Cloning And Expression Of Human Brain-Derived Neurotrophic Factor (hBDNF)

Human brain-derived neurotrophic factor (hBDNF) is a soluble protein, can not only promote and enhance survival and differentiation of multiple nerve cells, but also repair the damaged nerve cells. So far some nerve diseases have no corresponding medicine such as Alzheimer's Disease (AD), Parkinson disease (PD) and diabetic neuropathy. The hBDNF protein was expressed by bioengineering, which provides a new means to cure these diseases.Firstly, cDNA of hBDNF was amplified using template synthesized from human blood total RNA and RT-PCR technique, simultaneously restricted enzyme sites, Ndel on the upstream and BamHI on the downstream, were introduced. Afterwards, the human BDNF cDNA was cloned into vector pMD18-T. PCR and restricted endonucleases were employed to identify recombinant pMD18-hBDNF. The sequence of recombinant is also in accord with the GenBank's. Prokaryotic expression vector was constructed via the target fragment (363bp), which was obtained by digestion of BamHI and Ndel from identified recombinant, was inserted into blanking vector pET32a. The recombinant pET32-BDNF was identified with BamHI and BamHI/Ndel. Escherichia coli transformed with recombinant plasmid, was induced with IPTG to express hBDNF protein after 4 hours culturing. SDS-PAGE gel analysis showed that the molecular weight was about 14kD. The specified protein expressed by E.coli accounted for 15.8% of the total protein of the bacteria. At last, the expressed protein was proved to own immunocompetence by Western blotting.In order to clone the hBDNF into eukaryotic expression vector pPIC9K, SnaBI on the upstream primer and EcoRI on the downstream primer were introduced by PCR using template pMD18-BDNF. To further verify the PCR results, the products were cloned into pMD18-T, then sequencing has been done. The results showed hundred-percent exactness. When constructing expression vectors, the similar method was used including clone and identification were done. The recombinant vector pPIC9K-BDNF was transformed into Pichia pastoris by electroporation. We got 21 identified recombinants with PCR from 23 samples, and screened out 3 recombinants with G418. The three recombinant strains of Pichia pastoris were cultured in the inducing medium about 96 hours. The expression of hBDNF cannot be observed distinctly by SDS-PAGE at the molecular weight 14.0 kD. In order to ensure the result, ELISA was insteaded. The result showed that 2 of 3 expressed hBDNF protein.