甲型肝炎病毒SYX1株在人二倍体细胞MRC-5上的适应性及遗传稳定性  

Adaptability and genetic stability of hepatitis A virus SYX1 strain in human diploid cell MRC-5

作　　者：李芳芳 廖辉 杨文腰 周荔葆 LI Fang-fang;LIAO Hui;YANG Wen-yao;ZHOU Li-bao(Liaoning Chengda Biotechnology Co.,Ltd.,Shenyang 110179,Liaoning Province,China)

机构地区：[1]辽宁成大生物股份有限公司,辽宁沈阳110179

出　　处：《中国生物制品学杂志》2023年第1期43-47,共5页Chinese Journal of Biologicals

基　　金：辽宁省科学技术计划项目(2014226004)。

摘　　要：目的探讨甲型肝炎病毒(hepatitis A virus,HAV)SYX1株在人二倍体细胞MRC-5上的适应性及遗传稳定性。方法将来源于甲肝患者粪便的HAV SYX1株接种至MRC-5细胞,连续传代至第28代,检测第1~26代病毒的抗原含量和病毒滴度;镜下观察第6代病毒的形态,并检测其理化性质。取第13~15代病毒进行病毒增殖动态研究,确定病毒增殖高峰期。选择第8、12、18、20、22、25、26和28代病毒,提取病毒基因组RNA进行序列测定,分析其遗传稳定性。建立HAV SYX1株主种子批和工作种子批,按照《中国药典》三部(2020版)要求进行检定。结果HAV SYX1株在MRC-5细胞上连续传至8代后,抗原含量稳定在160~320 EU/mL之间,病毒滴度维持在7.3~8.3 lgCCID_(50)/mL;病毒颗粒有空心和实心两种类型,直径为27~32 nm,呈球形,无包膜,表面无突起;可耐受低pH及乙醚。确定病毒增殖高峰期为10 d,抗原含量达160 EU/mL以上,病毒滴度达7.0 lgCCID_(50)/mL以上。HAV SYX1株为HAV IB亚型,传代过程中所有结构蛋白编码区基因序列未发生突变。HAV的主种子批和工作种子批的各项检定结果均符合相关要求。结论HAV SYX1株在MRC-5上传代具有良好的适应性及遗传稳定性,可用于甲肝灭活疫苗的研究及生产。Objective To investigate the adaptability and genetic stability of hepatitis A virus(HAV)SYX1strain in human diploid cell MRC-5.Methods HAV SYX1strain isolated from feces of patients with hepatitis A was continuously propagated in MRC-5 cells for 28 passages,of which the 1st~26th passages were determined for antigen contents and virus titers,the6th passage was observed for the morphology under microscope and detected for physicochemical properties,and the 13th~15th passages were studied for virus proliferation dynamics to determine the peak yield of virus proliferation.Genomic RNA was extracted from the 8th,12th,18th,20th,22nd,25th,26th and 28th passages and sequenced to analyze the genetic stability.The main seed batch and working seed batch of HAV SYX1strain were established and verified according to the requirements of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).Results The antigen content of HAV SYX1was stable at 160~320 EU/mL and the titer was maintained at 7.3~8.3 lgCCID_(50)/mL after the 8th passages in MRC 5 cells;Virus particles showed two types:hollow and solid,with a diameter of 27~32 nm,spherical,without envelope and protrusions on the surface,which tolerated low pH value and ether.The peak period of virus proliferation was 10 d with an antigen content of more than 160 EU/mL and a virus titer of more than 7.0 lgCCID_(50)/mL.HAV SYX1was a subtype of HAV IB,and no mutation in the coding region of all structural proteins during passage was observed.The verification results of main seed batch and working seed batch of HAV all met the relevant requirements.Conclusion HAV SYX1strain showed good adaptability and genetic stability in MRC-5,which might be used for the development and production of inactivated hepatitis A vaccine.

关 键 词：甲型肝炎病毒SYX1株 人二倍体细胞MRC-5 适应性 遗传稳定性 

分 类 号：R392-33[医药卫生—免疫学]