CN105695424B - Adapted strain and its vaccine of the Japanese Encephalitis Vaccine,Live strain SA14-14-2 on human diploid cell 2BS - Google Patents

Adapted strain and its vaccine of the Japanese Encephalitis Vaccine,Live strain SA14-14-2 on human diploid cell 2BS Download PDF

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CN105695424B
CN105695424B CN201610180176.9A CN201610180176A CN105695424B CN 105695424 B CN105695424 B CN 105695424B CN 201610180176 A CN201610180176 A CN 201610180176A CN 105695424 B CN105695424 B CN 105695424B
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李玉华
俞永新
余凝盼
刘欣玉
徐宏山
贾丽丽
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Abstract

The adapted strain and its vaccine of a kind of Japanese Encephalitis Vaccine,Live strain SA14-14-2 on human diploid cell 2BS, its main feature is that, by the way that epidemic encephalitis B virus SA14-14-2 is inoculated on human diploid cell, pass through different mode continuous passages, it well adapt to virus can on cell and stable proliferation, obtain the virus compared with high titre, establish the three-level Virus seed library of virus, by measuring to Virus seed library complete genome sequence, its whole genome sequence is obtained.Validity and security inspection are carried out to virus, meet the standard of defined Japanese Encephalitis Vaccine,Live.Encephalitis B seed culture of viruses virus titer of the present invention is higher, safety meets that regulation, immunogenicity be good and inheritance stability, meet requirement of " Chinese Pharmacopoeia " the third portion (2010 editions) about the safety of encephalitis B strain and validity, is suitable as the production seed culture of viruses of Vaccinum Encephalitis B.

Description

Japanese Encephalitis Vaccine,Live strain SA14-14-2 is suitable on human diploid cell 2BS Answer strain and its vaccine
Technical field
The invention belongs to viral vaccine fields, especially highly attenuated with biology techniques breeding and immunogenicity is good Encephalitis B live vaccine strain.Japanese Encephalitis Vaccine,Live strain SA14-14-2 is inoculated with 2BS by way of single layer, suspension Cell, plaque method measures virus titer after adaptation of virus, determines the best harvest time of virus and inoculum concentration.Choose virus titer compared with High three generations establishes original seed culture of viruses, main generation seed culture of viruses and work Virus seed library, to Virus seed library virus according in " Chinese Pharmacopoeia " three into Row sterility test, mycoplasma inspection, immunogenicity inspection, Virulence detection in mouse brain, subcutaneous infection enters brain test, suckling mouse passes For comprehensive calibrating of the projects such as reversion test, immune protective test.In addition also it is carried out complete genome sequence measurement and with The sequence of the vaccine strain SA14-14-2 vaccine strain of primary hamster kidney cell (PHK) passage of listing is compared.
Background technique
Japanese Type-B encephalitis (Japanese encephalitis, encephalitis) is the viral brain of Asia one of the most common type Inflammation is a kind of Amphixenosis through killing propagation.Encephalitis B virus category flaviviridae Flavivirus has packet for single-stranded positive RNA Film virus, overall length 11kb or so.Encephalitis B virus has 5 genotype, and the popular virus of the encephalitis in China is III type of gene and base at present Because of I type.It is estimated that encephalitis B virus at least causes 50,000 clinical case every year, wherein most is 10 years old or less children, cause About 10,000 people is dead, separately has about 15, there are long-term neuro-psychiatric sequelae for 000 case.Encephalitis is almost all There is generation in Asian countries, and no matter it is located at temperate zone, subtropical zone or the torrid zone.In recent decades, encephalitis is before some without place Property popular area there is outburst.The whole world has nearly 3,000,000,000 population lives in encephalitis Endemic Area, the annual newborn in these Endemic Areas About 7,000 ten thousand.Infection and encephalitis B virus infection is by mosquitoes spread, and mosquito is infected from the animal that has viremia virusemia, people.Do not have also at present There is the specificity antivirus treatment method for encephalitis, vaccine inoculation is still unique most important control measure.
Currently, there are three types of the Vaccinum Encephalitidis Epidemicaes of large-scale use: (1) Nakayama plants or Beijing strain purifying mouse brain inactivation epidemic disease Seedling, several Asian countries's productions such as main Japan；(2) Beijing P3 plants of Japanese encephalitis inactivated vaccine of Vero cell culture；(3) primaryly The SA14-14-2 strain attenuated live vaccine of hamster kidney cell culture.The shortcomings that purifying mouse brain Vaccinum Encephalitidis Epidemicae has: inducing protective immunity Duration it is too short, need multi-agent time inoculation；For majority state, the price of every vaccinating agent is relatively high.Cell culture It once in domestic product and was widely used before vaccine nineties in last century.1989, SA14-14-2 plants of attenuated live vaccine listings Afterwards, because of it safely, effectively, the advantage that immunity inoculation number is few, guard time is long, large area is inoculated at home, occupation rate of market More than ten countries such as 90% or more is had reached, and exports South Korea, India, Thailand, Nepal.
Human diploid cell refers to the cell that normal human tissue is cultivated in vitro, derives from normal human embryonic, is passing Keep diploid karyotype during generation, the pollution of no exogenous factor, no oncogenicity, without lacking for passage cell and primary zooblast Point, is worth very big in terms of culture virus and preparation virus type vaccine, and it is thin for the ideal of vaccine for man production to be that WHO is advocated Born of the same parents.Human diploid cell at the beginning of the sixties comes out, and the seventies is used for a variety of production of vaccine.Recent years become Aimmugen, varicella epidemic disease The Cells for production of the products such as seedling, ervevax, polio vaccine, fiblaferon.WHO thinks epidemic disease The production of seedling should be considered as humanized's diploid cell first, because it is compared with other animal passage cells, theoretically not There are oncogenic risk, the safety and efficacy for producing vaccine is much better than the vaccine of other cellular matrixs preparation.
Japanese Encephalitis Vaccine,Live is China's independent intellectual property right vaccine, ratifies production listing through the Ministry of Public Health from 1989 Since, it has used more than so far 600,000,000 doses times, has significantly reduced the disease incidence and case fatality rate of Endemic Area encephalitis, be children Health provide effective immanoprotection action.On October 9th, 2013, Japanese Encephalitis Vaccine,Live pass through world health group Vaccine pre-authentication is knitted, the first vaccine product that old China hand crosses World Health Organization's pre-authentication is become.But Vero cell and primary The advantages of hamster kidney cell is all animal derived cell, Vero cell is that its quality and exogenous factor can be controlled highly, numerous It is fast to grow speed, the duration is long, is easy to marking, but Vero cell DNA remaining in vaccine is to the potential carcinogenicity wind of inoculator Danger, therefore must be strictly controlled residual content during vaccine preparation.And primary hamster kidney cell derives from suslik, suslik kind Group is not only difficult to control, relatively high to manufacturing technique requirent, and is easy by the dirt of the allogenic materials such as bacterium, virus, helminth The Japanese Encephalitis Vaccine,Live of dye, at present Co., Ltd, Chengdu Inst. of Biological Products, China production is thin using SPF suslik kidney Born of the same parents can preferably solve the problems, such as the above-mentioned exogenous factor that fears are entertained that pollution, but increase as production of vaccine cellular matrix Vaccine cost.Therefore, many vaccine enterprises are also actively researching and developing vaccine for man using human diploid cell now.The application Proposed adoption human diploid cell researches and develops Japanese Encephalitis Vaccine,Live, further increases vaccine quality.
Summary of the invention
Human diploid cell 2BS cell strain is established by 3 monthly age female child lung tissues of one miscarriage of China in 1973 One plant of human embryonic lung diploid fibroblast, limited life is 63-65 generation, has that source is clear, passage history understands, without exogenous factor The characteristics of pollution and China ratify one of the two people's diploid cell strains that can be used for vaccine for man production at present.Two times of people Body cell 2BS cell strain has been used to the production of the vaccines such as people's nettle rash vaccine, chicken pox vaccine, connects by decades and a large amount of human bodies It kind uses, it was demonstrated that the cell is safety, good cellular matrix.
The Vaccinum Encephalitidis Epidemicae used on domestic market is mainly primary suslik kidney (PHK) cell vaccine and Vero cell epidemic disease Seedling.PHK cell is easy to be polluted by allogenic materials such as bacterium, virus, helminths, is relatively difficult to control, and Vero cell is to inoculation people Group has potential carcinogenicity.In order to further increase the quality of Vaccinum Encephalitidis Epidemicae, more international markets can be exported to, allow more by The crowd threatened to encephalitis B virus especially children can health growth, we are by epidemic encephalitis B virus SA14-14-2 Human diploid cell 2BS is adapted to, virus is enable to well adapt to and rise in value on human diploid cell, and it is raw to study the vaccine The process for stabilizing of production is the global people in particular by B-mode to improve the safety and validity of Vaccinum Encephalitidis Epidemicae The health service of the harm crowd of encephalitis.
The purpose of the present invention is to provide that can reach, the highly attenuated of WHO standard, immunogenicity are good, genetic stability is high Japanese encephalitis virus strain and its breeding method, provide production of vaccine seed culture of viruses, preparation encephalitis B is attenuated human diploid cell Live vaccine.
The object of the present invention is achieved like this: Japanese Encephalitis Vaccine,Live strain is suitable on human diploid cell 2BS Answer the selection of strain, it is characterised in that: select JE attenuated live vaccine SA14-14-2 strain in China's as parent This strain, incubation step include at least:
(1) adaptability passage of the encephalitis B virus SA14-14-2 on human diploid cell 2BS；
(2) every generation Strain of harvest is titrated；
(3) the three-level Virus seed library that the highest Strain of titre establishes virus is filtered out, three-level Virus seed library is respectively original poison Kind library: Strain is SA14-14-2-2BS P34, and virus titer is 7.00lgPFU/mL；Main generation Virus seed library: Strain is SA14-14-2-2BS P36, virus titer are 7.25lgPFU/mL；Work Virus seed library: Strain SA14-14-2-2BS P38, virus titer are 7.15lgPFU/mL；
(4) whole genome sequence measurement is carried out to three-level Virus seed library, obtains whole genome sequence；SA14-14-2-2BS P34 plants, P36 plants of SA14-14-2-2BS, P38 plants of complete genome sequences of SA14-14-2-2BS such as SEQ ID NO:1, SEQ ID Shown in NO:2, SEQ ID NO:3.
(5) safety calibrating and immunoassays are carried out to three-level Virus seed library.
The selection of adapted strain of the Japanese Encephalitis Vaccine,Live strain on human diploid cell 2BS, can also be in this way It realizes: being inoculated with 2BS cell by way of single layer, suspension, plaque method measures virus titer after adaptation of virus, determines that virus is best Harvest time and inoculum concentration.
Specific inoculation propagating method is:
SA14-14-2-PHKC5 is inoculated in 2BS cell suspension by 0.2MOI, obtains P1；
Virus is harvested after culture 5 days, band poison cell continuous passage to the 5th generation obtains P2-P5；
Continue culture 5 days, will be obtained with poison cell suspension and normal cell suspension mixed in equal amounts continuous passage to the 10th generation P6-P10；
10th generation virus is suspended in 2BS cell suspension by MOI0.1, obtains P11；
It is further cultured for 4 days, will be passaged to for the 50th generation with poison cell suspension and normal cell suspension mixed in equal amounts, obtain P12- P50；
It filters out highest P34, P36, P38 virus of titre and establishes original Virus seed library, main generation Virus seed library and work poison respectively Kind library.
What the selection of adapted strain of the Japanese Encephalitis Vaccine,Live strain on human diploid cell 2BS obtained Three-level Virus seed library, including original Virus seed library: Strain SA14-14-2-2BS P34 complete genome sequence such as SEQ ID NO:1；Main generation Virus seed library: Strain SA14-14-2-2BS P36, complete genome sequence such as SEQ ID NO:2；Work Virus seed library: Strain SA14- 14-2-2BS P38, complete genome sequence is as shown in SEQ ID NO:3.
The selection of adapted strain of the Japanese Encephalitis Vaccine,Live strain of the present invention on human diploid cell 2BS The Japanese Encephalitis Vaccine,Live SA14-14-2-2BS that the three-level Virus seed library of acquisition is used to prepare out.
The present invention is by being inoculated in people two for epidemic encephalitis B virus SA14-14-2 (JEV SA14-14-2-PHKC) On times body cell, by different mode continuous passages, it well adapt to virus can on cell and stable proliferation, obtain Obtain the virus compared with high titre.According to " Chinese Pharmacopoeia " three (2010 editions), validity and security inspection are carried out to virus, met The standard of defined Japanese Encephalitis Vaccine,Live.Meanwhile having studied the life for having been adapted to the encephalitis B virus of human diploid cell Object characteristic, including immunogenicity, immune protective efficiency and weak virulence stability etc., it is final to obtain safely and effectively quality controllable people Diploid somatic cell encephalitis B attenuated live vaccine.
Advantages of the present invention and significant benefit: human diploid cell is normal karyotype cell, and two are kept in succeeding generations Times body caryogram, no exogenous factor pollution, non-carcinogenesis, the shortcomings that without passage cell and primary zooblast, the height having is exempted from Epidemic focus and good tolerance are the ideal cells for vaccine for man production that WHO is advocated, have been successfully applied at present The production of a variety of vaccines, such as Aimmugen, varicella, polio vaccine.Endemic Area of the China as encephalitis B, encephalitis The research and development of vaccine are on the forefront, although the safety of Live attenuated vaccine and validity have been confirmed, primaryly Hamster kidney cell vaccine is easy to be polluted by allogenic materials such as bacterium, virus, helminths there are animal, it is difficult to control.Although China adopts Use SPF hamster kidney cell as Japanese Encephalitis Vaccine,Live Cells for production matrix, and the WHO of in September, 2013 has announced China Chengdu Japanese Encephalitis Vaccine,Live produced meets WHO requirement, enters international procurement catalogue, can be in the world especially It is the national children's health service seriously threatened by encephalitis, but developed country is B-mode using hamster kidney cell production to China Encephalitis attenuated live vaccine is still suspected, and worries the exogenous factor pollution that Cells for production is not yet known at present.So originally grinding Study carefully the vaccine safety problem for JEV SA14-14-2-PHKC being adapted in 2BS cell, these someone worry can be solved, both Without different DNA and the remaining hidden danger of foreign protei, and can be to avoid other animal derived viruses and harmful microorganism to the mankind's It influences.Therefore, safety promotes technological innovation, is expected to make up better than non-human is primary and the vaccine of passage cell preparation Blank of the China in terms of human diploid cell Vaccinum Encephalitidis Epidemicae, serves Chinese Japanese Encephalitis Vaccine,Live in the world More population healths.
Detailed description of the invention
Fig. 1 is SA14-14-2-2BS Strain screening process figure；
Fig. 2 is SA14-14-2-2BS pnca gene group structural schematic diagram；
Indicate that number respectively indicates from top to bottom in figure:
Full-length genome (10976)；
One long open reading frame nucleotide (96~10394)；
Amino acid number that structural region and non-structural region are separately encoded (127,167,500,415,164,131,619, 267,137,905)；
Have 3 structural proteins at its 5 ' end, i.e. capsid protein C, premembrane proteins prM and envelope protein E, 3 ' ends have 7 it is non- Structural proteins, i.e. NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5.
Fig. 3 is to observe lesion situation of the 10th generation SA14-14-2 on 2BS cell under the microscope；
Show that SA14-14-2 virus adapts to P10 on 2BS cell in figure, pathological change form (A) and normal 2BS at 4 days Cell controls (B)；
Fig. 4 is titration curve of the P10 virus on 2BS cell under different MOI and incubation time.
Specific embodiment
With reference to the accompanying drawing, by specific embodiment, the invention will be further described, but does not limit this in any way The protection scope of invention.
One, adaptation passage of the JEV (encephalitis attenuated vaccine strain SA14-14-2) on 2BS cell
1. cell and seed culture of viruses source
Cell used in this work is 2BS cell (27~37 generation), is protected for National Institute for Food and Drugs Control's cell Hiding center provides.Cell culture fluid is Eagle ' the s MEM containing 10% fetal calf serum.Encephalitis attenuated vaccine strain SA14-14-2 disease Poison is encephalitis B virus (JEV) SA14-14-2-PHKC5 that this room saves.
2.SA14-14-2 the adaptation on 2BS cell is passed on
(1) the 2BS cell of single layer has been formed, and with 0.25% trypsin digestion with Eagle ' s MEM-10% tire ox blood Cell is resuspended in clear culture solution, and by 0.2MOI inoculum concentration virus inoculation in suspension cell, virus and cell are sufficiently mixed uniformly, It is cultivated in 37 DEG C of incubators, draws 0.5ml virus liquid daily and survey its titre.After 5 days, harvest culture supernatant is that encephalitis B virus 2BS is thin Born of the same parents' adapted strain --- SA14-14-2-2BS 1st generation is denoted as P1, freezes spare in -70 DEG C of refrigerators.It will continue former bottle with poison cell Passage, passage in every 5 days is primary, in this way by viral continuous passage 5 times, harvests SA14-14-2-2BS P5, each to withhold the disease obtained Venom freezes in -70 DEG C of refrigerators.
After (2) the 5th generation virus liquids harvest, band poison cell presses 1:1 in passage, with the fresh 2BS cell of uninfecting virus Co-passage is placed in 37 DEG C of incubators and cultivates, and encephalitis B virus the 6th generation of adapted strain on 2BS cell, band poison cell are harvested after 5 days It is passed on the fresh 2BS cell suspension mixed in equal amounts of uninfecting virus, passage in every 5 days is primary, and virus liquid was reached for the 10th generation.It passes During generation, 0.5ml virus liquid is drawn daily and surveys its titre, each virus liquid obtained of withholding freezes in -70 DEG C of refrigerators.
Lesion situation of the 10th generation SA14-14-2 of observation on 2BS cell is shown in Fig. 1 under the microscope.Fig. 1 shows SA14- 14-2 virus adapts to P10 on 2BS cell, pathological change form (A) and normal 2BS cell controls (B) at 4 days.
(3) each virus obtained of withholding is titrated with plaque ethods:
BHK-21 cell inoculation is spaced 48h and waits for that cell grows up to single layer, by 10 times of Strain to be measured in 6 porocyte culture plates It is serially diluted, from 10-1~10-5, take the viral 0.2mL after diluting to be inoculated into 6 orifice plates cell, each dilution does a pair Hole, 37 DEG C of absorption 60min add final concentration of 1% methylcellulose nutrition covering, suck and cover after cultivating 5 days in 37 DEG C of incubators Cover material, violet staining 30min, tap water, which rinses, counts plaque number, calculates plaque slip.
(4) the virus titer measurement of P1~P10 harvest.
Calculation method is average × 5 (because every hole is inoculated with 0.2mL, the logarithm of every mL is obtained after × 5) of two hole plaque numbers, That is virus titer=(logarithm of the dilution)+(logarithms of average spot number × 5) lgPFU/mL.
P1~P10 virus titer see the table below 1.
The titre for the preceding 10 generation virus that table 1 harvests
(5) in the 10th generation of selection, adapts to the MOI that strain (SA14-14-2-2BS P10) presses 0.1,0.01,0.001,0.0001 Virus quantity is inoculated in 2BS cell suspension and carries out Virus culture, takes viral supernatants respectively at 1d, 2d, 3d, 4d, 5d, 6d, uses plaque Method detects virus titer.Different MOI, 2 be see the table below without virus titer under incubation time.
Titre of the P10 virus on 2BS cell under 2 difference MOI of table and incubation time
Titration curve of the P10 virus on 2BS cell under difference MOI and incubation time referring to fig. 2.
(6) the 2BS cell for growing up to single layer is digested with 0.25% pancreatin and is dispersed, take the encephalitis B virus SA14-14-2- of harvest In the 10th generation of 2BS, is added by 0.1MOI virus quantity into 2BS cell, and 37 DEG C adsorb 60 minutes, is placed in 37 DEG C of incubators and is cultivated, and 4 days After will be with being passed on after poison cell suspension and healthy cell suspension mixed in equal amounts, 37 DEG C of cultures；Viral supernatants are harvested after 4 days, are marked For P11, continue to harvest for the 12nd generation after cultivating 4 days with poison cell suspension and the passage of healthy cell suspension mixed in equal amounts, 37 DEG C again Virus culture supernatant.Band poison cell and healthy cell co-passage are repeated to 50 generations, until harvest viral supernatants P50.P11~ The virus titer of P50 harvest see the table below 3.
The titre for P11~P50 virus that table 3 harvests
Two, the foundation of three-level Virus seed library
This research passes through band poison cell continuous passage, band by the way that (JEV) SA14-14-2 to be seeded on suspension 2BS cell Poison cell and the method for healthy cell mixing continuous passage are adapted to encephalitis B virus stabilization on 2BS cell, filter out encephalitis disease Malicious SA14-14-2 diploid cell 2BS adapted strain, and virus reaches higher titre, from 20 generations to 50 generations, virus titer can be steady It is scheduled on compared with high titre range.By analyzing adaptation of virus analysis and summary of the virus on 2BS cell, we have chosen adapted strain ((7.00lgPFU/mL) is as original malicious library, the 36th generation virus for the higher generation of virus titer, i.e. the 34th generation virus (7.25lgPFU/mL) is used as main generation seed culture of viruses, and the 38th generation virus (7.15lgPFU/mL) is used as work seed culture of viruses.
Three, the research of the security inspection and biological characteristics of three-level Virus seed library
1. intracerebral Virulence detection:
(1) to be inoculated with the Kunming 12~14g (KM) respectively with the 34th, 36,38 generation virus stock solution used of (JEV) SA14-14-2-2BS small It mouse 20, every intracerebral injection 0.03mL, observes 14 days.Died disregards that (animal dead quantity should must not surpass in 3 days after inoculation The 20% of overtesting animal number).If any mouse invasion after 3 days, brain is taken after should putting to death, and measures pathogenicity.Virulence in mouse brain Not higher than 3.0lgLD50/0.03mL, while with 10-1 virus brain suspension subcutaneous injection weight for 10~12g mouse 10, every 0.1mL is observed 14 days.
(2) result: after 14 days, no dead mouse.
2. subcutaneous infection enters brain test:
(1) it is inoculated with 10~12g kunming mice 10 respectively with the 34th, 36,38 generation virus stock solution used of (JEV) SA14-14-2-2BS Only, every subcutaneous injection 0.1mL, while right side intracerebral sky pierces, and observes 14 days.
(2) result: after 14 days, all mouse are all strong to be deposited.
3. reverse mutation test in suckling:
(1) it is inoculated with 3~5 age in days Kunming suckling mouses 10 respectively with the 34th, 36,38 generation virus stock solution used of (JEV) SA14-14-2-2BS Only, every intracerebral injection 0.02ml.The suckling mouse of morbidity is put to death 3, solution takes brain, surveys it with weight 12~14g kunming mice Pathogenicity, intracerebral virulence should be not higher than 3.0lgLD50/0.03ml, while body is subcutaneously injected with the morbidity suckling rat brain suspension of 10-1 Weight is 10~12g kunming mice 10, every 0.1ml, is observed 14 days.
(2) result: after 14 days, all mouse are strong to be deposited.
4. the immune protective efficiency of three-level Virus seed library
(1) it is immunized: taking Vaccinum Encephalitidis Epidemicae SA14-14-2 (6.5lgPFU/mL), P34 (7.0lgPFU/mL), P36 (7.25lgPFU/mL), P38 (7.15lgPFU/mL) are diluted to 10 respectively3PFU/0.1mL、102PFU/0.1mL、10PFU/ 0.1mL (i.e. 104PFU/mL、103PFU/mL、102PFU/mL), subcutaneous 10~12g KM mouse, 0.1mL/ are only, each dilute respectively Degree of releasing is 10 immune.Set dilution immune group as control group simultaneously, it is parallel with the above method to be immunized one group, for immunoprotection Observation.
(2) it attacks poison: after 14 days immune, attack poison P3 velogen strain being diluted to 10-6(its virulence is not less than 1000LD50), abdomen Chamber attacks immune group and control group, and 0.3mL/ only, while carrying out encephalocoele sky thorn to every mouse, to destroy blood-brain barrier.Inoculation Death is disregarded in 3 days afterwards, determines that the attack control group mice death rate is not lower than as a result, comparing its protection after attack 14 days 80%.
(3) result:
Protective rate is calculated according to the following formula:
Protective rate=(the control group mice death rate-experimental mice death rate)/control group mice death rate
Subcutaneously it is immunized 103PFU virus quantity protects P3 strain lethal hit up to 100%, with vaccine SA14-14-2 Protective rate it is identical, see the table below 4.
Table 4 tests virus, immunizing dose, dead mouse situation and protective rate
5. the serum neutralizing antibody of three-level Virus seed library is horizontal
(1) it is immunized: taking Vaccinum Encephalitidis Epidemicae SA14-14-2 (6.5lgPFU/mL), P34 (7.0lgPFU/mL), P36 (7.25lgPFU/mL), P38 (7.15lgPFU/mL) are diluted to 10 respectively5PFU/0.1mL、103PFU/0.1mL, subcutaneous inoculation 10 ~12g kunming mice, 0.1mL/ is only.Every plant of each dilution 10.Immune dilution mouse 10 only compares in parallel simultaneously, uses In blood sampling, it is horizontal to measure Serum Antibody.
(2) take a blood sample: 14 days after immune, every mouse is collected in 1.5mL sterile centrifugation from eye 0.5~1mL of blood sampling respectively Guan Zhong, 4 DEG C save overnight.
(3) separation of serum: separating serum (100~200 μ L) from every mouse, the independent pipe of every mice serum, no It merges.It is spare that -20 DEG C of refrigerators are stored in after 56 DEG C of water-bath inactivation 30min.
(4) Serum Antibody level detection (PRNT): BHK-21 cell inoculation is spaced 48h and waits in 24 porocyte culture plates Cell grows up to single layer, and P3 Strain is diluted to the virus quantity of about 25 plaques in every hole, and mice serum presses 1:5,1:10,1 respectively: 20,1:40 dilutes, and serum mixes in equal volume with virus, after 37 DEG C of water-bath effect 90min, serum-virus mixture 0.1mL is taken to connect For kind into 24 orifice plate cells, each dilution does a secondary orifices, and 37 DEG C of absorption 60min add final concentration of 1% methylcellulose to seek Covering to be supported, covering, violet staining 30min are sucked after cultivating 5 days in 37 DEG C of incubators, tap water, which rinses, counts plaque number, Calculate serum neutralization titer.
(5) result:
PRNT test in, can make plaque number reduce 50% serum dilution be the serum neutralization titer.
Subcutaneously it is immunized 103Or 105When PFU virus quantity, all 100% sun of all mice serums turns.Immunizing dose is 103PFU When, viral geometric mean titer 1:65,1:71,1:71, SA14-14-2 1:71；Immunizing dose is 105When PFU, virus and with The serum neutralizing antibody level of vaccine SA14-14-2 reaches 1:80, see the table below 5.
5 experimental group virus of table, immunizing dose, geometric mean titer and Conversion rate
Four, three-level Virus seed library genome sequencing
1. design of primers and synthesis
It is soft using Primer Premier 5.0 according to the whole genome sequence of the SA14-14-2 delivered in Genbank Part designs 7 sections of primers, and by Shanghai, Ying Jun Bioisystech Co., Ltd is synthesized, and see the table below 6.
6 SA14-14-2 of table segmentation sequencing amplification primers
2. the extraction of viral RNA, RT-PCR
The cell conditioned medium virus harvest liquid frozen is taken, illustrates to extract disease by the virus RNA extraction kit of TIANGEN company Malicious total serum IgE.CDNA is synthesized with Reverse Transcription System, then carries out purpose with each fragment-specific primer The PCR amplification of segment.Reaction condition are as follows: 95 DEG C of initial denaturation, 15min；94 DEG C of denaturation, 30s；52 DEG C of annealing, 30s；Extend 72 DEG C, 3.5min, after 27 recycle, 72 DEG C of extension 10min.Amplified production uses Gel after agarose gel electrophoresis is identified Extraction Kit purification and recovery.
3. clone's connection and bacterium solution sequencing
PCR glue recovery purifying product is connected in pGEM-T Easy carrier, transformed competence colibacillus E.coli DH5 α is selected The sequencing of Hai Yingjun Bioisystech Co., Ltd is served after positive colony Zengjing Granule, each segment surveys 5-8 clone's result.
3. sequence assembly and bioinformatics compare analysis
With the SeqMan function in DNAstar software by each fragment assembly；Gene order and ammonia are carried out with MegAlign The comparison of base acid sequence is analyzed.
4. result
(1) gene structure result
It is one long open read that SA14-14-2-2BS Strain, which is terminated since the 96th, the end 5' to 10392, the end 3', Frame, about 10.3kb, energy transcription and translation go out a polyprotein precursor, and this polyprotein passes through virus and host cell again The effect of protease and divide to form multiple protein, main includes 3 structural proteins (capsid protein, premembrane proteins and coating sugar Albumen) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).
(2) gene mutation site is analyzed
It is compared with Japanese Encephalitis Vaccine,Live strain SA14-14-2 complete genome sequence, SA14-14-2-2BS P34, SA14- 14-2-2BS P36, the variation of SA14-14-2-2BS P38 nucleotide are as follows:
7 vaccine strain of table is in 2BS cell culture P34, P36, P38 compared with attenuated live vaccine nucleotide
The results show that Japanese Encephalitis Vaccine,Live strain SA14-14- in SA14-14-2 P34, P36, P38 and Genbank 2 E gene protein nucleotide is identical, and full genome one shares 12 nucleotide sites and changes.
It can prove that by above-mentioned experiment
1. background understands, source is clear, proliferation is good for the japanese encephalitis virus SA14-14-2-2BS passage that the present invention screens And assay approval, reach the requirement as production of vaccine seed culture of viruses.
2. showing through safety, the verifying of immunogenicity, three aspect of genetic stability: SA14-14-2-2BS P34, P38 plants of safeties of SA14-14-2-2BS P36, SA14-14-2-2BS meet wanting for " Chinese Pharmacopoeia " third portion (2010 editions) It asks；And immunogenicity is good, can effectively immune attack protection and induction protection neutralizing antibody generate.
It is risen 3. according to the literature, Live attenuated vaccine SA14-14-2 and E protein gene are attenuated with it with immunogenicity Most important functions, gene order-checking shows SA14-14-2-2BS P34, SA14-14-2-2BS P36, SA14- in this research P38 plants of full-length genomes of 14-2-2BS are compared with parent plant SA14-14-2, and E gene nucleotide series are completely the same, full length gene Comparison the variation of 12 nucleotide has occurred altogether.

Claims (4)

1. adapted strain Virus seed library of the Japanese Encephalitis Vaccine,Live strain on human diploid cell 2BS, it is characterised in that: original poison Kind library: Strain SA14-14-2-2BS P34 complete genome sequence is as shown in SEQ ID NO:1.
2. adapted strain Virus seed library of the Japanese Encephalitis Vaccine,Live strain on human diploid cell 2BS, it is characterised in that: main generation poison Kind library: Strain SA14-14-2-2BS P36, complete genome sequence is as shown in SEQ ID NO:2.
3. adapted strain Virus seed library of the Japanese Encephalitis Vaccine,Live strain on human diploid cell 2BS, it is characterised in that: work poison Kind library: Strain SA14-14-2-2BS P38, complete genome sequence is as shown in SEQ ID NO:3.
4. it is malicious to be worked with adapted strain of the Japanese Encephalitis Vaccine,Live strain as claimed in claim 3 on human diploid cell 2BS The Japanese Encephalitis Vaccine,Live that kind library is prepared.
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