Vero细胞培养流感病毒的低血清培养基的筛选
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摘要
目的筛选Vero细胞生长的最适低血清培养基,用于流感病毒的细胞培养。方法分别以MEM、M199、DMEM/F12、DMEM/F12与MEM混合物(1∶1)为基础培养基,添加5%、2%、1%的小牛血清传代培养Vero细胞,通过细胞形态观察、细胞计数及MTT法检测细胞的增殖活力筛选最适基础培养基;将流感病毒接种低血清适应的Vero细胞,检测病毒收获液的血凝效价及残留BSA含量,电子显微镜观察病毒形态。结果以DMEM/F12与MEM混合物(1∶1)为基础培养基,添加2%小牛血清培养的Vero细胞长势良好,细胞数量较多,增殖活力最强。低血清适应的Vero细胞培养的流感病毒血凝效价最高可达1∶512,平均值为1∶384;病毒液中的残留BSA含量约为正常血清培养条件下的1/18;病毒形态完整。结论成功筛选出Vero细胞培养流感病毒的低血清培养基,为更具生物安全性的流感疫苗的研发奠定了基础。
Objective To screen the low serum medium for culture of Vero cells and use for culture of influenza virus. Methods MEM,M199,DMEM / F12 and mixture of DMEM / F12 and MEM(1 ∶ 1)were used as basal media,in which 5%,2% and 1% calf sera were added separately,and Vero cells were cultured. The cultured cells were observed for morphology,counted and determined for proliferative activity,based on which the optimal basal medium was screened. Influenza virus was inoculated into the Vero cells adapted in low serum medium,and the HA titer and residual BSA content in harvest were determined,while the virus morphology was observed by electron microscopy. Results The mixture of DMEM / F12 and MEM(1 ∶ 1) was used as a basal medium and added with 2% calf serum,in which Vero cells grew well in a large quantity and showed high proliferative activity. The HA titer of influenza virus cultured in low serum-adapted Vero cells reached 1 ∶ 512 at most,with a mean of 1 ∶ 384. The residual BSA content in virus liquid was about 1 / 18 of that cultured in normal serum medium. The virus particles in low serum medium were intact,of which the morphology showed no significant difference with those in normal serum medium. Conclusion Low serum medium for culture of influenza virus in Vero cells was screened,which laid a foundation of development of influenza vaccine with higher biosafety.
Objective To screen the low serum medium for culture of Vero cells and use for culture of influenza virus. Methods MEM,M199,DMEM / F12 and mixture of DMEM / F12 and MEM(1 ∶ 1)were used as basal media,in which 5%,2% and 1% calf sera were added separately,and Vero cells were cultured. The cultured cells were observed for morphology,counted and determined for proliferative activity,based on which the optimal basal medium was screened. Influenza virus was inoculated into the Vero cells adapted in low serum medium,and the HA titer and residual BSA content in harvest were determined,while the virus morphology was observed by electron microscopy. Results The mixture of DMEM / F12 and MEM(1 ∶ 1) was used as a basal medium and added with 2% calf serum,in which Vero cells grew well in a large quantity and showed high proliferative activity. The HA titer of influenza virus cultured in low serum-adapted Vero cells reached 1 ∶ 512 at most,with a mean of 1 ∶ 384. The residual BSA content in virus liquid was about 1 / 18 of that cultured in normal serum medium. The virus particles in low serum medium were intact,of which the morphology showed no significant difference with those in normal serum medium. Conclusion Low serum medium for culture of influenza virus in Vero cells was screened,which laid a foundation of development of influenza vaccine with higher biosafety.
引文
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[13]Gu M,Shao GB,Gu Q,et al.Study on culture of Vero cells with low concentration calf serum[J].Chin J Biologicals,1995,8(1):28-30.(in Chinese)顾鸣,邵盖斌,顾勤,等.低浓度牛血清培养Vero细胞的研究[J].中国生物制品学杂志,1995,8(1):28-30.
[14]Tang J,Ma L,Zhou FY,et al.Study on the heat stability of ha antigen in influenza virus rapid diagnosis kit[J].J Anhui Agri Sci,2012,40(16):8935-8936.(in Chinese)唐静,马磊,周芳烨,等.流感病毒快速诊断试剂中HA抗原热稳定性的研究[J].安徽农业学报,2012,40(16):8935-8936.
[15]Lu Y,Song SH,Cai W,et al.Detection of a Vero cell-adapted H3N2 influenza virus strain by ELISA method[J].Prog Modern Biomed,2013,13(10):1811-1815.(in Chinese)卢勇,宋绍辉,蔡玮,等.ELISA法检测H3N2亚型流感病毒Vero细胞适应株[J].现代生物医学进展,2013,13(10):1811-1815.
[16]Peschel B,Frentzel S,Laske T,et al.Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line[J].Vaccine,2013,31(48):5693-5699.
[2]WHO.Human infection with influenza A(H7N9)virus in China[J].N Engl J Med,2013,368:1888-1897.
[3]Zeng XX,Li KS.The pandemic history of influenza:review and revelation of the influenza pandemic in last century[J].Med Soci,2010,23(11):4-6.(in Chinese)曾祥兴,李康生.流感百年:20世纪流感大流行的回顾与启示[J].医学与社会,2010,23(11):4-6.
[4]Mendelman PM,Cordova J,Cho I.Safety,efficacy and effectiveness of the influenza virus vaccine,trivalent,types A and B,live,cold-adapted(CAIV-T)in healthy children and healthy adults[J].Vaccine,2001,19(17-19):2221-2226.
[5]Nerome K,Kumihashi H,Nerome R,et al.Evaluation of immune responses to inactivated influenza vaccines prepared in embryonated chicken eggs and MDCK cells in a mouse model[J].Dev Biol Stand,1999,98(1):53-63,73-74.
[6]Wu JD,Zhu FC.Advances in influence vaccine[J].Jiangsu J Prev Med,2008,19(2):83-85.(in Chinese)吴俊东,朱凤才.流感疫苗研究的进展[J].江苏预防医学,2008,19(2):83-85.
[7]Kistner O,Barrett PN,Mundt W,et al.Development of a mammalian cell(Vero)derived candidate influenza virus vaccine[J].Vaccine,1998,16(9-10):960-968.
[8]Tree JA,Richardson C,Fooks AR,et al.Comparison of largescale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains[J].Vaccine,2001,19(25-26):3444-3450.
[9]Barrett PN,Mundt W,Kistner O,et al.Vero cell platform in vaccine production:moving towards cell culture-based viral vaccines[J].Expert Rev Vaccines,2009,8(5):607-618.
[10]Liu Z,Sun MB,Gao JX,et al.Optimization of condition for culture of influenza virus in Vero cells in low serum medium[J].Chin J Biologicals,2009,22(6):593-595.(in Chinese)刘铮,孙明波,高菁霞,等.低血清培养Vero细胞和流感病毒条件的优化[J].中国生物制品学杂志,2009,22(6):593-595.
[11]Luo ZW,Qiu F,Wei XL,et al.Adaptability of influenza virus in Vero cells[J].Chin J Biologicals,2008,21(4):304-307.(in Chinese)罗振武,邱丰,魏晓露,等.流感病毒在Vero细胞上的适应性[J].中国生物制品学杂志,2008,21(4):304-307.
[12]Job ER,Bottazzi B,Gilbertson B,et al.Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses[J].PLoS One,2013,8(3):e59623.
[13]Gu M,Shao GB,Gu Q,et al.Study on culture of Vero cells with low concentration calf serum[J].Chin J Biologicals,1995,8(1):28-30.(in Chinese)顾鸣,邵盖斌,顾勤,等.低浓度牛血清培养Vero细胞的研究[J].中国生物制品学杂志,1995,8(1):28-30.
[14]Tang J,Ma L,Zhou FY,et al.Study on the heat stability of ha antigen in influenza virus rapid diagnosis kit[J].J Anhui Agri Sci,2012,40(16):8935-8936.(in Chinese)唐静,马磊,周芳烨,等.流感病毒快速诊断试剂中HA抗原热稳定性的研究[J].安徽农业学报,2012,40(16):8935-8936.
[15]Lu Y,Song SH,Cai W,et al.Detection of a Vero cell-adapted H3N2 influenza virus strain by ELISA method[J].Prog Modern Biomed,2013,13(10):1811-1815.(in Chinese)卢勇,宋绍辉,蔡玮,等.ELISA法检测H3N2亚型流感病毒Vero细胞适应株[J].现代生物医学进展,2013,13(10):1811-1815.
[16]Peschel B,Frentzel S,Laske T,et al.Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line[J].Vaccine,2013,31(48):5693-5699.