鼠源Bif-1蛋白7种选择性剪接异构体真核表达质粒的构建及鉴定

Construction and identification of eukaryotic expression vectors for seven alternatively spliced isoforms of mus musculus Bax-interacting factor-1

目的构建鼠源Bif-1(Bax-interacting factor-1)蛋白7种选择性剪接异构体(alternatively spliced isoforms)真核表达质粒,并在BHK和N2a细胞中表达。方法以提取的N2a细胞总RNA为模板,采用RT-PCR、重叠延伸PCR等方法扩增目的基因Bif-1(transcript variant 1、transcript variant 2、transcript variant 3、transcript variant x1、transcript variant x2、transcript variant x3、e),并将目的基因连入真核表达载体p IRES2-EGFP,构建重组质粒。重组质粒经鉴定正确后经脂质体介导转染BHK和N2a细胞,采用荧光显微镜观察及Western blot法检测转染细胞中Bif-1蛋白的表达。结果琼脂糖凝胶电泳可见与预期大小相符的核酸条带;重组质粒经PCR、双酶切、测序鉴定证明构建正确;重组质粒转染BHK或N2a细胞后荧光显微镜下可见绿色荧光;Westen blot可检出相应大小的目的条带。结论成功构建了鼠源Bif-1蛋白7种选择性剪接异构体真核表达质粒,并在BHK和N2a细胞中获得表达。为进一步探索Bif-1蛋白不同选择性剪接异构体的生物学功能,研究其在细胞自噬、细胞凋亡以及抗肿瘤等方面的作用奠定了基础。

Objective To construct the eukaryotic expression vectors for seven alternatively spliced isoforms of mus musculus Bax-interacting factor-1（Bif-1） and express in BHK and N2 a cells. Methods Target genes Bif-1（transcript variant 1）, Bif-1（transcript variant 2）, Bif-1（transcript variant 3）, Bif-1（transcript variant x1）, Bif-1（transcript variant x2）, Bif-1（transcript variant x3） and Bif-1（e） were amplified by RT-PCR and SOE PCR using the total RNA extracted from N2 a cells as templates, and inserted into eukaryotic expression vector p IRES2-EGFP. BHK cells and N2 a cells were transfected with the constructed recombinant plasmids in mediation of liposomes, and determined for expression of Bif-1by fluorescent microscopy and Western blot. Results Nucleic acid bands at the expected lengths were observed on agarose gel electrophoretic profile. Recombinant plasmids were constructed correctly as proved by PCR, restriction analysis and sequencing. Green fluorescence was observed in BHK and N2 a cells after transfection with the recombinant plasmids by fluorescent microscopy, while the target bands with the corresponding relative molecular masses were observed by Western blot. Conclusion Eukaryotic expression vectors for seven alternatively spliced isoforms of mus musculus Bif-1were constructed correctly, and expressed successfully in BHK and N2 a cells, which laid a foundation of further study on biological function of different alternatively spliced isoforms of Bif-1 as well as their roles in autophagy, apoptosis and antitumor therapy.