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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 expressing the expression and secretion of extracellular domain - activated T cells of transforming growth factor β receptor (TβR Ⅱ) and to identify its expression in tumor cells. Methods The extracellular domain of TβRⅡ and RANTES gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The fusion gene of TBRⅡ extracellular domain-RANTES was amplified by overlap PCR. The fusion gene was cloned into pDC316 vector, The recombinant adenovirus vector was constructed by AdMax Adenovirus Vector Creation kit. The murine adenocarcinoma L795 cell line was infected with the fusion gene recombinant adenovirus at a ratio of moi = 100: 1. The protein expression of the tumor cells was detected by Western Blot . Results The 440 bp TβRⅡ extracellular domain and 244 bp RANTES gene were correctly amplified by RT-PCR. The 745 bp TpRⅡ fusion gene was amplified by overlap PCR, and the recombinant plasmid fusion gene-pDC316 was identified by restriction enzyme digestion And the recombinant adenovirus expressing the fusion gene was co-transfected with the pJM17 double plasmid. The virus titer was 8 × 10 10 pfu / ml, and the relative molecular mass of the infected LA795 cells was 33,000. Conclusion The recombinant adenovirus vector expressing the extracellular domain of RANTES fusion gene of TBRⅡ was successfully constructed, which could create conditions for the study of anti-tumor gene therapy in vivo.