SLC-Her-2eu-P53-Fc融合基因重组腺病毒表达载体的构建及检测

摘要

Objective To construct a recombinant adenovirus expression vector with fused gene of SLC, Her-2eu, P53 and Fc fragment. Method The fused gene SLC-Her-2eu-P53 was inserted into the plasmid of adenovirus shuttle vector pShuttle-CMV-sig-Fc. The shuttle plasmid pShuttle-CMV-SLC-HP-Fc and the backbone plasmid pAdEasy-1 were recombinated homologously in E.coli BJ5183 cells by the modified 2-step transfection method. Then fusion gene eukaryotic expression vector AdEasyTM-SLC-HP-Fc were constructed. After amplified in E.coli DH5a, the purified recombinant plasmid was transfected into 293 cells by Lipofectamine 2000 to obtain recombinant adenovirus AdEasyTM-SLC-HP-Fc. PCR was used to identify the expression of fusion gene SLC-HP in 293 cells in-fected with AdEasyTM-SLC-HP-Fc. Result The fusion gene SLC-Her-2eu-P53 was successfully cloned to the shut-tle plasmid pShuttle-CMV-sig-Fc. Restriction analysis and the PCR conformed that correct recombinant adenoviral plasmid was constructed, and expression of fusion gene SLC-Her-2eu-P53 could be detected in the 293 cells infect-ed with AdEasyTM-SLC-HP-Fc. Conclusion The success of construction, amplification and purification of the adenovi-ral vector furnishes the rationale and experimental evidence for further study on the anti-tumor function of SLC-Her-2eu-P53-Fc.%目的：构建携带融合基因SLC-Her-2eu-P53-Fc（SLC-HP-Fc）的重组腺病毒AdEasyTM-SLC-HP-Fc表达载体。方法提取SLC-HP基因及含Fc段基因的质粒构建携带目的基因的腺病毒穿梭载体pShuttle-CMV-SLC-HP-Fc ，经酶切线性化后转入含有pAdEasy-1质粒的E.coli BJ5183中进行同源重组，得到重组腺病毒真核表达载体AdEasyTM-SLC-HP-Fc。线性化后的病毒表达载体通过脂质体Lipofectamine 2000转染293细胞，通过包装扩增并经PCR鉴定后得到大量复制病毒并纯化保存。结果得到重组腺病毒AdEasyTM-SLC-HP-Fc ，成功转染293细胞出现细胞病变反应，经酶切及PCR法确定目的基因的表达。结论成功构建了SLC-Her-2eu-P53-Fc融合基因重组腺病毒表达载体，并建立了重组腺病毒AdEasyTM-SLC-HP-Fc种子病毒库和工作病毒库，为进一步研究该融合基因与肿瘤相关作用奠定了实验基础。