凋亡抑制蛋白Livin两种异构体真核表达载体的构建及表达鉴定

Construction of eukaryotic expression vectors for Livin α and β

目的构建凋亡抑制蛋白Livin两种异构体(Livinα和β)真核细胞表达载体pcDNA3.1-Livinα和pcDNA3.1-Livinβ。方法利用分子克隆技术,首先设计合成扩增Livin基因异构体全长cDNA序列的特异性PCR引物,以肺腺癌SPC-A1细胞总RNA为模板,RT-PCR获得Livin基因异构体全长cDNA序列,TA克隆将Livin全长cDNA序列克隆到T载体上,用限制性内切酶HindⅢ和XbaⅠ双酶切取所需目的片段,然后将该片段插入真核表达载体pcDNA3.1的多克隆位点,最后对产生的重组子进行双酶切、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(pcDNA3.1-Livin)。电穿孔法获得稳定表达Livinα和β的A549细胞克隆,Westernblot验证Livin蛋白在转染细胞中的表达情况。结果成功获得Livinα和Livinβ全长cDNA序列,并克隆到真核表达载体pcDNA3.1上;基因转染后,阳性细胞分别表达Livinα和β。结论Livin基因两种异构体真核表达载体的构建及在细胞中的表达鉴定,为进一步研究Livin异构体功能及在肺癌细胞中的抗凋亡效应奠定了基础。

Objective To construct eukaryotic expression vectors for Livin α＆β, two isoforms of a novel inhibitor of apoptosis protein family member. Methods Total RNA of SPC-A1 was extracted. The full-length cDNA of Livin isoforms was gained by RT-PCR, then inserted into T vector by T-A cloning. Positive recombinants were gained. Sequencing was performed to guarantee correct sequence insertion. The inserted sequence was subcloned into pcDNA3.1 to get expression vectors for the isoforms Livin α＆β. A549 cell line was transfected with pcDNA3.1-Livin α＆ pcDNA3.1-Livin βrespectively by electroporation. The positive clones were gained by G418-resistance screening. Western blotting was performed to assess the expression of Livin in transfected cells. Results Full-length cDNA of Livin α＆β was cloned respectively and subcloned into pcDNA3.1 successfully. After gene transfection, Livin α＆β were expressed respectively in positive cells. Conclusion The construction of eukaryotic expression vector for Livin α＆β and validation of expression in cells provide basis for further research on functions of Livin α＆β and their anti-apoptosis effect in lung cancer cell.