The Tandem Expression Of The CCPs In Porcine Erythrocyte CR1-like Gene And Their Activity In Vitro

Objective:Pichia pastoris expression system was established successfully to expresses the porcine erythrocyte CRl-like complement control protein domain by using genetic engineering methods. To lay the foundation for the further study on the mechanism of the porcine CRl-like.Methods:The sequence of amino acid of porcine CRl-like CCPs was bioinformatically analyzed, and the gene segments of CCPs were selected. The CCPs sequence were synthesized artificially based on the codon usage bias of Pichia pastoris and cloned into pwPICZalpha vector. Pichia pastoris was used for recombinant protein expression, and analysed the supernatant of expression by SDS-PAGE and Western blotting. The recombinant proteins were purified with Ni-NTA resin and strong anion exchange resin, respectively. Immune adherence reaction of both opsonized Bacillus pumilu which was dealt with rabbit serum and no-opsonized Bacillus pumilu with recombinant protein was observed by confocal laser scanning microscopy. After co-incubation of CRl-like McAb and recombinant protein, the mixture mixed with porcine erythrocyte to observe whether blocked reaction of immunity could work.Results:The results of bioinformatics analysis showed that the porcine CR1-like gene contains 19 CCP domains. Among them, CCP 3 to CCP 6 and CCP 8 to CCP11 were selected to express. The supernatant expressed from Pichia pastoris was verified by SDS-PAGE, and result showed an expected size of band in gel. Western blotting analysis using mouse porcine E-CR1-like McAb further confirmed the expression of recombinant. After purification with both Protein Pure Ni-NTA resin and strong anion exchange resin, the final concentrations of CCP36 and CCP811 were 0.828 mg/ml and 0.945mg/ml, respectively. The results of immune adherence assay and antibody blocking assay showed that recombinant protein could interact with opsonized Bacillus pumilu, could block the adherence of CRl-like McAb binding to red cell.Conclusion:Two Pichia pastoris strains of CRl-like CCPs with heredity stability were obtained. The recobinant protein had activities of complement fixation, immune adherence receptor function.