[Construction of a multi-copy Pichia expression vector and expression of human brain-derived neurotrophic factor in Pichia pastoris].

A pPICZ alpha1 vector was reconstructed with pPICZ alpha vector and PCR product of His4-Kan sequence from pPIC9K vector. The sequence encoding human brain-derived neurotrophic factor (hBDNF) was inserted into pPICZ alpha1. The expression vectors carrying 1, 2, 3, 6 copies of expression cassettes were constructed and transformed into Pichia pastoris GS115 respectively. The transformants were screened with G418/Zeocin and induced by 0.5% methanol, and the results of SDS-PAGE confirmed that the expression protein was about 14 kD and the percentage of the protein was about 20% of the total secreted protein. ELISA and Western blot analysis demonstrated that the protein could be specifically combined with chicken antibodies to human-BDNF.