甲型肝炎减毒活疫苗(H2株)快速繁殖株不同代次全基因序列比较

Sequencing and Comparison of Two-passage Genomes of Fast-growth Isolates of Attenuated Hepatitis A Virus(H2 Strain) in KMB17 Cells

为探索甲型肝炎减毒活疫苗 (H2株 )细胞适应的分子机制 ,将甲型肝炎减毒活疫苗毒株(HAVH2K7)在人胚肺二倍体细胞KMB17上快速连续系列传代增强适应 ,繁殖周期由原来的 2 8d缩短为 14d ,连续传代后抗原滴度和感染性滴度不断增加 ,传至 2 2代抗原滴度达 1∶10 2 4 ,感染性滴度lgCCID50 (每ml)为 7 83 .分别将第 6代和第 2 2代病毒用AC PCR法和PCR法扩增 .扩增片段分别与pGEM T载体连接得到重组质粒 ,测定cDNA插入片段的序列 .对 2个不同代次全基因序列及氨基酸序列比较分析表明 ,HAVH2K7适应至第 6代时 ,整个基因组有 6个核苷酸变异 ,全部位于编码区内 ,变异率为 0 0 7% ,导致 3个氨基酸变化 ,分别位于VP2 (A S) ;2C(N H) ;3A(R C) .适应至第 2 2代时 ,整个基因组出现 18个核苷酸突变 ,变异率为 0 2 4 % ,13个是该代次产生的 ,变异最大区域 5′端非编码区 (5′UTR)有 5个核苷酸变异 .编码区突变导致 7个氨基酸变化 ,其中 4个氨基酸变化是该代次在 6代基础上特有的变异 (2C ,Q P ;3A ,A S ;3C ,T A ;3D ,V G) .2C区是编码区变异最多区域 ,共有 4个核苷酸突变 ,在 6代变异基础上出现 3个新突变 ,导致 1个氨基酸变异 .说明 5′UTR的2C区变异对病毒的翻译效率、感染性滴度提高具有重要作用 .

The complete genomes of two passage isolates of live attenuated hepatitis A virus(HAV,H2 Strain) in KMB17 cells were sequenced and compared in order to study the mechanism of cellular adaptation of HAV.During 22 passage adaptation ,the replicating cycle was shortened from 28 days to 14 days,antigenic and infectious titer increased with increasing passages.At passage 22 antigenic titer and infectious titer (lgCCID 50 per ml) reached 1∶1024 and 7.83 ,respectively.The sequence alignment of passages 6 (HAVH2K7P6) and 22 (HAVH2K7P22) with original passage (HAVH2K7) showed that mutations of genomes increased with increasing passages,and most of the mutations were concentrated in 5′ untranslated region (5′UTR) and 2C coding region.HAVH2K7P6 showed only difference of 0.07% with HAVH2K7,and only six nucleotide mutations in the whole genome,all in open reading frame (ORF),resulting in the three amino acids exchanges,A to S exchange in structural protein VP2;N to H exchange in nonstructural protein 2C;R to C exchange in nonstructural protein 3A.However,there were eighteen nucleotide mutations in the genome of HAVH2K7P22,five mutations in 5′UTR,thirteen in ORF,resulting in seven amino acids exchanges,four mutations were new in addition to three common mutations with HAVH2K7P6,one Q to P exchange in 2C protein;A to S in 3A;T to A in 3C;V to G in 3D. In addition to 5′UTR,the 2C coding region was more mutating region with four nucleotide mutations,resulting in one amino acid exchange.The analysis showed that 5′UTR and 2C coding region play an important role in adaptation of HAVH2K7 in KMB17 cells.