重组人禽流感H7N9疫苗株在Vero细胞上的适应性和传代稳定性研究  

A Study on the Adaptability and Stability of Recombinant Human Avian Influenza H7N9 Vaccine Strains on Vero Cells

作　　者：崔兆海 赵卫忠 马磊[1] 高菁霞[1] 朱文勇[1] 李卫东[1] 廖国阳[1] CUI Zhaohai;ZHAO Weizhong;MA Lei;GAO Jingxia;ZHU Wenyong;LI Weidong;LIAO Guoyang(Institute of Medical Biology,Peking Union Medical College,Chinese Academy of Medical Sciences,Kunming 650018,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,昆明650018

出　　处：《激光生物学报》2020年第3期268-273,共6页Acta Laser Biology Sinica

基　　金：中国医学科学院医学与健康科技创新工程经费资助项目(2016-I2M-3-026);云南省应用基础研究计划项目(2017FB039);国家自然科学基金青年科学基金项目(31500011)。

摘　　要：本文主要研究重组人感染高致病性禽流感H7N9株作为主工作种子批在Vero细胞连续传代中的高产特性和传代稳定性,为疫苗开发提供前期研究。将重组获得的H7N9流感病毒Vero细胞适应株(A/Anhui/1/2013Va)按照病毒感染复数(MOI)0.01的病毒量接种于Vero细胞,并连续传15代。血凝试验检测每代病毒血凝效价,并绘制病毒在Vero细胞上的生长曲线;每代病毒用MDCK细胞检测病毒的感染性滴度;通过单向免疫扩散试验对重组病毒H7N9的第1及第16代进行型别鉴定;取第1、5及16代病毒,用鸡胚半数感染量及致死量检测病毒在传代过程中毒力变化;分别提取第1和16代H7N9流感病毒的RNA,反转录及克隆后测序,对比病毒在连续传代过程中血凝素(HA)和神经氨酸酶(NA)基因的变化。试验结果表明:在连续传代过程中,病毒效价越来越趋于稳定,血凝效价稳定维持在384~448;病毒感染性滴度维持在7 Log10TCID50/mL左右;病毒在传代过程中毒力未发生显著变化;HA和NA在连续传代过程中并未出现因基因变异导致的编码氨基酸改变的状况。H7N9安徽株病毒在Vero细胞传代过程中不仅产量稳定,其毒力和基因也很稳定,可以将其作为H7N9流感疫苗候选株。This paper mainly studies the high-yield characteristics and passage stability of recombinant human infection with highly pathogenic avian influenza H7N9 strain as the main working seed batch in the continuous passage of Vero cells,providing preliminary research for vaccine development.The H7N9 influenza virus Vero cell adaptation group(A/Anhui/1/2013Va)was inoculated into Vero cells at MOI=0.01,and passaged for 15 consecutive generations.Hemagglutination test was used to detect the hemagglutination titer of each generation of virus was and conducted the virus growth curve on Vero cells was drawn;MDCK cells are used for each generation of virus to detect the infectious titer of the virus;type identification of recombinant viruses H7N9 passage 1 and passage 16 was done by single radial immunodiffusion;take the 1st,5th and 16th generation viruses were taken,and half the amount of infection and lethality of chicken embryos to detect the virulence of the virus during the passage;RNA from H7N9 influenza viruses of the 1st and 16th generation were extracted,reverse-transcribed and sequenced after cloning to compare changes in hemagglutinin(HA)and neuraminidase(NA)genes of the virus during successive passages.The test results showed that the virus titer became more and more stable during successive passages,and the titer remained stable at 384~448;the infectious titer of the virus was maintained at 7 Log10TCID 50/mL;virus virulence did not change significantly during the passaging process;HA and NA did not show changes in the encoded amino acids due to gene mutations during continuous passaging.The H7N9 Anhui strain virus has not only a stable yield during the passage of Vero cells,but also its virulence and genes are stable.It can be used as a candidate strain of H7N9 influenza vaccine.

关 键 词：H7N9 Vero细胞适应株 病毒滴度 生长曲线 HA及NA序列 

分 类 号：R373.13[医药卫生—病原生物学]