Expression, Identification, Purification And Polyclonal Antibody Preparation Of Human EGFL6

Objectives: The recent research showed that EGFL6was closely related to tumor genesisand development because of its angiogenesis promotion and over expression in most tumortissues and cells. In this study, the full-length EGFL6gene and EGFL6gene fragment werecloned and expressed in E.coli. The recombinant EGFL6fragment protein was purified andanti-EGFL6polyclonal antibody was prepared for detecting EGFL6in tumor cell lines, whichwill make foundation for EGFL6function study.Methods:(1).The sub cloning and prokaryotic expression of human full-length EGFL6:The full-length EGFL6gene was cloned by PCR amplification using pre-conserved EGFL6vector plasmid as a template, and then was constructed into pMDTM18-T followed byidentification with DNA sequencing and double enzyme digestion. The correct EGFL6gene wasthen constructed into expression vector pET32a (+) to express the full-length EGFL6proteinwith IPTG inducing. The EGFL6expression was analyzed by the SDS-PAGE and Western blot.(2)The detection of human EGFL6gene expression in cell lines: Real-time fluorescentquantitative PCR was used to detect EGFL6gene expression in6tumor cell strains and2normalcell strains.(3). The cloning of human EGFL6gene from tumor cells and expression in E.coli:The EGFL6gene fragment was cloned from the tumor cell with the high EGFL6expression, andconstructed in Prokaryotic expression vector pET32a (+) through gene recombination technology.The recombinant EGFL6was expressed with IPTG inducing, and the protein expression wereanalyzed by SDS-PAGE and Western blot.(4) The polyclonal antibody preparation and detectionof human EGFL6: The soluble EGFL6protein fragment was purified with Ni-NTA affinitychromatography. Kunming mices were vaccinated with the purified recombinant EGFL6and theanti-EGFL6polyclonal antibodies were prepared. The sensitivity and specificity of polyclonalantibody were analyzed by ELISA and Western blot. The antibody was purified through theCaprylic acid and ammonium sulfate precipitation for the further Western blot analysis in tumorcell lysates.Results:(1) The sub cloning and prokaryotic expression of human full-length EGFL6: Theresults of DNA sequencing and double enzyme digestion showed that the EGFL6full-lengthprotein expression vector pET32a (+)-EGFL6was constructed successfully. SDS-PAGE andWestern blot results showed that EGFL6whole protein was successfully expressed inRosetta-gami B stain of E. coli. The recombinant EGFL6was expressed mainly in inclusion body form, which was more than90%in the whole EGFL6protein.(2) The detection of humanEGFL6gene expressed in different cell strains: qRT-PCR detection results showed that EGFL6was highly expressed in tumor cell strains of A375and SKOV3.(3) The cloning of humanEGFL6gene from tumor cells and expression in E.coli: RT-PCR results showed that the EGFL6gene fragments were cloned successfully from A375cell strain, and the EGFL6expressionvector pET32a (+)-EGFL6was constructed successfully. SDS-PAGE and Western blot resultsshowed that the soluble EGFL6target protein was expressed at the size of38kD.(4) Thepolyclonal antibody preparation and detection of human EGFL6: Anti-EGFL6polyclonalantibody was prepared in Kunming mice and the Western blot analysis results showed that thepolyclonal antibody could recognize not only the recombinant full-length EGFL6or proteinfragment but also the nature EGFL6in cell strains. The western-blot results with polyclonalantibody showed that the EGFL6was highly expressed in A375and SKOV3and lowlyexpressed in HUVEC, which was corresponding to the qRT-PCR results.Conclusion: In this study, A375and SKOV3cell strains were selected to be the tumor cellstrains with high expression of EGFL6in8cell strains, and EGFL6gene fragment was clonedfrom A375cell. Full-length EGFL6and EGFL6protein fragment could be both expressed athigh level in E.coli. The specific polyclonal antibodies against EGFL6could be obtained bymice vaccination and used to identify and analyze EGFL6expression in cell lines, which can beused for further function studies of EGFL6.