Recombinant expression,polyclonal antibody preparation and preliminary application of human EGFL7

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.