The Construction Of Pichia Pastoris Expression System Of Human Neuritin And Function Research Of Expression Product

Objects: To construct Pichia pastoris secretary system of neuritin, and to obtain Neuritin protein and study its function, which was the basis of making a further research on function and mechanism of neuritin.Methods: The ORF of neuritin was amplified by PCR, and then the fragment was inserted into shuttle plasmid pPIC9K, The plasmid pPIC9K-neuritin was transformed into DH5α, positive clone was screened by PCR and sequencing, and then plasmid pPIC9k-neuritin was extracted and transformed into Pichia pastoris GS115 by electrization, Pichia pastoris recombinant with stable expression was screened by G418. The recombinant was identified by PCR analysis and sequencing, then induced the recombinant to express Neuritin by methanol. The expression products were analyzed by SDS-PAGE and, at the same time, selectted the optimal expression time. Neuritin protein was purified from yeast culture supernatant through nickel iron affinity chromatography, and the purified protein was identified by Western-blot. Finally, the bioactivity of Neuritin was analyzed via PC12 cell and chicken dorsal root ganglia culture analysis.Results:1. The recombinant shuttle plasmid pPIC9K-neuritin constructed was identified by PCR and sequencing, and the results showed that neuritin gene was successfully inserted into the vector.2. The GS115-neuritin Pichia pastoris recombinant obtained was verified by PCR and sequencing, and the results showed that neuritin gene successfully recombined with Pichia pastori's genome.3. The GS115-neuritin Pichia pastoris recombinant was induced by methanol to express Neuritin protein. SDS-PAGE analysis showed that Neuritin protein was secretated into yeast culture supernatant, the molecular weight of recombinant Neuritin protein was about 12KDa, and the optimal time of expression was 72h.4. The Neuritin protein was purified from yeast culture supernatant through nickel iron affinity chromatography, SDS-PAGE analysis showed Neuritin protein got an effective purification, and Western-blot analysis verified the purified protein was Neuritin.5. The function research of Neuritin showed that the recombinant Neuritin protein not only can promote neurite outgrowth of PC12 cell and cause a neuron-like change; but also can promote neurite outgrowth of DRG, and prolong its survival time.Conclusion:1. We successfully constructed Pichia pastoris secretary system of neuritin, and purified Neuritin protein.2. Neuritin that we obtained from Pichia pastoris has a good neurobiology:. Neuritin can promote neurite outgrowth of PC12 cell and cause a neuron-like change;. Neuritin can promote neurite outgrowth of DRG, and prolong its survival time.The research made a good basis for further study of neuritin's function and application of clinical treatment of nervous system diseases.