突变N-ras基因重组痘苗病毒rV-N-ras/61的构建

目的：构建Ｎ－ｒａｓ基因第６１位密码子突变基因（Ｎ－ｒａｓ／６１）的重组痘苗病毒，通过痘苗病毒载体所产生的蛋白的强烈免疫原性来提高变异Ｎ－ｒａｓ蛋白有肿瘤抗原性。方法：利用双ＰＣＲ方法得到第６１位突变的Ｎ－ｒａｓ／６１基因片段（５００ｂｐ），将该基因生段插入痘苗病毒表达载体Ｐ１１０８，通过Ｃｅｌｌ ＦＥＣＴＩＮ转染ＣＶ－１细胞，以ＰＣＲ，Ｗｅｓｔｅｒｎ ｂｌｏｔ方法鉴定重组痘苗病毒。结果：通过ｔｋ＾－筛选、ＰＣＲ鉴定，得了第６１位突变Ｎ－ｒａｓ／６１基因的重组痘苗病毒ｒＶ－Ｎ－ｒａｓ／６１，Ｗｅｓｔｅｒｎ ｂｌｏｔ检测其在ＣＶ－１细胞中有表达。结论：目前对突变ｒａｓ基因的研究多着重于机理方面，本研究对研制有效的突变ｒａｓ基因疫苗是一个有益的尝试，其免疫治疗作用有待于进一步研究。

Construction of Mutant N-ras/61 Gene Recombinant Vaccinia Virus

Objective: To construct the recombinant vaccinia virus of mutant N ras/61 gene and enhance the immunogenecity of mutant N ras/61 protein produced by the recombinant vaccinia virus. Methods: N ras/61 gene was inserted into P1108 and transfected into CV 1 cell infected with vaccinia virus by Cell FECTIN. PCR and Western blot were used to identify the recombinants. Results: We get recombinant vaccinia virus rV N ras/61 by PCR and tk - selecting. The expression of N ras gene was detected by Western blot. Conclusion: This study is a test for studing effective vaccine of mutant N ras/61 gene.The efficacy in vivo of the N ras/61 vaccine in immunotherapy is under investgation.