单纯疱疹病毒I型SM_(44)株胸苷激酶基因的克隆及序列测定

Cloning and sequencing of the thymidine kinase gene of herpes simplex virus type I strain SM_(44)

为了分离、克隆单纯疱疹病毒Ｉ型（ＨＳＶ－Ｉ） ＳＭ_(４４)株胸苷激酶基因（ＴＫ），采用原代兔婴肾细胞培养ＨＳＶ－ＩＳＭ_(４４) 株病毒，提取病毒核酸作为模板，设计了 ＨＳＶ－ＩＴＫ基因的上、下游引物，在引物的 ５’端分别引入 ＢａｍＨＩ和 ＥｃｏＲ Ｉ 限制性内切酶位点，通过ＰＣＲ方法扩增出 ＴＫ基因，经ＢａｍＨＩ／ＥｃｏＲＩ双酶切与 ＰＵＣ１９质粒载体相连接，构建重组 体转化受体菌ＪＭ１０９，通过筛选和酶切鉴定，获得ＨＳＶ－ＩＳＭ_(４４) ＴＫ基因阳性克隆，行全序列测定分析，与ＨＳＶ－ＩＣＬ１０１ 株 ＴＫ基因同源性为 ９９．０２％，存在 １１个核苷酸和 ７个氨基酸的差异。 ＨＳＶ－ＩＴＫ基因的克隆成功，为该基因应用于恶 性肿瘤的自杀基因治疗奠定了基础。

In order to isolating and cloning the HSV-I TK gene, HSV-I strain SM_44 virus were proliferated by culturing primary rabbit fetal kidney cells, and the virus nucleic acid was extracted as template. The 5'terminals of the primers to amplify TK gene were incorporated restriction endonuclease sites of BamH I and EcoR I. The PCR products of TK gene fragment were inserted into PUC 19 plasmid vector at corresponding sites, then transformed into E.coli JM109.By restriction enzyme cleavage characterization, the recombined clone of TK gene was obtained and named PTKs. The nucleotide sequences were deterimined. Comparing with the TK gene of HSV-I strain CL101, HSV-I SM_44 TK gene have homology of 99.2% for nucleotide sequence, and 98. 12% for amino acid sequence. The clone of HSV-I SM_44 TK gene might contribute to using in the suicide gene therapy of cancer.