重组人白细胞介素-2痘苗病毒的构建与鉴定

Construction and Identification of Recombinant Human Interleukin-2 in Vaccinia Virus

目的：构建表达人白细胞介素－２（ｈＩＬ－２）的重组痘苗病毒，为肿瘤免疫基因治疗作必要准备。方法：应用分子克隆基因重组技术构建重组ｈＩＬ－２痘苗病毒真核表达载体ＰＭＪ６０１（ＭＪ６０１ｈｌＬ－２）。在此基础上，应用分子病毒学同源重组技术构建能表达ｈＩＬ－２的重组痘苗病毒（ＶＭＪ６０１ｈＩＬ－２）。制备ｈＩＬ－２探针，用ＤＮＡ杂交技术鉴定ＶＭＪ６０１ｈＩＬ－２，并用聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）检测ＶＭＪ６０１ｈＩＬ－２的基因产物。结果：成功构建了ＭＪ６０１ｈＩＬ－２和ＶＭＪ６０１ｈＩＬ－２，ＶＭＪ６０１ｈＩＬ－２感染的ＳＧＣ７９０１胃癌细胞能分泌具有生物活性的ｈＩＬ－２蛋白。结论：ＶＭＪ６０１ｈＩＬ－２的构建与鉴定为进一步实施体内恶性肿瘤免疫基因治疗打下了坚实的基础。

BackgroundiAims: To construct the recombinant vaccinia virus expressing human interleukin-2 (hIL-2) to form the essential prerequisite of gene therapy for cancer. Methods: Recombinant hIL-2 pMJ6Ol cukaryotic expression vector of vaccinia virus (MJ6O1hIL-2) was constructed using recombi- nant DNA technique with molecular cloning, and recombinant vaccinia virus expressing hIL-2 (VMJ 6O1hIL-2) was constructed by homologous recornbination using molecular virology. Then hIL-2 probe was prepared to detect VMJ6O1hIL.2 using DNA hybridization technique. Eventually the recombinant gene product was detected by polvacrylamide gel electrophoresis (SDS.PAGE). Results: MJ6O1hIL-2 and VMJ6O1hIL-2 were successfully constructed and hIL-2 protein with biological activity could be secreted by SGC79O1 gastric cancer cells infected by VMJ601hIL-2. Conclusions: It is one of the crucial steps in the construction and identification of VMJ6O1hJL-2 since it is the firm basis for further in vivo gene therapy in the treatment of malignant tumor.