CN104862336A - Building method of mouse model for expressing human NTCP (Na+/taurocholate Cotransporting Polypeptide) as hepatitis b virus receptor - Google Patents

Abstract

The invention discloses a building method of a mouse model for expressing human NTCP (Na+/taurocholate Cotransporting Polypeptide) as a hepatitis b virus receptor. A human NTCP gene carried micro ring carrier is built and introduced into a mouse body in a hydrodynamic transfection type transgenic mode so as to enable human NTCP genes to be expressed in a mouse liver. According to the building method of the mouse model for expressing the human NTCP as the hepatitis b virus receptor, the defect that the existing experimental mouse cannot be naturally infected with an HBV (Hepatitis B Virus) is overcome due to the mouse model, the mouse model comprising the HNV functional receptor is built, the HBV in-vivo infection can be supported, the normal immune function is achieved, and accordingly the clinical HBV occurrence, development and chronic process can be really and fully reflected and the ideal animal model is provided for the further research on a pathogenic mechanism of the HBV, the pathological changes of the engine body after the HBV infection and an action mechanism of an engine body immune system in the process of antiviral infection and immune pathogenesis.

Description

A kind of expression hepatitis B virus acceptor---the construction process of the mouse model of people NTCP

Technical field

The invention belongs to animal model preparing technical field, be specifically related to a kind of expression hepatitis B virus acceptor---the construction process of the mouse model of people NTCP.

Background technology

It is the significant problem affecting human health in global range that hepatitis B virus (hepatitis B virus, HBV) infects.The chronic progressive external inflammation pathology caused by HBV infection, usually develops into liver cirrhosis, liver cancer also finally causes death.According to World Health Organization, the whole world about 2,000,000,000 people infected HBV, about had 3.5 hundred million people to be HBV chronic infection at present, and these people progress is that the risk of liver cirrhosis, liver failure or liver cancer is very high.In global range, the liver cirrhosis of 30% is with the liver cancer more than 50% because HBV infection causes, and the death toll caused because of HBV infection is every year about 1,000,000-200 ten thousand.The hepatic diseases that HBV infection causes remains a great public health problem urgently to be resolved hurrily.All the time, China is all the district occurred frequently that HBV infects, and hepatitis B sickness rate entirety is in rising trend, and based on chronic HBV infection.Data shows: the existing Patients with Chronic HBV Infection of China is about 9,300 ten thousand examples, and wherein chronic hepatitis B patient about 2,000 ten thousand example, accounts for the last 1/5 of Patients with Chronic HBV Infection.Guangdong is the hotspot of viral hepatitis always, Monitoring on epidemic situation of infectious diseases information displaying: Guangdong Province's hepatitis B surface antigen carrying rate is 11. 10%, belongs to hepatitis B height Endemic Area, especially based on chronic adult's hepatitis B infection.As can be seen here, the task of preventing and treating of China's hepatitis B especially chronic hepatitis B is still very arduous.

Patients with Chronic HBV Infection and chronic hepatitis B patient, owing to carrying a large amount of HBV, are the main contagium of HBV.China all drops into a large amount of funds and manpower physical resources every year for the treatment of such crowd with stop the further propagation of HBV in crowd.But, still lack the means of effective treatment chronic HBV infection at present.An important reason is that the chronically infected mechanism of HBV is failed clearly so far.And lack suitable animal model for a long time, seriously hinder the progress of HBV.

Mankind HBV belongs to Hepadnaviridae, has stronger species specificity, therefore sets up the animal model not a duck soup of energy widespread use.People, non-human primates (as chimpanzee etc.) and tree shrew is only infected under HBV natural condition.Chimp model is applied because its volume is comparatively large, cost is high and limit it by ethics constraint etc.; Tree shrew model is then low due to its efficiency of infection, only can cause of short duration low-grade infection, and virus titer is very low, and is difficult to apply.Investigator also once established the small animal model of other hepadnavirus that HBV is correlated with to carry out correlative study, as duck hepatitis-B model, woodchuck hepatitis model, ground squirrel hepatitis model etc., but these models are very different with the mankind in pharmacokinetics and immunology genetic background, the ideal model for people HBV correlative study therefore all cannot be become.

In recent years, due to heredity and immune background clear, be easy to the advantages such as raising, mouse receives the concern of researchist gradually, becomes the optimal selection of structure HBV animal model.Researchist also establishes people-mouse liver mosaic HBV mouse model, gene transfection HBV mouse model and HBV transgene mouse model etc.But, owing to constantly copying in vivo after HBV infection human body, infection cycle liver cell, and cause a series of immune responses of body thus, or the monitoring of escape from immune system and removing, thus long-term existence and cause the chronicity of hepatitis B in vivo, therefore understanding HBV mechanism of causing a disease being had to a clear and definite is wanted, and then find out effective treatment means, researchist has intact immune system in the urgent need to one, and can simulate the animal model of people HBV in the course of infection of human liver.Because mouse is natural not by HBV infection, and current set up hepatitis B mouse model is all failed to overcome mouse liver cell and is infected this difficult problem of HBV, thus also cannot meet the requirement of HBV research to animal model.As, people-mouse liver mosaic HBV mouse model can realize the infection duplication process of HBV, but this model is the mouse based on immune deficiency, therefore can not be used for the immune response of Study Mouse, and manufacture difficulty is large, cost is high, success ratio is low, is difficult to extensively promote the use.Gene transfection HBV mouse model, in preparation process, HBV enters the immune response that can cause host in Mice Body, produce the cellular immunization for HBV and humoral immunization, but due to the antigenicity of carrier and HBV self, and HBV can not infecting mouse liver cell, causes HBV of short durationly to secrete, is not suitable for the research of chronic HBV infection and chronic hepatitis B.Although the secretion HBV virus that current existing HBV transgenic mice can be stable and antigen; without the need to carrying out transfection or transplanting at every turn; but because model is set up not by by the approach of HBV infection, therefore cannot be used for carrying out HBV and enter infected body and the distribution research of HBV in body; On the other hand, current existing HBV transgenic mice is in immune tolerance state to HBV virus antigen, and body can not produce the immune response to HBV, does not also produce hepatitis B pathology.And the acquisition of transgenic mice needs expensive microinjection equipment and complicated operation, very time and effort consuming.Above reason result in the disappearance of the animal model that can be used for chronic HBV infection and the nonimmune tolerance of chronic hepatitis B, objectively limits the correlative study to chronic hepatitis B pathogenesis, immunopathogenesis, medicine and immunotherapy.

How to set up a kind of cheap, easy to operate, have intact immune system, the key issue that HBV need become urgently to be resolved hurrily at the small animal model of the course of infection of human liver to meet HBV institute can be simulated.

HBV enters the acceptor that hepatocellular door is virus.Research shows, HBV has strict host susceptibility and addicted to Hepatocellular.Infect the antigen particular peptide section being present in HBV adventitia in early days to combine with specific receptors identification on liver cell, mediate retroviral enters cell, the replicative cycle of startup HBV.Scholars report in succession can active hepatitis B virus acceptor, as polyerized human serum albumin, the plain II of interior connection, Apolipoprotein H, IL-6, Glyceraldehyde 3-phosphate dehydrogenase, metalloprotease, the additional element of human liver cell etc., but until do not determine the acceptor played a decisive role in HBV infection the end of the year 2012 yet.

Recently, the achievement in research that China scientist is up-to-date confirms that the sodium ion taurocholate polypeptide (Na+/taurocholate Cotransporting Polypeptide, NTCP) that cotransports is the critical functionality acceptor of hepatitis B virus.NTCP wide expression, in liver cell basolateral membrane, is topmost sodium ion dependency taurocholate absorption system in liver cell, is also most important bile acid adsorbent system in liver, plays an important role in enterohepatic circulation.The Pre-S1 region specific recognition NTCP of HBV, combines and infected liver cell with it.Research shows, by suppressing the NTCP of the endogenous generation of liver cell, the expression of NTCP is reduced, reduces the content of the NTCP in liver cell, result considerably reduces HBV to people and the hepatocellular infection of tree shrew.If NTCP reduces in the HepaRG cell of enable infection HBV, these cell infections HBV just can be reduced.Usually can not infect Huh7 and the HepG2 cultured cell in vitro system of hepatitis B virus, after importing NTCP gene, produce corresponding protein, these cells just can infect by HBV.Research shows simultaneously: although NTCP all has expression in multiple species such as mouse, rat, tree shrew, chimpanzee, people, and have higher homology, but a few amino acid whose difference in particular combination territory just can cause different plant species NTCP to identify HBV and the greatest differences of binding ability, and this is also one of reason that HBV host range is narrow.The NTCP of cynomolgus monkey usually can not in conjunction with hepatitis B virus, but experiment shows, as long as the region that its NTCP the preceding paragraph that suddenlys change is minimum, makes it, by the corresponding aminoacid replacement of people NTCP, just can become effective HBV acceptor, mediates HBV to the hepatocellular infection of cynomolgus monkey.For this reason, people NTCP is considered to the hepatitis B virus acceptor of the most potential quality found up to now, cause great interest and the concern of scholars, and be greatly prized by, this achievement in research also brings valuable opportunity for setting up desirable HBV infection model simultaneously.

But have not yet to see the method that people NTCP is proceeded to the mouse model of construction expression people NTCP in Mice Body by report in prior art so far, reason may be due to: 1.NTCP is just proved to be hepatitis B functional receptor recently, therefore not yet has the report that correlation model mouse builds up; 2., in animal model preparation, the importing form of foreign gene and introduction method directly have influence on the existence of foreign gene in animal and expression.All will through groping in different animal model preparation process.For this problem, prior art does not also have effective solution.

Summary of the invention

The technical problem to be solved in the present invention is to overcome the mouse model lacking in prior art and express people NTCP, especially lack continue, the defect of the mouse model of stably express people NTCP, provide a kind of and express hepatitis B virus acceptor---the construction process of the mouse model of people NTCP.

Object of the present invention is achieved by the following technical programs:

Express a construction process for the mouse model of people NTCP, comprise the steps:

S1. from plasmid pcDNA6-hNTCP, people NTCP gene eucaryon expression frame is obtained through pcr amplification;

Between attB and the attP site of S2. people NTCP gene eucaryon expression frame being inserted plasmid ZY781 by molecule clone technology, obtain the Wei Huan parental plasmid ZY781-CMV-hNTCP of carrier NTCP gene eucaryon expression frame;

S3. Wei Huan parental plasmid ZY781-CMV-hNTCP is proceeded in E.coli ZYCY10P3S2T competence, through kalamycin resistance screening positive clone, gained recombinant plasmid obtains the minicircle dna (pMC-CMV-hNTCP) of carrier NTCP eukaryotic expression cassette after pectinose induction recombinase Ф C31 effect;

S4. by minicircle dna (pMC-CMV-hNTCP) by hydrodynamic coefficients method, to import in Mice Body to obtain gene transfection mouse, gene transfection mouse, through PCR, namely can be used as model after the detection of groupization detection and related biological index and uses and correlative study.

Described in S3, the acquisition condition of the minicircle dna of carrier NTCP eukaryotic expression cassette is: 300 μ l micro-ring induction mother liquor is inoculated 50ml and contains in the TB nutrient solution of 50 μ g/ml kantlex, 37 DEG C, 250 rpm/min cultivate after 12 ~ 18h, isopyknic induced liquid is added in nutrient solution, 32 DEG C, 250 rpm/min cultivate 5 h, receive bacterium, extract minicircle dna; Described induced liquid contains the LB nutrient solution of 50 ml, the NaOH solution of 2 ml 1N and 300 μ l 20% L-arabinose.

Preferably, the preparation method of described micro-ring induction mother liquor is: parental plasmid ZY781-CMV-hNTCP transforms e.colizYCY10P3S2T competence, the single positive colony of picking is inoculated in TB nutrient solution, 37 DEG C, 250 rpm/min, cultivates 15 ~ 18h to OD ₆₀₀when value is about 4 ~ 5, namely can be used as induction mother liquor and carry out induction generation minicircle dna.

Described hydrodynamic coefficients method steps is: choose 6 ~ 8 weeks, the ICR mouse of body weight 26 ~ 28g, is dissolved in 2.5ml physiological saline by the minicircle dna of 15 μ g carrier NTCP eukaryotic expression cassettes, is injected in Mice Body through mouse tail vein in 5 ~ 8s.The present invention also does control group and the test of blank group with pcDNA6 and physiological saline simultaneously respectively.

People NTCP proceeded in Mice Body if the present invention imagines on the Research foundation of forefathers and make it express, so people NTCP can in conjunction with people HBV, thus enable mouse liver cell by HBV infection, this mouse also has normal immunity system simultaneously, therefore this mouse model can overcome existing mouse can not the defect of natural infection hepatitis B virus, realize the farthest clinical hepatitis B of simulation to occur, develop and lapse to process, for studying the course of infection of hepatitis B virus, the immunotherapy finding new drug target and exploration hepatitis B provides desirable model.And how people NTCP is successfully proceeded in Mice Body, and make its lasting, stably express in Mice Body, be the Important Problems that will solve.The mouse model that the present invention compares bearer type by optimizing, method of gene introduction draws a kind of sustainable, stably express people NTCP.Selected carrier is micro-ring carrier ZY781; Micro-ring carrier ZY781 is transformed again by Shenzhen Institutes of Advanced Technology, Chinese Academy of Science Chen Zhi English etc., micro-ring carrier ZY781 is except the characteristic having general micro-ring carrier and have, micro-ring carrier ZY781 also one step can obtain from bacterium, and do not need to be further purified, output is up to 99%.Selected method of gene introduction is hydrodynamic coefficients technology, hydrodynamic coefficients technology is a kind of simple and effective living gene rotaring dyeing technology, utilize the injection liquid scale of construction injected in the short period of time and be equivalent to Mouse Weight about 8%, solution pressure and capacity is caused to exceed mouse systolic pressure and cardiac output, plasmid is made to be gathered in postcava, and the rich blood vessel be attached thereto directly is entered through postcava, the liver with expansible structure contacts with liver cell, thus avoid plasmid DNA through circulation of blood degrade by nuclease, substantially increase transfection efficiency, thus realize the high expression of foreign gene in Mice Body.And, hydrodynamic coefficients method has good liver targeting, with this method import gene in body in liver expression level compared with high by 100 ~ 1 000 times in other organs, can up to 40 ~ 90% to transfection efficiency hepatocellular in body, so be particularly useful for hepatitis virus experimental animal model and relevant functional study thereof.

The invention has the beneficial effects as follows:

The present invention, by building micro-ring carrier of carrier NTCP gene, takes the transgenosis mode of hydrodynamic coefficients to be imported in Mice Body, makes people NTCP gene at liver expression.This model can not the defect of natural infection HBV by overcoming experiment mice, builds up one and has hepatitis B virus functional receptor, can support HBV In vivo infection and have the mouse model of normal immunological function.Thus the generation of clinical hepatitis B, development and chronicity process can be reflected truly, all sidedly, for studying the mechanism of causing a disease of HBV further, after HBV infection, the pathological change of body and the mechanism of action of body immune system in anti-virus infection and immune pathogenic course provide desirable animal model.The foundation of this model will fill up the blank lacking this type of animal model both at home and abroad, make the research means of being correlated with abundanter, also for the research and development of anti-hbv drug provide more advanced appraisement system and technology platform.

Desirable animal model should be repeatably, or even can be standardized.The present invention expresses hepatitis B virus acceptor preparing---and during the mouse model of people NTCP, except consider will by people NTCP channel genes Mice Body, the more important thing is and will consider to take what mode to keep people NTCP gene continues in Mice Body, stably express.Therefore, the mouse model that the present invention compares bearer type by optimizing, method of gene introduction draws a kind of sustainable, stably express people NTCP.

figure of description

Fig. 1. pcDNA6-hNTCP plasmid map.

Fig. 2 .ZY781 empty carrier collection of illustrative plates.

Fig. 3. ZY781-CMV-hNTCP Wei Huan parental plasmid collection of illustrative plates.

Fig. 4. pMC-hNTCP micro-ring carrier for expression of eukaryon collection of illustrative plates.

Fig. 5. the schema of the mouse model of construction expression hNTCP.

Fig. 6 .hNTCP gene eucaryon expression frame pcr amplification electroresis appraisal figure.

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 transfection mouse PCR qualification result figure.

Figure 10. gene transfection murine liver tissue hNTCP immunohistochemical methods qualification result figure.

Embodiment

The present invention is further described below in conjunction with Figure of description and specific embodiment.Unless stated otherwise, the present invention adopts reagent, equipment are the art conventional reagent and equipment.

PcDNA6-hNTCP plasmid is so kind as to give by Beijing Life Sciences Institute Li Wenhui researcher, and its structure iron as shown in Figure 1; e.colizYCY10P3S2T and ZY781 empty carrier is so kind as to give by Shenzhen Xianjin Technology Academe of Chinese Academy of Sciences professor Chen Zhiying, and ZY781 empty carrier structure iron as shown in Figure 2.Construction expression hepatitis B virus acceptor of the present invention---the schema of the mouse model of people NTCP as shown in Figure 5.

embodiment 1

Round pcr is adopted from pcDNA6-hNTCP, to obtain people NTCP gene eucaryon expression frame and introduce BglII-SalI double enzyme site, between attB and the attP site being then inserted into plasmid zy781, after pectinose induction recombinase Ф C31 effect, obtain the minicircle dna (pMC-CMV-hNTCP) of carrier NTCP eukaryotic expression cassette.

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

Forward primer F:5 '-GAAGATCTCCCGATCCCCTAT-3 '

Reverse primer R:5 '-ACGCGTCGACCCATAGAGCCCACC-3 '

PCR reaction system and response procedures as follows:

Reaction conditions:

After PCR reaction terminates, reclaim PCR primer, obtain the CMV-hNTCP-BGHpolyA gene fragment of 2260bp, CMV-hNTCP-BGHpolyA gene fragment is through BglII(NEB) and SalI(NEB) double digestion obtain CMV-hNTCP-BGHpolyA(2248bp), i.e. hNTCP gene eucaryon expression frame.The qualification of PCR result agarose gel electrophoresis as shown in Figure 6.

S2. by between attB and the attP site of people NTCP gene eucaryon expression frame insertion plasmid ZY781, obtain the Wei Huan parental plasmid ZY781-CMV-NTCP of carrier NTCP gene order, Wei Huan parental plasmid ZY781-CMV-NTCP, as shown in SEQ ID NO:2, is transformed into by its sequence e.coliin ZYCY10P3S2T competence, after pectinose induction recombinase Ф C31 effect, obtain the minicircle dna of carrier NTCP eukaryotic expression cassette.Concrete steps are:

Enzyme cuts enzyme used: hNTCP gene eucaryon expression frame and ZY781 empty carrier all use BglII(NEB) and SalI(NEB) carry out double digestion.

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

Digestion products purification process: cut glue and reclaim, adopts glue to reclaim test kit (TaKaRa, DV805A) purifying.

Digestion products connects: adopt and connect test kit (NEB, M2200S), linked system is as follows:

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

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

ddH ₂O 6.93μl ；

2x quick ligation buffer 10μl；

Quick T4 ligase enzyme 1 μ l.

After configuring solution by above linked system, thoroughly react 5min at 25 DEG C after mixing, after cooled on ice, for conversion or in-20 DEG C of preservations.

The product that success connects is ZY781-CMV-hNTCP plasmid, Ji Wei ring parental plasmid; The plasmid map figure of ZY781-CMV-hNTCP plasmid as shown in Figure 3.Successful connection can tentatively be determined by next step conversion and kalamycin resistance screening, cuts qualification confirm further subsequently through enzyme.

Transform: adopt Calcium Chloride Method preparation e.colizYCY10P3S2T competent cell, proceeds to the ZY781-CMV-hNTCP plasmid of structure e.coliin ZYCY10P3S2T competence, the kalamycin resistance preliminary screening of being carried by parental plasmid obtains positive colony, extracts plasmid, BglII-SalI double digestion electroresis appraisal.Electroresis appraisal result is as Fig. 7.(foreign gene 2248bp, carrier 4066bp)

S3. the preparation of the minicircle dna pMC-CMV-hNTCP of carrier NTCP eukaryotic expression cassette.The minicircle dna of carrier NTCP eukaryotic expression cassette is obtained after pectinose induction recombinase Ф C31 effect.Concrete steps are as follows: after Wei Huan parental plasmid transformed competence colibacillus, screening positive clone, and positive colony is inoculated in TB nutrient solution, 37 DEG C, 250 rpm/min, cultivate 12 ~ 18h to OD ₆₀₀value obtains micro-ring induction mother liquor when being about 4 ~ 5, can carry out the generation of micro-ring plasmid of inducing carrier NTCP gene eucaryon expression frame.The generating step of micro-ring plasmid of carrier NTCP gene eucaryon expression frame is: 300 μ l micro-ring induction mother liquor is inoculated 50 ml containing kantlex (50 μ g/ml) TB nutrient solution in, 37 DEG C, after 250 rpm/min cultivate 18h, the induced liquid (LB of 50 ml and the NaOH solution of 2 ml 1N and 300 μ l 20% L-arabinose) of same volume is added again in nutrient solution, 32 DEG C, 250 rpm/min cultivate 5 h, receive bacterium, employing goes toxin plasmid to extract test kit (E.Z.N.A. Endotoxin Free Plasmid Isolation System, D6915) micro-ring plasmid DNA (pMC-hNTCP) of carrier NTCP gene eucaryon expression frame is extracted, its nucleotides sequence is classified as shown in SEQ ID NO:3.Identify through NdeI single endonuclease digestion.(parental plasmid cuts out 4411bp, 1903bp two band, and micro-ring cutting goes out 2315bp mono-band) electroresis appraisal result as shown in Figure 8.The collection of illustrative plates of hNTCP gene micro-ring carrier for expression of eukaryon pMC-hNTCP as shown in Figure 4.

Micro-ring Production conditions of the present invention is that contriver obtains through a large amount of exploratory experiments summary, only has under these conditions, just can prepare the micro-ring carrier of a large amount of restructuring, and the purity of carrier is very high, and assorted carrier is as the pollution of parental plasmid.If change condition, be not that amount vector can not be guaranteed, the purity being exactly carrier cannot ensure, only has above-mentioned condition to meet simultaneously.

embodiment 3

The present invention also uses the carrier for expression of eukaryon of this area routine to carry out Mediated Human NTCP gene preparation animal model simultaneously, such as by recombinant eukaryotic expression plasmid pcDNA6-hNTCP also by carrying out the mouse model of preparation table intelligent NTCP gene in hydrodynamic coefficients technological sourcing ICR Mice Body.But found that: when carrying out Mediated Human NTCP gene with conventional carrier for expression of eukaryon, the mouse model of preparation can not continuous expression people NTCP gene, can not meet the quality requirement of animal model.And adopt the plasmid ZY781 and engineering bacteria that mention in the present invention e.colizYCY10P3S2T prepares micro-ring carrier for expression of eukaryon of carrier NTCP, and optimize the micro-ring Production conditions of induction, a final step obtains micro-ring carrier for expression of eukaryon of carrier NTCP, reduces step, removes leaching process from, saves cost.

embodiment 4with the mouse model of micro-ring pMC-hNTCP preparation table intelligent NTCP of carrier NTCP gene eucaryon expression frame.

1, hydrodynamic coefficients technology prepares people NTCP gene transfection mouse: the ICR mouse choosing 6 ~ 8 weeks, body weight 26 ~ 28g.Be divided into three groups: experimental group, control group, blank group.The minicircle dna 15 μ g of carrier NTCP eukaryotic expression cassette is dissolved in 2.5ml physiological saline by experimental group, pcDNA615 μ g is dissolved in 2.5ml physiological saline by control group, blank group is 2.5ml physiological saline, is all injected in Mice Body in 5-8s through mouse tail vein.

2, PCR Screening and Identification gene transfection mouse: respectively at the 1st, 14 day, often group is got 2 mouse and is got hepatic tissue, extracts hepatic tissue DNA respectively and carries out PCR detection.

Forward primer F:5 '-CCTGGTGGCACAGTATGG-3 '

Reverse primer R:5 '-TTTGGATTTGAGGACGAT-3 '

PCR reaction system and response procedures as follows:

Reaction conditions:

Above-mentioned PCR primer is carried out 1.5%(w/v) agarose gel electrophoresis, as shown in Figure 9, the positive is the specific band of a 346bp to result, shows that hNTCP gene successfully proceeds in Mice Body.

3. immunohistochemical methods identified gene transfected: in the 14th day, often group is got 2 mouse and is got hepatic tissue, puts 10% formaldehyde and fixes 12 ~ 14h, make tissue slice according to a conventional method, and immunohistochemical methods detects the expression of hNTCP in hepatic tissue, the results are shown in Figure 10.(Anti-SLC10A1 antibody antibody 1:200 dilutes, Sigma, HPA042727).

SEQUENCE LISTING

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

<120> mono-kind expresses hepatitis B virus acceptor---the construction process 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 Wei Huan 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-ring 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 (4)

1. express a construction process for the mouse model of people NTCP, it is characterized in that, comprise the steps:

S1. amplification obtains people NTCP gene eucaryon expression frame;

Between attB and the attP site of S2. people NTCP gene eucaryon expression frame being inserted plasmid ZY781 by molecule clone technology, obtain the Wei Huan parental plasmid ZY781-CMV-hNTCP of carrier NTCP gene eucaryon expression frame;

S3. Wei Huan parental plasmid ZY781-CMV-hNTCP is proceeded to e.coliin ZYCY10P3S2T competence, screening positive clone, gained positive colony obtains the minicircle dna pMC-CMV-hNTCP of carrier NTCP eukaryotic expression cassette after pectinose induction recombinase Ф C31 effect;

S4. minicircle dna is passed through hydrodynamic coefficients method, import in Mice Body and obtain gene transfection mouse, gene transfection mouse, through PCR, namely can be used as model after the detection of groupization detection and related biological index and uses and correlative study.

2. the construction process of mouse model according to claim 1, it is characterized in that, described in S3, the acquisition condition of minicircle dna pMC-CMV-hNTCP is: 300 μ l micro-ring induction mother liquor is inoculated 50ml and contains in the TB nutrient solution of 50 μ g/ml kantlex, 37 DEG C, 250 rpm/min cultivate after 12 ~ 18h, isopyknic induced liquid is added in nutrient solution, 32 DEG C, 250 rpm/min cultivate 5 h, receive bacterium, extract minicircle dna; Described induced liquid contains the LB nutrient solution of 50 ml, the NaOH solution of 2 ml 1N and 300 μ l 20% L-arabinose.

3. construction process according to claim 2, it is characterized in that, the preparation method of described micro-ring induction mother liquor is: parental plasmid ZY781-CMV-hNTCP transforms e.colizYCY10P3S2T competence, the single positive colony of picking is inoculated in TB nutrient solution, 37 DEG C, 250 rpm/min, cultivates 15 ~ 18h to OD ₆₀₀when value is about 4 ~ 5, namely can be used as induction mother liquor and carry out induction generation minicircle dna.

4. the construction process of mouse model according to claim 1, it is characterized in that, described hydrodynamic coefficients method steps is: choose 6 ~ 8 weeks, the ICR mouse of body weight 26 ~ 28g, the minicircle dna of 15 μ g carrier NTCP eukaryotic expression cassettes is dissolved in 2.5ml physiological saline, is injected in Mice Body in 5 ~ 8s through mouse tail vein.