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

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23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(nt25-7418)。L-A-1株开放读码框架(ORF)长6675个核苷酸，编码2225个氨基酸。比较分析表明，该株与国际代表株MBB株和HM175野毒株在核苷酸水平上同源性分别为98．00％和94．00％，在氨基酸水平上同源性分别为98．51％和98．65％。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-152、591、646、687处的突变和180-181处插入11个核苷酸以及2B区的m3889(aa1052-Val)处的突变是病毒适应宿主细胞的分子基础；2C区的m4185(aa1152-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(nt5020-5025)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LA-1株的基因序列。得出其适应细胞和减毒的核苷酸位点。

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

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.