CN105695424A - Adaptive strain of Japanese encephalitis vaccine strain SA14-14-2 in human diploid cells 2BS and vaccine of adaptive strain - Google Patents
Adaptive strain of Japanese encephalitis vaccine strain SA14-14-2 in human diploid cells 2BS and vaccine of adaptive strain Download PDFInfo
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Abstract
The invention provides an adaptive strain of a Japanese encephalitis vaccine strain SA14-14-2 in human diploid cells 2BS and a vaccine of the adaptive strain. The adaptive strain is characterized in that epidemic encephalitis b virus SA14-14-2 is inoculated to a human diploid cell and is subjected to continuous passage by virtue of different methods, so that the virus can propagate in the cell with favorable adaptability and stability, relatively high-titer viruses are obtained, and a three-level virus seed library of viruses is established; by virtue of measuring whole-genome sequence of the virus seed library, the whole-genome sequence thereof is obtained. Effectiveness and safety inspection is performed on the viruses, and the viruses conform to specified standards of Japanese encephalitis vaccines. The Japanese encephalitis strain is relatively high in virus titer, has safety conforming to specification, is favorable in immunogenicity and stable in heredity, conforms to requirements on safety and effectiveness of Japanese encephalitis strains in the third version (2010 edition) of Chinese Pharmacopoeia and is suitable for serving as a strain for producing encephalitis b vaccines.
Description
Technical field
The invention belongs to viral vaccine field, particularly with biology techniques selection-breeding is highly attenuated and also encephalitis B live vaccine strain that immunogenicity is good。By the mode of monolayer, suspendible, Japanese Encephalitis Vaccine,Live strain SA14-14-2 is inoculated 2BS cell, and after adaptation of virus, plaque method measures virus titer, it is determined that the best harvest time of virus and inoculum concentration。Choose the higher three generations of virus titer and set up original seed culture of viruses, main generation seed culture of viruses and work Virus seed library; Virus seed library virus is carried out sterility test, mycoplasma inspection, immunogenicity inspection according in " Chinese Pharmacopoeia " three; Virulence detection in mouse brain, subcutaneous infection enters comprehensive calibrating of the projects such as brain test, reverse mutation test in suckling, immune protective test。Additionally also it is carried out complete genome sequence mensuration and the sequence of vaccine strain SA14-14-2 vaccine strain that goes down to posterity with the primary hamster kidney cell (PHK) listed is compared。
Background technology
Epidemic encephalitis type B (Japaneseencephalitis, encephalitis b) is the modal a kind of viral encephalitis in Asia, is a kind of Amphixenosis through killing propagation。Encephalitis b virus belongs to flaviviridae Flavivirus, for single-stranded positive RNA envelope virus, about total length 11kb。Encephalitis b virus has 5 genotype, and the popular virus of encephalitis b of current China is gene III type and genotype Ⅰ。According to estimates, encephalitis b virus at least causes 50,000 example clinical cases every year, and wherein majority is less than 10 years old child, causes that about 10,000 people are dead, separately has about 15,000 example cases to leave long-term neuro-psychiatric sequela。Encephalitis b almost has generation in all of Asian countries, and no matter it is positioned at temperate zone, subtropical zone or the torrid zone。In recent decades, encephalitis b occurred in that outburst without endemoepidemic area before some。There are nearly 3,000,000,000 population lives in the whole world in encephalitis b Endemic Area, the annual neonate in these Endemic Areas about 7,000 ten thousand。Infection and encephalitis B virus infection passes through mosquitoes spread, and mosquito obtains infection from ill toxemic animal, people。There is presently no the specificity antivirus Therapeutic Method for encephalitis b, vaccination is still unique most important control measure。
Several Asian countries such as at present, the Vaccinum Encephalitidis Epidemicae of large-scale use has three kinds: (1) Nakayama strain or Beijing Strain purification Mus brain inactivated vaccine, main Japan produce；(2) Beijing P3 strain Japanese encephalitis inactivated vaccine that Vero cell is cultivated；(3) the SA14-14-2 strain attenuated live vaccine that primary hamster kidney cell is cultivated。The shortcoming of purification Mus brain Vaccinum Encephalitidis Epidemicae has: the persistent period of inducing protective immunity is too short, needs multi-agent time inoculation；For majority state, the price of every vaccinating agent is of a relatively high。Once at domestic product and widely use before vaccine nineties in last century that cell is cultivated。1989; after the listing of SA14-14-2 strain attenuated live vaccine, because of it safely, effectively, immunity inoculation number of times is few, the advantage of guard time length, large area inoculation at home; market share has reached more than 90%, and exports more than ten countries such as Korea S, India, Thailand, Nepal。
Human diploid cell refers to normal human tissue cultured cells in vitro, it derives from normal human embryonic, succeeding generations keeps diploid karyotype, pollute without exogenous factor, without oncogenicity, there is no passage cell and the shortcoming of primary zooblast, be worth very big in cultivating virus and preparation virus type vaccine, be that WHO advocates the desirable cell produced for vaccine for man。The beginning of the sixties, human diploid cell came out, and the seventies is used for multiple production of vaccine。Recent years becomes the Cells for production of the products such as hepatitis A vaccine, chickenpox vaccine, attenuated rubella live vaccine, poliomyelitis vaccine, fiblaferon。WHO thinks that first the production of vaccine should be considered as humanized's diploid cell, because they are compared with other animal passage cells, is absent from oncogenic danger in theory, and its safety and efficacy producing vaccine is much better than vaccine prepared by other cellular matrixs。
Japanese Encephalitis Vaccine,Live is China's independent intellectual property right vaccine; from 1989 since Ministry of Public Health approval produces listing; up to now already with more than 600,000,000 doses times, significantly reducing sickness rate and the case fatality rate of Endemic Area encephalitis b, the health for child provides effective immanoprotection action。On October 9th, 2013, Japanese Encephalitis Vaccine,Live passes through World Health Organization's vaccine pre-authentication, becomes old China hand and crosses the first vaccine product of World Health Organization's pre-authentication。But, Vero cell and primary hamster kidney cell are all animal derived cells, the advantage of Vero cell is in that its quality and exogenous factor can Altitude control, reproduction speed is fast, persistent period is long, it is prone to markization, but Vero cell DNA remaining in vaccine is to the potential carcinogenecity risk of inoculator, therefore must strictly control residual content in vaccine preparation process。And primary hamster kidney cell derives from suslik, suslik population is not only difficult to control, manufacturing technique requirent is higher, and be easily subject to the allogenic materials such as antibacterial, virus, parasite and pollute, the Japanese Encephalitis Vaccine,Live that company limited of Chengdu Inst. of Biological Products of China produces at present adopts SPF hamster kidney cell as production of vaccine cellular matrix, can better solve the problem that the above-mentioned exogenous factor that fears are entertained that pollutes, but be the increase in vaccine cost。Therefore, present many vaccine enterprises are also at actively application human diploid cell research and development vaccine for man。The application intends adopting human diploid cell research and development Japanese Encephalitis Vaccine,Live, improves vaccine quality further。
Summary of the invention
Human diploid cell 2BS cell strain is the strain human embryonic lung diploid fibroblast set up by 3 monthly age female child lung tissues of one miscarriage of China in 1973, limited life is 63-65 generation, having the advantages that source is clear, the history that goes down to posterity is clear, pollute without exogenous factor, Ye Shi China ratifies one of two people's diploid cell strains that can be used for vaccine for man production at present。Human diploid cell 2BS cell strain has been used for the production of the vaccines such as people's rubella vaccine, chickenpox vaccine, uses through decades and a large amount of human vaccination, it was demonstrated that this cell is safety, good cellular matrix。
The Vaccinum Encephalitidis Epidemicae used on domestic market is mainly primary ground Ren Mus (PHK) cell vaccine and Vero cell vaccine。PHK cell is easily polluted by allogenic materials such as antibacterial, virus, parasites, is relatively difficult to control, and inoculation crowd is had potential carcinogenecity by Vero cell。In order to improve the quality of Vaccinum Encephalitidis Epidemicae further, more international market can be exported to, allow and more be subject to the growth that crowd particularly child that encephalitis b virus threatens can be healthy, epidemic encephalitis B virus SA14-14-2 is adapted to human diploid cell 2BS by us, make virus can well adapt on human diploid cell and rise in value, and study the process for stabilizing of this production of vaccine, to improve safety and the effectiveness of Vaccinum Encephalitidis Epidemicae, for the global people in particular by encephalitis B harm crowd health service。
It is an object of the invention to provide highly attenuated, immunogenicity good, hereditary stability the is high encephalitis b virus strain and breeding method thereof that can reach WHO standard, it is provided that production of vaccine seed culture of viruses, prepare encephalitis B attenuation human diploid cell live vaccine。
The object of the present invention is achieved like this: the selection of Japanese Encephalitis Vaccine,Live strain adapted strain on human diploid cell 2BS, it is characterized in that: selecting China's JE attenuated live vaccine SA14-14-2 strain as parent plant, incubation step includes at least:
(1) encephalitis b virus SA14-14-2 adaptability on human diploid cell 2BS goes down to posterity；
(2) the every generation Strain titration to results；
(3) filter out the highest Strain of titre and set up three grades of Virus seed library of virus, three grades of Virus seed library respectively original Virus seed library: Strain is SA14-14-2-2BSP34, and virus titer is 7.00lgPFU/mL；Main generation Virus seed library: Strain is SA14-14-2-2BSP36, and virus titer is 7.25lgPFU/mL；Work Virus seed library: Strain is SA14-14-2-2BSP38, and virus titer is 7.15lgPFU/mL；
(4) three grades of Virus seed library are carried out whole genome sequence mensuration, it is thus achieved that whole genome sequence；SA14-14-2-2BSP34 strain, SA14-14-2-2BSP36 strain, SA14-14-2-2BSP38 strain complete genome sequence are such as shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3。
(5) three grades of Virus seed library are carried out safety calibrating and immunoassays。
The selection of Japanese Encephalitis Vaccine,Live strain adapted strain on human diploid cell 2BS, can also be achieved in that: inoculate 2BS cell by the mode of monolayer, suspendible, after adaptation of virus, plaque method measures virus titer, it is determined that the best harvest time of virus and inoculum concentration。
Concrete inoculation propagating method is:
SA14-14-2-PHKC5 is inoculated in 2BS cell suspension by 0.2MOI, it is thus achieved that P1；
Results virus after cultivating 5 days, band poison cell continuous passage is to the 5th generation, it is thus achieved that P2-P5；
Continue to cultivate 5 days, by band poison cell suspension and normal cell suspension mixed in equal amounts continuous passage to the 10th generation, it is thus achieved that P6-P10；
10th generation virus is suspended in 2BS cell suspension by MOI0.1, it is thus achieved that P11；
It is further cultured for 4 days, band poison cell suspension and normal cell suspension mixed in equal amounts was passaged to for the 50th generation, it is thus achieved that P12-P50；
Filter out the highest P34, P36, P38 virus of titre and set up original Virus seed library, main generation Virus seed library and work Virus seed library respectively。
Three grades of Virus seed library that the selection of described Japanese Encephalitis Vaccine,Live strain adapted strain on human diploid cell 2BS obtains, including original Virus seed library: Strain SA14-14-2-2BSP34 complete genome sequence such as SEQIDNO:1；Main generation Virus seed library: Strain SA14-14-2-2BSP36, complete genome sequence is SEQIDNO:2 such as；Work Virus seed library: Strain SA14-14-2-2BSP38, complete genome sequence is such as shown in SEQIDNO:3。
The three grades of Virus seed library that the selection of the Japanese Encephalitis Vaccine,Live strain of the present invention adapted strain on human diploid cell 2BS the obtains Japanese Encephalitis Vaccine,Live SA14-14-2-2BS for preparing。
The present invention is by being inoculated on human diploid cell by epidemic encephalitis B virus SA14-14-2 (JEVSA14-14-2-PHKC), by different mode continuous passages, virus is enable to well adapt on cell and stable propagation, it is thus achieved that the virus of higher titre。According to " Chinese Pharmacopoeia " three (2010 editions), virus is carried out effectiveness and security inspection, meets the standard of the Japanese Encephalitis Vaccine,Live of regulation。Meanwhile, have studied the biological characteristics of the encephalitis b virus having been adapted to human diploid cell, including immunogenicity, immune protective efficiency and weak virulence stability etc., the human diploid cell Japanese Encephalitis Vaccine,Live that final acquisition is safely and effectively quality controllable。
Advantages of the present invention and notable benefit: human diploid cell is normal karyotype cell, succeeding generations keeps diploid karyotype, pollute without exogenous factor, non-carcinogenesis, there is no passage cell and the shortcoming of primary zooblast, the high immunogenicity having and good toleration, be the WHO desirable cell produced for vaccine for man advocated, it is successfully applied to the production of multiple vaccine at present, such as hepatitis A vaccine, chickenpox, poliomyelitis vaccine etc.。China is as the Endemic Area of encephalitis B, the research and development of Vaccinum Encephalitidis Epidemicae are on the forefront, although the safety of Live attenuated vaccine and effectiveness are confirmed, but primary hamster kidney cell vaccine exists animal easily to be polluted by allogenic materials such as antibacterial, virus, parasites, it is difficult to control。Although China adopts SPF hamster kidney cell as Japanese Encephalitis Vaccine,Live Cells for production substrate, and in JIUYUE, 2013 WHO has announced that the Japanese Encephalitis Vaccine,Live that China Chengdu produces meets WHO requirement, enter international procurement catalogue, it can be the national children's health service being especially subject to encephalitis b serious threat in the world, but China is adopted hamster kidney cell to produce Japanese Encephalitis Vaccine,Live by developed country still suspects, and worries that Cells for production is polluted by the exogenous factor not yet known at present。So, JEVSA14-14-2 PHKC is adapted to and can solve these vaccine safety sex chromosome mosaicism having people to worry in 2BS cell by this research, both the hidden danger remained without different DNA and foreign protein, can avoid again other animal derived viruses and the harmful microorganism impact on the mankind。Therefore, safety is better than the vaccine that non-human is primary and prepared by passage cell, promote technological innovation, be expected to the blank making up China in human diploid cell Vaccinum Encephalitidis Epidemicae, made China's Japanese Encephalitis Vaccine,Live can serve more population health in the world。
Accompanying drawing explanation
Fig. 1 is SA14-14-2-2BS Strain screening process figure；
Fig. 2 is SA14-14-2-2BS pnca gene group structural representation；
Figure indicating, numeral represents from top to bottom respectively:
Full-length genome (10976)；
One long open reading frame nucleotide (96～10394)；
The amino acid number (127,167,500,415,164,131,619,267,137,905) that structural region and non-structural region are separately encoded；
Have 3 structural protein at its 5 ' end, i.e. capsid protein C, premembrane proteins prM and envelope protein E, 3 ' ends have 7 non-structural proteins, i.e. NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5。
Fig. 3 examines under a microscope the 10th generation SA14-14-2 pathological changes situation on 2BS cell；
Figure showing, SA14-14-2 virus adapts to P10 on 2BS cell, pathological change form (A) when 4 days and normal 2BS cell controls (B)；
Fig. 4 is P10 virus titration curve on 2BS cell under different MOI and incubation time。
Detailed description of the invention
Below in conjunction with accompanying drawing, by specific embodiment, the invention will be further described, but the protection domain not limited the present invention in any way。
One, the JEV (encephalitis b attenuated vaccine strain SA14-14-2) adaptation on 2BS cell is gone down to posterity
1. cell and seed culture of viruses source
The cell used in this work is 2BS cell (27～37 generation), provides for National Institute for Food and Drugs Control's cyropreservation center。Cell culture fluid is the Eagle ' sMEM containing 10% hyclone。Encephalitis b attenuated vaccine strain SA14-14-2 virus is encephalitis b virus (JEV) SA14-14-2-PHKC5 that this room preserves。
2.SA14-14-2 adaptation on 2BS cell is gone down to posterity
(1) the 2BS cell of monolayer has been formed with 0.25% trypsinization, and with Eagle ' sMEM-10% hyclone culture fluid re-suspended cell, by 0.2MOI inoculum concentration virus inoculation in suspendible cell, virus and cell are sufficiently mixed uniformly, 37 DEG C of incubators are cultivated, draws 0.5ml virus liquid every day and survey its titre。After 5 days, results culture supernatant is encephalitis b virus 2BS cell adapted strain SA14-14-2-2BS 1st generation, is denoted as P1, frozen standby in-70 DEG C of refrigerators。Band poison cell being continued former bottle go down to posterity, within every 5 days, go down to posterity once, so by virus continuous passage 5 times, results SA14-14-2-2BSP5, each to withhold the virus liquid obtained all frozen in-70 DEG C of refrigerators。
After (2) the 5th generation virus liquid results, band poison cell is when going down to posterity, 1:1 co-passage is pressed with the fresh 2BS cell of uninfecting virus, it is placed in 37 DEG C of incubators and cultivates, gather in the crops encephalitis b virus adapted strain the 6th generation on 2BS cell after 5 days, fresh 2BS cell suspension mixed in equal amounts with poison cell Yu uninfecting virus goes down to posterity, and within every 5 days, goes down to posterity once, virus liquid reached the 10th generation。In succeeding generations, drawing 0.5ml virus liquid every day and survey its titre, each to withhold the virus liquid obtained all frozen in-70 DEG C of refrigerators。
Examine under a microscope the 10th generation SA14-14-2 pathological changes situation on 2BS cell and see Fig. 1。Fig. 1 shows that SA14-14-2 virus adapts to P10 on 2BS cell, pathological change form (A) when 4 days and normal 2BS cell controls (B)。
(3) with plaque ethods, each virus obtained of withholding is carried out titration:
BHK-21 cell is inoculated in 6 porocyte culture plates, and interval 48h treats that cell grows up to monolayer, by 10 times of serial dilutions of Strain to be measured, from 10-1～10-5Take the viral 0.2mL after dilution and be inoculated in 6 orifice plate cells, each dilution factor does a secondary orifices, 37 DEG C of absorption 60min, add final concentration of 1% methylcellulose nutrition covering, after 37 DEG C of incubators are cultivated 5 days, suck covering, violet staining 30min, tap water counting plaque number, calculates plaque slip。
(4) virus titer of P1～P10 results measures。
Computational methods are average × 5 (because 0.2mL is inoculated in every hole, draw the logarithm of every mL after × 5) of holes plaque number, i.e. virus titer=(this dilution logarithm value)+(logarithm value of average speckle number × 5) lgPFU/mL。
P1～P10 virus titer is shown in table 1 below。
The titre of the front 10 generation viruses of table 1 results
(5) selected for the 10th generation adaptation strain (SA14-14-2-2BSP10) be inoculated in 2BS cell suspension by the MOI virus quantity of 0.1,0.01,0.001,0.0001 and carry out Virus culture, take viral supernatants respectively at 1d, 2d, 3d, 4d, 5d, 6d, detect virus titer with plaque ethods。Different MOI, see table 2 below without virus titer under incubation time。
P10 virus titre on 2BS cell under the different MOI of table 2 and incubation time
Referring to P10 virus titration curve on 2BS cell under Fig. 2 difference MOI and incubation time。
(6) the 2BS cell growing up to monolayer is disperseed with 0.25% trypsinization, get encephalitis b virus SA14-14-2-2BS the 10th generation obtained by 0.1MOI virus quantity addition to 2BS cell, 37 DEG C adsorb 60 minutes, it is placed in 37 DEG C of incubators and cultivates, go down to posterity after band poison cell suspension and healthy cell suspension mixed in equal amounts after 4 days, 37 DEG C of cultivations；Gather in the crops viral supernatants after 4 days, be labeled as P11, continue band poison cell suspension and healthy cell suspension mixed in equal amounts to be gone down to posterity again, 37 DEG C cultivate 4 days after results the 12nd generation Virus culture supernatant。Repeat band poison cell and healthy cell co-passage to 50 generations, until results viral supernatants P50。The virus titer of P11～P50 results is shown in table 3 below。
The titre of P11～P50 virus of table 3 results
Two, the foundation of three grades of Virus seed library
This research is by being seeded on suspendible 2BS cell by (JEV) SA14-14-2, by making encephalitis b virus stably be adapted on 2BS cell with poison cell continuous passage, method with poison cell and healthy cell mixing continuous passage, filter out encephalitis b virus SA14-14-2 diploid cell 2BS adapted strain, and virus reaches higher titre, from 20 generations to 50 generations, virus titer can stably in higher titre scope。By virus adaptation of virus analysis and summary on 2BS cell is analyzed, we have chosen the generation that adapted strain virus titer is higher, namely ((7.00lgPFU/mL) is as original poison storehouse for the 34th generation virus, in 36th generation virus (7.25lgPFU/mL), in the 38th generation virus (7.15lgPFU/mL), was as work seed culture of viruses as main generation seed culture of viruses。
Three, the research of the security inspection of three grades of Virus seed library and biological characteristics
1. Virulence detection in brain:
(1) 12～14g Kunming (KM) mice 20 is inoculated respectively with (JEV) SA14-14-2-2BS the 34th, 36,38 generation virus stock solution used, every intracerebral injection 0.03mL, observes 14 days。Inoculate died in latter 3 days and disregard (animal dead quantity should must not exceed the 20% of experimental animal sum)。If any mouse invasion after 3 days, should locate after death to take brain, measure pathogenicity。In mouse brain, virulence is not higher than 3.0lgLD50/0.03mL, simultaneously with 10-1 virus brain suspension subcutaneous injection body weight for 10～12g mice 10, every 0.1mL, observes 14 days。
(2) result: after 14 days, without dead mouse。
2. subcutaneous infection enters brain test:
(1) inoculating 10～12g kunming mice 10 respectively with (JEV) SA14-14-2-2BS the 34th, 36,38 generation virus stock solution used, every subcutaneous injection 0.1mL, empty thorn in the brain of right side, observes 14 days simultaneously。
(2) result: after 14 days, all mices are all strong deposits。
3. reverse mutation test in suckling:
(1) 3～5 age in days Kunming neonatal rat 10 is inoculated respectively with (JEV) SA14-14-2-2BS the 34th, 36,38 generation virus stock solution used, every intracerebral injection 0.02ml。The neonatal rat of morbidity is put to death 3, and solution takes brain, surveys its pathogenicity with body weight 12～14g kunming mice, in brain, virulence should not higher than 3.0lgLD50/0.03ml, simultaneously with the morbidity suckling mouse brain suspension subcutaneous injection body weight of 10-1 for 10～12g kunming mice 10, every 0.1ml, observes 14 days。
(2) result: after 14 days, all mices are all strong deposits。
4. the immune protective efficiency of three grades of Virus seed library
(1) immunity: take 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 (namely 104PFU/mL、103PFU/mL、102PFU/mL), subcutaneous 10～12gKM mice respectively, 0.1mL/, each dilution factor immunity 10。Set diluent immune group as matched group simultaneously, parallel with said method immune one group, for the observation of immunoprotection。
(2) counteracting toxic substances: after immune 14 days, is diluted to 10 by attack poison P3 virulent strain-6(its virulence is not less than 1000LD50), immune group and matched group are attacked in abdominal cavity, and every mice only, is carried out encephalocoele sky thorn, to destroy blood brain barrier by 0.3mL/ simultaneously。Inoculate death in latter 3 days to disregard, result of determination after attacking 14 days, compare its protection, attack control group mice mortality rate and be not lower than 80%。
(3) result:
Protective rate is calculated by following equation:
Protective rate=(control group mice mortality rate-experimental mice mortality rate)/control group mice mortality rate
Through subcutaneous inoculation 103P3 strain lethal hit is protected by PFU virus quantity up to 100%, identical with the protective rate of vaccine SA14-14-2, sees table 4 below。
Table 4 tests virus, immunizing dose, dead mouse situation and protective rate
5. the serum neutralizing antibody level of three grades of Virus seed library
(1) immunity: take 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。The each dilution factor of every strain 10。Parallel immunity diluent mice 10 only compares simultaneously, is used for taking a blood sample, measures Serum Antibody level。
(2) blood sampling: latter 14 days of immunity, every mice, from eye blood sampling 0.5～1mL, is collected in 1.5mL sterile centrifugation tube respectively, and 4 DEG C overnight preserve。
(3) separation of serum: separate serum (100～200 μ L) from every mice, every independent pipe of mice serum, do not merge。It is saved in-20 DEG C of refrigerators standby after 56 DEG C of water-bath inactivation 30min。
(4) Serum Antibody horizontal detection (PRNT): BHK-21 cell is inoculated in 24 porocyte culture plates, interval 48h treats that cell grows up to monolayer, P3 Strain is diluted to the virus quantity of about 25 plaques in every hole, mice serum presses 1:5 respectively, 1:10, 1:20, 1:40 dilutes, serum mixes with virus equal-volume, after 37 DEG C of water-bath effect 90min, take serum-virus mixture 0.1mL and be inoculated in 24 orifice plate cells, each dilution factor does a secondary orifices, 37 DEG C of absorption 60min, add final concentration of 1% methylcellulose nutrition covering, covering is sucked after 37 DEG C of incubators are cultivated 5 days, violet staining 30min, tap water counting plaque number, calculate in serum and titer。
(5) result:
In PRNT tests, the serum dilution that plaque number reduces 50% can be made to be the neutralization titer of this serum。
Through subcutaneous inoculation 103Or 105During PFU virus quantity, all mice serums all 100% sun turn。Immunizing dose is 103During PFU, virus geometric mean titer 1:65,1:71,1:71, SA14-14-2 is 1:71；Immunizing dose is 105During PFU, virus and the serum neutralizing antibody level with vaccine SA14-14-2 all reach 1:80, see table 5 below。
Table 5 experimental group virus, immunizing dose, geometric mean titer and Conversion rate
Four, three grades of Virus seed library genome sequencings
1. design of primers and synthesis
Whole genome sequence according to the SA14-14-2 delivered in Genbank, applies 7 sections of primers of PrimerPremier5.0 software design, Shanghai Ying Jun Bioisystech Co., Ltd synthesize, see table 6 below。
Table 6SA14-14-2 segmentation order-checking amplification primers
2. the extraction of viral RNA, RT-PCR
Take frozen cell conditioned medium virus harvest liquid, extract test kit by the viral RNA of TIANGEN company and illustrate to extract virus total RNA。Synthesize cDNA with ReverseTranscriptionSystem, then carry out the pcr amplification of purpose fragment with each fragment-specific primer。Reaction condition is: denaturation 95 DEG C, 15min；Degeneration 94 DEG C, 30s；Anneal 52 DEG C, 30s；Extending 72 DEG C, 3.5min, after 27 circulations, 72 DEG C extend 10min。Amplified production purifies recovery with GelExtractionKit after agarose gel electrophoresis is identified。
3. clone connects and bacterium solution order-checking
PCR glue is reclaimed purified product and is connected in pGEM-TEasy carrier, transformed competence colibacillus E.coliDH5 α, serve the order-checking of Hai Ying fine horse Bioisystech Co., Ltd after selecting positive colony Zengjing Granule, each fragment is surveyed 5-8 and is cloned result。
3. sequence assembly and bioinformatics comparison are analyzed
By the SeqMan function in DNAstar software by each fragment assembly；MegAlign is used to carry out the comparison analysis of gene order and aminoacid sequence。
4. result
(1) gene structure result
SA14-14-2-2BS Strain is a long open reading frame from the beginning of the 96th, 5' end to 10392 terminations of 3' end, about 10.3kb, transcription and translation can go out a polyprotein precursor, this polyprotein divides formation multiple protein again through the effect of virus and host cell proteins enzyme, mainly includes 3 structural protein (capsid protein, premembrane proteins and envelope glycoprotein) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5)。
(2) gene mutation site analysis
Contrasting with Japanese Encephalitis Vaccine,Live strain SA14-14-2 complete genome sequence, SA14-14-2-2BSP34, SA14-14-2-2BSP36, SA14-14-2-2BSP38 nucleotide changes such as following table:
Table 7 vaccine strain is cultivated P34, P36, P38 at 2BS cell and is compared with attenuated live vaccine nucleotide
Result shows, in SA14-14-2P34, P36, P38 and Genbank, the E gene protein nucleotide of Japanese Encephalitis Vaccine,Live strain SA14-14-2 is identical, and full genome one has 12 nucleotide sites and changes。
Be can prove that by above-mentioned experiment
1. the encephalitis b virus SA14-14-2-2BS of present invention screening goes down to posterity, and background is clear, source is clear and definite, propagation is good and assay approval, reaches the requirement as production of vaccine seed culture of viruses。
2. through safety, immunogenicity, hereditary stability three aspect checking show: SA14-14-2-2BSP34, SA14-14-2-2BSP36, SA14-14-2-2BSP38 strain safety meets the requirement of " Chinese Pharmacopoeia " the 3rd (2010 editions)；And immunogenicity is good, can protect and induce protection neutralizing antibody to produce by effective immune attack。
3. according to bibliographical information, Live attenuated vaccine SA14-14-2 plays most important functions with E protein gene with its attenuation and immunogenicity, in this research, gene order-checking shows, SA14-14-2-2BSP34, SA14-14-2-2BSP36, SA14-14-2-2BSP38 strain full-length genome is compared with parent plant SA14-14-2, and E gene nucleotide series is completely the same, and the comparison of full length gene there occurs 12 nucleotide changes altogether。
Claims (5)
1. the selection of Japanese Encephalitis Vaccine,Live strain adapted strain on human diploid cell 2BS, it is characterised in that: selecting China's JE attenuated live vaccine SA14-14-2 strain as parent plant, incubation step includes at least:
(1) encephalitis b virus SA14-14-2 adaptability on human diploid cell 2BS goes down to posterity；
(2) the every generation Strain titration to results；
(3) filter out the highest Strain of titre and set up three grades of Virus seed library of virus, three grades of Virus seed library respectively original Virus seed library: Strain is SA14-14-2-2BSP34, and virus titer is 7.00lgPFU/mL；Main generation Virus seed library: Strain is SA14-14-2-2BSP36, and virus titer is 7.25lgPFU/mL；Work Virus seed library: Strain is SA14-14-2-2BSP38, and virus titer is 7.15lgPFU/mL；
(4) three grades of Virus seed library are carried out whole genome sequence mensuration, it is thus achieved that whole genome sequence；SA14-14-2-2BSP34 strain, SA14-14-2-2BSP36 strain, SA14-14-2-2BSP38 strain complete genome sequence are such as shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3。
(5) three grades of Virus seed library are carried out safety calibrating and immunoassays。
2. the selection of the Japanese Encephalitis Vaccine,Live strain according to claim 1 adapted strain on human diploid cell 2BS, it is characterized in that: inoculate 2BS cell by the mode of monolayer, suspendible, after adaptation of virus, plaque method measures virus titer, it is determined that the best harvest time of virus and inoculum concentration。
3. the selection of the Japanese Encephalitis Vaccine,Live strain according to claim 1 and 2 adapted strain on human diploid cell 2BS, it is characterised in that: concrete inoculation propagating method is:
SA14-14-2-PHKC5 is inoculated in 2BS cell suspension by 0.2MOI, it is thus achieved that P1；
Results virus after cultivating 5 days, band poison cell continuous passage is to the 5th generation, it is thus achieved that P2-P5；
Continue to cultivate 5 days, by band poison cell suspension and normal cell suspension mixed in equal amounts continuous passage to the 10th generation, it is thus achieved that P6-P10；
10th generation virus is suspended in 2BS cell suspension by MOI0.1, it is thus achieved that P11；
It is further cultured for 4 days, band poison cell suspension and normal cell suspension mixed in equal amounts was passaged to for the 50th generation, it is thus achieved that P12-P50；
Filter out the highest P34, P36, P38 virus of titre and set up original Virus seed library, main generation Virus seed library and work Virus seed library respectively。
4. three grades of Virus seed library that the selection of the adapted strain on human diploid cell 2BS of the Japanese Encephalitis Vaccine,Live strain described in claim 1 obtains, it is characterised in that: original Virus seed library: Strain SA14-14-2-2BSP34 complete genome sequence such as SEQIDNO:1；Main generation Virus seed library: Strain SA14-14-2-2BSP36, complete genome sequence is SEQIDNO:2 such as；Work Virus seed library: Strain SA14-14-2-2BSP38, complete genome sequence is such as shown in SEQIDNO:3。
5. the Japanese Encephalitis Vaccine,Live SA14-14-2-2BS that the three grades of Virus seed library obtained with the selection of the adapted strain on human diploid cell 2BS of the Japanese Encephalitis Vaccine,Live strain described in claim 1 are prepared。
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