甲型肝炎病毒L-A-1株在人胚肺二倍体细胞2BS株上的遗传稳定性  

Genetic stability of hepatitis A virus L-A-1 strain in 2BS cells

作　　者：徐艳玲 李昊堃 王艺博 夏青娟 岳立广 刘令九 XU Yan-ling;LI Hao-kun;WANG Yi-bo;XIA Qing-juan;YUE Li-guang;LIU Ling-jiu(Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2021年第8期898-903,共6页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(201603007YY,20190404001YY)。

摘　　要：目的探讨甲型肝炎病毒(hepatitis A virus,HAV)L-A-1株在人胚肺二倍体细胞2BS株(简称2BS细胞)上的遗传稳定性。方法将HAV L-A-1株在2BS细胞上连续传代培养至32代,取23、24、26、28和32代次病毒,检测抗原含量和病毒滴度。RT-PCR扩增各代病毒HAV1~HAV7基因序列片段,采用DNAMAN软件进行拼接,得到全基因组核苷酸序列,分析不同代次病毒基因组核苷酸和氨基酸同源性;并与GenBank中登录的国内外主要HAV毒株进行同源性分析,同时比对VP1和VP3区氨基酸序列。各代次病毒液经腹腔免疫ICR小鼠,0.5 m L/只,于免疫后28 d,经心脏采血,分离血清,ELISA法检测血清抗体效价。结果HAV L-A-1株23、24、26、28和32代次病毒抗原含量在10240~20480 EU/mL之间,病毒滴度为7.00~7.50 lgCCID50/mL。不同代次HAV L-A-1株病毒全基因组核苷酸和氨基酸序列同源性均为100%;与国内外主要HAV毒株的核苷酸序列同源性为91.40%~99.70%,氨基酸序列同源性为98.20%~99.69%,与L-A-1参考株(AF314208)核苷酸和氨基酸序列同源性最高,分别为99.70%和99.69%;与国内外主要HAV毒株VP1区有5处氨基酸不一致,VP3区有2处氨基酸不一致,与L-A-1参考株(AF314208)和H2减毒株(H2K25株)VP1和VP3区氨基酸序列完全一致。不同代次病毒液免疫ICR小鼠后阳转率均为100%,血清抗体效价在684.69~997.84 mIU/mL之间,各代病毒间差异无统计学意义(P>0.05)。结论HAV L-A-1株在2BS细胞上连续传代后,具有良好的遗传稳定性。Objective To investigate the genetic stability of hepatitis A viru(HAV)L-A-1 strain in 2 BS cells.Methods HAV L-A-1 strain was subcultured in 2 BS cells for passages,from which the samples of passages 23,24,26,28 and 32 were taken and determined for antigen content and virus titer.The HAV1~HAV7 gene sequences of each passage were amplified by RT-PCR and spliced by using DNAMAN software to obtain the nucleotide sequence of complete genome.The homologies of nucleotide and amino acid sequences of various passages as well as the homologies to the major virus strains at home and abroad in GenBank were analyzed,and the amino acid sequences of VP1 and VP3 regions were compared.ICR mice were immunized i.p.with HAV of various passages,0.5 mL for each,of which the serum samples were collected 28 d after immunization and determined for antibody titer by ELISA.Results The antigen contents of L-A-1 strain of passages 23,24,26,28 and 32 were 10240~20480 EU/mL,while the virus titers were7.00~7.50 lgCCID50/mL.All the homologies of nucleotide and amino acid sequence of L-A-1 strain of various passages were 100%.The homologies of nucleotide and amino acid sequences to those of the major virus strains at home and abroad were 91.40%~99.70%and 98.20%~99.69%respectively.However,L-A-1 strain showed the highest homologies of nucleotide and amino acid sequences to those of reference strain(AF314208),which were 99.70%and99.69%respectively.There were variations of five amino acids in VP1 region and two in VP3 region as compared with those of major virus strains at home and abroad,while the amino acid sequences in the two regions of L-A-1 strain were completely consistent with those of the reference strain(AF314208)and attenuated H2 K25 strain.All the antibody positive conversion rates of ICR mice immunized with the virus of various passages were 100%,while the serum antibody concentrations were 684.69~997.84 mIU/mL,which showed no significant difference(P>0.05)between the passages.Conclusion HAV L-A-1 strain showed high genetic stability a

关 键 词：甲型肝炎病毒 L-A-1株 人胚肺二倍体细胞2BS株 遗传稳定性 

分 类 号：R373.21[医药卫生—病原生物学]