CN105062979A - Method for cultivating hepatitis e virus - Google Patents
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Abstract
The invention discloses a method for cultivating hepatitis e virus. The method comprises the steps that a serum of a later pregnant woman is added into an HEB strain obtained through separation of an epidemic disease investigation for in-vitro culture, the virus can be replicated massively, and immunofluorescence and Western Blot analysis are utilized for revealing that the serum of the later pregnant woman can promote massive replication of HEV progeny virus. According to the method, great breakthrough is made in the aspect of HEV in-vitro culture, and a good foundation is laid for further researching the biological and immunological characteristics of HEV and the HEV infection and pathogenic mechanism and screening and identifying anti-HEV drugs.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of hepatitis E virus strain and successfully cultivate in vitro and the novel method improving progeny virus duplicating efficiency.
Background technology
Hepatitis E virus (HepatitisEVirus, HEV) is a kind of through intestinal transmitted viral hepatitis pathogenic agent, can across interspecific infection people and many animals.HEV is without cyst membrane single strand plus RNA virus, and ball-type virus granular size is 27 ~ 34nm, and there is projection on surface and incises, and internal density is uneven, and full-length genome is about 7.3kb, is made up of 3 open reading frame.Whole world HEV mainly contains 4 kinds of genotype, mainly common with IV type HEV strain in China.HEV route of transmission, mainly through fecal oral route, also propagates by other approach, comprise blood born, contact transmission, vertical transmission, and organ transplantation is propagated.
HEV is the pathogenic agent causing many developing countries hepatitis E fulminant popular, and after HEV infects, general patient self-healing within 4-6 week, immune deficiency patient and the elderly easily develop into chronic hepatitis.Estimate according to the World Health Organization, HEV is infected in the whole world every year nearly 2,010 ten thousand people, cause 700,000 people's death and the death of 3000 fetal in utero, the non-pregnant woman and the pregnant patient's mortality ratio that infect HEV are respectively 1.9% and 19.8% infection of pregnant women HEV, and sickness rate is high, be in a bad way, especially the most serious with third trimester of pregnancy, case fatality rate can up to 25%, the premature labor of normal generation fetus, miscarriage, the symptoms such as stillborn foetus and pregnant woman's acute severe hepatitis in postpartum.The prevention and control situation of hepatitis E is extremely urgent, and the urgency that developing effective hepatitis E vaccine has become relation public health is urgent problem, and virus culture is most important in this course.
Virus culture is research viral biology characteristic, the basis of pathogenesis and vaccine development.HEV virion is observed under nineteen eighty-three first Electronic Speculum, apart from the present more than 30 years, all the time, relate to the research of HEV in a large number, the vitro culture of virus is one of the focus and difficult point of research always, this also just seriously hinders the research of virus replicanism in cell, and then has blocked the research and development of HEV vaccine.Until Tanaka in 2007 etc., attempt cultivating HEV by 21 kinds of clones, HEV breeds very fast and is easy to detected in A549 and PLC/PRF/5 cell, Okamoto and Tanaka in 2011 etc., A549 and PLC/PRF/5 clone is utilized successfully to establish HEV cell culture system further, by inoculating the virus (10 of high titre
7) after obtain higher titre progeny virus (inoculate and reach 10 after 50 days
8).Solid basis is established in be established as follow-up HEV replicanism, the antiviral research and development etc. of HEV culture system in vitro.But HEV virus replication is still limited, thus must optimize.
Summary of the invention
The object of the present invention is to provide a kind of hepatitis E virus (HepatitisEVirus, the method of HEV) cultivating in vitro, the method is cultivated in vitro by the HEV strain of adding the higher titre that pregnant late period, serum pop disease investigation was separated to, viral massive duplication can be made, for further investigation HEV biology, immunological characteristic and pathogenicly provide requisite cause of disease material, for the research of the diagnosis of hepatitis E, drug screening and evaluation and vaccine provides effective in vitro models.
Hepatitis E virus extracorporeal culturing method of the present invention, adopts following steps to carry out:
(1) by HEV viral solution after 0.22 μm of membrane filtration is degerming, add the dual anti-solution of viral solution volume 1 ~ 2%, 4 DEG C process 1h ,-80 DEG C save backup, and measuring its viral copy number through Real-timeqPCR is 2 × 10
5~ 2 × 10
6copy number/mL, wherein dual anti-solution is the solution containing 400U/mL penicillin and 1000U/mL Streptomycin sulphate;
(2) A549 cell is pressed 2 × 10
5/ hole is seeded in 6 orifice plates, with the DMEM substratum containing mass percent 10% foetal calf serum at 37 DEG C, 5%CO
2static gas wave refrigerator cell in incubator, until cell grows up to individual layer;
(3) be seeded in monolayer cell by the HEV viral suspension of step (2) by every hole 100 μ L, put 37 DEG C and hatch 1 hour, every 15min slightly shakes up once, and virus is fully adsorbed onto on cell; Abandon supernatant, 1 × PBS washed cell 3 times, add the DMEM substratum containing mass percent 2 ~ 5% serum in pregnant late period, add 0.01mMMgCl simultaneously
2the protection integrity of virion and the adsorption of enhanced virus and cell, be placed in 37 DEG C, 5%CO
2continue in incubator to cultivate;
(4) inoculate HEV virus after 6 days A549 cell there is pathology phenomenon, multigelation, collect sick cell and nutrient solution ,-80 DEG C of preservations are stand-by.
Described pregnant late period, serum was up to pregnant woman's serum in late period or animal serum in pregnant late period, adopted ordinary method collection.
Compared to prior art, the present invention adopts hepatitis E virus to cultivate in A549 cell, adds pregnant woman's serum in late period and viral replication efficiency can be made to be increased to 2 × 10
8copy number/ml.
Adopt the inventive method to the strain HEV virus be separated from the positive swine excrement sample of Chinese yunnan Kunming HEVRNA, genotype is 4 types, GenBankNo.JF747598; This strain can be cultivated in vitro, reaches 2 × 10 in culture supernatant without concentrated virus titer
8copy number/ml, virus is constant in-80 DEG C of Refrigerator stores 3 years biological activitys, still can infect human lung carcinoma cell (A549) strain, in vitro can be more than continuous passage to 9 generations.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the A549 cell infection HEV used in the present invention; Wherein A figure is normal A549 cell, and B figure is the A549 cell that HEV infects after 6 days, and C figure is the electromicroscopic photograph of A549 cell infection HEV;
Fig. 2 is that in the present invention, RT-nPCR detects HEVRNA schematic diagram in culture supernatant; Wherein 1 is pregnancy serum cultivation A549 cell; 2 is calf serum cultivation A549 cell; The 3 A549 cells infected for pregnancy serum cultivation HEV; The 4 A549 cells infected for calf serum cultivation HEV;
Fig. 3 is that in the present invention, RT-qPCR detection pregnant woman serum in late period promotes that HEVmRNA's copies schematic diagram;
Fig. 4 is that in the present invention, Westernblot detects HEVORF2 proteose intention; Wherein 1 is A549 cell; The 2 A549 cells infected for calf serum cultivation HEV; The 3 A549 cells infected for pregnancy serum cultivation HEV.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail, but scope is not limited to described content; The main cell biology method adopting routine in embodiment, these methods are well known to those of ordinary skill in the art.According to following examples, be not difficult slightly modified and conversion and successful implementation the present invention as the case may be, and these amendments and conversion all drop in the scope of the application's claim.
Embodiment 1:A549 cell cultures
In liquid nitrogen, take out the warm water that A549 cell (buying in U.S. ATCC, numbering CCL – 185) puts into rapidly 37 DEG C, make cell suspension fast melt; Then add and buy in GIBCOInvitrogenCorporation company containing 10% new-born calf serum DMEM() nutrient solution, pH to 7.2 is adjusted to cultivate about 4h in 37 DEG C, discard whole nutrient solution, add fresh nutrient solution to continue to cultivate, after growing up to fine and close individual layer, buy in GIBCOInvitrogenCorporation company with PBS() wash cell, Yi Mei – EDTA(buys in GIBCOInvitrogenCorporation company) digestion, add above-mentioned nutrient solution, be placed in 37 DEG C, 5%CO
2continue in incubator to cultivate, shown in A549 cell as normal in Figure 1A.
Embodiment 2: with the DMEM culture medium culturing HEV adding pregnant woman's serum in late period
1, the PBS(pH7.4 of weightmeasurement ratio 10% is prepared by the HEV the being used for HEV separation positive ight soil collected) fecal suspension, concuss, make ight soil emulsification, through 4 DEG C, the centrifugal 10min of 12000g, collect supernatant, 0.22 μm of membrane filtration is degerming, adds the dual anti-solution (400U/mL penicillin and 1000U/mL Streptomycin sulphate) of viral solution volume 2%, 4 DEG C of process 1h,-80 DEG C save backup, and measuring its viral copy number through Real-timeqPCR is 2 × 10
6copy number/mL;
Be separated from the positive swine excrement sample of Chinese yunnan Kunming HEVRNA, genotype is 4 types, and GenBank database accession number is No.JF747598;
2, by cell in embodiment 1 (A549 cell) by 2 × 10
5/ hole is seeded in 6 orifice plates, with the DMEM substratum containing mass percent 10% foetal calf serum at 37 DEG C, 5%CO
2static gas wave refrigerator cell in incubator, when cell grows to 70 ~ 80% of individual layer, abandon supernatant substratum, every hole adds 100 μ LHEV viral suspensions, puts 37 DEG C and hatches 1 hour, every 15min slight wobble once, virus is fully adsorbed onto on cell, abandons supernatant, 1 × PBS washed cell 3 times, add the DMEM substratum containing mass percent 2% pregnant woman serum in late period, add final concentration is 0.01mMMgCl simultaneously
2the integrity of protection virion, is placed in 37 DEG C, 5%CO
2continue in incubator to cultivate; As shown in Figure 1B, HEV infects shown in latter 6 days, and when cytopathy reaches more than 75%, multigelation makes cell come off completely, results virus.
Corresponding carry out following system identification by the HEV virus that above-mentioned cultural method obtains: 1. use RT-PCR to detect in culture supernatant to have the RNA(of HEV qualitative) (see Fig. 2); 2. utilize electron microscopic observation, confirm that HEV forms progeny virus (see Fig. 1 CHEV electromicroscopic photograph, shown in arrow) in A549 cell; 3. detect pregnant woman's serum in late period with RT-qPCR and promote copying (see Fig. 3) of HEVmRNA; 4. use HEVORF2 monoclonal antibody, detect through Westernblot, again prove that pregnant woman's serum in late period can promote the synthesis (see Fig. 4) of HEV albumen compared with calf serum.In a word, the quantitative and qualitative analysis qualification of different levels has been carried out from the filial generation HEV of aspect to culture supernatant such as the existence of the genomic amplification of HEV, progeny virion, progeny virus biological activity and infectivities; Result confirms, pregnant woman's serum in late period of interpolation 2% compared with the calf serum of 2% significantly can promote virus replication (see Fig. 2 and Fig. 3).
Embodiment 2:
(1) HEV virus (is separated from the positive swine excrement sample of Chinese yunnan Kunming HEVRNA, genotype is 4 types, GenBank database accession number is No.JF747598) solution is after 0.22 μm of membrane filtration is degerming, add the dual anti-solution of viral solution volume 1%, 4 DEG C of process 1h,-80 DEG C save backup, and measuring its viral copy number through Real-timeqPCR is 2 × 10
5copy number/mL, wherein dual anti-solution is the solution containing 400U/mL penicillin and 1000U/mL Streptomycin sulphate;
(2) A549 cell is pressed 2 × 10
5/ hole is seeded in 6 orifice plates, with the DMEM substratum containing mass percent 10% foetal calf serum at 37 DEG C, 5%CO
2static gas wave refrigerator cell in incubator, until cell grows up to individual layer;
(3) be seeded in monolayer cell by the HEV viral suspension of step (2) by every hole 100 μ L, put 37 DEG C and hatch 1 hour, every 15min slightly shakes up once, and virus is fully adsorbed onto on cell; Abandon supernatant, 1 × PBS washed cell 3 times, add the DMEM substratum containing mass percent 5% calver serum in late period, add final concentration is 0.01mMMgCl simultaneously
2the protection integrity of virion and the adsorption of enhanced virus and cell, be placed in 37 DEG C, 5%CO
2continue in incubator to cultivate;
(4) inoculate HEV virus after 6 days A549 cell there is pathology phenomenon, multigelation, collect sick cell and nutrient solution ,-80 DEG C of preservations are stand-by.
(5) utilize RT-qPCR to detect to find, the virus titer adding 5% pregnant woman's serum in late period is 6.02 × 10
8, and the virus titer adding 5% calf serum is only 4.25 × 10
6.
Result confirms, the calver serum in late period of interpolation 5% compared with interpolation 5% calf serum can significantly promote copying of HEV.
Embodiment 3:(1) HEV virus (is separated from the positive swine excrement sample of Chinese yunnan Kunming HEVRNA, genotype is 4 types, GenBank database accession number is No.JF747598) solution is after 0.22 μm of membrane filtration is degerming, add the dual anti-solution of viral solution volume 1%, 4 DEG C of process 1h,-80 DEG C save backup, and measuring its viral copy number through Real-timeqPCR is 2 × 10
6copy number/mL, wherein dual anti-solution is the solution containing 400U/mL penicillin and 1000U/mL Streptomycin sulphate;
(2) A549 cell is pressed 2 × 10
5/ hole is seeded in 6 orifice plates, with the DMEM substratum containing mass percent 10% foetal calf serum at 37 DEG C, 5%CO
2static gas wave refrigerator cell in incubator, until cell grows up to individual layer;
(3) be seeded in monolayer cell by the HEV viral suspension of step (2) by every hole 100 μ L, put 37 DEG C and hatch 1 hour, every 15min slightly shakes up once, and virus is fully adsorbed onto on cell; Abandon supernatant, 1 × PBS washed cell 3 times, add the DMEM substratum containing mass percent 3% pregnant woman serum in late period, add final concentration is 0.01mMMgCl simultaneously
2the protection integrity of virion and the adsorption of enhanced virus and cell, be placed in 37 DEG C, 5%CO
2continue in incubator to cultivate;
(4) inoculate HEV virus after 6 days A549 cell there is pathology phenomenon, multigelation, collect sick cell and nutrient solution ,-80 DEG C of preservations are stand-by.
(5) utilize RT-qPCR to detect to find, the virus titer adding 3% pregnant woman's serum in late period is 3.57 × 10
8, and the virus titer adding 3% calf serum is only 7.34 × 10
6.
Result confirms, compared with the calf serum of interpolation 3%, the pregnant woman's serum in late period adding 3% can significantly promote that HEV copies.
Claims (1)
1. cultivate a method for hepatitis E virus, it is characterized in that carrying out as follows:
(1) by HEV viral solution after 0.22 μm of membrane filtration is degerming, add the dual anti-solution of viral solution volume 1 ~ 2%, 4 DEG C process 1h ,-80 DEG C save backup, and measuring its viral copy number through Real-timeqPCR is 2 × 10
5~ 2 × 10
6copy number/mL, wherein dual anti-solution is the solution containing 400U/mL penicillin and 1000U/mL Streptomycin sulphate;
(2) A549 cell is pressed 2 × 10
5/ hole is seeded in 6 orifice plates, with the DMEM substratum containing mass percent 10% foetal calf serum at 37 DEG C, 5%CO
2static gas wave refrigerator cell in incubator, until cell grows up to individual layer;
(3) be seeded in monolayer cell by the HEV viral suspension of step (2) by every hole 100 μ L, put 37 DEG C and hatch 1 hour, every 15min slightly shakes up once, and virus is fully adsorbed onto on cell; Abandon supernatant, 1 × PBS washed cell 3 times, add the DMEM substratum containing mass percent 2 ~ 5% serum in pregnant late period, add final concentration is 0.01mMMgCl simultaneously
2the protection integrity of virion and the adsorption of enhanced virus and cell, be placed in 37 DEG C, 5%CO
2continue in incubator to cultivate;
(4) inoculate HEV virus after 6 days A549 cell there is pathology phenomenon, multigelation, collect sick cell and nutrient solution ,-80 DEG C of preservations are stand-by.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105671004A (en) * | 2016-03-23 | 2016-06-15 | 昆明理工大学 | Method for cultivating hepatitis E viruses |
| CN106148289A (en) * | 2016-08-10 | 2016-11-23 | 昆明理工大学 | A kind of method of purification hepatitis E virus |
| CN109652384A (en) * | 2019-02-21 | 2019-04-19 | 昆明理工大学 | A kind of method of in vitro culture Hepatitis E virus |
| CN109769748A (en) * | 2019-02-21 | 2019-05-21 | 昆明理工大学 | The construction method of Hepatitis E virus chronicity mouse model |
| CN109943536A (en) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | A kind of Hepatitis E virus, the preparation method of cultural method and its inactivated vaccine |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105671004A (en) * | 2016-03-23 | 2016-06-15 | 昆明理工大学 | Method for cultivating hepatitis E viruses |
| CN106148289A (en) * | 2016-08-10 | 2016-11-23 | 昆明理工大学 | A kind of method of purification hepatitis E virus |
| CN109652384A (en) * | 2019-02-21 | 2019-04-19 | 昆明理工大学 | A kind of method of in vitro culture Hepatitis E virus |
| CN109769748A (en) * | 2019-02-21 | 2019-05-21 | 昆明理工大学 | The construction method of Hepatitis E virus chronicity mouse model |
| CN109769748B (en) * | 2019-02-21 | 2021-07-06 | 昆明理工大学 | Construction method of hepatitis E virus chronic mouse model |
| CN109652384B (en) * | 2019-02-21 | 2021-10-26 | 昆明理工大学 | Method for culturing hepatitis E virus in vitro |
| CN109943536A (en) * | 2019-03-26 | 2019-06-28 | 昆明理工大学 | A kind of Hepatitis E virus, the preparation method of cultural method and its inactivated vaccine |
| CN109943536B (en) * | 2019-03-26 | 2021-09-14 | 昆明理工大学 | Method for culturing hepatitis E virus and method for preparing inactivated vaccine thereof |
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