Construction of Multiple Copy Expression Cassette for Expression of Human Interleukin-10 in Pichia pastoris

Objective To construct the multiple copy expression cassette for human interleukin-10(IL-10) gene and improve the expression level of the gene in Pichia pastoris.Methods Construct recombinant plasmid αIL-10/pAO815 and digest with BamHⅠ and BglⅡ to obtain expression cassette AOX-αIL-10 for target gene,then link to BglⅡ site of αIL-10/pAO815 to construct multiple copy expression vector n(AOX-αIL10)/pAO815.Digest plasmid pPIC9K with NdeⅠ and SalⅠ to obtain Kan-resistant gene and insert into n(AOX-αIL-10)/pAO815.Transform expression vector n(AOXαIL-10)/pAO815 to Pichia pastoris by electrotransformation,and screen the transformants carrying IL-10 gene by PCR for expression under induction of methanol.The transformants with high expression level were further transformed to Pichia pastoris by electrotransformation using expression vector n(AOX-αIL10)/pAO815-Kan,and new transformants were screened with G418 at a higher concentration for expression under induction of methanol.Determine the content of expressed IL-10 by ELISA,and the activity with MC/9 cells.Results Eight copy expression cassette 8(AOX-αIL-10)/pAO815 and four copy expression cassette 4(AOX-αIL-10)/pAO815-Kan were constructed.The highest expression level of IL-10 was(8.25±1.65)mg/L,and the specific activity was 1.465×10~5 U/mg.Conclusion Multiple copy expression cassettes for IL-10 were successfully constructed,and the expression level of IL-10 in Pichia pastoris was significantly improved.