GOLPH2的原核表达、纯化、抗体制备和初步应用

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DHSa,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.%Recombinant expression of G0LPH2 and preparation of anti-G0LPH2 polyclonal antibody that may be a serologic marker of hepatocellular carcinoma(HCC) and clinical significance of the feasibility of the basis, golphl 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-jS-D-galactoside (IPTG). The recombinant protein G0LPH2 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 ELJSA respectively. The prokaryotic expression plasmid pET21a (+)-TRX-G0LPH2 was successfully constructed. The recombinant protein TRX-G0LPH2 could be expressed in abundance in the soluble form and got the purified purpose 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 protein, 73 kD cell lysates and serum protein specifically by Western blot analysis. The polyclonal antibody a-gainst G0LPH2 protein was successfully prepared, and can be used for follow-up examination.