CN1216985C - Hepatitis A virus strain, method for preparing hepatitis A inactivated vaccine and obtained vaccine - Google Patents
Hepatitis A virus strain, method for preparing hepatitis A inactivated vaccine and obtained vaccine Download PDFInfo
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- CN1216985C CN1216985C CN021069859A CN02106985A CN1216985C CN 1216985 C CN1216985 C CN 1216985C CN 021069859 A CN021069859 A CN 021069859A CN 02106985 A CN02106985 A CN 02106985A CN 1216985 C CN1216985 C CN 1216985C
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- 208000005252 hepatitis A Diseases 0.000 title claims abstract description 27
- 229940031551 inactivated vaccine Drugs 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 24
- 229960005486 vaccine Drugs 0.000 title claims abstract description 21
- 241000700605 Viruses Species 0.000 claims abstract description 14
- 210000003501 vero cell Anatomy 0.000 claims abstract description 14
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- 239000011159 matrix material Substances 0.000 claims description 11
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- 230000029087 digestion Effects 0.000 claims description 5
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- 238000012258 culturing Methods 0.000 claims description 3
- 230000001524 infective effect Effects 0.000 claims description 3
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- 108010009736 Protein Hydrolysates Proteins 0.000 claims 1
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- 239000000427 antigen Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
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- 229930006000 Sucrose Natural products 0.000 description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 8
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- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 2
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- 229940124724 hepatitis-A vaccine Drugs 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
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- 208000028774 intestinal disease Diseases 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 229960003127 rabies vaccine Drugs 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a new hepatitis A virus strain YN5, a method for preparing hepatitis A inactivated vaccines by using the virus strain YB5 through Vero cells and a vaccine prepared by using the method. The present invention also relates to a method for breeding the hepatitis A virus strain.
Description
Technical field
The present invention relates to a kind of new hepatitis A virus strain, utilize its method for preparing hepatitis A inactivated vaccine, and the vaccine that makes of method thus.The present invention especially relates to a kind of method of the Vero of utilization cell preparation hepatitis A inactivated vaccine and the vaccine that makes of method thus.The invention still further relates to the selection of hepatitis A virus strain.
Background technology
Hepatitis A is to infect a kind of acute infectious intestinal disease that human body causes by hepatitis A virus.Hepatitis A be have now found that have a strong impact on one of six kinds of viral hepatitis of human health, and infection rate is for the highest.China is the high popular district of hepatitis A, all has every year popular or breaks out.1988 during the spring the sea break out hepatitis A and be very popular, the infected morbidity of nearly 300,000 people causes serious social influence and enormous economic loss.For hepatitis A so far people still do not have effective medicine and treatment means, vaccination is the effective and the most most economical measure of human so far this disease of control.
From the end of the seventies, carry out the development of hepatitis A vaccine both at home and abroad in succession, American-European developed country adopts the reliable inactivated vaccine technological line of development security, but manufacturing cost is high; In country's " the Seventh Five-Year Plan " brainstorm subject was listed the Attenuated HAV development in 1985 by China, obtained the trial production certification to 1992, and put on market.Being extensive use of of Attenuated HAV, for reducing the sickness rate of China's hepatitis A, the control outbreak of epidemic has played important role.
Yet Attenuated HAV exists significant disadvantages, and the most outstanding is virulence reversion and time propagation problem, and inoculator and Close contacts thereof are constituted the potential potential safety hazard; Secondly to immunologic hypofunction or handicapped's highly dangerous; Attenuated live vaccine also exists poor heat stability, transportation storage requirement to require shortcomings such as harshness.Above-mentioned shortcoming has not existed in inactivated vaccine, and because of inactivated vaccine purity of protein height, so good immune effect is safe, this also is the major reason that hav inactivated vaccine is only used in developed country and area so far.Inactivated vaccine is the important step of generally acknowledging preparation polyvalent vaccine, combined vaccine in addition, and combined vaccine is vaccine developing direction and strategy that The World Health Organization (WHO) and various countries health authority are advocated.
The cell matrix that the hepatitis A strain of existing hav inactivated vaccine (comprising attenuated vaccine) is adapted to is human diploid cell or human fibroblasts, reproductive efficiency is not high to be its common feature, and the preparation inactivated vaccine needs the virus antigen of large-scale purification, and this is the high basic reason of hepatitis A deactivation vaccine manufacturing cost.Therefore method for preparing hepatitis A inactivated vaccine and vaccine that still being necessary to develop can large-scale industrialized production, saved time, saved manpower and reduce cost.
Summary of the invention
One aspect of the present invention relates to a kind of new hepatitis A virus strain, and called after YN5, this strain are preserved in Chinese typical culture collection center April 20 calendar year 2001, and preserving number is CCTCC NO:V200104.Hepatitis A virus strain of the present invention has the characteristic that goes up efficient stable propagation at Vero cell (ATCC NO:CCL-81), proliferate efficiency is that other hepatitis A strain is at more than 10 times of human diploid cell propagation, and animal experiment has proved that the YN5 strain has good immunogenicity, can be as the seed culture of viruses of producing inactivated vaccine.
Another aspect of the present invention relates to a kind of method for preparing hepatitis A inactivated vaccine, and this method comprises the steps:
(a) static or three-dimensional absorption culturing cell matrix Vero cell in nutrient solution makes cell concn reach 10
5-10
7/ ml;
(b) be that 0.01-0.2 is inoculated into hepatitis A virus strain YN5 of the present invention in the cell matrix nutrient solution of above-mentioned cell concn with infective dose (M.O.I.), cultivated 14-21 days;
(c) obtain virus-cell cultures suspension with pancreas enzyme-EDTA digestion;
(d) purified virus prepares vaccine.
The method that relates in one aspect to a kind of seed selection hepatitis A virus seed culture of viruses more of the present invention is characterized in that comprising the steps:
(a) separation obtains virus from clinical hepatitis A patient's ight soil, confirms as hepatitis A virus through the biological characteristics inspection;
(b) carrying out viral cell cultures through following method adapts to:
(b1) elder generation's blind passage on diploid cell adapts to 5-6 generation, cultivates then to adapt to 35-40 days on Vero, reduces incubation time later on gradually, up to breeding the peak at 14-21 days;
(b2) directly in the cell adapted cultivation of Vero, originally incubation time is 35-40 days, reduces incubation time later on gradually, up to breeding the peak at 14-21 days;
(c) select the virus after step (b) cell cultures adapts to test the back as the seed culture of viruses of producing vaccine.
Than prior art, the present invention adopts hepatitis A virus YN5 strain and YN5-Vero cell culture system, can carry out the production of new and effective refining hav inactivated vaccine (Vero cell), and, the virus yield height, be existing more than 10 times of diploid cell hepatitis A strain productive rate, for reducing production costs significantly, preparing effective, cheap hav inactivated vaccine provides guarantee.In addition, vaccine of the present invention is the high purity inactivated vaccine, and the security of hav inactivated vaccine, validity are guaranteed.
Embodiment
Immunogenic production process of the present invention makes and can utilize Vero cell large-scale industrialization to produce hepatitis A inactivated vaccine, fundamentally solved the problem that hepatitis A viral antigen is difficult to obtain in a large number from technology.Adopt hepatitis A virus YN5 strain of the present invention, Vero cell matrix and corresponding method can obtain hepatitis A virus (HAV) antigen in a large number.Compare with other hepatitis A strains, cell matrix and Technology thereof, the cell matrix of viral proliferation is the Vero cell in the inventive method, is the accurate fair cell matrix that is used to prepare the human biological products of The World Health Organization (WHO).Compare with diploid cell, the Vero cell has easy cultivation, and can satisfy with clear superiorities such as fermentor tank (microcarrier) scale operation, the Vero cell now has been widely used in upgraded products such as producing the refining rabies vaccine of human, Vaccinum Encephalitidis Epidemicae both at home and abroad.In addition, YN5 strain of the present invention has the characteristic of efficient stable propagation on the Vero cell, and proliferate efficiency is that other hepatitis A virus (HAV) strain is at more than 10 times of human diploid cell propagation; Animal experiment has proved that the YN5 strain has good immunogenicity.
In the method for preparing hepatitis A inactivated vaccine of the present invention, the described cell matrix of step (a) is the Vero cell, this cell for example can be available from ATCC, nutrient solution is to have the serum nutrient solution for example to contain the MEM nutrient solution of 10% serum, culture temperature is well known to those skilled in the art, and is generally 36.5-37.5 ℃.
The nutrient solution of step (b) is to have the serum nutrient solution for example to contain the MEM nutrient solution of 2% serum, and culture temperature is well known to those skilled in the art, and is generally 34.5-35.5 ℃, does not occur cytopathy (CPE) in the culturing process.
The purified virus of step (d) prepares the method that vaccine can adopt this area routine, for example can adopt following method to carry out: at first, virus-cell cultures suspension ultrasonic grinding cell reaches more than 95% to cell crashing ratio.Use chloroform extracting cell 3-5 time then, the 20-40min that at every turn vibrates, the centrifugal 20-40min of 3000rpm extracts supernatant liquor then, removes foreign protein.Then with 10%PEG (molecular weight: 6000) and the NaCl solution concentration of 0.4M, the centrifugal 50-70min collecting precipitation of 10000rpm.The precipitation of collecting is suspended with PBS, through glycerine saccharose gradient (concentration range: begin 0.5ml 80% glycerine (100mM NaCl, 100mM Tris pH7.4) from bottom; 1.8ml30% sucrose; 1.8ml 20% sucrose; 1.8ml 10% sucrose (containing 10%SDS)).The centrifugal 6hr of 37000rpm.Column chromatography is further removed residual DNA and foreign protein afterwards.The virus formalin-inactivated that obtains is surveyed antigen titre, and being diluted to every milliliter of whole antigen titre is 1: 1600.Every milliliter adds adjuvant 0.5-1.25mg aluminium hydroxide, makes hepatitis A inactivated vaccine thus.
Below with embodiment the present invention is described in more detail.
Embodiment 1: the selection of hepatitis A virus strain YN5
(a) separation obtains virus from clinical hepatitis A patient's ight soil, confirms as hepatitis A virus through the biological characteristics inspection;
(b) in diploid cell 2BS (source: go up blind passage Nat'l Pharmaceutical ﹠ Biological Products Control Institute) and adapted to for 6 generations;
(c) on Vero, cultivate adaptation 40 days then, reduce incubation time later on gradually, up to breeding the peak at 14 days.
Wherein, each cultivate expiration after, obtain virus-cell cultures suspension with pancreas enzyme-EDTA digestion, it is standby to put-60 ℃ to-80 ℃ preservations.Use the ultrasonic grinding cell before the inoculation, reach more than 95% to cell crashing ratio.With infective dose (M.O.I.) is that 0.01-0.2 inoculates.Nutrient solution is that the MEM nutritive medium that contains 2% new-born calf serum, 0.20% lactoalbumin hydrolysate (is used 7%NaHCO
3Transfer pH to 7.4).
Final acquisition one strain has the required hepatitis A virus strain of expected characteristics, called after YN5, and this strain is preserved in Chinese typical culture collection center April 20 calendar year 2001, and preserving number is CCTCC NO:V200104.
Embodiment 2: the preparation of hepatitis A inactivated vaccine
(source: Nat'l Pharmaceutical ﹠ Biological Products Control Institute), centrifugal 10 minutes of 1000rpm discards former preservation liquid, adds the MEM nutritive medium that contains 10% new-born calf serum, 0.20% lactoalbumin hydrolysate and (uses 7%NaHCO to take out Vero cell ampoule in liquid nitrogen vessel
3Transfer pH to 6.8) put 37 ℃ of cultivations, treat that cell grows up to individual layer after, with the PBS washing, pancreas enzyme-EDTA digestion adds above-mentioned nutritive medium, with 1: 2 minute kind rate, goes down to posterity 1 time in per 5 days, until obtaining to produce in batches the enough cells that need usefulness.
Above-mentioned cell cultures is reached 10 in concentration
6During/ml, the branch kind rate of pressing 0.05MOI in the nutrient solution of pH7.4 adds the hepatitis A virus strain YN5 that embodiment 1 obtains, and static absorption was cultivated 14 days.Change 2 times therebetween and keep liquid.After cultivating expiration, obtain virus-cell cultures suspension with pancreas enzyme-EDTA digestion.This virus-cell cultures suspension is through ultrasonication and crash cells, reaches more than 95% to cell crashing ratio.Use the chloroform extracting afterwards 4 times, the 40min that at every turn vibrates, the centrifugal 40min of 3000rpm extracts supernatant liquor then, removes foreign protein.The NaCl solution concentration of gained supernatant 10%PEG (molecular weight 6000) and 0.4M, the centrifugal 70min collecting precipitation of 10000rpm.The precipitation of collecting is suspended the different gradient centrifugation (concentration ranges: begin 0.5ml 80% glycerine (100mM NaCl, 100mM Tris pH7.4) of glycerine sucrose from bottom with PBS; 1.8ml30% sucrose; 1.8ml 20% sucrose; 1.8ml 10% sucrose (containing 10%SDS).) the centrifugal 6hr of 37000rpm.The Sepharose-4FF gel is crossed post and is further removed residual DNA and foreign protein.With 1: 4000 formalin-inactivated 12 days, the packing finished product was that every milliliter of whole antigen titre is 1: 1600 with the virus behind the purifying.Every milliliter adds adjuvant 1mg aluminium hydroxide, makes vaccine.
Related experiment data of the present invention are seen following tabulation.Proof YN5 strain has the characteristic of efficient stable propagation on the Vero cell, and proliferate efficiency is that other hepatitis A virus (HAV) strain is at more than 10 times of human diploid cell propagation; Animal experiment has proved that the YN5 strain has good immunogenicity.
The different generation propagation of table 1.YN5 strain situation:
| Generation | Antigen titre (EIA) | Infection titer (lgCCID 50/ml) |
| 4 6 10 14 18 20 23 | 1∶2 1∶8 1∶128 1∶640 1∶2560 1∶5120 1∶5120 | Do not do 4.23 6.5 7.33 8.23 8.50 8.67 |
Table 2.YN5 strain continuous 3 one step growths of 23 generations:
Table 3.YN5 strain continuous 3 propagation situations of 23 generations:
| Number of times | Antigen titre | Infection titer (lgCCID 50/ml) |
| 1 2 3 | 1∶5120 1∶5120 1∶5120-10240 | 8.33 ≥8.5 ≥8.5 |
Table 4.YN5 strain hepatitis A strain and other known strain propagation are renderd a service relatively
| Strain | YN5 | TZ-84 | L8 |
| Cell matrix vaccine type antigen titre proliferating cycle | Vero deactivation vaccine 14-21 days＞1: 5000 | 2BS deactivation vaccine 28 days 1: 256-512 | KMB17 deactivation vaccine 28 days 1: 256-512 |
Table 5.YN5 strain hav inactivated vaccine small white mouse potency test result
| Test group | Dosage (EU/ml) | The sun revolution | Sun rate of rotation (%) | MIU |
| 1 2 3 4 | 3200 1600 800 400 | 10/10 10/10 8/10 3/10 | 100％ 100％ 80％ 30％ | 2500 2000 1750 1250 |
| SmithKline seedling Al (OH) 3Contrast | 1440 0 | 10/10 0/10 | 100％ 0 | 1750 0 |
Claims (4)
1, hepatitis A virus strain, called after YN5, this strain preserving number is CCTCC NO:V200104.
2, a kind of method for preparing hepatitis A inactivated vaccine, this method comprises the steps:
(a) static or three-dimensional absorption culturing cell matrix Vero cell in nutrient solution makes cell concn reach 10
5-10
7/ ml;
(b) being 0.01-0.2 with infective dose M.O.I. is inoculated into the hepatitis A virus strain YN5 of claim 1 in the cell matrix nutrient solution of above-mentioned cell concn, cultivates 14-21 days;
(c) obtain virus-cell cultures suspension with pancreas enzyme-EDTA digestion;
(d) purified virus prepares vaccine.
3, method as claimed in claim 2, wherein said nutrient solution are the MEM nutritive medium that contains 2-10% new-born calf serum, 0.20-0.30% lactoalbumin hydrolysate, and this nutritive medium pH is 6.8-7.4.
4, the hepatitis A inactivated vaccine that makes of method according to claim 2.
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| CN021069859A CN1216985C (en) | 2002-03-12 | 2002-03-12 | Hepatitis A virus strain, method for preparing hepatitis A inactivated vaccine and obtained vaccine |
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| CN021069859A CN1216985C (en) | 2002-03-12 | 2002-03-12 | Hepatitis A virus strain, method for preparing hepatitis A inactivated vaccine and obtained vaccine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570566B (en) * | 2008-04-30 | 2013-02-20 | 上海泽润生物科技有限公司 | Vero cell cracked protein, preparation method and application thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101440372B (en) * | 2007-11-23 | 2013-01-16 | 上海泽润生物科技有限公司 | 18 type human papilloma virus major capsid protein L1 gene |
| CN101525597B (en) * | 2008-03-08 | 2012-07-11 | 江苏先声卫科生物制药有限公司 | New hepatitis A inactivated vaccine virus strain and method for culturing same |
| CN105349500B (en) * | 2015-11-26 | 2019-02-22 | 中国疾病预防控制中心病毒病预防控制所 | A kind of novel hepatitis A virus strain and its application |
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2002
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101570566B (en) * | 2008-04-30 | 2013-02-20 | 上海泽润生物科技有限公司 | Vero cell cracked protein, preparation method and application thereof |
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Address after: No. 333 Edison Road, Zhangjiang, Shanghai, Pudong Patentee after: Shanghai Wison-Bio Co., Ltd. Address before: No. 333 Edison Road, Zhangjiang, Shanghai, Pudong Patentee before: Shanghai Huisheng Biological Engineering Co., Ltd. |
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Effective date of registration: 20210224 Address after: No. 83, Dongfeng South Road, high tech Zone, Yuxi City, Yunnan Province Patentee after: Yuxi Walvax Biotechnology Co.,Ltd. Address before: 201203, Zhangjiang Road, Zhangjiang, Pudong, No. 333, Edison Road, Shanghai Patentee before: SHANGHAI ZERUN BIOTECHNOLOGY Co.,Ltd. |
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Granted publication date: 20050831 |