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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 isolate and clone HSV-I SM_ (44) thymidine kinase gene (TK), HSV-ISM_ (44) strain of virus was cultured in primary rabbit neonatal kidney cells and the viral nucleic acid was extracted as a template. The upstream and downstream primers of HSV-ITK gene were designed. The BamHI and EcoR I restriction enzyme sites were introduced into the 5 ’end of the primer respectively. The TK gene was amplified by PCR and digested with BamHI / EcoRI and ligated with PUC19 The recombinant plasmid was transformed into E. coli JM109. The positive clones of HSV-ISM_ (44) TK gene were obtained by screening and restriction enzyme digestion. The full-length HSV-ISM_ (44) 99.02%, there is a difference of 11 nucleotides and 7 amino acids. The successful cloning of HSV-ITK gene laid the foundation for suicide gene therapy of malignant tumors.