家蚕膜蛋白基因(BmTmC27)的序列分析及组织特异性和原核表达

Sequence Analysis,Tissue Specific and Prokaryotic Expression of a Transmembrane Protein Gene(BmTmC27) from Silkworm,Bombyx mori

从家蚕蛹cDNA文库中筛选获得一条家蚕膜蛋白基因序列,通过编码氨基酸序列的同源性比对表明其可能是家蚕的未知功能序列,命名为BmTmC27(GenBank登录号:DY231073.1)。生物信息学分析结果表明该基因全长833 bp,由558 bp的开放阅读框、102 bp的5'端非翻译区和173 bp的3'端非翻译区组成,编码蛋白由185个氨基酸组成且含有4个跨膜区,预测蛋白分子质量为20.94 kD,等电点为7.38。将该基因片段与BmNPV的polh基因连接后克隆到原核表达载体pGEX-4T-1中,构建原核融合表达载体pGEX-4T-1-polh-BmTmC27,经IPTG诱导、SDS-PAGE分析表明目的蛋白以包涵体形式在E.coli BL21(DE3)中表达。利用多角体蛋白可溶于碱性环境的性质,采用调节pH值的方法纯化融合表达的重组蛋白GST-Polh-BmT-mC27,然后经TEV酶消化获得BmTmC27蛋白。荧光定量PCR分析BmTmC27在家蚕5龄幼虫不同组织中的表达存在明显差异,其中在脂肪体、中肠和气管中的转录水平较高。利用GST沉降技术研究了BmTmC27的互作蛋白,与对照组相比,发现了4条特异性互作蛋白条带。研究结果有助于进一步探究BmTmC27的生物学功能,也为膜蛋白的表达和纯化提供了一种新方法。

The sequence of a membrane protein gene was obtained from silkworm pupa cDNA library.Sequence alignment of amino acids encoded by this gene indicated that it was a nucleotide sequence of unknown function from Bombyx mori,and was named BmTmC27（GenBank accession No.DY231073.1）.Bioinformatics analysis showed that the full length of BmTmC27 was 833 bp,containing a 558 bp open reading frame,102 bp 5′ untranslated region and 170 bp 3′ untranslated region.BmTmC27 encoded a 185 amino acid protein with 4 transmembrane domains,a predicted molecular weight of 20.94 kD and an isoelectric point of 7.38.In this study,BmTmC27 gene fragment was ligated with polh gene from BmNPV,and then cloned into the prokaryotic expression vector pGEX-4T-1 to construct recombinant expression vector pGEX-4T-1-polh-BmTmC27.SDS-PAGE analysis showed that the target protein was expressed in the form of inclusion body after induced by IPTG in E.coli BL21（DE3）.Because polyhedra are soluble in the alkaline environment,recombinant fusion protein GST-Polh-BmTmC27 was purified by adjusting pH value of the solution,and then BmTmC27 was obtained by digestion with TEV protease.Fluorescent quantitative PCR analysis revealed that transcriptional levels of BmTmC27 in various tissues of the 5th instar larva were obviously different,and were higher in fat body,midgut and trachea.GST pull-down technique was used to investigate the proteins that interacted with BmTmC27,and 4 specific protein bands had been found compared with the control group.These results laid a foundation for further study of BmTmC27 biological functions,and provided a new method for expression and purification of membrane proteins.