重组单纯疱疹病毒-胸苷激酶和人白介素-2基因痘苗病毒真核表达载体的构建

Construction and identification of recombinant HSV-tk and human interleukin-2 gene vaccinia expression vector pMJ601

目的 构建含单纯疱疹病毒 胸苷激酶 (HSV tk)和人白介素 2 (hIL 2 )基因的痘苗病毒真核表达载体 pMJ6 0 1,为进一步实施胃癌的基因治疗作必要的准备。 方法 利用目的基因与载体的连接、感受态细胞的制备及转化、质粒抽提、琼脂糖凝胶电泳、酶切、DNA序列分析等多种基因工程技术 ,将纯化回收的HSV tk和hIL 2分别与 pMJ6 0 1进行连接、转化并鉴定。 结果 分别用BamHⅠ HindⅢ和SalⅠ BamHⅠ酶切位点 ,将HSV tk与hIL 2DNA成功地克隆到pMJ6 0 1真核表达载体上。结论 重组HSV tk +hIL 2基因 pMJ6 0 1的构建 ,为进一步研究胃癌的基因治疗打下坚实的基础。

Objective To construct vaccinia expression vector pMJ601, which contains HSV-tk and human interleukin-2(hIL-2) genes fo r gene therapy of gastric cancer. Methods Genetic engineering tech niques such as ligation, preparation of competent cells and transformation, plas m id extraction, agarose gel electrophoresis, restriction analysis, DNA sequence analysis and so on were used to make HSV-tk, and hIL-2 genes cloned to pMJ 601 in the BamHⅠ-HindⅢ and SalⅠ-BamHⅠ multiple cloning site and then identified. Results hIL-2 DNA fragments from pLXSN and HSV-tk from X-pP N T with restriction enzymes digest were apparently shown by agarose gel electrop horesis. In addition, HSV-tk and hIL-2 DNA were successfully cloned to pMJ601. Conclusion It is one of the crucial steps to construct pMJ601 coexpressing HSV-tk and hIL-2 gene as it is the firm basi s of gene therapy for gastric cancer.