人类肥胖相关新基因LYRM1原核表达载体的构建及其在大肠杆菌中的表达

Construction of Prokaryotic Expression Vector of LYR Motif Containing 1,A Novel Gene Related with Human Obesity,and Its Expression in Escherichia Coli

目的构建人类肥胖相关新基因LYRM1原核表达载体,并获取LYRM1重组蛋白。方法采用RT-PCR技术从人网膜脂肪组织中扩增出LYRM1基因的完整编码框,将其亚克隆到原核表达载体pGEX-6P-1,转化大肠杆菌BL21(DE3),用异丙基-β-D-硫代板乳糖苷(IPTG)诱导LYRM1融合蛋白的表达,利用变性聚丙烯酰胺凝胶电泳(SDS-PAGE)及免疫蛋白印迹(Western blot)鉴定其表达。结果PCR、酶切鉴定及测序结果表明重组质粒构建正确。LYRM1基因在原核细胞中成功表达,经SDS-PAGE鉴定融合蛋白相对分子质量约为40000,主要以不可溶的包涵体形式存在于菌体沉淀中,Western blot分析表明融合蛋白具有免疫原性。结论成功扩增和克隆了人类肥胖相关新基因LYRM1,并通过构建其原核表达载体,使其在大肠杆菌中高效表达,为进一步研究该基因的功能奠定了基础。

Objective To construct the prokaryotic expression vector of LYR motif containing 1（LYRM1）,a novel gene related with human obesity,and to obtain the recombinant protein.Methods The complete coding sequence of LYRM1 gene was amplified by reverse transcriptase-polymerase chain reaction（RT-PCR） from human omental adipose tissue and subcloned into the prokaryotic expression vector pGEX-6P-1.The recombinant plasmid was transformed into competence E.coli BL21（DE3）.After induction with isopropyl-β-D-thiogalactopyranoside（IPTG）,the expression of LYRM1 fusion protein was identified by means of sulfate polyacrylamide gelelectropheresis（SDS-PAGE） and Western blot analysis.Results The recombinant plasmid was confirmed by PCR,restriction enzyme digestion and sequencing.LYRM1 gene was successfully expressed in prokaryotic cells.The result of SDS-PAGE showed that the fusion protein with relative molecular weight of 40 000 was expressed successfully and the protein existed mainly in the deposition of bacterial in the form of undissolved inclusion body.The result of Western blot indicated the immunogenicity of the fusion protein.Conclusions LYRM1 gene was amplified and cloned successfully.The prokaryotic expression vector of LYRM1 was constructed successfully and the fusion protein was expressed highly and effectively.These results provide solid foundation for further studies on the function of LYRM1.