表达转化生长因子βⅡ型受体胞外区-活化T细胞表达和分泌的调节因子融合基因重组腺病毒的构建与鉴定  

Construction and identification of a recombinant adenovirus vector containing extracellular domain of TβⅡ-RANTES fusion gene

作　　者：王旭东[1] 刘虹[1] 曹水[1] 李慧[1] 任秀宝[1] 郝希山[1] 

机构地区：[1]天津医科大学附属肿瘤医院生物治疗中心,300060

出　　处：《中华实验外科杂志》2007年第4期423-425,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目(30300438)

摘　　要：目的构建表达转化生长因子βⅡ型受体(TβRⅡ)胞外区-活化T细胞表达和分泌的调节因子(RANTES)融合基因重组腺病毒并鉴定其在肿瘤细胞的表达。方法采用逆转录-聚合酶链式反应(RT-PCR)方法扩增小鼠TβRⅡ胞外区和RANTES基因,重叠PCR法扩增TBRⅡ胞外区-RANTES融合基因,将融合基因克隆至pDC316载体,采用AdMax Adenovirus Vector Creation试剂盒构建融合基因重组腺病毒载体,融合基因重组腺病毒按moi=100:1的比例体外感染小鼠肺腺癌L795细胞系,Western Blot方法检测感染后肿瘤细胞的蛋白表达。结果RT-PCR正确扩增了440 bp TβRⅡ胞外区、244 bp RANTES基因,重叠PCR正确扩增了745 bp TpRⅡ胞外N-RANTES融-合基因,重组质粒融合基因-pDC316经酶切鉴定正确,与pJM17双质粒共转染获得表达融合基因重组腺病毒,病毒滴度为8×10^(10)pfu/ml,感染后的LA795细胞有相对分子质量为33000的蛋白表达。结论成功构建表达TBRⅡ胞外区-RANTES融合基因重组腺病毒载体,为进行体内抗肿瘤基因治疗的研究创造条件。Objective To construct a recombinant adenovirus vector containing extracellular domain of TβRⅡ -RANTES fusion gene and identify its expression. Methods Mouse origin extracellular domain of TβRⅡ and RANTES gene were amplified by RT-PCR method; Extracellular domain of TβRⅡ- RANTES fusion gene was amplified by overlapping PCR method; Extracellular domain of TβRⅡ-RANTES fusion gene was cloned into pDC316 vector,and recombinant adenovirus vector containing the fusion gene was constructed by the AdMax Adenovirus Vector Creation System;Recombinant adenovirus was transfected into LA795 cells, and the expression of recombinant adenovirus was detected by Western blot. Results Extracellular domain of TβRⅡ and RANTES gene were amplified by RT-PCR; Extracellular domain of TβR Ⅱ -RANTES fusion gene was amplified by overlapping PCR; Recombinant adenovirus vector containing fusion gene was constructed successfully and its titer was 8 × 10^10 pfu/ml;33 000 fusion proteins was detected in LA795 cells infected with recombinant adenovirus containing fusion gene. Conclusion Recombinant adenovirus vector containing extracellular domain of TβRⅡ -RANTES fusion gene was constructed successfully, and it may be used as a novel agent for gene therapy in anti-tumor.

关 键 词：转化生长因子-Β 融合基因 重组腺病毒 

分 类 号：R73-3[医药卫生—肿瘤]