CN102517255B - Stably-copied cell line containing hepatitis B viruses, preparation method and application - Google Patents

Abstract

The invention provides a new stably-copied cell line containing hepatitis B viruses (HBV), a preparation method for the cell line and application of the cell line in screening of anti-HBV medicaments. The cell contains Chinese special HBV genes and can secrete a large quantity of E antigens and S antigens, so that research of Chinese anti-HBV patient medicaments has an accurate target.

Description

A kind of new hepatitis B virus that contains is stablized clone, the Preparation method and use that copies

Field that the present invention belongs to:

The invention belongs to the genetically engineered field, specifically, the present invention relates to a kind of stable cell, the method for preparing this clone and this cell purposes that copies of hepatitis virus that contain.

Background technology:

It is an interior important public hygiene problem of global range that hepatitis B virus (HBV) infects.According to the World Health Organization, by 2008 only, the whole world has and surpasses 2,000,000,000 people and infected hepatitis B virus, wherein also has 3.5 hundred million demographic transitions of surpassing to become chronic carrier, dies from the hepatic fibrosis that caused by hepatitis B and the number of primary hepatocarcinoma every year and almost is accumulated to 600,000.China is high incidence of hepatitis b.Be 10% reckoning according to national serum of hepatitis B epidemiology survey in 1992 general population HBV infection as a result positive rate, the hepatitis b virus carrier of the existing Chronic Asymptomatic of China surpasses 100,000,000, and annual hundreds thousand of patients die from the hepatitis B relative diseases such as liver cirrhosis, liver cancer.

The pathogenesis of hepatitis B is still not fully aware of at present.The relation of hepatitis B virus genotypes and hepatitis genesis causes scholar's attention day by day in recent years, is the hot issue of studying both at home and abroad.Hepatitis B virus genotypes is distributed with obvious geographic regional property, and differ greatly, clinical manifestation after hepatitis b virus infected and cause a disease process except having substantial connection with host immune state, infection opportunity etc. also may be in close relations with the genotype that infects HBV.In addition, along with being widely used of antiviral and carrying out in a large number of clinical study, increasing evidence shows the relation that has of the antiviral effect of medicine and HBV genotypic difference.

Preventing and treating hepatitis B need to have more deep fundamental research to HBV, but the investigator usually be transferred in hepatoma cell line the HBV genome of self-replicating is instantaneous, with copying and pathogenesis of research HBV.The genome of transient transfection is free in cell, be not incorporated on karyomit(e), generally after 1-4 days with regard to the harvested cell analyzing samples.The persistence that transient transfection HBV genome and HBV itself are incorporated in karyomit(e) has the essence difference, therefore contains the stable clone that copies of HBV and has more practical significance for research, and result is also more accurate and reliable.The cell model that is used for now hepatitis B research in worldwide mainly is based on the constructed HepG2.2.15 of a D type Hepatitis B virus-DNA (Sells MA of separation, Chen ML, Acs G.Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.Proc.Natl.Acad.Sci.USA 1987; 84:1005-1009).The D type genotype of the HBV that HepG2.2.15 is contained is popular in the world, is especially the preponderant genotype of the north, Africa, Mediterranean Zone, the Middle East and India.In China, the genotypic HBV of B and C has accounted for the overwhelming majority, and the D genotype distributes seldom in China, and this just makes the HepG2.2.15 cell in the HBV of China fundamental research, significant limitation be arranged.The more important thing is, the HepG2.2.15 Growth of Cells is slower, is secreted in cell conditioned medium E antigen and S antigen very low, can't guarantee effective differentiation and the accuracy of result.In the control of China's hepatitis B virus, lack the cell research model of the distinctive hepatitis B virus of China, make and explore new antiviral without accurate target, can't reverse hepatitis B infected high incidence and the high case fatality rate present situation of present China.

The invention technology contents:

An object of the present invention is to provide a kind of new hepatitis B virus that contains and stablize the cell that copies, this cell contains Chinese distinctive hepatitis B virus gene, can secrete in a large number E antigen and S antigen, makes Effect of Anti China hepatitis B patient medicine that accurate target be arranged.

Another object of the present invention is to provide a kind of method for preparing this clone.

The present invention also provides the purposes of a kind of this cell in the anti-hepatic-B virus medicine screening.

The invention discloses a kind of new hepatitis B virus that contains and stablize the cell that copies, it is characterized in that, this cell contains the Chinese hepatitis B virogene that manually changes over to, and hepatitis B virogene has the nucleotide sequence shown in SEQ ID NO:1, this cell is human liver cell Huh7.37 cell, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC C201185.

The Huh7.37 cell is to change the HBV gene over to Huh7 clone, and obtains the stable clone of a strain through G418 pressure screening and culturing.This clone can be in common DMEM substratum stable growth, hepatitis B gene group DNA replication dna can be detected in the long-term cultivation process, and can express sustainedly and stably S antigen and E antigen is secreted to cell culture fluid.

The method for preparing Huh7.37 clone that discloses of the present invention.The method step is as follows:

1) the complete genomic clone of HBV: extract the HBV viral DNA from Chinese B-type hepatitis human serum, and amplification is to the full genome of HBV;

2) structure of HBV infectious clone: use overlap extension pcr to build HBV1.3 times of body, then HBV1.3 times of body is inserted on pBlueScrip II KS (+) carrier that contains the NEO gene, is built into the pBlue-HBV1.3-NEO infectious clone;

3) transfection and screening: transfection plasmid pBlue-HBV1.3-NEO is to Huh7 clone, and the G418 pressurized screen is selected the stable clone that copies HBV.

The invention also discloses preferably, effectively preparation contains the method for the stable clone that copies of HBV, and the method comprises the steps:

1) the complete genomic clone of HBV: extract the HBV viral DNA from Chinese B-type hepatitis human serum, and amplification is to the full genome of HBV.

2) structure of HBV infectious clone: 1.3 times of bodies of whole HBV have been divided into four sections sequence constructs, are respectively fragments 1,1050-1822; Fragment 2,1823-3215; , fragment 3,1-1822 and fragment 4,1823-1990; Adopting overlapping extension PCR (SOE PCR) technical point three-wheel to complete HBV1.3 times of body builds; In first round PCR, obtain above four different fragments; Then, through the lap over extension, 1,2 fragments and 3,4 fragments are stitched together respectively, obtain Segment A and fragment B; In third round PCR, allow Segment A, B put up a bridge and extend 1.3 times of bodies of whole HBV; G418 resistant gene NEO is connected in carrier pBluescript II KS (+) obtains pBlue-NEO, cut by enzyme HBV1.3 times of body is connected on pBlue-NEO, obtain infectious clone pBlue-HBV1.3-NEO.

3) transfection and screening: adopt the transfection of liposome transfection method to contain the infectious clone pBlue-HBV1.3-NEO of resistant gene, then detect protein marker E antigen in supernatant and the expression of S antigen by ELISA, determine the transfection positive cell; The digestion all cells, then evenly distribute is in a plurality of cell flat boards, and the fraction of coverage that guarantees cell in each hole is 30%; Add and contain G418 selection substratum, changed a not good liquor in 3-5 days, after 15 days, remaining cell forms some white colonies, cell colony is chosen to 96 orifice plates after digestion, cell covers with the expression of rear detection associated protein, obtains the clone of high expression level albumen and HBVDNA by the limiting dilution method, finally builds up the stable genomic Huh7.37 clone of Type B HBV that copies.

This cell called after " Huh7.37 cell ", contain the Chinese hepatitis B virogene that manually changes over to, and hepatitis B virogene has the nucleotide sequence shown in SEQ ID NO:1, this cell is human liver cell Huh7.37 cell, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC C201185, and preservation date is on August 20th, 2011.

The invention also discloses the application of Huh 7.37 cells in the anti-hepatic-B virus medicine screening.

Plasmid vector pBlueScrip II KS (+) in the present invention can obtain by commercially available business biotech firm, as companies such as Thremo Fisher Scientific inc., Huh7, HepG2, the HepG2.2.15 cell can obtain by commercially available business biotech firm or Spawn preservation organization, the center preservation has this cell as the Chinese Typical Representative culture collection, and the investigator can ask for to this center.

In the present invention, the applicant discloses the specific anti-hbv drug method of screening, and the method comprises the following steps:

1) cell is prepared: cultivate the Huh7.37 cell with ordinary method, keep the good growth conditions of cell, paving 96 porocyte plates are as for containing 5%CO ₂, 37 degree cell culture incubators cultivate, until Growth of Cells to fraction of coverage greater than 90%;

2) processing of medicine to be screened: be ready to drug solution to be screened, and set corresponding feminine gender and positive control, 3 multiple holes are done in every group of experiment;

3) collect supernatant: treated with medicaments is collected the cells and supernatant preservation each hole in after appropriate time;

4) detect E and S antigen: adopt enzyme-linked immunosorbent assay to measure HBV protein level E and the variation of S antigen presentation in above-mentioned cell culture fluid supernatant;

5) detect the DNA of HBV: use the DNA level variation that Real-Time Fluorescent Quantitative PCR Technique detects HBV in supernatant;

6) interpretation of result: be secreted into albumen and the DNA level of cells and supernatant from HBV according to the data of 5 and 6 steps, analyze the effect of medicine hepatitis B virus resisting.

Advantage of the present invention: compare in prior art, the present invention has the following advantages:

1) Huh7.37 cell provided by the invention can be in containing the DMEM substratum of 10% foetal calf serum stable growth, and expression-secretion E antigen and S antigen sustainedly and stably.For the HepG2.2.15 cell, the Huh7.37 cell has the speed of growth faster, more convenient cultivation and use; And Huh7.37 emiocytosis significantly improves than HepG2.2.15 cell to E antigen and the S antigen in cells and supernatant, and particularly S antigen has reached more than 2 times, has reflected higher the copying and translation skill of HBV in Huh7.37 clone.In anti-HBV drug screening, use Huh7.37 and can shorten the screening cycle and improve the discrimination of experimental data and the accuracy of result.

2) in Huh7.37 cell provided by the invention, contained HBV viral genome is separated from Chinese B-type hepatitis human serum, belongs to the popular wider B genotype in China.The structure of Huh7.37 cell more meet China in the urgent need to.The screening that the Huh7.37 cell is used for anti-hepatic-B virus medicine is more pointed, can for China clinical study and the medical science control of hepatitis B virus more practicable research platform is provided.

Description of drawings

Fig. 1 is carrier pBlueScript II KS schematic diagram

Fig. 2 is infectious clone pBlue-HBV1.3-NEO building process schematic diagram

Fig. 3 is the full Genomic PCR collection of illustrative plates of HBV.M is DNA molecular Marker, and swimming lane 1-7 is 7 different samples

Fig. 4 is SOE PCR first round electrophorogram

M is DNA molecular Marker, and fragment 1 is 773bp, 1050nt-1822nt; Fragment 2 is 1393bp, 1823nt-3215nt; Fragment 4 is 168bp, 1823nt-1990nt; Fragment 3 is 1822bp, 1nt-1822nt)

Fig. 5 is that SOE PCR second takes turns electrophorogram

M is DNA molecular Marker, amplifies Segment A by fragment 1 and fragment 2; Amplify fragment B by fragment 3 and fragment 4

Fig. 6 is SOE PCR third round electrophorogram

M is DNA molecular Marker, and Segment A and fragment B amplify 1.3 times of bodies of HBV

Fig. 7 is HBV1.3 times of body schematic diagram

Fig. 8 is that infectious clone pBlue-HBV1.3-NEO enzyme is cut evaluation figure

M is DNA molecular Marker, and 1,2 swimming lanes are the restriction enzyme mapping of positive colony

Fig. 9 is that the Huh7.37 cell routine is cultivated 1-6 month cell conditioned medium E, S antigen and HBVDNA dynamic change

Figure 10 is that in the stable Huh7.37 cell culture fluid of cultivating, E antigen secretion level detects

Figure 11 is that in the stable Huh7.37 cell culture fluid of cultivating, S antigen secretion level detects

Figure 12 is that the mRNA relative level of Huh7.37 intracellular Ca2+ plectin (CRT) detects

(the * representative has significant difference)

Figure 13 is that the mRNA relative level of HepG2.2.15 intracellular Ca2+ plectin (CRT) detects

(the * representative has significant difference).

Embodiment

Further set forth the present invention below in conjunction with specific embodiment.Should be appreciated that these embodiment only are used for explanation the present invention, and can not limit protection scope of the present invention.

Embodiment 1 sets up the genomic Huh7.37 clone of stable transfection HBV

According to illustrated in figures 1 and 2, construction step is as follows:

1.1 the extraction of viral DNA: the broken virus of viral DNA out reagent that adopts sky damp genetically engineered company limited to produce, releasing virus DNA.

(1) add 480 μ l viral DNA out solution at 1.5mL screw-cap plastic centrifuge tube.

(2) add 160 μ l serum samples, after the 30s mixing that thoroughly vibrates, room temperature is placed 10min.

(3) the 30s mixing that vibrates again adds 560 μ l Virahols, and it is unanimously outside that vibration 30s, the centrifugal 15min of 15000g room temperature, band pipe cover the direction of handle.

(4) first abandon supernatant with the 1mL liquid-transfering gun, draw at twice (note not touching precipitation or allow the liquid contamination rifle that picks up), stay a small amount of liquid, ofer short duration centrifugal 1min, abandon carefully all residual liquids with 100-200 μ l liquid-transfering gun, foreign protein in liquid is removed in this step effect as far as possible.

(5) add the ethanol of 800 μ l 70%, softly put upside down and mix 3 times, do not shake loose bottom settlings, the centrifugal 5min of 15000g room temperature then, the method according to 4 is abandoned supernatant at twice, and this step effect is to remove residual isopropanol and salinity.

(6) 8min that uncaps makes residual ethanol evaporation, thereby allows DNA be easier to be dissolved in water, adds 80 μ LTris damping fluids this moment, waits for 2-3min, makes precipitation become loose, and the membranaceous precipitation with liquid-transfering gun piping and druming tube wall and the pipe end makes its dissolving.

(7) get the appropriate PCR of being used for, all the other-20 degrees centigrade of preservations after the centrifugal 1min of 15000g.

1.2HBV the amplification of full-length gene group: reference report primer sequence carries out HBV amplification (S G ü nther, B C Li, S Miska, D H Kr ü ger, H Meisel and H Will.A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.J.Virol.1995,69 (9): 5437.).

Normal chain primers F 1:5 '-CCGGA AAGCTT GAGCTC TTCTTT TTCACC TCTGCC TAATCA-3 ' (SEQ ID NO:2)

Minus strand primer R1:5 '-CCGGA AAGCTT GAGCTC TTCAAA AAGTTG CATGGT GCT GG-3 ' (SEQ ID NO:3)

With the HBV genomic templates of above-mentioned Auele Specific Primer and extraction, with the full genome of archaeal dna polymerase amplification HBV of high-fidelity.Agarose gel electrophoresis separates PCR product (Fig. 3), enzyme cuts back to close through order-checking and learns that size is 3215bp, learns that by BLAST sequence alignment in NCBI it is to belong to the HBV genome, and with the highest with the HBV genome similarity of Type B, reached 92%, therefore determine that its genotype is Type B;

1.3HBV1.3 the structure of body doubly: 1.3 times of bodies of whole HBV have been divided into four sections sequences, are respectively 1050-1822 (fragment 1), 1823-3215 (fragment 2), 1-1822 (fragment 3) and 1823-1990 (fragment 4).We adopt the SOE round pcr to build HBV1.3 times of body.In first round PCR (Fig. 4), we obtain above four different fragments; Then through the lap over extension, 1,2 fragments and 3,4 fragments are stitched together respectively, obtain Segment A and fragment B (Fig. 5); In third round PCR, we just can allow Segment A, B bridging extend 1.3 times of bodies (Fig. 6) of whole HBV at last.HBV1.3 times of body schematic diagram as shown in Figure 7.We have also introduced the restriction enzyme site of EcoRI and Sal I simultaneously, prepare HBV1.3 times of body connected into carrier pBluescript II KS (+).

1.4 the structure of infectious clone: due to the repetition of restriction enzyme site, therefore need first G418 resistant gene NEO to be connected in carrier pBluescript II KS (+) by XhoI and KpnI to obtain pBlue-NEO, and then by the double digestion of EcoR I and Sal I, HBV1.3 times of body is connected in the pBlue-NEO carrier, obtained the pBlue-HBV1.3-NEO plasmid clone, HBV infectious clone (Fig. 7) namely, we carry out double digestion with Kpn I and Xho I and identify, pick out correct clone, and check order and confirm sequence correct (Fig. 8).

1.5 transfection and screening: the Huh7 cell of 3 times of going down to posterity after recovering is with tryptic digestion, with the DMEM substratum furnishing 5 * 10 that contains 10% (volume/volume) foetal calf serum ⁵The density of individual/mL is inoculated in the culture dish that several diameters are 35mm, 37 ℃, 5%CO in gnotobasis ₂Cultivated 24 hours under condition, when cytogamy to 90%, carry out the cell transfecting operation according to Lipofectamine 2000 process specificationss, after 6 hours, replacing has serum, unparalleled anti-DMEM substratum to continue to cultivate.After transfection 2 days, begin the G418 pressure screening with 300 μ g/mL concentration, until most necrocytosis, this process was approximately wanted about 2 weeks.To obtain cell and adopt the Method of Limited Dilution method to reach 96 porocyte culture plates, be used alternatingly conventional medium and G418 (300 μ g/mL) culture medium culturing individual cells, grow single clone to it.Pick out optimum cell clone and continue to cultivate, and constantly expand numerously, finally build up the genomic Huh7.37 clone of stable transfection HBV.Build the method for cell bank according to general clone, the cell that the cultured HBV of containing is stablized replicator step by step the frozen transgenic cell storehouse set up with standby.This cell called after " Huh7.37 cell " is preserved in Chinese Typical Representative culture collection center on August 20th, 2011, and deposit number is CCTCC C201185.

1.6Huh7.37 the detection of cell expressing E, S antigen and supernatant DNA: employing contains 10% foetal calf serum and 1% pair of anti-DMEM substratum is cultivated, continue 6 months, cell can be stablized Fast Growth, time segment gathers cells and supernatant, adopt respectively enzyme-linked immunosorbent assay and fluorescent quantitative PCR experiment to detect the DNA of E, S antigen and HBV in supernatant, result shows expression E, the S antigen that the Huh7.37 cell can be continual and steady and secretes HBV to cells and supernatant (Fig. 9), shows that HBV energy stable existence is among the purchase clone of building.

The cultivation of embodiment 2Huh7.37 cell, frozen and recovery

Consistent with normal Huh7 cell culture processes, can buy the DMEM substratum, or be equipped with conventional DMEM substratum voluntarily.Add the two anti-and mixings of penicillin-Streptomycin sulphate of 10% foetal calf serum and 1% in the DMEM substratum, namely can be used for cultivating the Huh7.37 cell.First with absorbing substratum, wash away remaining substratum with the PBS after sterilization during passage, add appropriate pancreatin, put into 37 degree cell culture incubator digestion 5 minutes.Until cell is loose take off wall after, can directly add and above-mentionedly contain serum and two anti-DMEM substratum stop digestion, and dispel into individual cells, be passaged to new cell bottle enlarged culturing.Growth of Cells to 90% abundance can further go down to posterity.Than the HepG2.2.15 cell, the Huh7.37 cell has growth velocity faster, and can go down to posterity average every day by 1: 2, is used for the drug screening application in the cell enlarged culturing and has good advantage.

The Huh7.37 cell can be frozen with the foetal calf serum of the DMSO that contains 10% (volume/volume).Stop digestion with substratum after peptic cell, centrifugal 5 minutes of 2000rpm absorbs supernatant in centrifuge tube, adds the foetal calf serum that contains 10% DMSO and dispels, and is drawn to cell cryopreservation tube.Cooling step by step, last frozen in liquid nitrogen container then as for-80 degrees centigrade of refrigerators 6 hours or spend the night, can steady in a long-termly preserve.During recovery Huh 7.37 cell, take out a Huh7.37 cell as for 37 degrees centigrade of water-baths from liquid nitrogen container, constantly jog is freeze-stored cell thermally equivalent recovery dissolving extremely fully in pipe, at last cells frozen storing liquid is added to and contains 10% foetal calf serum and 1% pair of anti-DMEM substratum, cell attachment after 6 hours, change fresh DMEM substratum and cultivate, growing to some amount can go down to posterity.

In embodiment 3Huh7.37 cell culture fluid, E antigen and S antigen secretion level detect

Take the clone HepG2.2.15 of the stable transfection HBV that is widely used at present as reference, detect the E antigen of our constructed Huh7.37 clone and the secretion level of S antigen.Adopt the ELISA detection method, buy hepatitis B virus E antigen and the medical detection kit of S antigen that Shanghai Kehua Bio-engineering Co., Ltd produces, concrete steps are as follows:

1 cell is prepared: contain 10% foetal calf serum and 1% pair of anti-DMEM substratum is cultivated respectively HepG2.2.15 cell and Huh7.37 cell with same.

2 bed boards: with HepG2.2.15 cell and Huh7.37 cell with trysinization after, with in appropriate above-mentioned DMEM substratum and digestion, and dispel into individual cells.Then, all with 3 * 10 ⁶The cell density of individual/mL spreads respectively each 2mL enchylema of 6 orifice plates.At the bottom of shaking up cell tiling plate, as for cultivating in cell culture incubator.

3 receive sample: the stable cultivation after 48 hours, and collect hole inner cell nutrient solution 4 degree and preserve.

4 test kits detect E, S antigen secretion level:

(1) add testing sample (comprising that transfection infectious clone, empty carrier and positive control are the HepG2.2.15 cell conditioned medium) 50 μ L in every hole, then establish one group of negative control;

(2) add enzyme conjugates 50 μ L (or 1) in every hole, fully shrouding after mixing, be placed in 37 degree and hatch 30min;

(3) by hand wash plate: discard the liquid hole in, washings is filled with each hole, standing 5sec, and drying pats dry after repeating 5 times;

(4) add developer A liquid, each 50 μ L (or 1) of B liquid in every hole, abundant mixing, shrouding is placed in 37 degree and hatches 15min;

(5) add stop buffer 50 μ L (or 1), mixing in every hole;

(6) use the microplate reader reading, get wavelength 450nm (when using the microplate reader colorimetric of dual wavelength, reference wavelength is 630nm), read each hole OD value;

5 interpretations of result: according to the standard that test kit is enumerated, negative control is less than 0.01, and positive control is greater than 1, so Huh7.37 emiocytosis is to E, S antigen result in cell culture medium positive (Figure 10 and Figure 11).

For HepG2.2.15, the E that the Huh7.37 cell is secreted under the same conditions, S higher level, particularly, the E antigen that the Huh7.37 cell is secreted under the same conditions is approximately 1.3 times of HepG2.2.15 cell, and the secretion level of S antigen has reached more than 2 times especially, show the relatively higher levels of hbv replication of Huh7.37 clone and translation skill, had more wide application prospect.

Embodiment 4 detects calprotectin mRNA level in the Huh7.37 cell

Calprotectin (CRT, Calreticulin) is a kind of multifunctional protein that is present in endoplasmic reticulum, and it has the critical functions such as molecular chaperones, calcium ion storage and Signal Regulation.Under some pathological conditions, endocytoplasmic reticulum forms a large amount of misfolded proteins stress causing endoplasmic reticulum pressure under situation, and calprotectin can be raised and form one of mode of resisting pathological conditions sometimes.Utilize protein translation system and the dubbing system of cell after HBV infects, reach the purpose of increment virion, during might regulate the calprotectin in host cell.We adopt the method for quantitative fluorescent PCR to remove to detect the intracellular calprotectin mRNA of Huh7.37 with respect to the change level of Huh7 cell, and with the HepG2.2.15 cell with respect to the change level of HepG2 cell as positive control, to obtaining identical regulative mode, thus the reliability of checking Huh7.37 cell.

Cultivate respectively Huh7 cell, Huh7.37 cell, HepG2 cell and HepG2.2.15 cell, get the 5x10 of equivalent ⁶Individual cell, with TRIzol (Invitrogen, Carlsbad, CA, USA) lysing cell, according to the highly purified RNA of normal process separation and Extraction that provides, then in the random primer system under 37 degree conditions reverse transcription obtained 4 parts of cDNA in 1 hour, be used for the mRNA level of fluorescence quantitative PCR detection calprotectin, detecting instrument is Light Cycler 480 (Roche, Indianapolis, IN).

The result demonstration, in the Huh7.37 cell, the calprotectin level has remarkable rising (Figure 12) for the Huh7 cell, shows that HBV can cause intracellular calprotectin mRNA level to change to host's stable infection.Similarly, as positive control, the calprotectin level in the HepG2.2.15 cell also has remarkable rising (Figure 13) for the HepG2 cell, and upper level-off is original 2-2.5 doubly.This a side illustration in the stable cell model that infects of HBV, the Huh7.37 cell has the stechiology feature identical with the HepG2.2.15 cell, has application prospect preferably.

The GAPDH gene is as internal reference, and primer sequence is as follows:

The positive strand primer of CRT: 5 '-TACAATGCTGACTATGGCTAC-3 ' (SEQ ID NO:4)

CRT minus strand primer: 5 '-ACTGATGCGTGAAGTGCTG-3 '; (SEQ ID NO:5)

The positive strand primer of GAPDH: 5 '-AAGGCTGTGGGCAAGG-3 ' (SEQ ID NO:6)

GAPDH minus strand primer: 5 '-TGGAGGAGTGGGTGTCG-3 '. (SEQ ID NO:7)

SEQUENCE LISTING

＜110〉Wuhan University

＜120〉a kind of new hepatitis B virus that contains is stablized clone, the Preparation method and use that copies

＜130〉a kind of new hepatitis B virus that contains is stablized clone, the Preparation method and use that copies

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 3215

<212> DNA

＜213〉people

<400> 1

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aaacagtatc tgaaccttta ccccgttgct cggcaacggc ctggtctgtg ccaagtgttt 2580

gctgacgcaa cccccactgg ttggggcttg gccatgggcc atcagcgcat gcgtgggacc 2640

tttgtgtccc ctctgccgat ccatactgcg gaactcctag ccgcttgttt tgctcgcagc 2700

aggtctgggg caaaactcat cgggactgac aattctgtcg tgctctcccg caagtataca 2760

tcatttccat ggctgctagg ctgtgctgcc aactggatcc tgcgcgggac gtcctttgtt 2820

tacgtcccgt cggcgctgta tcccgcggac gacccctccc ggggccgctt ggggctctac 2880

cgcccgcttc tccgcctgtt gtaccgaccg acctcggggc gcacctctct ttacgcggac 2940

tccccgtctg tgccttctca tctgccggac cgtgtgcact tcgcttcacc tctgcacgtc 3000

gcatggaaac caccgtgaac gtccacggga atctgcccaa ggtcttgcat aagaggactc 3060

ttggactttc agcaatgcca acgaccgacc ttgaggcata cttcaaagac tgtgtgttta 3120

atgagtggga ggagttgggg gaggaggtta ggttaaaggt ctttgtacta ggaggctgta 3180

ggcataaatt ggtgtgttca ccagcaccat gcaac 3215

<210> 2

<211> 41

<212> DNA

＜213〉synthetic

<400> 2

ccggaaagct tgagctcttc tttttcacct ctgcctaatc a 41

<210> 3

<211> 40

<212> DNA

＜213〉synthetic

<400> 3

ccggaaagct tgagctcttc aaaaagttgc atggtgctgg 40

<210> 4

<211> 21

<212> DNA

＜213〉synthetic

<400> 4

tacaatgctg actatggcta c 21

<210> 5

<211> 19

<212> DNA

＜213〉synthetic

<400> 5

actgatgcgt gaagtgctg 19

<210> 6

<211> 16

<212> DNA

＜213〉synthetic

<400> 6

aaggctgtgg gcaagg 16

<210> 7

<211> 17

<212> DNA

＜213〉synthetic

<400> 7

tggaggagtg ggtgtcg 17

Claims (3)

1. one kind contains the stable clone that copies of hepatitis B virus, it is characterized in that, this cell contains the Chinese hepatitis B virogene that manually changes over to, and hepatitis B virogene is the nucleotide sequence shown in SEQ ID NO:1, this cell is human liver cell Huh7.37 cell, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC C201185.

2. the preparation method of the described clone of claim 1, is characterized in that comprising the steps:

1) the complete genomic clone of HBV: extract the HBV viral DNA from Chinese B-type hepatitis human serum, and amplification is to the full genome of HBV;

2) structure of HBV infectious clone: 1.3 times of bodies of whole HBV have been divided into four sections sequence constructs, are respectively fragments 1,1050-1822; Fragment 2,1823-3215; , fragment 3,1-1822 and fragment 4,1823-1990; Adopting overlapping extension PCR (SOE PCR) technical point three-wheel to complete HBV1.3 times of body builds; In first round PCR, obtain above four different fragments; Then, through the lap over extension, 1,2 fragments and 3,4 fragments are stitched together respectively, obtain Segment A and fragment B; In third round PCR, allow Segment A, B put up a bridge and extend 1.3 times of bodies of whole HBV; G418 resistant gene NEO is connected in carrier pBluescript II KS (+) obtains pBlue-NEO, cut by enzyme HBV1.3 times of body is connected on pBlue-NEO, obtain infectious clone pBlue-HBV1.3-NEO;

3) transfection and screening: adopt the transfection of liposome transfection method to contain the infectious clone pBlue-HBV1.3-NEO of resistant gene, then detect protein marker E antigen in supernatant and the expression of S antigen by ELISA, determine the transfection positive cell; The digestion all cells, then evenly distribute is in a plurality of cell flat boards, and the fraction of coverage that guarantees cell in each hole is 30%; Add and contain G418 selection substratum, changed a not good liquor in 3-5 days, after 15 days, remaining cell forms some white colonies, cell colony is chosen to 96 orifice plates after digestion, cell covers with the expression of rear detection associated protein, obtains the clone of high expression level albumen and HBV DNA by the limiting dilution method, finally builds up the stable genomic Huh7.37 clone of Type B HBV that copies.

3. the application of the clone described in claim 1 in the anti-hepatic-B virus medicine screening.

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