甲肝减毒活疫苗L-A-1疫苗株的核酸序列测定与比较分析  

Genomic sequence of hepatitis A virus L-A-1 vaccine strain

作　　者：姜春来[1] 王鹏富[2] 刘景晔[2] 张华远[3] 万宗举[3] 

机构地区：[1]吉林大学生命科学学院,长春130062 [2]长春生物制品研究所疫苗二室 [3]中国药品生物制品检定所疫苗二室

出　　处：《中华实验和临床病毒学杂志》2004年第4期360-362,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的　测定甲型肝炎 (甲肝 )减毒活疫苗龙甲 1株 (L A 1)核酸序列 ,了解其减毒和适应二倍体细胞的分子机制 ,并与其他甲肝病毒株进行比较 ,得出L A 1株的一些特点。方法　通过抗原捕获聚合酶链反应 (AC PCR)扩增甲肝病毒减毒活疫苗L A 1株 2 3代的片段 ,产物纯化后克隆至T载体并进行序列测定 ,应用生物信息学软件分析比较。结果　得到了甲肝减毒活疫苗L A 1株的基因序列 (nt 2 5～ 74 18)。L A 1株开放读码框架 (ORF)长 6 6 75个核苷酸 ,编码 2 2 2 5个氨基酸。比较分析表明 ,该株与国际代表株MBB株和HM 175野毒株在核苷酸水平上同源性分别为 98.0 0 %和 94 .0 0 % ,在氨基酸水平上同源性分别为 98 5 1%和 98 6 5 %。通过与其他细胞适应株、细胞病变株以及减毒株的比较得出 :L A 1株 5′非编码区 ( 5′NTR)nt 15 2、5 91、6 4 6、6 87处的突变和 180～ 181处插入 11个核苷酸以及 2B区的nt3889(aa 10 5 2 Val)处的突变是病毒适应宿主细胞的分子基础 ;2C区的nt 4 185 (aa115 2 Lys)处的突变可能参与病毒的减毒过程 ;由于 3A区缺失 6个核苷酸 (nt 5 0 2 0～ 5 0 2 5 )从而导致两个氨基酸 (Asn、Asp)缺失是病毒适应细胞快速增殖的分子基础。结论　分析了L A 1株的基因序列 。Objective To study the genome sequence of hepatitis A virus L-A-1 strain which has been applied for live attenuated vaccine production in China, to compare with other HAV strains, to understand some characteristics of L-A-1 strain, and to find the mechanism of attenuation and cell adaptation. Methods Genome fragments were prepared by antigen-capture PCR from infected cell (2BS), PCR products were cloned into T vector, sequenced and analyzed by using bioinformatics program. Results Analysis of the genomic sequences (nt 25- 7 418 ) showed that the open reading frame contains 6 675 nucleotides in length encoding 2 225 amino acids. Sequence homology comparison showed 98.00% and 94.00% homology at nucleotide level, and 98.51% and 98.65% homology at amino acid level with international strains MBB and HM 175, respectively. Through comparison with other attenuated, cell adapted and cytopathic effect (CPE) strains, L-A-1 strain had mutation at nt 152,591,646,687 and insertion at nt 180-181 in 5′NTR and had mutation at nt 3 889 (aa 1 052-Val) in 2B region, these mutations and insertion are molecular basis for cell adaptation; mutation at nt 4 185(aa 1 152-Lys) in 2C region should be attenuated marker; deletion in 3A region (nt 5 020-5 025) that caused two amino acids deletion is virus fast growth basis. Conclusion Through analyzing L-A-1 strain genomic sequence, certain sites related to cell adaptation and attenuation were found.

关 键 词：甲肝减毒活疫苗 核苷酸 甲肝病毒 细胞 疫苗株 适应 核酸序列 产物纯化 同源性 氨基酸 

分 类 号：R346[医药卫生—基础医学]