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目的 :构建N ras基因第 6 1位密码子突变基因 (N ras/ 6 1)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蛋白的肿瘤抗原性。方法 :利用双PCR方法得到第 6 1位突变的N ras/ 6 1基因片段 (5 0 0bp) ,将该基因片段插入痘苗病毒表达载体P110 8,通过CellFECTIN转染CV 1细胞 ,以PCR ,Westernblot方法鉴定重组痘苗病毒。结果 :通过tk-筛选、PCR鉴定 ,得了第 6 1位突变N ras/ 6 1基因的重组痘苗病毒rV N ras/ 6 1,Westernblot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。
OBJECTIVE: To construct a recombinant vaccinia virus with codon 16 (N ras / 6 1) of N ras gene and to enhance the tumor antigenicity of mutant N ras protein by the strong immunogenicity of the protein produced by vaccinia virus vector. Methods: The N ras / 6 1 gene fragment (500kb) was amplified by double PCR. The gene fragment was inserted into the vaccinia virus expression vector P1108 and transfected into CV1 cells by CellFECTIN. PCR, Western blot Methods Identification of recombinant vaccinia virus. Results: The recombinant vaccinia virus rV N ras / 6 1 with mutant N ras / 6 1 gene was obtained by tk-screening and PCR. The expression of rV N ras / 6 1 was detected by Western blot in CV 1 cells. CONCLUSION: At present, the research on mutant ras gene mostly focuses on the mechanism. This study is a beneficial attempt to develop an effective mutant ras gene vaccine, and its immunotherapy needs further study.