CN101298605A - Hepatitis C virus in vitro replication model, construction method and use thereof - Google Patents
Hepatitis C virus in vitro replication model, construction method and use thereof Download PDFInfo
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- CN101298605A CN101298605A CNA2007100972963A CN200710097296A CN101298605A CN 101298605 A CN101298605 A CN 101298605A CN A2007100972963 A CNA2007100972963 A CN A2007100972963A CN 200710097296 A CN200710097296 A CN 200710097296A CN 101298605 A CN101298605 A CN 101298605A
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Abstract
The invention discloses an HCV in vitro reconstructed model, a method for establishing the reconstructed model and an application. The invention aims to provide an HCV in vitro infection cell model which has high HCV reproducing rate and can culture generation by generation and is close to the natural infection state. Meanwhile, the invention provides a method for establishing the in vitro reconstructed model, that is a plurality of HCV infection positive serums are mixed to inoculate the cell line cultured in vitro by adopting the centrifugal method; the concentration of the iron ion inside the HCV culture medium is changed to reconstruct the HCV in a high level; the HCV in vitro reconstructed model is obtained by a plurality of numbers of times of virus subculturing. The immunology method and the nucleic acid hybridization method are adopted to detect. The model can be applied to the establishment of an HCV animal infection model, the research on the HCV reconstructing mechanism, the drug screen and the vaccine development.
Description
Technical field
The present invention relates to hepatitis C virus in vitro replication model and construction process thereof and application, belong to virusology technology and medical biotechnology field.
Background technology
(Hepatitis C Virus is called for short: HCV) be the sub-thread positive chain RNA virus, belong to flaviviridae family hepatitis C virus.The hepatitis C that HCV causes is a kind of transmissible disease of serious threat human health, and world statistics has 1.7 hundred million people to be infected, and annual newly-increased 300～4,000,000 routine patients.Because HCV makes a variation easily, also there is not vaccine to come out so far, this makes that the harm of hepatitis C is even more serious than hepatitis B in a sense.
In the prior art, the about 9.6kb of hepatitis c virus gene group leader is made of relatively long 5 ' NCR (341nt), big opening code-reading frame, short 3 ' NCR and polyA or polyU.5 ' the NCR of HCV is the most conservative zone in all HCV strain isolateds, belongs to non-coding region, contains inherent IRES, and it may mediate the cap dependent/non-dependent translation of polyprotein precursor, belongs to the regulation and control unit; 3 ' the NCR that belongs to non-coding region together plays an important role to duplicating of HCV.Structural protein and Nonstructural Protein are contained in the coding region, and an initiator codon is only arranged, and translate into a polyprotein precursor, respectively this albumen are cut by virus and host's enzyme; Wherein, the NS3 virus enzyme from host's enzyme cutting ns3 coding is cut other albumen by viral enzyme again; The NS5B albumen of ns5b genes encoding virus has the RdRp activity, is the key enzyme that HCV duplicates.HCV is to be that template duplicating goes out its intermediate minus strand with normal chain intracellular duplicating, and be that template duplicating goes out its normal chain again with the minus strand, so the existence of HCV minus strand is the sign that HCV duplicates.
In the prior art, HCV concentration in infected tissue and serum is lower, lacks the suitable cell culture system practical animal model of unifying for a long time.
HCV Infection in Vitro cell model is very important instrument for the evaluation of deeply being familiar with HCV etiology characteristics, virus replication mechanism and pathogenesis, HCV protective antigen, vaccine development and specificity anti-HCV drug screening etc.For this reason, Chinese scholars has been done a large amount of explorations, mainly contains HCV cells infected model and HCV transgenic cell model two big classes.HCV is a kind of liver venereal disease poison of having a liking for, and people at first select for use liver source sexual cell to set up model; In addition, find that clinically HCV can also invade extrahepatic tissue such as peripheral blood cells, nephrocyte, spermatid etc.But in these HCV reconstructed models, the ubiquity replication rate is low, the cycle long, can not detect virus replication with the conventional nucleic acid hybridization technique, and only depends on RT-PCR to measure the synthetic of positive and negative chain.In addition, people attempt to strengthen to duplicate the HCV reconstructed model that the method that adopts gene transfection makes up does not mostly have sure virion to form.Therefore improve the infection rate of HCV in cultured cell in vitro, especially the strain that the adaptation cell in vitro that obtaining to go down to posterity cultivates is cultivated is one of problem that the researchist is badly in need of solving in this area for HCV replicanism, drug screening and vaccine development provide the desirable cell model near the natural infection state.
Summary of the invention
One object of the present invention is to provide a kind of hepatitis C virus in vitro replication model near the natural infection state.
Hepatitis C virus in vitro replication model provided by the invention is the cultured cell in vitro that has infected the viruses of human hepatitis C, and hepatitis C virus wherein is from hepatitis C patients serum.This hepatitis C virus in vitro replication model is the cells infected with higher replication rate that the viruses of human hepatitis C repeatedly activates on host cell, goes down to posterity and obtains.Described host cell is a cells of mamma animals, comprises liver source cell, lymph source cell, kidney-derived cells, spermatid etc.Wherein preferred host cell is kidney-derived cells, liver source cell, lymph source cell.
Another object of the present invention is to provide a kind of method that makes up hepatitis C virus Infection in Vitro reconstructed model.
The construction process of hepatitis C virus in vitro replication model provided by the invention is achieved through the following technical solutions:
1. select a kind of virus inoculation method,, contact, adsorb, cultivate with centrifuging with the jolting method with 1 and above infection with hepatitis C virus patients serum mixing.
2. select a kind of substratum, this substratum contains the iron ion that promotes hepatitis c viral replication.
3. select cells infected cracking supernatant liquor repeated infection normal host cell for use, through repeatedly cultivating the hepatitis C virus poison strain that has higher infection rate, can cause the cellular form change to obtain.
The construction process of hepatitis C virus Infection in Vitro model of the present invention specifically has the following steps:
(1) screening inoculum;
(2) screen responsive host cell strain;
(3) carry out virus inoculation and cultivation;
(4) optimize the virus culture condition;
(5) cell cultures adapts to the acquisition of strain;
Wherein said step (1) is that one or more infection with hepatitis C virus patients serum is mixed; Responsive host cell in the described step (2) is a cells of mamma animals, comprises a kind of of liver source cell, lymph source cell, kidney-derived cells, spermatid; Described step (3) is to inoculate with jolting method normal temperature, and after the absorption, centrifuging is cultivated; Described step (4) is the iron ion that contains 10-300 μ M in the conventional cell cultures based component; Described step (5) is a HCV cells infected cracking supernatant liquor repeated infection normal host cell, and cultivates the adaptation strain through repeatedly cultivating to obtain cell in vitro.
The invention has the advantages that: hepatitis C virus in vitro replication model of the present invention is to adopt existing virusology technology, and on this basis kind, inoculation method, the medium component of virus inoculation are transformed, thereby obtain to carry out subculture in vitro separately, and can keep the hepatitis C virus in vitro replication model of higher duplicating efficiency, and obtain cell in vitro cultivation adapted strain.Can adopt immunocytochemistry and nucleic acid hybridization technique to detect the expression of the antigenic expression of hepatitis C virus specific and the positive and negative chain of hepatitis C virus in this reconstructed model.
The construction process of hepatitis c viral replication model of the present invention has designed above-mentioned (1) first---and (4) important step: screening contains the HCV-RNA positive serum of high titre, adopt centrifugal culture method, the first Infection in Vitro lysis supernatant liquor that obtains infects the normal host cell again, and changes in the HCV nutrient solution iron concentration to improve replication rate.Above-mentioned (1) that the present invention designs first---(4) step, its method is easy, and is practical; Inoculum selection mode in this step, inoculation method can shorten the HCV infection time; The above-mentioned hepatitis C virus substratum of She Ji this has increased the levels of replication of hepatitis C virus in the cell in vitro first.The cell of the present invention in above-mentioned steps (2) can be liver source cell, lymph source cell, kidney-derived cells and spermatid.
Reconstructed model of the present invention is tested and appraised, and utilizes it to carry out anti-hepatitis C virus drug screening and vaccine research.
Description of drawings
Fig. 1 is hepatitis C virus (HCV) cells infected RT-PCR analysis chart.
M:DNA Marker, DL2000; 1: the RT-PCR of positive chain RNA; 2: the RT-PCR of strand RNA;
3,4: the RT-PCR of control cells RNA
Fig. 2 is hepatitis C virus (HCV) positive serum Infection in Vitro Vero cell in-situ results of hybridization figure
A: normal chain probe in situ hybridization 10 *, B: normal chain probe in situ hybridization 25 *
C: minus strand probe in situ hybridization 10 *, D: minus strand probe in situ hybridization 25 *
Fig. 3 is that hepatitis C virus (HCV) positive and the external inoculation of negative serum Vero cellular immunization cytochemistry detect figure.
A: negative control Vero cell; B: vero cells infection
Fig. 4 HCV positive serum Infection in Vitro HepG2 cellular immunization cytochemistry detects figure
Fig. 5 infects normal Vero cell in-situ results of hybridization figure for cells infected cracking supernatant
A: normal chain probe in situ hybridization 10 *, B: normal chain probe in situ hybridization 25 *
C: minus strand probe in situ hybridization 10 *, D: minus strand probe in situ hybridization 25 *
Fig. 6 infects normal Vero cellular immunization cytochemistry detection figure for cells infected cracking supernatant.
Fig. 7 detects figure for the Vero cellular immunization cytochemistry that recovery the 8th generation HCV infects
Fig. 8 is the form of the adapted strain HCV vero cells infection of acquisition
A: be normal short shuttle type Vero cell, B: for infecting long shuttle type Vero cell
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail:
Embodiment 1. hepatitis C viruss (HCV) positive serum is selected
Screening hospital outpatient acute hepatitis C patients serum detects negatively through HAV, HBV, HDV, HIV, do not carry out antiviral therapy, and fluorescence quantitative PCR detection HCV-RNA tires＞and 10
6The 2-3 person-portion serum of copy/ml mixes, and packing after the filtration sterilization is deposited for-80 ℃.
The inoculation of embodiment 2. host cells:
1. experiment material
(1) cell strain: Vero cell, HepG2 cell, MA104 cell, HEK293 cell.Inoculum: HCV infected patient positive serum, from No. 302 Hospital, CPLA Viral Laboratory, 3 person-portions, index and detection by quantitative are as described in the embodiment 1.The normal serum contrast, through HAV, HBV, HCV, HDV, HIV index detect negative.
(2) used substratum of experiment and reagent: cell culture fluid: the DMEM substratum that contains 10%FBS; Keep liquid, contain the DMEM substratum of 2-5%FBS; Infect nutrient solution for to contain the cell culture fluid of different concns ferrous ion (ferrous sulfate, iron protochloride or ferrous ammonium sulphate etc.) or to keep liquid.
2. experimental technique
(1) the logarithmic phase cell is with 0.25% pancreatin+0.02%EDTA digestion, and transferring cell concn is 1 * 10
6Individual/ml, 2ml adds (built-in cover glass is equipped with immunocytochemical determination and nucleic acid hybridization mensuration is used) in 35mm culture dish or the 6 porocyte culture plates, 5%CO
224-48h in the incubator.
(2) virus inoculation: after cell covers with 80%, remove substratum, and, dilute positive pooled serum with the DMEM substratum, final concentration 10-20% with DMEM substratum washing 2 times, the inoculating cell surface, every hole or ware 0.5ml, room temperature jog 30min, 37 ℃ hatch after, add the substratum (keeping liquid) that contains 2%FBS, centrifugal cultivation.Abandon inoculum, use the infection nutrient solution that contains 10%FBS instead.5%CO
2, hatch 2-5d for 37 ℃.If negative serum contrasts and does not add iron ion and contrasts.
(3) experiment condition: respectively from centrifugal speed, the HCV infection rate is investigated in iron concentration three aspects in adsorption time, the substratum.Rotating speed is established 0g/min, 600g/min, 1000g/min, 1500g/min, 2000g/min respectively; 37 ℃ of adsorption times are established 0min, 30min, 60min, 90min, 120min respectively; The concentration of ferrous ion is selected 0 μ M respectively for use, 10 μ M, 50 μ M, 75 μ M, 100 μ M, 150 μ M, 200 μ M, 300 μ M.
(4) adopt embodiment 5 methods to measure
The result shows: rotating speed is at 600-2000g/min, and 37 ℃ of incubation times are at 30-120min, all can shorten time that HCV infects and the amount that penetrates host cell and the state of not obvious change cell itself; Ferrous ion can increase duplicating of HCV at 10-300 μ M.Optimum condition is rotating speed 600-2000g/min, and incubation time is at 60-90min, and ferrous ion concentration is 50-150 μ M.With this understanding, all obtain higher infection rate at selected cell strain.
Embodiment 3. nido RT-PCR (nRT-PCR) methods are measured the expression of the positive and negative chain of HCV in hepatitis C virus (HCV) the Infection in Vitro model:
Cell is through the PBS washed twice of trysinization, ice precooling, with the PBS suspension of 100 μ l.The TRIZOL method is extracted total RNA of serum and cells infected.
' cDNA of NCR is stencil design medial and lateral primers, is template with total RNA, obtains cDNA first chain with outside downstream primer R1 reverse transcription, again with interior survey upstream and downstream primers F 2 and R2 amplification, obtains the RT-PCR product of positive chain RNA with the conserved regions 5 of HCV; With RNA is that template obtains cDNA first chain with outside upstream primer F1 reverse transcription, again with interior survey upstream and downstream primers F 2 and R2 amplification, obtains the RT-PCR product of strand RNA.
If HCV does not duplicate, there is not strand RNA among total RNA of extraction, then can't use F1 reverse transcription cDNA first chain, promptly do not detect the RT-PCR product of strand RNA, vice versa.The results are shown in accompanying drawing 1 and description of drawings thereof.
Embodiment 4. in situ hybridization methods detect the expression of the positive and negative chain of hepatitis C virus (HCV) Infection in Vitro model HCV:
With the plasmid that has HCV-cDNA is template, is equipped with single stranded DNA with the asymmetric PCR legal system, carries out mark, the positive and negative chain probe of preparation DIG mark with the digoxigenin labeled test kit of German Bao Ling Man.Cell climbing sheet carries out in situ hybridization and measures after Paraformaldehyde 96 is fixing, and microscopically is observed and taken a picture, and positive signal is a blue-purple granule in the cell.The results are shown in accompanying drawing 2 and description of drawings thereof.
Embodiment 5. immunocytochemical methods are measured the antigenic expression of HCV in hepatitis C virus (HCV) the Infection in Vitro model
After cell climbing sheet is fixing, be one anti-with anti-HCV-NS3 monoclonal antibody, sheep anti mouse HGP-IgG is two anti-, and the SABC method is carried out immunocytochemical determination, and microscopically is observed, and positive signal is a brown particle in the cell.The results are shown in accompanying drawing 3 and accompanying drawing 4 and description of drawings thereof.
Embodiment 6. continuous passage methods are measured going down to posterity of hepatitis C virus (HCV) cells infected model HCV:
Use embodiment 2 methods to obtain external first-generation replication-competent virus, after the amplification of going down to posterity, with the cells infected cracking, centrifugal back supernatant infects normal adherent back cell with the MOI of 10FFU/ cell, uses with embodiment 2 identical HCV and infects culture medium culturing, this is the s-generation, and the like.In situ hybridization and immunocytochemical determination infection rate, method is with embodiment 4 and embodiment 5.The result: the virus that this method obtains can pass more than 10 generations.See accompanying drawing 5, accompanying drawing 6 and accompanying drawing 7 and description of drawings thereof.
Embodiment 7. agar culture methods are measured the HCV infection rate of hepatitis C virus (HCV) cells infected model cracking supernatant liquor:
3-4 cracking cell of cells infected multigelation, getting supernatant after centrifugal is HCV liquid, and to keep liquid 10 doubling dilution viruses, inoculation grows up to the cell of individual layer, and inoculation method is with embodiment 2, and extent of dilution is 10
1～10
8, cultivate 24h.
Preparation soft solid DMEM substratum is poured above-mentioned cell into before solidifying, hatch for 37 ℃ and continue 48h.Rinse out agar, with the PBS washing, carry out immunocytochemical determination, method is with embodiment 5, and positive findings is made the sxemiquantitative record, and mirror is counted the quantity of positive cell in 200 cells down.Result: the HCV virus quantity (MOI)＞10 in the cells infected cracking supernatant
12FFU/ml (multiplicity ofinfection, MOI; Focus forming units, FFU), promptly every milliliter of viral liquid can form kitchen range sex clone number greater than 10
12
Embodiment 8. hepatitis C viruss (HCV) cell cultures adapted strain obtains
Virus inoculation also goes down to posterity after 6-8 time, replacing contains 2-5%FBS, the DMEM substratum of 50-150 μ M ferrous ion is kept cultivation, this strain cells infected of continuous passage, obtain the HCV cell in vitro and cultivate adapted strain, this strain virus causes morphological change to host cell, and promptly cell becomes elongated shuttle type by short shuttle type.Method is with embodiment 4 and embodiment 5.The results are shown in accompanying drawing 8 and description of drawings thereof.
Embodiment 9. antiviral are to the restraining effect of hepatitis C virus (HCV) replication in vitro
Use embodiment 2 methods, obtain the HCV infection model, select cell inoculation 96 orifice plates of third generation virus infection for use, behind the cell attachment, choose virazole, γ-INF, α
2The sulfo-antisense oligonucleotide of β-INF and 5 ' non-coding region carries out drug study as trial-product with each medicine non-toxic concn, acts on termination in 7 days, changes liquid therebetween once.Use embodiment 4, embodiment 5 methods to carry out the evaluation of pesticide effectiveness, gray scale scanning quantitative assay drug effect; Measure the antigen presentation of HCV with original position cell ELISA method and streaming instrument; The result shows that virazole can reach 28% to the inhibiting rate of HCV, and virazole and γ-INF drug combination can reach 40%, and the sulfo-antisense oligonucleotide can reach more than 55%.
The application of embodiment 10. hepatitis C virus in vitro replication model in vaccine development
The viral liquid that this model is obtained carries out purifying with sucrose density gradient centrifugation, with 37 ℃ of deactivation 10d of 1/3000 formaldehyde, after PEG2000 concentrates, measure protein content, be diluted to 1mg/ml with PBS, mixed with 1: 1, immune C57/BL mouse with the Fu Shi Freund's complete adjuvant, select back, intracutaneous, multi-point injection, each some 0.1ml.7d behind the initial immunity, with the amount booster immunization once.Muscle or vein, every some 0.1-0.5ml, the ELISA method is measured serum titer.Get that immune serum carries out virus neutralization experiment and cell killing, virus suppresses experiment, with the humoral immunization protection and the cellular immunization protective effect of estimation immune serum.The humoral immunization protective capability that the result shows HCV a little less than, promptly the virus neutralizing capacity of serum is relatively poor, but can induce stronger cell immune response.
The structure of embodiment 11. hepatitis C virus animal infection modals
Utilize the rhesus monkey of the viral vena axillaris injection of purifying, tracking and measuring monkey body inner virus amount through x radiation x and excessive iron ion processing; Getting positive monkey serum continues to infect of the same race through pre-treated animal.Serological identification is inoculated transaminase, year poison amount of animal, and liver puncture is measured the pathological change of hepatic tissue and duplicating of HCV, and the result shows that the HCV that utilizes this replication in vitro model to prepare can infection animal.
Claims (7)
1. hepatitis C virus in vitro replication model is characterized in that infecting the cultured cell in vitro of hepatitis C virus.
2. hepatitis C virus in vitro replication model according to claim 1 is characterized in that: host cell is a cells of mamma animals, comprises liver source cell, lymph source cell, kidney-derived cells, spermatid.
3. the construction process of a hepatitis C virus in vitro replication model as claimed in claim 1 or 2 may further comprise the steps:
(1) screening inoculum;
(2) screen responsive host cell strain;
(3) carry out virus inoculation and cultivation;
(4) optimize the virus culture condition;
(5) cell cultures adapts to the acquisition of strain;
Wherein said step (1) is that one or more infection with hepatitis C virus patients serum is mixed; Responsive host cell strain in the described step (2) is a cells of mamma animals, comprises a kind of of liver source cell, lymph source cell, kidney-derived cells, spermatid; Described step (3) is to inoculate with jolting method normal temperature, and after the absorption, centrifuging is cultivated; Described step (4) is the iron ion that contains 10-300 μ M in the conventional cell cultures based component; Described step (5) is a HCV cells infected cracking supernatant liquor repeated infection normal host cell, and cultivates the adaptation strain through repeatedly cultivating to obtain cell in vitro.
4. the purposes of a hepatitis C virus in vitro replication model as claimed in claim 1, this model is used to screen the anti-hepatitis c virus medicine.
5. the purposes of a hepatitis C virus in vitro replication model as claimed in claim 1, this model is used for the hepatitis c viral replication Mechanism Study.
6. the purposes of a hepatitis C virus in vitro replication model as claimed in claim 1, this model is used to prepare inoculum, makes up the hepatitis C virus animal infection modal.
7. the purposes of a hepatitis C virus in vitro replication model as claimed in claim 1, this model is used to prepare the hepatitis C virus immune vaccine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864397A (en) * | 2009-04-14 | 2010-10-20 | 中国医学科学院基础医学研究所 | Establishment of in vitro cell model supporting HCV 1b sub-genome duplication |
| CN108998405A (en) * | 2018-06-15 | 2018-12-14 | 翁炳焕 | A kind of preparation of HCV-RNA detection monoclonal Quality Control Reference Strains |
| CN110669739A (en) * | 2019-09-30 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of novel hepatitis A virus antigen |
| WO2023051607A1 (en) * | 2021-09-29 | 2023-04-06 | 上海行深生物科技有限公司 | Virus culture method |
-
2007
- 2007-04-30 CN CNA2007100972963A patent/CN101298605A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864397A (en) * | 2009-04-14 | 2010-10-20 | 中国医学科学院基础医学研究所 | Establishment of in vitro cell model supporting HCV 1b sub-genome duplication |
| CN108998405A (en) * | 2018-06-15 | 2018-12-14 | 翁炳焕 | A kind of preparation of HCV-RNA detection monoclonal Quality Control Reference Strains |
| CN110669739A (en) * | 2019-09-30 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Preparation method of novel hepatitis A virus antigen |
| WO2023051607A1 (en) * | 2021-09-29 | 2023-04-06 | 上海行深生物科技有限公司 | Virus culture method |
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