Research On Expression Of HBDNF And Function Of Protein Expressed In E.coli And COS-7 Cells

objective:To clone and express hBDNF gene and study the function of protein expressed in E.coli and COS-7 cells to lay a foundation for producing recombinat hBDNF protein and gene therapy vector.Methods: The cDNA fragment was amplified from genomic DNA of human white blood cells by polymerase chain reaction .It was cloned into pUCm-T vector and sequenced.Then the identified insert fragment from pUCm-T-BDNF was directedly ligated with linearized pBV220 vector and pTracer?EV/V5-His vector with the compatible termini. Recombinat vectors were identified and expressed in E.coli and COS-7 cells.By using of ELISA, Dot-ELISA, MTT and observing the growth of PC 12 cells to detected the function of protein expressed in E.coli and COS-7 cells.Results: The sequence of cloned DNA fragment was compatiable with that reported by GeneBank, Prokaryotic and eukaryotic expression vectors pBV220-hBDNF and pTracer?EV/V5-His-hBDNF was constructed correctly and expressed successfully in E.coli and COS-7 cells . The Prokaryotic expression rate is about 21%0 The activity proteine was obtained after renaturation. The renaturated protein and products expressed inCOS-7cells have good biological activity and antigenicity.Conclusion: The target gene is obtained and successfully expressed in E.coli and COS-7 cells and has good function.This method provides an approach for producing recombant hBDNF protein and gene therapy vector.