【摘要】 目前发现GOLPH2蛋白与肝癌密切相关,将golph2基因进行克隆、表达并制备多克隆抗体,为进一步研究其功能奠定基础.应用RT-PCR技术,从人肝癌细胞系HepG2细胞中扩增得到golph2 cDNA,将其克隆到原核表达载体pET21a(+)-TRX内、转化大肠杆菌DH5a,用IPTG诱导其在大肠杆菌BL21(DE3)中表达.His-tag磁珠纯化试剂盒纯化重组蛋白GOLPH2,SDS-PAGE鉴定.将纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,采用ELISA、Western blot方法检测抗体的灵敏度和特异性,并测定临床血清标本中GOLPH2蛋白水平.成功地构建了表达TRX-GOLPH2融合蛋白的原核表达质粒pET21a(+)-TRX-GOLPH2.并在大肠杆菌BL21(DE3)内得以高效表达,且以可溶性的形式存在.SDS-PAGE和Western blot证实,重组GOLPH2蛋白质与预期结果一致.抗血清能够特异地识别52 kD重组蛋白、73 kD细胞裂解液和血清蛋白质.成功制备了GOLPH2蛋白质和多克隆抗体,能用于后续研究.更多还原

【Abstract】 Recombinant expression of GOLPH2 and preparation of anti-GOLPH2 polyclonal antibody that may be a serologic marker of hepatocellular carcinoma(HCC) and clinical significance of the feasibility of the basis.golph2 was amplified from HepG2 cells and cloned into the prokaryotic expression vector pET21a(+)-TRX,then expressed in E.coli BL21(DE3) which was induced by isopropylthio-β-D-galactoside(IPTG).The recombinant protein GOLPH2 was purified by His-tag magnetic bead purification kit and detected by SDS-PAGE.Polyclonal antibodies was developed by immunizing BALB/c mice with purified recombinant protein,The specificity and titer of the antibody in anti-sera were determined by Western blot and ELISA respectively.The prokaryotic expression plasmid pET21a(+)-TRX-GOLPH2 was successfully constructed.The recombi-nant protein TRX-GOLPH2 could be expressed in abundance in the soluble form and got the purified pur-pose protein of 52 kD.SDS-PAGE and Western blot analysis showed that GOLPH2 protein was successfully expressed in BL21(DE3).It was showed that antiserum can specifically identify the 52 kD recombinant pro-tein,73 kD cell lysates and serum protein specifically by Western blot analysis.The polyclonal antibody a-gainst GOLPH2 protein was successfully prepared,and can be used for follow-up examination.更多还原