单纯疱疹病毒I型Stocker株gD膜外区基因的克隆与序列测定

Cloning and sequencing of the gD gene of herpes simplex virus type I strain Stocker

目的 :为了克隆单纯疱疹病毒I型 (HSV -I) gD膜外区基因。方法 :采用Vero细胞培养HSV -IStocker株 ,并提取病毒DNA作为PCR模板。PCR扩增出的gD片段经EcoRI和PstI双酶切 ,插入质粒pBV220 相应位点 ,转化E.coliDH5α。重组质粒经PCR、酶切鉴定命名为pBV220-gD。对克隆的HSV -IStocker株gD基因进行序列分析 ,并与其他HSV -I、Ⅱ gD基因相应部分进行比较。结果 :核苷酸序列同源性分别为99.77 %、86.55 % ,氨基酸序列同源性分别为99.31 %、88.28%。结论 :HSV -IgD基因的克隆为表达该基因 ,进一步研制重组抗原诊断试剂、研究亚单位疫苗及gD、gD受体的结构功能奠定了基础。

Objective: To clone HSV-I gD ectodomain gene HSV-I strain Stocker. Methods:HSV-I strain Stocker was proliferated in Vero cells (Africa green monkey kidney cells) and the virus nucleic acid was extracted as template of PCR. After the restriction endonucleases EcoRI and PstI were dealt with, the PCR products of gD gene fragment were inserted into pBV220 plasmid vectors at corresponding sites then transferred into E.coli DH5α. By PCR and restriction enzyme analysis the recombinant clone of gD gene was obtained and named pBV220-gD. The nucleotide sequences of gD gene were determined by sequencing. Results:Comparing the clone with other HSV-I strains and HSV-Ⅱ we verified that they have homology of 99.77%, 86.55% respectively for nucleotide sequence and 99.31%, 88.28% for amino acid sequence. Conclusion:The clone of HSV-I gD gene might contribute to the expression of gD gene, preparation of recombinant gD as diagnostic antigen and subunit vaccine as well as be a meams of study for the structure and function of gD or gD receptor.