Research On Expression Of HBDNF And Function Of Protein Expressed In E.coli And CHO Cells

Objective: To recombinate the gene of BDNF (Brain-derived neurotrophic factor) into both procaryon and eukaryotic expressing systems, then induce them to express and detect the function of the hBDNF protein so as to establish the base of producing recombinant hBDNF protein and gene therapy vector.Methods: To transfect the eukaryotic expression vector of the hBDNF gene: pTracer^TM-EV/V5-His- hBDNF, which is kept by our department, into CHO cells; then cleave the hBDNF gene from the plasmid of pBV220-BDNF kept by our department and recombinate it into another more high-efficience prokaryotic expression vector pET3.0a+. Recombined vectors were identified and expressed in E.coli and CHO cells. By SDS-PAGE,RT-PCR,Western-blotting,MTT assay and observing the growth of PC12 cells to detected the function of protein expressed in E.coli and CHO cells.Results: The prokaryotic vector was successfully recombinated and constructed. Both prokaryotic and eukaryotic expression vectors pET3.0a+-hBDNF and pTracer^TM-EV/V5-His-hBDNF expressed successfully in E.coli and CHO cells . The Prokaryotic expression rate is about 37.9%。The active protein was obtained after renaturation. The renaturated proteins and products expressed in CHO cells have good biological activity and antigenicity and also can promote PC12 cells'growth.Conclusion:The hBDNF has been successfully expressed in E.coli and CHO cells with good biologic activity and antigenicity. This method...