CN100528227C - Vaccine for virus of encephalitis B and preparation method - Google Patents
Vaccine for virus of encephalitis B and preparation method Download PDFInfo
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- CN100528227C CN100528227C CNB2004100379480A CN200410037948A CN100528227C CN 100528227 C CN100528227 C CN 100528227C CN B2004100379480 A CNB2004100379480 A CN B2004100379480A CN 200410037948 A CN200410037948 A CN 200410037948A CN 100528227 C CN100528227 C CN 100528227C
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Abstract
A vaccine of the encephalitis B virus for preventing viral encephalitis B is prepared through inoculating said virus into human diploid cells, culturing, naturalizing, adapting, amplifying, concentrating and purifying.
Description
Technical field
The present invention relates to a kind of encephalitis b virus vaccine of cultivating preparation through human diploid cell.Particularly, the present invention relates to a kind ofly encephalitis b virus is inoculated in human diploid cell cultivates, through taming, adapt to and the amplification PROCESS FOR TREATMENT, encephalitis b virus is breeding in a large number in human diploid cell, is mixed with a kind of encephalitis b virus vaccine through concentrated, purification again.This viral vaccine can prevent infant, child, teenager and adult to infect the encephalitis B disease that causes because of encephalitis b virus.The invention still further relates to the preparation method of this viral vaccine.
Background technology
Encephalitis B is the infectious disease of central nervous system's acute viral infection, and up to 10～1,00/,100,000, nearly 3,000,000,000 populations are lived in the popular district of encephalitis B at popular regional annual morbidity, and the annual neonate in this area just surpasses 7,000 ten thousand.In the viral encephalitis in Asia, encephalitis B is the most serious disease.At least cause that 50,000 clinical cases and 10,000 examples die of illness every year, the overwhelming majority betides among the child.In decades recently, several outbreak of epidemic of encephalitis B are more and more to right and wrong popular area expansion once, follow very high case fatality rate and serious persistency nervous system sequela simultaneously, thus in the Asia a lot of countries and regions, encephalitis B becomes serious public health problem.
Epidemic encephalitis B virus (Japanese Encephalitis Virus) is called for short encephalitis b virus, and virion is the icosahedron symmetrical structure, diameter 45-50nm, and virion is made up of core, peplos and furcella, belongs to a kind of of banzi virus.Gene is the normal chain single-stranded RNA, and the RNA bag is formed the core of virion by in the capsid C of polypeptide.Glycosylated protein E and non-glycosylated protein prM/M are arranged in its peplos.
Mosquito is the main communication media of encephalitis b virus in the encephalitis B route of infection, and pig is main intermediate host, and the people is because of being infected by the mosquito bite of band poison.Also do not treat at present the specific medicament of encephalitis B disease, the effect of controlling its propagation from the environment aspect is undesirable.The immunity inoculation of Vaccinum Encephalitis B is the most cost-effective measure of control encephalitis B disease.
Vaccinum Encephalitis B has 3 classes in large-scale application, through murine brain cultivate preparation inactivated vaccine, Ren Mus primary cell or the inactivated vaccine of Vero cell culture preparation and the attenuated live vaccine of ground Ren Mus primitive cell culture preparation.
Because the Vaccinum Encephalitis B fundamental immunity starts from the healthy infants at 6 monthly ages, this must guarantee at first that with regard to requiring vaccine enough safety is precondition.Which kind of cultivating system no matter above-mentioned Vaccinum Encephalitis B adopt, and all is to make through animal derived tissue or animal cell culture.The shortcoming of these vaccines is: at first, can't thoroughly remove the pollution of animal derived exogenous factor.Secondly, in human body, produce higher immune response, immunoprotection higher, more of a specified duration is provided thereby can't further increase antigenic content.Its former because as when further increasing virus antigen content, the animal derived material of following such as foreign protein, nucleic acid and lipid etc. also increase thereupon, cause serious allergy, so the vaccine side reaction just strengthens.For example, the inactivated vaccine of cultivating through murine brain is about 20% with pain, local side reaction rubescent, swelling, and the systemic reaction that comprises headache, heating, myalgia, discomfort and gastrointestinal symptom is 10～30%.1989 and nineteen ninety are also found 31 example and 7 routine urticaria and angioedematous serious allergy respectively in Denmark and Australia.Surpass 38 ℃ heating up to 12% through the inactivated vaccine of ground Ren Mus primitive cell culture, after having reduced the Ox blood serum residual quantity, surpass 38 ℃ heating reduce to 6% (Tsai, Theodore F, Japanese encephalitis vaccines, http: //
Www.cdc.gov.publication date:01/01/1990).And attenuated live vaccine be with non-cleaning level ground Ren Mus primary cell as the vaccine culture matrix, this is difficult to be avoided the pollution of the exogenous factor in Mus source, this pollution that may cause is again to be difficult to detect with existing biological method.Therefore, can not make vertification regulation by World Health Organization's vaccine and be admitted (the Ze literary composition is far away, planned immunization, Shanghai scientific and technical literature publishing house, calendar year 2001,484 pages).Though the safety with the inactivated vaccine of Vero cell culture preparation has had the significance raising, but the Vero cell has tumorigenesis phenomenon (Levenbook IS after surpassing 232 generations in nude mouse, etal, Tumorigenicity of vero cells.J Biol Stand 1984Oct; 12 (4): 391-8).Therefore, U.S. FDA requires the vaccine with the Vero cells produce, the technology of removing cell will be arranged in the production process, must guarantee not have remaining cell in the finished product (FDA letterto vaccine manufacturers concerning the use of Vero cells.http: //www.fda.gov/cber/letters.htm).
In view of the foregoing, need be to carry out transforming to existing encephalitis b virus vaccine bebcell revolutionaryly, seek new Virus culture cell line, avoid above-mentioned murine brain, the potential safety hazard that exists of Ren Mus primary cell and Vero cell line, prepare can satisfy and start from the Vaccinum Encephalitis B that infant is inoculated.
Confirm that the diploid cell that comes from the people comprises donor's age, sex, race, region, physical ability and health status, tissue or organ, the cell generation number etc. of cell direct sources because its source is quite clear.Human diploid cell system is through the research of many decades, and the characteristic of its growth characteristics, inheritance stability, exogenous factor (antibacterial, fungus, mycoplasma and virus) pollute characteristics such as checking, cause the tumor feminine gender and fully verified.The what is more important human diploid cell itself is the cell that comes from human body, and can not to become allergy former for the remaining composition of human diploid cell when being used for vaccine and making, and can not cause inoculator's allergy substantially.Therefore, from the angle of quality control, be compared to above-mentioned murine brain, Ren Mus primary cell and Vero cell line, human diploid cell does not have potential hidden danger, is preparation vaccine ideal cell line.
Further confirm, the personnel selection diploid cell line prepares vaccine and is more and more used, for example with the rubella virus vaccine of MRC-5 and/or the preparation of WI-38 cell system, also be human diploid cell cultivation vaccine with the hepatitis A virus (HAV) vaccine of MRC-5 cell line preparation, with the Rotavirus Vaccine of FrhL-2 cell line preparation, Varivax, the generally acknowledged in the world goldstandard rabies vaccine for preparing with MRC-5 cell line.Therefore, prepare the quality that encephalitis b virus vaccine energy significance ground improves existing encephalitis b virus vaccine with human diploid cell.
Core content of the present invention is, adopt human diploid cell system to cultivate the cellular matrix of breeding as encephalitis b virus, simultaneously encephalitis b virus is tamed adaptation in human diploid cell, and virus and cell all there is good stable, and then is prepared into the encephalitis b virus vaccine.
In sum, encephalitis b virus vaccine emphasis of the present invention has solved, with human diploid cell substitute murine brain, Ren Mus primary cell and Vero cell carry out encephalitis b virus and cultivate, further make vaccine again.But with the harm of vaccine significance ground reduction vaccine side reaction of human diploid cell preparation especially anaphylaxis and animal derived virus, remove potential safety hidden danger, guaranteed the safety that vaccine uses in the adult of child and teenager and Pest-or disease-free area.
In order to overcome the deficiencies in the prior art part, the object of the present invention is to provide a kind of new encephalitis b virus vaccine and preparation method thereof.
Summary of the invention
In order to finish purpose of the present invention, the invention provides a kind of encephalitis b virus vaccine, it is characterized in that the contained encephalitis b virus antigen of this vaccine is that encephalitis b virus obtains through human diploid cell cultivation, breeding, purification, extraction.Described encephalitis b virus vaccine is an inactivated vaccine, also comprise attenuated live vaccine, split vaccine and/or subunit vaccine and with the combined vaccine of other vaccine.
Described encephalitis b virus vaccine is by encephalitis b virus SA
14Strain, SA
14-14-2Strain, P
3Arbitrary strain prepares in other separated strain of strain, Nakayama strain and encephalitis b virus.
Described human diploid cell is the arbitrary strain in 2BS, MRC-5, KMB-17, WI-38 or the HL cell line.
The invention still further relates to a kind of with encephalitis b virus domestication, adapt to and increase in the method for human diploid cell.
In other words, the invention provides and encephalitis b virus is inoculated in human diploid cell cultivates, through domestication, adapt to and the amplification PROCESS FOR TREATMENT, encephalitis b virus is breeding in a large number in human diploid cell, again through deactivation, concentrate, purification is mixed with a kind of encephalitis b virus vaccine.The method that the present invention also provides encephalitis b virus to tame in human diploid cell, adapt to and increase.This encephalitis b virus vaccine with the human diploid cell preparation can prevent infant, child, teenager and adult to infect the encephalitis B disease that causes because of encephalitis b virus respectively.
Notion that emphasis is introduced among the present invention and method are that existing encephalitis b virus vaccine is transformed and upgrading makes it to reach better safety and immune effect.The present invention be with human diploid cell substitute murine brain, Ren Mus primary cell and Vero cell be used for encephalitis b virus and cultivate, the viral liquid of results through deactivation, concentrate, purification is after preparation becomes the encephalitis b virus vaccine.This encephalitis b virus vaccine with the human diploid cell preparation, except that containing as the immunogenic virus antigen existence, do not contain any remaining composition that comes from animal tissue or zooblast, fundamentally got rid of animal proteinum, nucleic acid and the lipid etc. that cause allergy.Because safety significance ground improves, and further makes vaccine can improve immunizing dose, increase the vaccine protection and render a service, thereby can overcome immune limitation and the postvaccinal untoward reaction that existing encephalitis b virus commercially available vaccine has.
The used encephalitis b virus of the present invention (Japanese Encephalitis Virus) seed culture of viruses is bought in Nat'l Pharmaceutical ﹠ Biological Products Control Institute.The seed culture of viruses name is called: SA
14, SA
14-14-2, P
3, Nakayama.Nat'l Pharmaceutical ﹠ Biological Products Control Institute is responsible for preparation and provides above-mentioned viral seed culture of viruses.
In one embodiment of the invention, encephalitis b virus is cultivated and can be adopted above-mentioned seed culture of viruses SA
14, SA
14-14-2, P
3, any strain among the Nakayama.If satisfy the demand, also can adopt other suitable seed culture of viruses, the seed culture of viruses that herein provides only is for illustrative the present invention.
Encephalitis b virus cultivating system of the present invention is the arbitrary strain in human diploid cell 2BS, MRC-5, KMB-17, WI-38 or the HL cell line.If satisfy the demand, also can adopt other suitable human diploid cell, the cell that herein provides only is for illustrative the present invention.
The invention provides encephalitis b virus is tamed, adapts to and increased in the method for human diploid cell.Particularly, the virus titer of encephalitis b virus in human diploid cell improves gradually, 〉=7.5lgPFU/ml, and trend towards stablizing.
The every dosage of encephalitis b virus vaccine of the present invention is for containing virus protein 5～90 μ g.As for the upper limit of consumption, those skilled in the art can finally determine according to vaccine human clinical trial data.
The encephalitis b virus vaccine of human diploid cell preparation of the present invention has good immunogenicity, and the encephalitis b virus vaccine of NAT 〉=hamster kidney cell and Vero cell preparation.
The encephalitis b virus vaccine of human diploid cell of the present invention preparation has excellent protection to be renderd a service, and renders a service the neutralization index encephalitis b virus vaccine of hamster kidney cell and Vero cell preparation above Ground.
Encephalitis b virus vaccine of the present invention can be formed encephalitis b-epidemic encephalitis combined vaccine with the combined vaccine that A, C group meningitis cocci polysaccharide and a protein carrier coupling form.
Encephalitis b virus vaccine of the present invention also can further be formed combined vaccine with other vaccine, as: acellular DPT vaccine-inactivated poliomyelitis vaccine-Hib vaccine-hepatitis B vaccine-meningococcus combined vaccine-encephalitis b virus vaccine, measles-encephalitis B combined vaccine, measles-rubella-parotitis-encephalitis B combined vaccine, yellow heat-encephalitis B combined vaccine etc.
The invention provides a kind of preparation method of encephalitis b virus vaccine, the used cell line of this vaccine Virus culture in preparation process is human diploid cell.Described in the method for the invention human diploid cell is the arbitrary strain that is selected from 2BS, MRC-5, KMB-17, WI-38 or the HL cell line.And described encephalitis b virus vaccine can be inactivated vaccine, and inactivated vaccine is by encephalitis b virus SA
14Strain, SA
14-14-2Strain, P
3Arbitrary strain prepares in strain or Nakayama strain or other the isolating encephalitis b virus strain.Described encephalitis b virus vaccine also can be attenuated live vaccine, purified vaccine, split vaccine/or subunit vaccine.
Described in addition encephalitis b virus vaccine can also be and the combined vaccine of other vaccine, comprise: measles-encephalitis B combined vaccine, encephalitis b-epidemic encephalitis combined vaccine, measles-rubella-parotitis-encephalitis B combined vaccine, yellow heat-encephalitis B combined vaccine.
Can prepare a kind of encephalitis b virus vaccine that more high immunogenicity and effectiveness, safer, easier vaccinate accept through method of the present invention.
The specific embodiment
Describe the present invention below in conjunction with embodiment, encephalitis b virus is tamed in human diploid cell, adapted to and increases, the viral liquid of gathering in the crops is concentrated and purification, again through suitably being mixed with the encephalitis b virus vaccine.Confirm that through animal experiment and laboratory verification this kind vaccine has reliable safety and good immunogenicity and remarkable protection and renders a service with the encephalitis b virus vaccine of diploid cell preparation.
Embodiment 1:
The preparation of human diploid cell
The preparation of human diploid cell is example with MRC-5: cell culture fluid is DMEM (also available other suitable cell nutrient solution such as MEM, 199 culture medium, 1640 culture medium etc.), the kanamycin (or gentamycin) of the hyclone of adding 4～10% (no exogenous factor pollutes) and concentration 100～200U/ml is transferred between pH to 7.0～8.0.Cell is incubated at 35～37 ℃ with Ke Shi bottle or rolling bottle, cultivate fine and close cell monolayer after 4～8 days after, with 1: 2 ratio amplification, when batch consumption is produced in amplification, cultivate through 3～5 days, cell grows up to fine and close cell monolayer, discards cell culture fluid.And, wash residual Ox blood serum off with the phosphate buffer drip washing cell surface of pH7.0～8.0.Preparation is used for the inoculation of virus.
Embodiment 2:
Encephalitis b virus is tamed in human diploid cell, is adapted to and increases
With human diploid cell is example with MRC-5: because the virus breeding titre of encephalitis b virus in human diploid cell is lower, and therefore must be with encephalitis b virus domestication in human diploid cell, adaptation gradually.Encephalitis b virus P3 strain other Strain such as (or) SA14 is inoculated in the human diploid cell that covers with monolayer, continues to cultivate in 30～37 ℃.Harvesting supernatant when cytopathy reaches 50～70% is the encephalitis b virus liquid of growth and breeding in human diploid cell.Carrying out a generation generation successively adapts to.Encephalitis b virus titre in the encephalitis b virus liquid of results reaches the seed virus that the above time conduct of 7.5lgPFU/ml is used for Vaccinum Encephalitidis Epidemicae production usefulness.Require after every test detection is qualified, promptly to can be used for the production preparation of vaccine by " Chinese biological goods rules " (Chemical Industry Press, 2000).
Embodiment 3:
Encephalitis b virus is mitotic stability in human diploid cell
Test procedure by embodiment 2 is carried out successively, and can be observed the virus titer of encephalitis b virus in human diploid cell and improve gradually, 〉=7.5lgPFU/ml, and trend towards stablizing, the virus titer of each generation sees Table 1.
Table 1. encephalitis b virus mitotic stability in human diploid cell
| Virus adapts to generation | Virus titer (lgpfu/ml) |
| 1 | 4.58 |
| 2 | 4.95 |
| 3 | 5.68 |
| 4 | 5.80 |
| 5 | 6.39 |
| 6 | 7.56 |
| 7 | 7.80 |
| 8 | 7.87 |
Embodiment 4:
The preparation of encephalitis b virus inactivated vaccine
Press embodiment 1 preparation human diploid cell, prepare the viral seed that production of vaccine are used according to embodiment 3.The generation of selecting virus titer to reach the production of vaccine requirement according to embodiment 3 is a production of vaccine work seed.To be that 0.1～0.00001 ratio inoculation encephalitis b virus is in human diploid cell with MOI with the cell of phosphate buffer drip washing.Cell maintenance medium is the DMEM cell nutrient solution that contains 0.1～0.5% human albumin, between the pH 7.0～8.0.30～37 ℃ of cultivations, the viral supernatant of results after 3 days.Viral liquid virus titer after (as cultivating and can gather in the crops continuously with microcarrier) results reaches more than the 7.5lgpfu/ml, and sterility test detects negative, the viral liquid after the results can be done suitable merging.Liquid after the merging is vaccinogen liquid.Add 1: 2000 formalin of suitable inactivator (or 1: 4000 beta-propiolactone, or other suitable inactivator only are example) herein.Obtain rough vaccine behind the inactivation of viruses.10～50 times of the ultrafilter concentrated vaccines of reuse interception 10～300,000 molecular weight carry out ion exchange column (DEAE-SepharoseFF) and solvent resistant column (Sepharose 4FF) through two step column chromatographies, obtain purified vaccine.Add the white protective agent of people, promptly can be made into vaccine after the dilution.
Embodiment 5:
The safety testing of encephalitis b virus vaccine
Safety to the encephalitis b virus vaccine of human diploid cell preparation is examined, and adopts mice, Cavia porcellus abnormal toxicity test to examine its safety respectively.Select the cleaning level Cavia porcellus of body weight 250～350g for use, every kind of vaccine is with 2 Cavia porcelluss, the encephalitis b virus vaccine of every lumbar injection 5.0ml, respectively at before the injection, injection weighed, and observes the vaccination afterreaction at any time in back 7 days.Select the cleaning level mice of body weight 18～20g for use, with 5 mices, the encephalitis b virus vaccine of every lumbar injection 0.5ml,, injection preceding respectively at injection weighed, and observed the vaccination afterreaction at any time in back 7 days.Result of the test is listed in table 2.
The test of table 2. encephalitis b virus vaccine safety
| The vaccine kind | Animal species and average weight (g) before the injection | The observation period reaction | Inject back 7 days average body weight (g) | Conclusion |
| Diploid inactivated vaccine (90 μ g) | Cavia porcellus, 278 | All be good for and deposit no abnormal reaction | 309 | Safety |
| Diploid inactivated vaccine (90 μ g) | Mice, 20 | All be good for and deposit no abnormal reaction | 23 | Safety |
| Diploid inactivated vaccine (5 μ g) | Cavia porcellus, 295 | All be good for and deposit no abnormal reaction | 326 | Safety |
| Diploid inactivated vaccine (5 μ g) | Mice, 19 | All be good for and deposit no abnormal reaction | 22 | Safety |
The animal safety test shows do not have dangerous acute toxicity factor in the encephalitis b virus vaccine with the human diploid cell preparation, can guarantee the safety that human body uses.
Embodiment 6:
The immunogenicity test of encephalitis b virus vaccine relatively
Every kind of vaccine selects 20 of 12～14g mices, every peritoneal immunity 0.5ml, immunity twice, 7 days at interval.Just exempt from blood sampling in 15 days, 2～8 ℃ are spent the night.Next day separation of serum, 56 ℃ of deactivations in 30 minutes.In reducing with plaque and the measuring NAT.In the encephalitis B and Strain be SA
14And P
3Strain.Result of the test sees Table 3.
The test of table 3. encephalitis b virus vaccine NAT
The encephalitis b virus vaccine of NAT test card person of good sense diploid cell preparation has good immunogenicity, and the encephalitis b virus vaccine of NAT 〉=hamster kidney cell and Vero cell preparation.
Embodiment 6:
The protection potency test of encephalitis b virus vaccine relatively
Adopt immune white mice neutralizing antibody algoscopy.Neutralizing antibody is measured and is adopted plaque to reduce test.With reference to vaccine (R
AAnd R
B) and the neutralization test positive serum available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Diluted respectively 1: 32 with vaccine to be measured with reference to vaccine (R), peritoneal immunity body weight 12～14g mice is 10 respectively, every 0.5ml, immunity 2 times, 7 days at interval.Back 7 days of the 2nd immunity blood sampling, separation of serum, mice serum mixed in equal amounts on the same group, in 56 ℃ of deactivations 30 minutes, dilute serum, virus (about 200PFU/0.4ml) mixed in equal amounts good respectively with dilution.To dilute the dilution in 1: 2 again of good virus simultaneously,, put 37 ℃ of water-bath effects 90 minutes, inoculate 6 orifice plate BHK as virus control
21Cell, every hole 0.4ml cultivated 90 minutes for 37 ℃, added the culture medium covering of methylcellulose, in CO
2Cultivated 5 days in the incubator, dyeing, plaque counting calculates the plaque slip.The plaque average of virus control group should be between 50～150, and the qualification determination standard is T 〉=(R
A+ R
B)/2-0.33, result of the test sees Table 4.
Table 4. encephalitis b virus vaccine protection potency test
| The vaccine kind | Neutralization index |
| Diploid inactivated vaccine (30 μ g) | 1.63 |
| Diploid inactivated vaccine (50 μ g) | 1.75 |
| Diploid inactivated vaccine (70 μ g) | 1.96 |
| Ground Ren Mus inactivated vaccine | 1.43 |
| Vero cell inactivated vaccine | 1.59 |
Potency test shows that the encephalitis b virus vaccine of human diploid cell preparation has excellent protection and renders a service, and renders a service the neutralization index encephalitis b virus vaccine of hamster kidney cell and Vero cell preparation above Ground.
Claims (10)
1. a method of utilizing human diploid cell to prepare the encephalitis b virus vaccine is characterized in that encephalitis b virus is to cultivate in human diploid cell, stable going down to posterity during virus titer 〉=7.51g PFU/ml and in human diploid cell.
2. method according to claim 1 is characterized in that described encephalitis b virus is the SA14 strain, SA14-14-2 strain, P3 strain or Nakayama strain.
3. method according to claim 1 is characterized in that described human diploid cell is 2BS, MRC-5, KMB-17, any in WI-38 or the HL cell line.
4. according to claim 1 or the prepared encephalitis b virus vaccine of 2 or 3 described methods.
5. encephalitis b virus vaccine according to claim 4 is characterized in that, described vaccine is inactivated vaccine, attenuated live vaccine, purified vaccine, split vaccine or subunit vaccine.
6. encephalitis b virus vaccine according to claim 5 is characterized in that described vaccine is an inactivated vaccine.
7. encephalitis b virus vaccine according to claim 5 is characterized in that described vaccine is an attenuated live vaccine.
8. encephalitis b virus vaccine according to claim 5 is characterized in that described vaccine is a purified vaccine.
9. encephalitis b virus vaccine according to claim 5 is characterized in that described vaccine is a split vaccine.
10. encephalitis b virus vaccine according to claim 5 is characterized in that described vaccine is a subunit vaccine.
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| CN1299768C (en) * | 2004-08-13 | 2007-02-14 | 崔栋 | Diploid cell cerebritis B vaccine and purified cerebritis B vaccine, dosage form freeze-drying and water injection |
| CN101732707B (en) * | 2008-11-21 | 2013-02-13 | 上海荣盛生物药业有限公司 | Mercury-free Japanese encephalitis inactivated vaccine composition and application thereof |
| CN101524536B (en) * | 2009-03-26 | 2012-10-10 | 成都康华生物制品有限公司 | Japanese encephalitis vaccine prepared by human embryonic lung fibroblasts and preparation method thereof |
| CN102343087A (en) * | 2011-10-21 | 2012-02-08 | 长春祈健生物制品有限公司 | Preparation of diploid cell attenuated live vaccine from measles long-47 strain and preparation process thereof |
| CN105695424B (en) * | 2015-04-01 | 2019-06-14 | 中国食品药品检定研究院 | Adapted strain and its vaccine of the Japanese Encephalitis Vaccine,Live strain SA14-14-2 on human diploid cell 2BS |
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