Expression And Analysis Of Biological Activity Of PTD-hBDNF

Objective To clone the PTD-hBDNF fusion gene , express it in E.coli, purify the target protein and study biological activity of PTD-hBDNF. Methods hBDNF gene was amplified by PCR from the genomic DNA and inserted into pUC19 and identified by sequencing. The recombinant plasmid was used as the template for PCR amplification of PTD-hBDNF fusion gene with a 5' primer containing PTD coding frame, then the PTD-hBDNF fusion gene was inserted into pUCm-T for sequencing. Then, the PTD-hBDNF fusion gene was cloned into temperature-inducing expression vector pJW2 and the recombinant plasmid was transformed into E. coli DH-5α. The PTD-hBDNF was mainly expressed as inclusion bodies. The extracted inclusion bodies was denatured and refolded , and then the refolded protein was purified by ion-exchange chromatography. The biological activity of the recombinant protein was tested by adding it to hippocampal cells method. Results The molecular weigh of PTD-hBDNF was confirmed as 15 kD by SDS-PAGE assay and the immunogenic assay was identified by Western-blot, and recombinant protein could be specifically recognized by monoclonal antibody against human BDNF. Bioassay in vitro showed that the proliferation of the central nerve cell was induced by purified PTD-hBDNF. Conclusion The pJW2-PTD-hBDNF vector was constructed. PTD-hBDNF was successfully expressed in E.coli and purified. The biological activity of purified PTD-hBDNF was good.