摘要
目的分析柯萨奇病毒A组10型毒株在人胚肺二倍体细胞(KMB17)上增殖的遗传稳定性。方法将V6-19/XY/CHN/2017毒株适应于KMB17细胞,并连续传代培养至15代。分别提取适应前及适应后第5、10、15代病毒RNA,分段对全基因组序列进行RT-PCR扩增、测序及拼接,利用Maga 7.0、Geneious 6.1.4等软件进行系统进化及同源性分析。结果命名该KMB17细胞适应株为K6-19/XY/CHN/2017,各子代病毒感染性滴度为7.25~7.75 lgCCID_(50)/mL。适应前后4个子代病毒的全基因核苷酸和氨基酸序列同源性分别为99.99%~100%和99.95%~100%。适应前与适应后第5、10代毒株均无核苷酸和氨基酸序列突变,第15代毒株VP4区域发生1个核苷酸突变(VP4172 A→G)及1个氨基酸突变(VP458 Ile→Val)。结论CV-A10能较好地适应KMB17细胞,K6-19/XY/CHN/2017在KMB17细胞上适应后可保持较高的病毒滴度和遗传稳定性,为后续疫苗研发奠定了实验基础。
Objective To analyze the genetic stability of a Coxsackie virus A10 strain in human embryonic lung diploid cells(KMB17).Methods V6-19/XY/CHN/2017 strain was adapted to KMB17 cells and subcultured consecutively to passage 15.Genomic RNAs of the strain before adaption and that of passages 5,10 and 15 were extracted,with which the complete genome sequences were amplified by RT-PCR,sequenced and spliced,and analysed for phyletic evolution and homology by using Maga 7.0 and Geneious 6.1.4 software.Results The KMB17 cell adapted strain was named as K6-19/XY/CHN/2017,of which the infectious titers of various passages were 7.25~7.75 lgCCID_(50)/mL.The homologies of nucleotide and amino acid sequences of four passages before and after adaption were 99.99%~100%and 99.95%~100%respectively.No mutations of nucleotide and amino acid sequences were observed in the strain before adaption and that of passages 5 and 10,while one nucleotide mutation(VP4172 A→G)and an amino acid mutation(VP458 Ile→Val)were observed in VP4 regin of strain of passage 15.Conclusion CV-A10 was adapted well to KMB17 cells,and adapted strain K6-19/XY/CHN/2017 showed high virus titer and genetic stability,which laid an experimental foundation of subsequent vaccine development.
作者
许丹菡
张捷
丛山日
张名
冯昌增
黄小琴
孙浩
杨昭庆
马绍辉
XU Dan-han;ZHANG Jie;CONG Shan-ri;ZHANG Ming;FENG Chang-zeng;HUANG Xao-qin;SUN Hao;YANG Zhao-qing;MA Shao-hui(Key Laboratory for Research and Development of Major Infectious Diseases Vaccine in Yunnan,Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第7期813-817,共5页
Chinese Journal of Biologicals
基金
云南省科技计划项目(2017FA006)。
