Cloning, Expression And Purification Of The CDNA For The Human Chorionic Gonadotropin Subunit α (HCGα)

Objective To clone human chorionic gonadotropin subunit a (HCG a ) cDNA, construct the expression vector, express and purify its product, prepared for further study of the actions of HCGa in tumors and abnormal pregnancies and for screening the antibody for HCGa by phage display techinology later. Methods The cDNA of HCG a was obtained by using RT-PCR method with total RNA extracted from the fresh human chorial tissue. Then it was clone into express ion vector PGEX4T-1. After proved to be correct by sequencing, the expression plasmid PGEX4T-1/HCGa was transformed into Escherichia coli (E. coli) BL21.The fusion protein GST-HCG was produced by IPTG induction. The fusion protein would be induced into supernatant by changing the growing conditions of E. coli BL21. After centrifuged the destroyed bacteria, the fusion protein in the supernatant was isolated by affirnity chromatography glutathione Sepharose 4B, the inclusion body was denatured, dialyzed and renatured. Then the products were analysed by SDS-PAGE electrophoresis and Western blot.Results Electrophoresis in agarose gels indicated that the amplified product by RT-PCR was about 280bp as expected.The results of SDS-PAGE electrophoresis and Western blot demonstrated that the IPTG induced bacteria expressed an extra band of 36KD in comparison with the uninduced ones and the expressed protein in the plasma is unsoluble mainly. The inclusion body decreased by changing the growing conditions of E. coli BL21.Conclusion The cDNA of HCG a is cloned successfully. The GST-HCG a is highly expressed in E. coli BL21 and the recombinant protein has a high special immunological activity.