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从杭州、兰州两地各一例乙型肝炎病毒 (HBV)表面抗原阳性血清中提取病毒DNA ,采取PCR技术扩增出前表面抗原 (preS)基因片段 ,重组到质粒载体上 ,对该基因进行了全序列测定 [GenBank索取号CpreS HZ :AF 32 5 6 74;preS LZ :AF 32 5 6 75 ]。克隆的HBVpreS基因杭州分离物 (preS HZ)和兰州分离物 (preS LZ)全长 5 2 2个核苷酸 ,编码174个氨基酸。preS HZ与已发表的HBVadr亚型上海分离物[8] 、北京分离物[9] 、日本分离物[5] 、HBVadw亚型[10 ] 和ayw亚型[11] preS基因的核苷酸序列同源性分别为 96 .7%、96 .2 %、97.3%、88.7%和 84.1% ,氨基酸序列同源性分别为 96 .0 %、94.9%、97.1%、85 .1%和 85 .4% ;preS LZ与相应序列的核苷酸序列同源性分别为 96 .4%、96 .2 %、96 .9%、88.7%、83 .7% ,氨基酸序列同源性分别为 94.9%、94.9%、96 .0 %、85 .1%、84.1%。分子进化分析 (DNASTAR ,1999)表明 ,克隆的两例HBVpreS基因属于adr亚型。preS LZ与preS HZ之间有两个核苷酸变异 (对应两个氨基酸变异 ) ,相对于以上报道的序列二者含有四个特异的氨基酸突变位点。在免疫保护区内二者具有较好的保守性 ,可用于表达乙肝重组亚单位疫苗。
The virus DNA was extracted from one case of hepatitis B virus (HBV) surface antigen-positive sera from Hangzhou and Lanzhou, and the preS gene fragment was amplified by PCR and recombined into the plasmid vector. Sequencing [GenBank Accession No. CpreS HZ: AF 32 5 6 74; preS LZ: AF 32 5 6 75]. The cloned HBVpreS gene Hangzhou isolate (preS HZ) and Lanzhou isolate (preS LZ) were 522 nucleotides in length and encoded 174 amino acids. PreS HZ is associated with the nucleotide sequence of the preS gene of the HBVadr subtype Shanghai isolate [8], Beijing isolate [9], Japanese isolate [5], HBVadw subtype [10] and ayw subtype [11] The homologies were 96.7%, 96.2%, 97.3%, 88.7% and 84.1%, respectively. The amino acid homologies were 96.0%, 94.9%, 97.1%, 85.1% and 85.4, respectively %; The homologies of the nucleotide sequence of preS LZ with the corresponding sequences were 96.4%, 96.2%, 96.9%, 88.7% and 83.7%, respectively, and the amino acid sequence homologies were 94.9% 94.9%, 96.0%, 85.1%, 84.1%. Molecular evolution analysis (DNASTAR, 1999) showed that two of the cloned HBV preS genes belong to the adr subtype. There are two nucleotide variations (corresponding to two amino acid variations) between preS LZ and preS HZ, which contain four specific amino acid mutation sites relative to the sequence reported above. Both in the immune protection zone has a good conservative, can be used to express hepatitis B recombinant subunit vaccine.