CN102174477A - Hepatitis A virus strain SH and diploid cell adaptation method thereof - Google Patents
Hepatitis A virus strain SH and diploid cell adaptation method thereof Download PDFInfo
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
- C12N2770/32421—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
- C12N2770/32434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
- C12N2770/32461—Methods of inactivation or attenuation
- C12N2770/32464—Methods of inactivation or attenuation by serial passage
Abstract
The invention provides a new hepatitis A vaccine virus strain SH and a separation method as well as an MRC-5 cell adaptation method thereof. The virus is separated from excrement of a hepatitis A acute infection patient and is transferred to a diploid cell MRC-5 for adaptation culture. The virus strain SH is proved to be a hepatitis A virus through methods such as gene sequencing, neutralization test and the like; and during adaptation, early-generation sub-culturing period is 35 days, and culturing period is shortened to be 24 days after the strain is sub-cultured for 8 generations. After the strain is continuously cultured for 8 generations, antigen titer can reach (1:512)-(1:1,024), and the virus infection titer can reach 7.0 to 8.01gCCID50/ml. Immunogenicity tests and cross protection tests show that the strain has a good immunogenicity protection effect during production of a hepatitis A inactivated vaccine, is suitable for industrially producing the hepatitis A inactivated vaccine strain on a large scale and is an ideal strain for producing the hepatitis A inactivated vaccine.
Description
Technical field
The present invention relates to microorganism field, specifically, relate to SH of MS-1 and diploid cell adaptive method thereof.
Background technology
Hepatitis A is called for short hepatitis A, is the acute infectious disease of a kind of serious harm human health of being caused by hepatitis A virus.Hepatitis A is mainly propagated by " excrement-mouth " approach, or the propagation in the individual human world, thereby or causes that hepatitis A breaks out because of water or the food that has polluted hepatitis A virus (HAV).After big-age-child and adult infect hepatitis A, be that clinical type infects more than 70%, case fatality rate is 0.3%～0.6%; Patient's case fatality rate is 1.8% more than 50 years old; After the chronic hepatopathy person infected hepatitis A, the danger that acute hepatic failure takes place raise.Along with the improvement of living condition, the grownup infects the hepatitis A number the trend of increasing, and clinical type hepatitis A ratio rises, thereby hepatitis A becomes more serious public health problem.
China is the hepatitis A district occurred frequently, has at least every year 240000 people to suffer from hepatitis A, causes enormous economic loss and social danger, and therefore, development and exploitation effectively prevent and the vaccine and the medicine of treatment hepatitis A become problem demanding prompt solution.
The hepatitis A inactivated vaccine of domestic production at present, because strain, cell matrix and production technique that each manufacturer uses are different, quality product is uneven.China is populous, and hepatitis A vaccine far can not satisfy domestic market demand.
Summary of the invention
The purpose of this invention is to provide a kind of new hepatitis A virus SH strain.
Another object of the present invention provides the method that hepatitis A virus SH strain adapts on diploid cell.
An also purpose of the present invention provide hepatitis A virus SH strain and go down to posterity after virus be used for preventing, treating the vaccine of hepatitis A, the application of medicine in preparation.
In order to realize the object of the invention, a kind of new hepatitis A virus SH strain of the present invention, now be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, Datun Road, Chaoyang District, Beijing City, address institute of microbiology of the Chinese Academy of Sciences, deposit number CGMCC NO.4501, preservation date on December 21st, 2010.
The separation method of this virus strain may further comprise the steps:
(a) get hepatitis A acute infection patient ight soil 30～50g, adding diameter is the PBS of 40 and 5 times volumes of 3mm granulated glass sphere, vibrates 10 minutes;
(b) 3000 leave the heart 20 minutes, collect supernatant;
(c) will precipitate and handle as stated above again 2 times, and collect and merge 3 times supernatant liquor;
(d) in supernatant liquor, add PEG600010g/100ml and NaCl 2.3g/100ml, treat that all dissolving is put a few hours for back 4 ℃;
(e) 8000 leave the heart 30 minutes, abandon supernatant;
(f) it is resuspended that throw out adds 50ml PBS, adds equal-volume chloroform vibration 20 minutes; 3000 left the heart 30 minutes, collected upper phase, added PEG600010g/100ml and NaCl2.3g/100ml, and dissolving is placed on 4 ℃ and spends the night;
(g) change 30 minutes centrifugal supernatants of abandoning with 8000 next day, throw out adds 10ml PBS and makes viral suspension.Dialysed 15～18 hours, and changed dialyzate PBS3 time, add 10 * MEM again;
(h) filtration sterilization adds 1% P.S..
Wherein, described MEM is 0.03%Glu, 0.08%NaHCO
3, 0.01% penicillin and 0.01% Streptomycin sulphate; P.S. be penicillin and Streptomycin sulphate, wherein the mol ratio of penicillin and Streptomycin sulphate is 1: 1.
The present invention also provides hepatitis A virus SH the method that strain adapts on diploid cell, comprise the steps:
1) the MRC-5 cell is cultivated through conventional method, cell is inoculated hepatitis A virus SH suspension, 40cm after growing up to individual layer
2Culturing bottle kind 1ml virus liquid, 37 ℃ adsorbed 2 hours, and add and keep liquid, 35 ℃ of cultivations, every interval was changed virus in 7 days and is kept liquid once, cultivated 35 days, washed the cell face 3 times with PBS, used trypsin digestion cell again, pressed 0.01ml/cm
2Culture area adds MEM suspension sedimentation cell, obtains the virocyte suspension, and the ultrasonication cell is prepared into viral suspension;
2) with the method for step 1), upload at the MRC-5 cell continuously and passed for 15～22 generations.
Wherein, the described liquid of keeping is that MEM adds 2% calf serum.
After virus after above-mentioned cell cultures adapts to is being tested, can be used as the alternative strain of production of vaccine.
The present invention separates the new hepatitis A virus SH strain of acquisition, and this virus antigen titre can reach 1: 512-1: 1024, and the virus infection titre can reach 7.0-8.0lgCCID
50/ ml; through immunogenicity and cross-protection test; produce hepatitis A inactivated vaccine with this strain and have good immunogenic protection effect; and this strain has the advantages that proliferating cycle is short on the MRC-5 cell, reproductive efficiency is high; being suitable as the hepatitis A inactivated vaccine seed culture of viruses of large-scale industrial production, is the desirable strain of producing hepatitis A inactivated vaccine.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The separation method of embodiment 1 hepatitis A virus strain SH
(a) gather acute phase ight soil 30～50g of four parts of clinical hepatitis A patients (patient that Shenyang City Sixth Man people hospital Hepatitis Dept. is accepted for medical treatment) altogether, adding diameter is the PBS of 40 and 5 times volumes of 3mm granulated glass sphere, vibrates 10 minutes;
(b) 3000 leave the heart 20 minutes, collect supernatant;
(c) will precipitate and handle as stated above again 2 times, and collect and merge 3 times supernatant liquor;
(d) in supernatant liquor, add PEG600010g/100ml and NaCl 2.3g/100ml, treat that all dissolving is put a few hours for back 4 ℃;
(e) 8000 leave the heart 30 minutes, abandon supernatant;
(f) it is resuspended that throw out adds 50ml PBS, adds equal-volume chloroform vibration 20 minutes; 3000 left the heart 30 minutes, collected upper phase, added PEG600010g/100ml and NaCl2.3g/100ml, and dissolving is placed on 4 ℃ and spends the night;
(g) change 30 minutes centrifugal supernatants of abandoning with 8000 next day, throw out adds 10ml PBS and makes viral suspension.Dialysed 15～18 hours, and changed dialyzate PBS 3 times, add 10 * MEM again;
(h) filtration sterilization adds 1% PS.
Wherein, described MEM is 0.03%Glu, 0.08%NaHCO
3, 0.01% penicillin and 0.01% Streptomycin sulphate.(MEM is available from the Beijing Qingdatianyi Bioisystech Co., Ltd, product batch number: 080302).P.S. be penicillin and Streptomycin sulphate, wherein the mol ratio of penicillin and Streptomycin sulphate is 1: 1.
Embodiment 2 adaptations of hepatitis A virus strain SH on the MRC-5 cell
Get the frozen pipe that contains the MRC-5 cell, put into 40 ℃ of water-baths rapidly and melt, join then in the MEM cell culture fluid of pre-temperature to 30～37 ℃, place 37 ℃ of cultivations, inoculation embodiment 1 isolating hepatitis A virus strain SH strain suspension after growing up to individual layer in 3-4 days, 40cm
2Culturing bottle kind 1ml viral suspension, 37 ℃ adsorbed 2 hours, and added 20ml and keep liquid (MEM adds 2% calf serum), place 35 ℃ of cultivations, every interval was changed virus in 7 days and is kept liquid once, cultivated 35 days, wash the cell face 3 times with PBS, use the 0.125w/v% trypsin digestion cell again, press 0.01ml/cm
2Culture area adds MEM suspension sedimentation cell, is the virocyte suspension, and the ultrasonication cell is prepared into viral suspension.
Press above method, embodiment 1 isolating hepatitis A virus strain SH is involved in a criminal case to continue at the MRC-5 cell uploaded for 15 generations, finally obtain the good hepatitis A virus strain of the cell adapted property of a strain MRC-5.
Hepatitis A virus strain SH of the present invention is characterized as:
Virion diameter: 27-32nm;
Acid resistance: under pH value 3.0 conditions, 2-8 ℃ acts on 8 hours, and infection titer does not have considerable change;
Alkali resistance: through ether 2-8 ℃ of effect 8 hours, infection titer did not have considerable change;
Neutralization test: neutralized by the HAV specific antibody;
Gene sequencing: with the gene order homology of the HM-175 of hepatitis A virus street strain up to 95.92%, belong to I B gene hypotype together.
Antigen titre: 1: 512-1: 1024;
Infection titer: 7.0-8.0lgCCID
50/ ml;
Rised in value peak period: 21-24 days;
The suitableeest culture temperature: 35 ℃-36 ℃;
Increment position: in the cell cytosol;
Has the hepatitis A virus (HAV) characteristic.
Different generation SH strain antigen titre of the present invention and virus infection titre are as shown in table 1.
Different generation antigen titres of table 1 SH strain and virus infection titre
SH strain 15 generations propagation peak period antigen titre of the present invention and virus infection titre test-results are as shown in table 2.
Table 2 SH strain 15 generations propagation peak period test-results
SH strain immune mouse test-results of the present invention is as shown in table 3.
The mouse immune originality test-results of table 3 SH strain
(the L-A-1 strain derives from China Sickness Prevention Control Center Virus Disease Prevention Control Institute for the contrast strain.)
SH strain of the present invention is as shown in table 4 to the cross-protection of other strain.
Table 4 SH strain is to the cross-protection of other strain
(above strain all derives from China Sickness Prevention Control Center Virus Disease Prevention Control Institute.)
Embodiment 3 hepatitis A virus strain SH are used for preventing, treating the vaccine of hepatitis A, the application of medicine in preparation
Get the cell factory cultured human embryo lung diploid cell (MRC-5) that covers with individual layer, through 0.125w/v% trypsinase-EDTA peptic cell, add the MEM cell culture fluid, make cell concn be every milliliter and contain ten thousand cells of 100-150, enchylema adds hepatitis A virus (HAV) by 0.05-0.1MOI to carry out under 20-30 ℃ of temperature mixing and absorption 30-90 minute.The cell of mixing and absorption-viral mixed solution is inoculated in 2-40 confluent monolayer cells factory propagation hepatitis A virus (HAV) with after 10 times of the MEM cell culture fluid dilutions that contains 10% calf serum by 0.5MOI, puts 35 ℃ ± 0.5 ℃ and leave standstill and cultivated 21-24 days.Treat the virus multiplication peak period,, add the PBS collecting cell virus mixed solution of 20 μ l 0.01M pH values 7.2 with every sq with the conventional peptic cell of the pancreatin of 0.125w/v%.Ultrasonication cell virus mixed solution, ultrasonication are output rating 1500W, and ultrasonic 5 minutes smudge cellses in ice bath are total to ultrasonic 5 times at every turn.Broken back 2000rpm got viral supernatant liquid in centrifugal 10 minutes, and viral liquid adds the chloroform extracting, and press 1: 2 volume ratio of trichloromethane and viral liquid and mix, jolting 20 minutes, with centrifugal 20 minutes of the speed of per minute 4500 commentaries on classics, absorption upper strata water; Albumen use again mutually extraction buffer (0.01M PBS, pH7.4) extracting is 4 times, extracting can be reclaimed most of virus for 4 times, merges the upper strata water.Extract is the ultra-filtration membrane ultrafiltration and concentration 10-40 back usefulness Phenyl Sepharose 6FF organophilic gel chromatography column purifying doubly of 50-100KD through molecular weight cut-off, with 0.9mol/L PB (pH6.8) as sample-loading buffer, with 0.01mol/L PBS (pH6.8) as gradient eluent, collect the elution of virus peak, hepatitis A venom for purifying, again through 0.2 μ m filter membrane Sterile Filtration, under aseptic condition, add final concentration and be 37 ℃ of deactivations of formaldehyde 12 days of 80 μ g/ml.The hepatitis A venom of deactivation is diluted to 640EU/ml through further after the assay was approved according to virus antigenicity titre level, makes work in-process through aluminum hydroxide adjuvant absorption, and work in-process are sub-packed in the aseptic cillin bottle, makes the finished product vaccine.
The finished product of making is carried out potency test in the mouse body, calculate the terminal point extent of dilution, observe its immunogenicity.The km mouse is available from pathogenic micro-organism institute of Chinese Military Medical Science Institute, male and female half and half, body weight 16-18g, random packet.Test is divided into 8 groups, and 10 every group, 4 groups of test seedlings: the vaccine of injecting embodiment 3 preparations respectively; Reference seedling group: external hepatitis A inactivated vaccine (available from Beijing Military Area Command disease prevention and control center, Ai Basu: lot identification mark: 3001424.02).With thinner for vaccine test seedling and reference seedling are carried out 4 times of serial dilutions, totally 4 extent of dilution are former times, 1: 4,1: 16,1: 64.Every mouse peritoneal injection 1.0ml, after immune 4 weeks, the blood sampling of the intraocular corner of the eyes, centrifugation serum ,-20 ℃ of preservations are standby.Adopt ELISA competition inhibition method to detect anti-HAV antibody, calculate the terminal point extent of dilution according to the Reed-Muench method.The result is as shown in table 5.
Table 5 hav inactivated vaccine mouse of the present invention potency test result
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (3)
1. hepatitis A virus SH strain, its preserving number are CGMCC NO.4501.
2. the method that adapts on diploid cell of the described hepatitis A virus SH of claim 1 strain is characterized in that, comprises the steps:
1) the MRC-5 cell is cultivated through conventional method, cell is inoculated the SH of MS-1 suspension, 40cm after growing up to individual layer
2Culturing bottle kind 1ml virus liquid, 37 ℃ adsorbed 2 hours, and add and keep liquid, 35 ℃ of cultivations, every interval was changed virus in 7 days and is kept liquid once, cultivated 35 days, washed the cell face 3 times with PBS, used trypsin digestion cell again, pressed 0.01ml/cm
2Culture area adds MEM suspension sedimentation cell, obtains the virocyte suspension, and the ultrasonication cell is prepared into viral suspension;
2) with the method for step 1), uploaded for 15～22 generations at the MRC-5 cell continuously;
Wherein, described MEM is 0.03%Glu, 0.08%NaHCO
3, 0.01% penicillin and 0.01% Streptomycin sulphate; The described liquid of keeping is that MEM adds 2% calf serum.
3. the described virus strain of claim 1, the described virus after going down to posterity of claim 2 are used for preventing, treating the vaccine of hepatitis A, the application of medicine in preparation.
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| CN2010106222587A CN102174477B (en) | 2010-12-28 | 2010-12-28 | Hepatitis A virus strain SH and diploid cell adaptation method thereof |
| PCT/CN2011/002181 WO2012088763A1 (en) | 2010-12-28 | 2011-12-26 | Hepatitis a virus strain sh and method for adapting same to diploid cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012088763A1 (en) * | 2010-12-28 | 2012-07-05 | 深圳康泰生物制品股份有限公司 | Hepatitis a virus strain sh and method for adapting same to diploid cells |
| CN102807972A (en) * | 2012-08-08 | 2012-12-05 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus purification method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994006446A1 (en) * | 1992-09-18 | 1994-03-31 | Smithkline Beecham Biologicals, S.A. | Hepatitis a virus vaccines |
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN102058882A (en) * | 2010-12-28 | 2011-05-18 | 北京民海生物科技有限公司 | Method of preparing hepatitis A inactivated vaccine |
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| CN102174477B (en) * | 2010-12-28 | 2012-11-21 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus strain SH and diploid cell adaptation method thereof |
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2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994006446A1 (en) * | 1992-09-18 | 1994-03-31 | Smithkline Beecham Biologicals, S.A. | Hepatitis a virus vaccines |
| CN101816786A (en) * | 2010-04-30 | 2010-09-01 | 长春生物制品研究所 | Inactivated hepatitis A vaccine and preparation method thereof |
| CN102058882A (en) * | 2010-12-28 | 2011-05-18 | 北京民海生物科技有限公司 | Method of preparing hepatitis A inactivated vaccine |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012088763A1 (en) * | 2010-12-28 | 2012-07-05 | 深圳康泰生物制品股份有限公司 | Hepatitis a virus strain sh and method for adapting same to diploid cells |
| CN102807972A (en) * | 2012-08-08 | 2012-12-05 | 深圳康泰生物制品股份有限公司 | Hepatitis A virus purification method |
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| CN102174477B (en) | 2012-11-21 |
| WO2012088763A1 (en) | 2012-07-05 |
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