【摘要】 目的:新近研究发现EGFL7蛋白与肝癌密切相关。本研究对基因egfl7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。方法:通过RT-PCR方法,从人肝癌细胞系HepG2细胞中扩增egfl7目的片段,利用基因重组技术构建pET21a(+)-TRX-EGFL7原核表达载体,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EGFL7,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EGFL7蛋白水平。结果:成功地构建了原核表达质粒pET21a(+)-TRX-EG-FL7。并在大肠杆菌BL21(DE3)内得以高效表达,且以包涵体的形式存在。SDS-PAGE和Western blot证实,重组EGFL7蛋白质与预期结果一致,抗血清能够特异地识别重组蛋白和血清蛋白质。结论:成功制备了EGFL7蛋白质和多克隆抗体,能用于后续研究。更多还原

【Abstract】 Objective:Recombinant expression of EGFL7 and preparation of anti-EGFL7 polyclonal antibody that may be a serologic marker of hepatocellular carcinoma(HCC) and for further study on the basis of its function.Methods:egfl7 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 EGFL7 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.Results:The prokaryotic expression plasmid pET21a(+)-TRX-EGFL7 was successfully constructed.The recombinant protein TRX-EGFL7 could be expressed in abundance in the form of inclusion bodies.We got the purified purpose protein of 52kDa.SDS-PAGE and Western blot analysis showed that EGFL7 protein was successfully expressed in BL21(DE3).It was showed that antiserum can specifically identify the recombinant protein and serum protein specifically by Western blot analysis.Conclusion:The polyclonal antibody against EGFL7 protein was successfully prepared,and can be used for follow-up study.更多还原