人GLIPR-2基因的克隆及在原核中可溶性表达

Cloning of human GLIPR-2 gene and its expression in Escherichia coli

目的构建pQE80L-GLIPR-2载体,并检测融合蛋白RGS·His-GLIPR-2在原核中的表达水平。方法采用RTPCR方法,将克隆人GLIPR-2(glioma pathogenesis related-2,GLIPR-2)基因全长编码区的cDNA连接到原核表达载体pQE80L中,经IPTG诱导原核表达,通过Western blot检测融合蛋白RGS·His-GLIPR-2的表达水平。超声破壁后,通过SDS-PAGE电泳分析表达产物的表达形式。结果成功构建pQE80L-GLIPR-2载体;经IPTG的诱导,GLIPR-2可与RGS·His以融合蛋白的形式高效表达。经Western blot鉴定,pQE80L-GLIPR-2在原核内表达产物相对分子质量为18000,与预期融合蛋白大小一致。对表达产物可溶性分析表明,表达蛋白在上清液中和包涵体中均有表达。结论成功构建的重组质粒能够在大肠杆菌中高效表达可溶性目的蛋白,为进一步研究GLIPR-2基因的功能和意义奠定了基础。

With the aim of setting a foundation for research of GLIPR2 gene function,we constructed pQE80L-GLIPR-2 vector and expressed the RGS·His-GLIPR-2 fusion protein in Escherichia coli DH5α.Firstly,the cDNA of GLIPR-2 was amplified from total RNA which was extracted from the peripheral blood by RT-PCR,and then the cDNA was inserted into pQE80L vector to construct pQE80L-GLIPR-2.Under the induction of IPTG,the GLIPR-2 and RGS·His expressed efficiently in the mode of RGS·His- GLIPR- 2 fusion protein and its expression level was analyzed by Western blotting.Cells were lysed by multiple rounds of sonication,and then the expression product was analyzed by using SDS-PAGE.The molecular weight of pQE80L- GLIPR-2 expressed in E.coli was 18 00,consistent with the expectation.The solubility analysis of expression product indicated that this recombinant protein could be expressed in both supernatant and inclusion bodies.All the result indicates that the constructed recombinant plasmid can express the soluble fusion protein efficiently in E.coli DH5α,which would be the first step for studying the GLIPR-2.