凋亡抑制蛋白Livin两种异构体原核表达载体的构建及融合表达  

Construction of Prokaryotic Expression Vectors for Livin Alpha and Livin Beta

作　　者：邹爱民[1] 沈建军[1] 高萍[1] 林芳[1] 张惠中[1] 

机构地区：[1]第四军医大学唐都医院中心实验室,西安710038

出　　处：《肿瘤防治研究》2007年第6期409-411,448,共4页Cancer Research on Prevention and Treatment

摘　　要：目的构建凋亡抑制蛋白Livin两种异构体(Livinα和β)的原核细胞表达载体pET32a(+)-livinα和pET32a(+)-livinβ。方法设计合成扩增Livin基因异构体全长cDNA序列的特异性PCR引物,以Hela细胞总RNA为模板,RT-PCR获得Livin基因异构体全长cDNA序列,用限制性内切酶BamHⅠ和HandⅢ双酶切取所需目的片段,并插入原核表达载体pET32a(+)的多克隆位点,经酶切、PCR鉴定,并经过测序证实后,构建成为Livin基因异构体表达载体(pET32a(+)-livinα、β)。将重组质粒转入表达菌株BL21,诱导表达收集菌液,超声碎菌,取其上清和沉淀分别进行SDS-PAGE电泳。结果成功获得Livinα和β全长cDNA序列,并克隆到原核表达载体pET32a(+)上;成功获得大小为55kd左右的融合蛋白。结论Livin基因两种异构体原核表达载体的构建及其融合蛋白的制备,为进一步研究Livin异构体功能及在肿瘤细胞中的抗凋亡效应奠定了基础。此蛋白也可用于进一步抗体制备、免疫鉴定和诊断等研究。Objective To construct prokaryotic expression vectors for Livinα and Livinβ and to obtained the fusion protein of pET32a（ ＋ ）-livina and pET32a（ ＋ ）-livinβ. Methods Total RNA of Hela cell was extracted. The full-length cDNA of Livin isoforms was gained by RT-PCR. Then inserted into pET32a（ ＋ ） vector and constructed the recombinant plasmids pET32a （ ＋ ）-livinα/DH5a and pET32a （ ＋ ）/livinβ/ DHfα, sequencing was performed to guarantee correct sequence insertion for the isoforms Livinα and Livinβ Reconstructed the recombinant plasmids pET32a （ ＋ ）-livinα/ BL21 and pET32a （ ＋ ）/livinβ/ BL21, the pET32a（ ＋ ）-livinα/BL21 and pET32a（ ＋ ）-livinβ/BL21 plasmids was induced after 4.5 hours by IPTG, and the value of A600nm was 0. 6 in LB medium. Expression of the pET32a（ ＋ ）-livinα/BL21 and pET32a（ ＋ ）-livinβ/BL21 were analyzed by SDS-PAGE. Results Full-length cDNA of Livinα and Livinβ was cloned respectively and subcloned into pET32a （ ＋ ） successfully. Fusion protein of positive recombinants were gained by pET32a（ ＋ ） prokaryotic expression system. Conclusion The construction of pro- karyotic expression vector for Livinα and Livinβ and validation of expression in cells provide basis for further research on functions of Livinα and Livinβ and their anti-apoptosis effect in cancer cell.

关 键 词：凋亡抑制蛋白 LIVIN 凋亡 原核表达 

分 类 号：Q781[生物学—分子生物学]