eucaryon expression frame is obtained through PCR amplification from plasmid pcDNA6-hNTCP；

S2., people's NTCP gene eucaryon expression frame is inserted into attB and attP of plasmid ZY781 by molecule clone technology Between point, the micro-loop parental plasmid ZY781-CMV-hNTCP of carrier's NTCP gene eucaryon expression frame is obtained；

S3. micro-loop parental plasmid ZY781-CMV-hNTCP is transferred in E.coli ZYCY10P3S2T competence, through card That chloramphenicol resistance screening positive clone, gained recombinant plasmid obtain carrier after arabinose induction recombinase Ф C31 effect The minicircle dna (pMC-CMV-hNTCP) of NTCP eukaryotic expression cassette；

S4. it by minicircle dna (pMC-CMV-hNTCP) by hydrodynamic coefficients method, imports in Mice Body and obtains gene transfection Mouse, gene transfect mouse through PCR, can be used as model after groupization detection and the detection of related biological index and ground with to related Study carefully.

The acquisition condition of the minicircle dna of carrier NTCP eukaryotic expression cassette described in S3 are as follows: by 300 μ l micro-loop induction mother liquors 50ml is inoculated with to contain in the TB culture solution of 50 μ g/ml kanamycins, 37 DEG C, 250 rpm/min cultivate 12~18h after, to Isometric induction liquid is added in culture solution, 32 DEG C, 250 rpm/min, 5 h of culture receive bacterium, extract minicircle dna；The induction Liquid contains 20% L-arabinose of the LB culture solution of 50 ml, the NaOH solution of 2 ml 1N and 300 μ l.

Preferably, the micro-loop induces with mother liquor the preparation method comprises the following steps: parental plasmid ZY781-CMV-hNTCP is convertedE.coliZYCY10P3S2T competence, the single positive colony of picking are inoculated into TB culture solution, 37 DEG C, 250 rpm/min, training Support 15~18h to OD₆₀₀When value about 4~5, it can be used as induction with mother liquor and carry out induction generation minicircle dna.

The hydrodynamic coefficients method and step are as follows: the ICR mouse for choosing 6~8 weeks, 26~28g of weight, by 15 μ g carriers The minicircle dna of NTCP eukaryotic expression cassette is dissolved in 2.5ml physiological saline, and Mice Body is injected into 5~8s through mouse tail vein It is interior.The present invention also does control group and blank control group test respectively simultaneously with pcDNA6 and physiological saline.

People NTCP is transferred in Mice Body and makes its expression, people if the present invention imagines on the Research foundation of forefathers NTCP can combine people HBV, to enable mouse liver cell by HBV infection, while this mouse also has and normally exempts from Epidemic disease system, therefore the defect that this mouse model will can overcome existing mouse to be unable to natural infection hepatitis B are realized maximum The simulation clinic hepatitis B of degree occurs, develops and lapse to process, for the course of infection for studying hepatitis B, finds new medicine target Point and the immunotherapy for exploring hepatitis B provide ideal model.And how people NTCP to be successfully transferred in Mice Body, and make its Continue in Mice Body, stablize expression, is the Important Problems to be solved.The present invention compares carrier type, channel genes by optimization Method obtains a kind of mouse model that is sustainable, stablizing expression people NTCP.Selected carrier is micro-loop carrier ZY781；Micro-loop Carrier ZY781 is transformed again by Shenzhen Institutes of Advanced Technology, Chinese Academy of Science's Chen Zhi English etc., micro-loop carrier ZY781 in addition to Except characteristic possessed by general micro-loop carrier, micro-loop carrier ZY781 one step can also be obtained from bacterium, not needed further Purifying, yield are up to 99%.Selected method of gene introduction is hydrodynamic coefficients technology, and hydrodynamic coefficients technology is a kind of letter Single effective living gene rotaring dyeing technology is equivalent to the injection scale of construction that Mice Body weighs about 8% using injection in the short time, causes Solution pressure and capacity are more than mouse systolic pressure and cardiac output, so that plasmid is gathered in inferior caval vein, and straight through inferior caval vein It taps into and is contacted into the rich blood vessel being attached thereto, the liver with expansible structure with liver cell, so as to avoid Plasmid DNA warp Blood circulation and degraded by nuclease, transfection efficiency is substantially increased, to realize foreign gene in the intracorporal efficient table of mouse It reaches.Moreover, hydrodynamic coefficients method has good liver targeting, intracorporal gene is imported with the method and is expressed in liver Level is high by 100 compared in other organs~and 1 000 times, 40~90% may be up to the transfection efficiency of internal liver cell, so especially suitable For hepatitis virus experimental animal model and its relevant functional study.

The beneficial effects of the present invention are:

The present invention by building carrier NTCP gene micro-loop carrier, take the transgenosis mode of hydrodynamic coefficients by its It imports in Mice Body, makes one NTCP gene in liver expression.This model will overcome experiment mice to be unable to lacking for natural infection HBV It falls into, building up one kind has hepatitis B functional receptor, can support HBV In vivo infection and the mouse with normal immunological function Model.It is the cause of further research HBV so as to really, comprehensively reflect clinical hepatitis B generation, development and chronicity process Interpretation of the cause, onset and process of an illness system, the pathological change of body and body immune system are in viral infection resisting and immune pathogenic course after HBV infection Mechanism of action provides ideal animal model.The foundation of this model lacks the blank of such animal model by filling up both at home and abroad, Keep relevant research means more abundant, also provides more advanced appraisement system for the research and development of anti-hbv drug and technology is flat Platform.

Preferably animal model should be repeatable, even can be standardized.The present invention expresses hepatitis B in preparation Virus receptor --- it, will be in people's NTCP channel genes Mice Body in addition to consideration when the mouse model of people NTCP, it is often more important that What consider that mode is taken to keep people's NTCP gene to continue in Mice Body, stablize expression.Therefore, the present invention passes through optimization ratio A kind of mouse model that is sustainable, stablizing expression people NTCP is obtained compared with carrier type, method of gene introduction.

Figure of description

Fig. 1 pcDNA6-hNTCP plasmid map.

Fig. 2 .ZY781 empty carrier map.

Fig. 3 ZY781-CMV-hNTCP micro-loop parental plasmid map.

Fig. 4 pMC-hNTCP micro-loop carrier for expression of eukaryon map.

The flow chart of the mouse model of Fig. 5 building expression hNTCP.

Fig. 6 .hNTCP gene eucaryon expression frame PCR amplification electrophoretic identification.

Fig. 7 .ZY781-CMV-hNTCP BglII-SalI double digestion qualification result figure.

Fig. 8 .pMC-hNTCP and ZY781-CMV-hNTCP NdeI single endonuclease digestion result figure.

Fig. 9 gene transfects mouse PCR qualification result figure.

Figure 10 gene transfects murine liver tissue hNTCP immunohistochemistry qualification result figure.

Specific embodiment

Present invention be described in more detail with specific embodiment with reference to the accompanying drawings of the specification.Unless stated otherwise, this hair The bright reagent used, equipment is the art conventional reagent and equipment.

PcDNA6-hNTCP plasmid is given by Beijing Life Sciences Institute Li Wenhui researcher, structure chart such as Fig. 1 It is shown；E.coliZYCY10P3S2T and ZY781 empty carrier is given by Shenzhen Xianjin Technology Academe of Chinese Academy of Sciences professor Chen Zhiying, ZY781 empty carrier structure chart is as shown in Figure 2.The present invention constructs expression hepatitis B receptor --- the stream of the mouse model of people NTCP Journey figure is as shown in Figure 5.

Embodiment 1

People's NTCP gene eucaryon expression frame is obtained from pcDNA6-hNTCP using round pcr and introduces BglII- SalI Double enzyme site is then inserted between the site attB and attP of plasmid zy781, induces recombinase Ф C31 through arabinose The minicircle dna (pMC-CMV-hNTCP) of carrier's NTCP eukaryotic expression cassette is obtained after effect.

S1. the acquisition of people NTCP gene eucaryon expression frame: with pcDNA6-hNTCP plasmid (plasmid sequence such as SEQ ID NO: Shown in 1) it is template, the forward direction (band BglII restriction enzyme site) and reverse primer (band of design amplification people NTCP gene eucaryon expression frame SalI restriction enzyme site):

Forward primer F:5 '-GAAGATCTCCCGATCCCCTAT -3 '

Reverse primer R:5 '-ACGCGTCGACCCATAGAGCCCACC -3 '

PCR reaction system and response procedures are as follows:

Reaction condition:

PCR after reaction, recycles PCR product, obtains the CMV-hNTCP-BGHpolyA genetic fragment of 2260bp, CMV- HNTCP-BGHpolyA genetic fragment is through BglII(NEB) and SalI(NEB) double digestion obtain CMV-hNTCP-BGHpolyA (2248bp), i.e. hNTCP gene eucaryon expression frame.The identification of PCR result agarose gel electrophoresis is as shown in Figure 6.

S2. by between the site attB and attP of people's NTCP gene eucaryon expression frame insertion plasmid ZY781, carrier is obtained The micro-loop parental plasmid ZY781-CMV-NTCP of NTCP gene order, sequence is as shown in SEQ ID NO:2, by micro-loop parent's matter Grain ZY781-CMV-NTCP is transformed intoE.coliIn ZYCY10P3S2T competence, make through arabinose induction recombinase Ф C31 The minicircle dna of carrier's NTCP eukaryotic expression cassette is obtained after.Specific steps are as follows:

Enzyme used in digestion: hNTCP gene eucaryon expression frame and ZY781 empty carrier use BglII(NEB) and SalI(NEB) Carry out double digestion.

Endonuclease reaction system and condition: 37 DEG C of reaction temperature, digestion 15h.Endonuclease reaction system is as follows:

Digestion products purification process: gel extraction is purified using plastic recovery kit (TaKaRa, DV805A).

Digestion products connection: using connection kit (NEB, M2200S), linked system is as follows:

The 1.4 μ l of PCR product (50ug/ μ l) of BglII-SalI double digestion；

The 1.67 μ l of ZY781 (30ug/ μ l) of BglII-SalI double digestion；

ddH₂O 6.93µl ；

2x quick ligation buffer 10µl；

1 μ l of Quick T4 ligase.

After having configured solution by the above linked system, after thoroughly mixing after 25 DEG C of reaction 5min, cooled on ice, it is used for Conversion is saved in -20 DEG C.

The product being successfully connected is ZY781-CMV-hNTCP plasmid, i.e. micro-loop parental plasmid；ZY781-CMV-hNTCP The plasmid figure spectrogram of plasmid is as shown in Figure 3.Whether successful connection can be first by the conversion of next step and kanamycin resistance screening Step determines, further confirms that subsequently through digestion identification.

Conversion: it is prepared using Calcium Chloride MethodE.coliZYCY10P3S2T competent cell, by the ZY781-CMV- of building HNTCP plasmid is transferred toE.coliIn ZYCY10P3S2T competence, tentatively sieved by the kalamycin resistance that parental plasmid carries Choosing obtains positive colony, extracts plasmid, BglII-SalI double digestion electroresis appraisal.Electroresis appraisal result such as Fig. 7.(foreign gene 2248bp, carrier 4066bp)

S3. the preparation of the minicircle dna pMC-CMV-hNTCP of carrier NTCP eukaryotic expression cassette.Arabinose induction recombination The minicircle dna of carrier's NTCP eukaryotic expression cassette is obtained after enzyme Ф C31 effect.Specific step is as follows: the conversion of micro-loop parental plasmid After competence, screening positive clone, positive colony is inoculated into TB culture solution, 37 DEG C, 250 rpm/min, cultivates 12~18h To OD₆₀₀Value obtains micro-loop induction mother liquor when being about 4~5, can carry out the micro-loop of induction carrier NTCP gene eucaryon expression frame The generation of plasmid.The generation step of the micro-loop plasmid of carrier's NTCP gene eucaryon expression frame are as follows: 300 μ l micro-loops are induced and are used Mother liquor is inoculated in the TB culture solution containing kanamycins (50 μ g/ml) of 50 ml, 37 DEG C, 250 rpm/min culture After 18h, added into culture solution same volume induction liquid (the NaOH solution of the LB of 50 ml and 2 ml 1N and 300 μ l, 20% L-arabinose), 32 DEG C, 250 rpm/min cultivate 5 h, bacterium are received, using going toxin plasmid extracts kit (E.Z.N.A. Endotoxin Free Plasmid Isolation System, D6915) extracts carrier NTCP gene The micro-loop Plasmid DNA (pMC-hNTCP) of eukaryotic expression cassette, nucleotides sequence are classified as shown in SEQ ID NO:3.Through NdeI single endonuclease digestion Identification.(parental plasmid cuts out 4411bp, two band of 1903bp, and micro-loop cuts out mono- band of 2315bp) electroresis appraisal result such as Fig. 8 It is shown.The map of hNTCP gene micro-loop carrier for expression of eukaryon pMC-hNTCP is as shown in Figure 4.

Micro-loop Production conditions of the present invention are that inventor summarizes to obtain by a large amount of exploratory experiment, only upper Under the conditions of stating, a large amount of recombination micro-loop carrier can just be prepared, and the purity of carrier is very high, without miscellaneous carrier such as parent's matter The pollution of grain.It is not that amount vector cannot be guaranteed if change condition, is exactly that the purity of carrier not can guarantee, only Above-mentioned condition could meet simultaneously.

Embodiment 3

Also carry out Mediated Human NTCP gene using the carrier for expression of eukaryon of this field routine prepares animal model to the present invention simultaneously, Such as recombinant eukaryotic expression plasmid pcDNA6-hNTCP is prepared into table also by hydrodynamic coefficients technological sourcing ICR Mice Body The mouse model of intelligent's NTCP gene.But as a result, it has been found that: when with conventional carrier for expression of eukaryon come Mediated Human NTCP gene, system Standby mouse model is unable to continuous expression people's NTCP gene, is not able to satisfy the quality requirement of animal model.And it uses in the present invention The plasmid ZY781 and engineering bacteria referred toE.coliZYCY10P3S2T prepares the micro-loop carrier for expression of eukaryon of carrier NTCP, Optimization induction micro-loop Production conditions, a final step obtain the micro-loop carrier for expression of eukaryon of carrier NTCP, reduce step, remove from and mentioning Process is taken, cost is saved.

Embodiment 4 is with the mouse of the micro-loop pMC-hNTCP preparation expression people NTCP of carrier's NTCP gene eucaryon expression frame Model.

1, hydrodynamic coefficients technology preparation people NTCP gene transfects mouse: the ICR mouse of selection 6~8 weeks, and weight 26~ 28g.It is divided into three groups: experimental group, control group, blank control group.Experimental group is by the minicircle dna of carrier's NTCP eukaryotic expression cassette 15 μ g are dissolved in 2.5ml physiological saline, and pcDNA615 μ g is dissolved in 2.5ml physiological saline by control group, and blank control group is 2.5ml physiological saline is injected into Mice Body in 5-8s through mouse tail vein.

2, PCR screening and identification gene transfects mouse: respectively at the 1st, 14 day, every group took 2 mouse to take hepatic tissue, mentioned respectively Hepatic tissue DNA is taken to carry out PCR detection.

Forward primer F:5 '-CCTGGTGGCACAGTATGG -3 '

Reverse primer R:5 '-TTTGGATTTGAGGACGAT -3 '

PCR reaction system and response procedures are as follows:

Reaction condition:

Above-mentioned PCR product is subjected to 1.5%(w/v) agarose gel electrophoresis, as a result as shown in Fig. 9, the positive is one The specific band of 346bp shows that hNTCP gene is successfully transferred in Mice Body.

3. immunohistochemistry identifies that gene transfects mouse: in the 14th day, every group took 2 mouse to take hepatic tissue, set 10% formaldehyde 12~14h is fixed, makes histotomy according to a conventional method, immunohistochemistry detects the expression of hNTCP in hepatic tissue, as a result sees figure 10.(Anti-SLC10A1 antibody antibody 1:200 dilution, Sigma, HPA042727).

SEQUENCE LISTING

<110>Hospital No.458 of P.L.A.

<120>a kind of expression hepatitis B receptor --- construction method of the mouse model of people NTCP

<130>

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 6074

<212> DNA

<213>pcDNA6-hNTCP plasmid sequence

<400> 1

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gccaccatgg aggcccacaa cgcgtctgcc ccattcaact tcaccctgcc acccaacttt 960

ggcaagcgcc ccacagacct ggcactgagc gtcatcctgg tgttcatgtt gttcttcatc 1020

atgctctcgc tgggctgcac catggagttc agcaagatca aggctcactt atggaagcct 1080

aaagggctgg ccatcgccct ggtggcacag tatggcatca tgcccctcac ggcctttgtg 1140

ctgggcaagg tcttccggct gaagaacatt gaggcactgg ccatcttggt ctgtggctgc 1200

tcacctggag ggaacctgtc caatgtcttc agtctggcca tgaaggggga catgaacctc 1260

agcattgtga tgaccacctg ctccaccttc tgtgcccttg gcatgatgcc tctcctcctg 1320

tacatctact ccagggggat ctatgatggg gacctgaagg acaaggtgcc ctataaaggc 1380

atcgtgatat cactggtcct ggttctcatt ccttgcacca tagggatcgt cctcaaatcc 1440

aaacggccac aatacatgcg ctatgtcatc aagggaggga tgatcatcat tctcttgtgc 1500

agtgtggccg tcacagttct ctctgccatc aatgtgggga agagcatcat gtttgccatg 1560

acaccactct tgattgccac ctcctccctg atgcctttta ttggctttct gctgggttat 1620

gttctctctg ctctcttctg cctcaatgga cggtgcagac gcactgtcag catggagact 1680

ggatgccaaa atgtccaact ctgttccacc atcctcaatg tggcctttcc acctgaagtc 1740

attggaccac ttttcttctt tcccctcctc tacatgattt tccagcttgg agaagggctt 1800

ctcctcattg ccatattttg gtgctatgag aaattcaaga ctcccaagga taaaacaaaa 1860

atgatctaca cagctgccac aactgaagaa acaattccag gagctctggg aaatggcacc 1920

tacaaagggg aggactgctc cccttgcaca gccaccgaga cctcccaggt ggcgcccgct 1980

tagaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg 2040

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 2100

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 2160

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 2220

gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 2280

agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt 2340

gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 2400

gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 2460

gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 2520

tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 2580

ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 2640

atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 2700

aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag 2760

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 2820

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 2880

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 2940

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 3000

aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 3060

gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc 3120

acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag 3180

gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac 3240

ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc 3300

tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc 3360

agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt 3420

cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct 3480

tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc 3540

agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg 3600

ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa 3660

ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 3720

tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 3780

aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 3840

gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 3900

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 3960

cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4020

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4080

agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4140

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4200

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4260

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4320

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4380

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 4440

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 4500

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 4560

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 4620

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 4680

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 4740

actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 4800

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 4860

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 4920

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 4980

tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 5040

caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 5100

cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 5160

agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 5220

acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 5280

gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 5340

ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 5400

tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 5460

ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 5520

tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 5580

attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 5640

agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 5700

ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 5760

ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 5820

cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 5880

gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 5940

tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 6000

tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 6060

tgccacctga cgtc 6074

<210> 2

<211> 6314

<212> DNA

<213>zy781-cmv-hntcp micro-loop parental plasmid sequence

<400> 2

acattaccct gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt 60

atccctagat gacattaccc tgttatccct agatgacatt taccctgtta tccctagatg 120

acattaccct gttatcccag atgacattac cctgttatcc ctagatacat taccctgtta 180

tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt 240

accctgttat ccctagatac attaccctgt tatcccagat gacataccct gttatcccta 300

gatgacatta ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct 360

gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta tcccagatga 420

cattaccctg ttatccctag atacattacc ctgttatccc agatgacata ccctgttatc 480

cctagatgac attaccctgt tatcccagat gacattaccc tgttatccct agatacatta 540

ccctgttatc ccagatgaca taccctgtta tccctagatg acattaccct gttatcccag 600

atgacattac cctgttatcc ctagatacat taccctgtta tcccagatga cataccctgt 660

tatccctaga tgacattacc ctgttatccc agataaactc aatgatgatg atgatgatgg 720

tcgagactca gcggccgcgg tgccagggcg tgcccttggg ctccccgggc gcgactagtg 780

aattcagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat 840

agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 900

aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 960

ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 1020

tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 1080

tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 1140

cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 1200

gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 1260

tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 1320

agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 1380

ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 1440

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 1500

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 1560

gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt 1620

actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgccacc 1680

atggaggccc acaacgcgtc tgccccattc aacttcaccc tgccacccaa ctttggcaag 1740

cgccccacag acctggcact gagcgtcatc ctggtgttca tgttgttctt catcatgctc 1800

tcgctgggct gcaccatgga gttcagcaag atcaaggctc actttggaag cctaaagggc 1860

tggccatcgc cctggtggca cagtatggca tcatgcccct cacggccttt gtgctgggca 1920

aggtcttccg gctgaagaac attgaggcac tggccatctt ggtctgtggc tgctcacctg 1980

gagggaacct gtccaatgtc ttcagtctgg ccatgaaggg ggacatgaac ctcagcattg 2040

tgatgaccac ctgctccacc ttctgtgccc ttggcatgat gcctctcctc ctgtacatct 2100

actccagggg gatctatgat ggggacctga aggacaaggt gccctataaa ggcatcgtga 2160

tatcactggt cctggttctc attccttgca ccatagggat cgtcctcaaa tccaaacggc 2220

cacaatacat gcgctatgtc atcaagggag ggatgatcat cattctcttg tgcagtgtgg 2280

ccgtcacagt tctctctgcc atcaatgtgg ggaagagcat catgtttgcc atgacaccac 2340

tcttgattgc cacctcctcc ctgatgcctt ttattggctt tctgctgggt tatgttctct 2400

ctgctctctt ctgcctcaat ggacggtgca gacgcactgt cagcatggag actggatgcc 2460

aaaatgtcca actctgttcc accatcctca atgtggcctt tccacctgaa gtcattggac 2520

cacttttctt ctttcccctc ctctacatga ttttccagct tggagaaggg cttctcctca 2580

ttgccatatt ttggtgctat gagaaattca agactcccaa ggataaaaca aaaatgatct 2640

acacagctgc cacaactgaa gaaacaattc caggagctct gggaaatggc acctacaaag 2700

gggaggactg ctccccttgc acagccaccg agacctccca ggtggcgccc gcttagaccg 2760

gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta 2820

gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 2880

ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 2940

attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 3000

gcaggcatgc tggggatgcg gtgggctcta tgggtcgacc catgggggcc cgccccaact 3060

ggggtaacct ttgagttctc tcagttgggg gtaatcagca tcatgatgtg gtaccacatc 3120

atgatgctga ttataagaat gcggccgcca cactctagtg gatctcgagt taataattca 3180

gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc 3240

gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt 3300

agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc 3360

agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac 3420

gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag 3480

cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg 3540

tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt 3600

atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgtag 3660

atgacatgga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca 3720

gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc 3780

gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc 3840

gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt 3900

gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca 3960

tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagagct tgatcccctg 4020

cgccatcaga tccttggcgg cgagaaagcc atccagttta ctttgcaggg cttcccaacc 4080

ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag 4140

tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt 4200

tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga 4260

ctggctttct acgtgctcga ggggggccaa acggtctcca gcttggctgt tttggcggat 4320

gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac 4380

agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag 4440

tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc 4500

aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt 4560

ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg 4620

aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt 4680

aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt 4740

tttctaaata cattcaaata tgtatccgct catgaccaaa atcccttaac gtgagttttc 4800

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4860

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4920

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4980

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 5040

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 5100

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 5160

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5220

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5280

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 5340

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 5400

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5460

gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5520

tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5580

cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 5640

tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 5700

tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg 5760

ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 5820

gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 5880

tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa 5940

tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg 6000

tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct 6060

tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag 6120

aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg 6180

gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta 6240

atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt 6300

gcgacgctgg cgat 6314

<210> 3

<211> 2315

<212> DNA

<213>pMC- hNTCP micro-loop carrier for expression of eukaryon sequence

<400> 3

cccaactggg gtaacctttg ggctccccgg gcgcgactag tgaattcaga tctcccgatc 60

ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc 120

tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa 180

ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc 240

ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 300

aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 360

cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 420

cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 480

tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 540

ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 600

actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 660

tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 720

accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 780

gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 840

atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta 900

atacgactca ctatagggag acccaagctg gctagcgcca ccatggaggc ccacaacgcg 960

tctgccccat tcaacttcac cctgccaccc aactttggca agcgccccac agacctggca 1020

ctgagcgtca tcctggtgtt catgttgttc ttcatcatgc tctcgctggg ctgcaccatg 1080

gagttcagca agatcaaggc tcactttgga agcctaaagg gctggccatc gccctggtgg 1140

cacagtatgg catcatgccc ctcacggcct ttgtgctggg caaggtcttc cggctgaaga 1200

acattgaggc actggccatc ttggtctgtg gctgctcacc tggagggaac ctgtccaatg 1260

tcttcagtct ggccatgaag ggggacatga acctcagcat tgtgatgacc acctgctcca 1320

ccttctgtgc ccttggcatg atgcctctcc tcctgtacat ctactccagg gggatctatg 1380

atggggacct gaaggacaag gtgccctata aaggcatcgt gatatcactg gtcctggttc 1440

tcattccttg caccataggg atcgtcctca aatccaaacg gccacaatac atgcgctatg 1500

tcatcaaggg agggatgatc atcattctct tgtgcagtgt ggccgtcaca gttctctctg 1560

ccatcaatgt ggggaagagc atcatgtttg ccatgacacc actcttgatt gccacctcct 1620

ccctgatgcc ttttattggc tttctgctgg gttatgttct ctctgctctc ttctgcctca 1680

atggacggtg cagacgcact gtcagcatgg agactggatg ccaaaatgtc caactctgtt 1740

ccaccatcct caatgtggcc tttccacctg aagtcattgg accacttttc ttctttcccc 1800

tcctctacat gattttccag cttggagaag ggcttctcct cattgccata ttttggtgct 1860

atgagaaatt caagactccc aaggataaaa caaaaatgat ctacacagct gccacaactg 1920

aagaaacaat tccaggagct ctgggaaatg gcacctacaa aggggaggac tgctcccctt 1980

gcacagccac cgagacctcc caggtggcgc ccgcttagac cggtcatcat caccatcacc 2040

attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 2100

tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 2160

aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 2220

gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 2280

cggtgggctc tatgggtcga cccatggggg cccgc 2315

Claims (3)

1. a kind of construction method for the mouse model for expressing people NTCP, which comprises the steps of:

S1. amplification obtains people's NTCP gene eucaryon expression frame；

S2. between the site attB and attP by people's NTCP gene eucaryon expression frame by molecule clone technology insertion plasmid ZY781, Obtain the micro-loop parental plasmid ZY781-CMV-hNTCP of carrier's NTCP gene eucaryon expression frame；

S3. micro-loop parental plasmid ZY781-CMV-hNTCP is transferred toE.coliIn ZYCY10P3S2T competence, positive gram of screening Grand, gained positive colony obtains the micro-loop of carrier's NTCP eukaryotic expression cassette after arabinose induction recombinase Ф C31 effect DNApMC-CMV-hNTCP；

S4. it by minicircle dna by hydrodynamic coefficients method, imports in Mice Body and obtains gene transfection mouse, gene transfects mouse warp PCR can be used as model use and correlative study after groupization detection and the detection of related biological index；

Wherein, the hydrodynamic coefficients method and step are as follows: the ICR mouse for choosing 6~8 weeks, 26~28g of weight carries 15 μ g The minicircle dna of people's NTCP eukaryotic expression cassette is dissolved in 2.5ml physiological saline, and mouse is injected into 5~8s through mouse tail vein In vivo.

2. the construction method of mouse model according to claim 1, which is characterized in that minicircle dna pMC-CMV- described in S3 The acquisition condition of hNTCP are as follows: 300 μ l micro-loop inductions are inoculated with the TB for containing 50 μ g/ml kanamycins into 50ml with mother liquor and are trained In nutrient solution, 37 DEG C, 250 rpm/min cultivate 12~18h after, isometric induction liquid, 32 DEG C, 250 are added into culture solution Rpm/min cultivates 5 h, receives bacterium, extracts minicircle dna；The induction liquid contains the LB culture solution of 50 ml, 2 ml 1N 20% L-arabinose of NaOH solution and 300 μ l.

3. construction method according to claim 2, which is characterized in that the micro-loop induces with mother liquor the preparation method comprises the following steps: parent This plasmid ZY781-CMV-hNTCP conversionE.coliZYCY10P3S2T competence, the single positive colony of picking are inoculated into TB training In nutrient solution, 37 DEG C, 250 rpm/min cultivate 15~18h to OD₆₀₀When value is 4~5, it can be used as induction and lured with mother liquor Minicircle dna is given birth in artificial delivery.