H5N1流感病毒疫苗株(NIBRG-14)在MDCK-G1细胞上的遗传稳定性  

Genetic stability of avian influenza H5N1 virus vaccine strain in NIBRG-14 in MDCK-G1 cells

作　　者：年悬悬 龚铮 李军英 邱冉 罗丹 孟子延 赵巍 王英 韩锡鑫 杨晓明 NIAN Xuan-xuan;GONG Zheng;LI Jun-ying;QIU Ran;LUO Dan;MENG Zi-yan;ZHAO Wei;WANG Ying;HAN Xi-xin;YANG Xiao-ming(National Institute of Engineering Technology Research in Combination Vaccines,Wuhan 430207,Hubei Province,China)

机构地区：[1]国家联合疫苗工程技术研究中心,湖北武汉430207 [2]武汉生物制品研究所有限责任公司,湖北武汉430207 [3]中国生物技术股份有限公司,北京100029

出　　处：《中国生物制品学杂志》2020年第2期121-125,130,共6页Chinese Journal of Biologicals

摘　　要：目的分析H5N1流感病毒疫苗株(NIBRG-14)在MDCK-G1细胞上的遗传稳定性。方法将H5N1种子毒株在MDCK-G1细胞上传代,收获病毒液作为P2病毒,继续传至P15,每隔5代[即P1(原代)、P5、P10、P15]进行测序。分别以含不同浓度(1、2、4μg/mL)TPCK-trypsin的培养基及不同病毒接种MOI(1、0. 1、0. 01、0. 001、0. 000 1、0. 000 01)在MDCK-G1细胞上培养P1 H5N1病毒,检测血凝滴度,计算CCID50,确定最适MOI及TPCK-trypsin浓度。将P1和P15 H5N1病毒接种鸡胚,收获尿囊液,经Sepharose 4 Fast Flow凝胶柱层析法纯化病毒后,进行电镜观察;采用培养法和指示细胞培养法(DNA染色法)检测支原体。结果 H5N1流感病毒疫苗株(NIBRG-14)在MDCKG1细胞传代过程中血凝滴度增加,从1∶128升至1∶1 024,P1、P5、P10和P15 H5N1种子病毒8条基因序列(PB1、PB2、PA、HA、NP、NA、M、NS)经DNAMAN比对结果一致。H5N1种子病毒株在MDCK-G1细胞传代的最适TPCKtrypsin浓度为4μg/mL,最适MOI为0. 001。两种方法检测P1、P15 H5N1种子病毒株均未被支原体感染。结论H5N1种子病毒株在MDCK细胞中传至P15时,病毒滴度增加但基因序列未发生改变,表明H5N1种子病毒株在MDCK-G1细胞中传代具有一定的遗传稳定性。Objective To analyze the genetic stability of influenza H5N1 virus vaccine strain NIBRG-14 in MDCK-G1 cells. Methods H5N1 virus seed was subcultured in MDCK-G1 cells,and the harvested viral liquid was served as the passage 2(P2)of virus and further subcultured to P15,and sequenced every five passages. The P1 of H5N1 virus was inoculated to the media containing various concentrations(1,2 and 4 μg/mL)of TPCK-trypsin at various MOIs(1,0. 1,0. 01,0. 001,0. 000 1 and 0. 000 01),cultured in MDCK-G1 cells and determined for hemagglutination titer,based on which the CCID50 was calculated,and the MOI and TPCKL-trypsin concentration were optimized. Chick embryo was inoculated with P1 and P15 of H5N1 virus,and the allantoic fluid was harvested,from which the virus was purified by Sepharose 4 Fast Flow gel column chromatography and observed by electron microscopy. The mycoplasma was examined by culture method in sterility test and indictor cell culture(DNA staining). Results The HA titer of influenza H5N1 virus vaccine strain NIBRG-14 increased from 1 ∶ 128 to 1 ∶ 1 024 during subculture in MDCK-G1 cells. The PB1,PB2,PA,HA,NP,NA,M and NS gene sequences of H5N1 virus of P1,P5,P10 and P15 were aligned by DNAMAN,and the results were in agreement. The optimal TPCK-trypsin concentration and MOI for subculture of H5N1 virus seed in MDCK-G1 cells were 4 μg/mL and 0. 001 respectively. The test results by two methods showed that the P1 and P15 of H5N1 virus were uninfected with mycoplasma. Conclusion After subculture in MDCK-G1 cells to P15,the titer of H5N1 virus increased,while the gene sequence showed no change,indicating a certain genetic stability of H5N1 virus seed during subculture in MDCK-G1 cells.

关 键 词：H5N1流感病毒 NIBRG-14株 疫苗 遗传稳定性 MDCK-G1细胞 

分 类 号：R183.3[医药卫生—流行病学]