Expression Of Human HSA-IFNα-2b Gene In Pichia Pastoris And The Analysis Of Expression Stablity

Objective The competent Pichia pastoris was transformed by the recombinant plasmid pPIC9k-HSA-IFNα-2b which was cut with restriction endonuclease SalI through electroporation. Then the target protein was induced to express by methanol. We made the preliminarily study on the expression conditions and the genetic stability of the positive transformants. Methods The integrate gene pPIC9k-HSA-IFNα-2b was identified by digesting with restrictive endonuclease EcoRI/BamHI. Then the recombinant plasmid pPIC9k-HSA-IFNα-2b was digested with restrictive endonuclease SalI. The lined recombinant plasmid DNA was transformed into the competent Pichia Pastoris SMD1168 through electroporation, then the Pichia Pastoris integrants was analyzed with PCR method and screened for the Mut phenotype of positive transformants on MM and MD plate. The positive transformants were induced to express by methanol and the expressed supernatant was identified by Western-Blot. The expression level of positive transformants was identified by ELISA and the level of its G418 resistence was measured on YPD-G418 plate. The anti-virus activity of the expressed rhHSA-IFNα-2b was measuredby Wish -VSV system. We studied the effect of methanol concentration and OD₆₀₀ value on the expression level of the positive transformants. Finally, we analyzed the genetic stability of the positive transformants. Results The lined recombinant plasmid DNA successfully transformed into the competent Pichia pastoris SMD1168 and integrated into SMD1168 chromosome through electroporation and the target protein was expressed in positive transformants and secreted into medium, which was induced by methanol. The expression level reaches as high as 300mg/L in medium through ELISA and the specific activity was...