Expression Of CR1-like CCP_（1-3） Domain In Porcine Erythrocytes And Detection Of Its Immune Adherence Activity

Objective: The aim of this study was to construct recombinant porcine erythrocyte CR1-like CCP_（1-3）protein and detect its biological function,so as to provide a theoretical basis for explaining the molecular mechanism of porcine erythrocyte CR1-like immune function.Methods:（1）According to the porcine erythrocyte CR1-like gene sequence registered by NCBI,the porcine erythrocyte CR1-like CCP_（1-3）gene sequence was screened by SMART and NCBI blast online software,and Xho I and Eco R I restriction sites were added at both ends of the sequence to design and synthesize the recombinant plasmid containing CCP_（1-3）target gene;（2）The recombinant plasmid p GEX-4T-1-CCP_（1-3）was constructed by connecting CCP_（1-3）target gene with expression vector p GEX-4T-1,and the recombinant plasmid p GEX-4T-1-CCP_（1-3）was sequenced and identified;（3）The recombinant plasmid p GEX-4T-1-CCP_（1-3）was transformed into the host strain BL21（DE3）p Lys S.when the concentration of Escherichia coli was OD600 to 0.6,the protein expression was induced by 1 mmol/L IPTG;（4）SDS-PAGE electrophoresis and Western Blot were used to detect the expression of recombinant protein CR1-like CCP_（1-3）;（5）The recombinant egg white CR1-like CCP_（1-3）was purified by Protein Iso（?） GST Resin column;（6）SDS-PAGE electrophoresis was used to verify the purification results,and BCA method was used to determine the protein concentration;（7）Immunoadhesion activity of CR1-like CCP_（1-3）protein to serum sensitized GFP-E.coli was detected by immunofluorescence and flow cytometry.Results:（1）The recombinant plasmid CCP_（1-3）was synthesized from the CR1-like CCP_（1-3）gene sequence of porcine erythrocytes by SMART and NCBI blast analysis;（2）The results of enzyme digestion of the synthesized recombinant plasmid CCP_（1-3）showed obvious bands at 582 bp,which were the same size as expected,indicating that the recombinant plasmid CCP_（1-3）was constructed correctly;（3）The sequencing results of the recombinant plasmid p GEX-4T-1-CCP_（1-3）were completely correct with those of NCBI gene bank,and could be used for subsequent experiments;（4）The recombinant plasmid p GEX-4T-1-CCP_（1-3）was transfected into BL21（DE3）p Lys S competent cells and identified by PCR,and there were bands at 582 bp,indicating that the recombinant Escherichia coli was successfully constructed;（5）After IPTG induced expression,the target band appeared at 46 KDa by SDS-PAGE electrophoresis,which preliminarily proved that the porcine erythrocyte CR1-like CCP_（1-3）protein was successfully expressed;（6）Western Blot showed that the target band appeared at 46 KDa,indicating that the recombinant porcine erythrocyte CR1-like CCP_（1-3）protein was successfully expressed;（7）SDS-PAGE electrophoresis showed that the purified protein had no heteroband,and the protein concentration determined by BCA was 1.23 mg/m L;（8）CR1-like CCP_（1-3）protein can bind to serum sensitized GFP-E.coli,thus reducing the binding level between porcine erythrocytes and sensitized GFP-E.coli.Conclusion:（1）The bioactive porcine erythrocyte CR1-like CCP_（1-3）protein was successfully expressed;（2）Porcine erythrocyte CR1-like CCP_（1-3）protein has the function of immunoadhesion to GFP-E.coli.