[Optimal expression and activity detection of recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies in P.pastoris].

AIM:To express the recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) using yeast cells secreting type carrier pPIC9K, and screen for optimal conditions of engineered yeast cells expressing cRFB4FC and cRFB4CH3. METHODS:The genes of cRFB4FC and cRFB4CH3 were cloned into P. pastoris eukaryotic expression vector pPIC9K to construct the recombinant plasmids pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3, then identified by DNA sequencing. The recombinant plasmid pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were transfected into P. pastoris SMD1163. The recombinant yeast cell line chosen by G418 resistance was identified by PCR. After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blotting, and biologic activity was identified by ELISA. Finally, orthogonal test was conducted to optimize the expression conditions of the recombinant yeast cell lines so as to increase the expression level of the antibodies. RESULTS:The secretory type yeast carriers pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were successfully constructed and chosen by G418 as well as identified by PCR to obtain the highly copied and integrated recombinant yeast cell line. The cRFB4FC and cRFB4CH3 proteins were expressed by yeast cells containing pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 induced by methanol. The relative molecular mass (M(r);) of cRFB4FC and cRFB4CH3 were about 326 000 and 295 000 Da. They could be identified by goat anti-human IgG(Fc) monoclonal antibody with Western blotting on SDS-PAGE, and higher biologic activity was confirmed by ELISA. Under the optimized expression conditions, the mean expression levels of cRFB4FC and cRFB4CH3 in recombinant yeast increased by 196.4% and 151.7%. CONCLUSION:The recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) proteins can be highly expressed in the P.pastoris SMD1163 expression system, and possess immunological activity.