CN103820392A - Drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and establishment method thereof - Google Patents
Drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and establishment method thereof Download PDFInfo
- Publication number
- CN103820392A CN103820392A CN201210469842.2A CN201210469842A CN103820392A CN 103820392 A CN103820392 A CN 103820392A CN 201210469842 A CN201210469842 A CN 201210469842A CN 103820392 A CN103820392 A CN 103820392A
- Authority
- CN
- China
- Prior art keywords
- hbv
- cell
- hepg2
- rts202g
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the fields of biotechnology and microbial animal cell lines, and in particular relates to a drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and an establishment method thereof. The method comprises the following steps: an HBV eukaryotic expression plasmid pREP-HBV-YVDD is adopted, the vector skeleton of the HBV eukaryotic expression plasmid pREP-HBV-YVDD is a pREP 10 vector, and an HBV replicon is HBV1.2 ploid containing a cp promoter and a complete HBV pregenome; after the HBV replicon is subjected to site-directed mutagenesis to become an entecavir drug-resistant genetype S202G, the pREP 10 excising an RSV promoter is inserted to establish an HBV eukaryotic expression plasmid pREP-HBV-S202G; after the plasmid is transfected to a HepG2 cell, hygromycin screening is conducted, and then HBV copy, the expression of specific antigen and the generation of virus particles in the cell are detected; after the cell is compared with a HepG2.2.15 cell strain, a drug-resistant HBV cell strain HepG2.HS202G is obtained, and the preservation number is CGMCC No.6393. The cell line can be used for HBV cell models for screening drug-resistance, researching HBV biological characteristics and interacting with cells.
Description
Technical field
The invention belongs to biotechnology, technical field of microbe cell line, relate to HBV cell model, be specifically related to based on HepG2 HBV rtS202G ETV resistance variant clone and establishment method thereof, this clone can be used for screening antiradiation drug, studies HBV Biological characteristics and the HBV cell model with cell-cell interaction thereof.
Background technology
Studies show that, HBV cell model is screening antiradiation drug, studies its Biological characteristics and the essential tool with cell-cell interaction thereof, in the screening of research pathogenesis, immunologic mechanism, antiviral, has important effect.HepG2.2.15 cell is the milestone of HBV cell model development research, it is with containing 2 HBV heads to the dimeric pDolt-HBV-1 recombinant vectors of tail transfection HepG 2 cell, after G418 screening, obtain a cell clone that produces high-level HBsAg and HBeAg, HBV-DNA inside and outside its born of the same parents, all can be detected and there is infective complete virion.Hep2.2.15 is still the HBV cell model being most widely used at present.The clinical application of the anti-HBV medicine of nucleoside analog class take lamivudine as representative is when chronic hepatitis B (CHB) patient brings glad tidings in recent years, also bring serious resistance problem, at present all can detect corresponding multidrug resistant disease strain for ucleosides (seemingly) medicine of application as lamivudine, Adefovir and Entecavir clinically.Due to the line medication that Entecavir is domestic Chronic Hepatitis B at present, although its high resistance barrier can suppress rapidly virus and resistance incidence is starkly lower than lamivudine and Adefovir, but using, Long-term clinical finds that entecavir resistant virus strain is also at increasing year by year, for the patient Ke Jia tynofovir continual cure of entecavir resistant, but domestic and do not go public and its price comparatively expensive.Generally can only increase Entecavir dosage with associating Adefovir continual cure for entecavir resistant patient clinically, therefore, obviously increase the weight of patient's economical load and treatment conformability.Become to attach most importance to for the Molecular Biology Mechanism of the resistance to Entecavir of HBV and the research of screening newtype drug at present.The genovariation of the known HBV of resistance to nucleoside medicine mainly concentrates on reverse transcription district (RT district) at present, the clinical Entecavir of resistance the most widely virus strain is to be, on YVDD variation basis, T184 occurs again at rtM204V, the variation of S202 or M250, can cause HBV obviously to decline for the susceptibility of lamivudine and Entecavir, because RT district nucleotide diversity affects the replication of the HBV of resistance to nucleoside medicine variant, the replication of resistance HBV virus strain is often lower than doubly left and right of the about 50-100 of wild-type, by also corresponding the increasing of difficulty of the gene constructed stably express HBV of HBV resistance variant variant clone.Only there is at present the foundation of the resistance to lamivudine of a small amount of bibliographical information and Adefovir HBV clone, be starkly lower than traditional HBV clone HepG2.2.15 but corresponding viral yield and viral protein are synthetic, and there is no the foundation report of stably express entecavir resistant clone.
Under the clinical settings rising year by year in the resistance to Entecavir virus strain of HBV, HepG2.2.15 can not meet the research for resistance HBV virus strain completely, for the screening of the Molecular Biology Mechanism of research HBV resistance and new anti-HBV medicine, the present invention intends building on the basis of resistance to lamivudine and Adefovir HBV clone in early stage, sets up a kind of hepatoma cell line that can the stably express HBV variant of resistance to Entecavir.
Summary of the invention
The object of the invention is to for meeting for the research of resistance HBV virus strain, for the screening of the Molecular Biology Mechanism of research HBV resistance and new anti-HBV medicine, provide based on HepG2 HBV rtS202G ETV resistance variant clone and establishment method thereof.
This laboratory built on wild-type, resistance to lamivudine and Adefovir anomaly clone basis and further builds the HBV of resistance to Entecavir cell strain in early stage, and this cell strain is especially applicable to laboratory study and uses.
The present invention adopts HBV eukaryon expression plasmid pREP-HBV-YVDD, (Medical University Of Fujian's molecule virus laboratory) its carrier framework is pREP10 carrier, and this carrier comparative advantage is that Totomycin Hygromycin resistant gene is conducive to cell screening work and EBNA-1 genetic transcription albumen can raise containing genetic transcription and expression in the eukaryon expression plasmid of OriP replication origin; Hbv replication is for containing cp promotor and the pregenomic HBV1.2 of complete HBV times body, and site-directed mutagenesis is that the pREP10 that inserts excision RSV promotor after entecavir resistant genotype S202G is built into HBV eukaryon expression plasmid pREP-HBV-S202G; By after this plasmid transfection HepG2 cell by detecting the expression of hbv replication in cell, specific antigens and the generation of virion after hygromycin selection, and after comparing with HepG2.2.15 cell strain, establish the foundation of resistance HBV cell clone.After testing, result shows that this resistance HBV cell clone pREP-HBVA181V can continuous expression HBsAg and HBeAg, has hbv replication, and can produce infectious HBV particle in born of the same parents, produces HBV gene order identical with institute transfection plasmid by gene sequencing.Compared with now widely used Hep2.2.15, this expression of cell lines HBsAg and HBeAg be apparently higher than Hep2.2.15, cell conditioned medium HBV particle level with a little less than HepG2.2.15.
This cell strain called after HepG2.HS202G, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, center preservation accession designation number CGMCC No.6393.
This Cell Line HepG2 .HS202G builds by following method,
1. plasmid transfection HepG2 cell
1. cell cultures: HepG2 cell is cultivated with DMEM nutrient solution;
2. cell transfecting: by the cell in growth logarithmic phase, through trysinization, after continuing to cultivate, by certain transfection amount, mix with transfection reagent, simultaneously mixed with the plasmid DNA of dilution, rear continuation is cultivated;
2. hygromycin selection cell clone
1. cell cultures enlarged culturing establish blank (untransfected HepG2 cell) after 24 hours after transfection;
2. after cell attachment, add Totomycin;
3. changing liquid cultivates;
4. observation of cell clonal growth situation after blank necrocytosis;
5. picking cell clone, continues to cultivate with Totomycin;
6. to 60-70%, survey HBsAg, HBeAg and the HBV-DNA level in supernatant until cell clone density;
7. obtain HBsAg, HBeAg and HBV-DNA positive clone strain, positive colony is reached to 6 well culture plates and continue to cultivate with Totomycin;
3. the limiting dilution of cell clone
Cell reached about 20 generations, and limiting dilution assay obtains purer and high level expression albuminous cell clone;
By the cell in growth logarithmic phase, trysinization, blow and beat into individual cells suspension, after counting, cell density dilution, inoculation is containing Totomycin substratum, continue to cultivate, to 60-70%, survey HBsAg, HBeAg and the HBV-DNA level in supernatant until cell density, obtain the cell clone of stably express virion and viral protein, and called after HepG2.HS202G.
4. the biological property of high expression level HBV YVDD variant and phenotypic resistance analysis
Cell conditioned medium HBsAg, HBeAg and HBV-DNA level detection
HBsAg and HBeAg accurate quantification test kit adopt the Archipct HBsAg Reagentkit of Abbott company and Archipct HBeAg Reagentkit, HBV-DNA detects and adopts Shanghai Ke Hua biotechnology company limited hepatitis B virus nucleic acid immue quantitative detection reagent box, detecting step is according to the test kit book that furnishes an explanation, real-timePCR amplification instrument adopts ABI-7300, and detected result (as shown in Figure 1) shows:
HepG2.HS202G cell strain of the present invention is stable going down to posterity for more than 20 generations, energy stably express virion and HBsAg and HBeAg viral protein, the about 200IU/ml of its HBsAg left and right, the about 200S/CO of HBeAg left and right, compared with doubly left and right of the high about 5-10 of HepG2.2.15, supernatant HBV-DNA approximately 10
4-10
5left and right, a little less than HepG2.2.15.
HepG2.HS202G cell strain of the present invention has carried out hbv replication situation in cell and has detected,
The total DNA extracting of cell, in 6 orifice plates, add 400ul cell pyrolysis liquid when cell 90% degree of converging, 37 ℃ add after spending the night equivalent phenol carry out DNA extracting, precipitation after DNA be dissolved in 20ul TE, 20XSSC transferring film after 1.3% gel electrophoresis, southern-blot hybridization, result shows HBV hybridization signal, shows to exist in cell hbv replication, but is low (as shown in Figure 2) compared with HepG2.2.15 cell.
This clone can meet the research for resistance HBV virus strain, can be used for screening antiradiation drug, studies HBV Biological characteristics and the HBV cell model with cell-cell interaction thereof.
Accompanying drawing explanation
To be that HepG2.HS202G cell strain is stable went down to posterity for more than 20 generations Fig. 1, can stably express virion and HBsAg and HBeAg viral protein.
Fig. 2 is hbv replication detected result in cell, wherein shows HBV hybridization signal, has hbv replication, but be low compared with HepG2.2.15 cell in prompting cell.
Embodiment
Embodiment 1 builds Cell Line HepG2 .HS202G
2 plasmid transfection HepG2 cells
1. cell cultures: HepG2 cell is cultivated with DMEM nutrient solution (Gibco company contains 10% calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, 0.03% glutamine)
2. cell transfecting: by the cell in growth logarithmic phase, with 0.25% trysinization, suck after pancreatin, blow and beat into individual cells suspension with cell culture fluid, count.By 5 × 10
5the concentration of cells/well is seeded to 6 well culture plates, cultivates after 18-24 hour, merges time in blocks until the about 80-90% of cell, renews the nutrient solution of fresh antibiotic-free, prepares transfection.According to the amount of every hole transfection 2 μ g, 50 μ l OptiDMEM nutrient solutions are mixed with 5 μ lLipofectamin2000 transfection reagents (Invitrogen), 2 μ g plasmid DNA are diluted in 50 μ l OptiDMEM nutrient solutions simultaneously, mix rear room temperature and leave standstill 5 minutes.By mixed to the Lipofectamin2000 of dilution and plasmid DNA, room temperature leaves standstill 20 minutes.Mixture is evenly added dropwise in 6 orifice plates, after 6 hours, cleans after 2 times and change fresh medium with PBS, continue to cultivate.
2) hygromycin selection cell clone
1. after transfection cell cultures after 24 hours enlarged culturing to 10cm culture dish, and establish blank (untransfected HepG2 cell)
2. after cell attachment, add Totomycin concentration to 300ug/ml
3. within every 3 days, change liquid once
4. about 14-21 days observation of cell clonal growth situations after the whole death of blank cell
5. picking cell clone to 24 well culture plate, continues to cultivate with Totomycin concentration 150ug/ml
6. to 60-70%, survey HBsAg, HBeAg and the HBV-DNA level in supernatant until cell clone density in 24 well culture plates
7. obtain altogether totally 2 strains of HBsAg, HBeAg and HBV-DNA positive colony, positive colony is reached to 6 well culture plates and continue to cultivate with Totomycin concentration 150ug/ml
3) limiting dilution of cell clone
In 4 strain positive colonies, 1 strain cell clone HBsAg, HBeAg and HBV-DNA level are higher, and after quantitatively, supernatant HBsAg is 120-150IU/ml left and right, HBeAg level 100 S/CO left and right, and HBV-DNA horizontal dimension is held in 10
4-10
5copies/ml.Cell reached about 20 generations, and limiting dilution assay obtains purer and high level expression albuminous cell clone.
By the cell in growth logarithmic phase, with 0.25% trysinization, suck after pancreatin, blow and beat into individual cells suspension with cell culture fluid, after counting, cell density dilution is 10cell/ml, inoculation 96 well culture plates, every hole 150ul substratum (containing Totomycin concentration 150ug/ml).The every porocyte number of micro-Microscopic observation, mark is only inoculated the culture hole of 1cell, continues to cultivate, and changes weekly liquid once.After 2-3 week, behind cell density approximately 60% left and right, reaching 24 well culture plates continues to cultivate.To 60-70%, survey HBsAg, HBeAg and the HBV-DNA level in supernatant until cell density in 24 holes, HBsAg, HBeAg and the higher cell clone of HBV-DNA level are continued to go down to posterity and observe virion and viral protein stably express situation, and called after HepG2.HS202G.
4) biological property of high expression level HBV YVDD variant and phenotypic resistance analysis
Cell conditioned medium HBsAg, HBeAg and HBV-DNA level detection
HBsAg and HBeAg accurate quantification test kit adopt the Archipct HBsAg Reagentkit of Abbott company and Archipct HBeAg Reagent kit, HBV-DNA detects and adopts Shanghai Ke Hua biotechnology company limited hepatitis B virus nucleic acid immue quantitative detection reagent box, detecting step is according to the test kit book that furnishes an explanation, real-timePCR amplification instrument adopts ABI-7300, detected result shows, HepG2.HS202G cell strain is stable to go down to posterity for more than 20 generations, energy stably express virion and HBsAg and HBeAg viral protein, the about 200IU/ml of its HBsAg left and right, the about 200S/CO of HBeAg left and right, compared with doubly left and right of the high about 5-10 of HepG2.2.15, supernatant HBV-DNA approximately 10
4-10
5left and right, a little less than HepG2.2.15(as shown in Figure 1),
The hbv replication situation in cell of carrying out detects, the total DNA extracting of cell, in 6 orifice plates, add 400ul cell pyrolysis liquid when cell 90% degree of converging, 37 ℃ add after spending the night equivalent phenol carry out DNA extracting, precipitation after DNA be dissolved in 20ul TE, 20XSSC transferring film after 1.3% gel electrophoresis, southern-blot hybridization, result shows HBV hybridization signal, show to exist in cell hbv replication, but be low (as shown in Figure 2) compared with HepG2.2.15 cell.
Claims (5)
1. based on HepG2 HBV rtS202G ETV resistance variant clone, it is characterized in that, this cell strain preservation accession designation number is CGMCC No.6393, called after HepG2.HS202G.
2. by claimed in claim 1 based on HepG2 HBV rtS202G ETV resistance variant clone, it is characterized in that, described HepG2.HS202G cell strain is stable to go down to posterity for more than 20 generations, stably express virion and HBsAg and HBeAg viral protein, its HBsAg200IU/ml, HBeAg200S/CO, compared with the high 5-10 of HepG2.2.15 doubly, supernatant HBV-DNA10
4-10
5.
3. it is characterized in that based on HepG2 HBV rtS202G ETV resistance variant clone by claimed in claim 1, there is hbv replication in cell in described HepG2.HS202G cell strain.
4. the construction process based on HepG2 HBV rtS202G ETV resistance variant clone of claim 1, is characterized in that, it comprises:
Adopt HBV eukaryon expression plasmid pREP-HBV-YVDD, its carrier framework is pREP10 carrier, hbv replication is for containing cp promotor and the pregenomic HBV1.2 of complete HBV times body, and site-directed mutagenesis is that the pREP10 that inserts excision RSV promotor after entecavir resistant genotype S202G is built into HBV eukaryon expression plasmid pREP-HBV-S202G; By after this plasmid transfection HepG2 cell by detecting the expression of hbv replication in cell, specific antigens and the generation of virion after hygromycin selection, and with HepG2.2.15 cell strain relatively after, establish the foundation of resistance HBV cell clone HepG2.HS202G.
Claim 1 based on HepG2 HBV rtS202G ETV resistance variant clone preparation screening antiradiation drug, research HBV Biological characteristics and with the HBV cell model of cell-cell interaction in purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210469842.2A CN103820392A (en) | 2012-11-18 | 2012-11-18 | Drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and establishment method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210469842.2A CN103820392A (en) | 2012-11-18 | 2012-11-18 | Drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and establishment method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103820392A true CN103820392A (en) | 2014-05-28 |
Family
ID=50755686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210469842.2A Pending CN103820392A (en) | 2012-11-18 | 2012-11-18 | Drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and establishment method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103820392A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111386126A (en) * | 2017-10-25 | 2020-07-07 | Nouscom股份公司 | Eukaryotic cell lines |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101988129A (en) * | 2009-08-03 | 2011-03-23 | 复旦大学附属华山医院 | Detection probe for hepatitis B virus adefovir dipivoxil medicament-resistant variant strains and application method thereof |
-
2012
- 2012-11-18 CN CN201210469842.2A patent/CN103820392A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101988129A (en) * | 2009-08-03 | 2011-03-23 | 复旦大学附属华山医院 | Detection probe for hepatitis B virus adefovir dipivoxil medicament-resistant variant strains and application method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 胡接力: "HBV逆转录酶特定变异对其药物敏感性的影响", 《中国博士学位论文全文数据库 医药卫生科技辑》, no. 2, 15 February 2011 (2011-02-15), pages 061 - 1 * |
| 黄清玲等: "乙型肝炎病毒在HepG2细胞中的稳定复制及表达", 《中华传染病杂志》, vol. 25, no. 9, 30 September 2007 (2007-09-30), pages 523 - 527 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111386126A (en) * | 2017-10-25 | 2020-07-07 | Nouscom股份公司 | Eukaryotic cell lines |
| CN111386126B (en) * | 2017-10-25 | 2024-01-30 | Nouscom股份公司 | Eukaryotic cell lines |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107080757B (en) | Humanized hepatitis B mouse model constructed by using stem cells and application | |
| CN102690796A (en) | Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme | |
| CN109897825A (en) | It is a kind of to be simple and efficient the cell system for generating hepatitis type B virus recombination cccDNA | |
| CN103820392A (en) | Drug-resistant variant cell line based on HepG2 HBV rtS202G ETV, and establishment method thereof | |
| CN104673760B (en) | A kind of purification process of procaryotic cell expression viruslike particle | |
| CN102807971B (en) | High-expression lamivudine-resistant hepatitis B virus (HBV) liver cancer cell strain | |
| CN103361314A (en) | Anti-ADV (adefovir) HBV (Hepatitis B virus) variant strain cell line capable of stably and efficiently replicating rtN236 and application of anti-ADV HBV variant strain cell line | |
| CN102816737A (en) | HepG2-based stable-expression HBV (hepatitis B virus) wild strain cell line and preparation method thereof | |
| CN105861442B (en) | Height transfer hepatoma cell strain and its construction method and application | |
| CN103361315A (en) | Drug-resistance variant strain cell line stably expressing HBV (Hepatitis B virus) and application of drug-resistance variant strain cell line | |
| CN102399749B (en) | Stable replication and expression cell line of Chinese patient C gene type multiple drug resistant HBV (Hepatitis B Virus) | |
| CN106754753A (en) | Virus culture process | |
| CN103436494B (en) | C genotype adefovir dipivoxil drug-resistant HBV (Hepatitis B Virus) stable replication and expression cell line | |
| CN102690801B (en) | Enzyme for synthesizing and metabolizing inosine monophosphate of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis and application thereof | |
| CN102102091B (en) | C genotype entecavir-resistant mutation HBV stable replication and expression cell line | |
| Ogura et al. | Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line | |
| CN101157910A (en) | Hybrid cell lines suitable for human hepatitis-B viral natural infective duplication and capable of subculturing | |
| CN100371022C (en) | Method for establishing anti-hepatitis C virus drug screening experimental animal model | |
| CN109055614A (en) | Primed probe group and kit based on RAA Fluorometric assay hepatitis type B virus | |
| CN102747057A (en) | Cordyceps sinensis hirsutella sinensis purine anabolism enzyme, gene thereof, and application thereof | |
| CN102690800B (en) | Enzyme for synthesizing and metabolizing xanthine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme | |
| CN103045574B (en) | Cordyceps sinensis hirsutella sinensis uridylic acid synthetase, as well as coding gene and application thereof | |
| CN1238508C (en) | Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation | |
| CN100500836C (en) | Method for culturing HCV virus in vitro | |
| CN102690797A (en) | Enzyme for synthesizing and metabolizing adenine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140528 |