Preparation And Application Analysis Of Canine Procalcitonin Polyclonal Antibody

At present,the abuse of antibiotics in animals has been widespread,which has caused great waste of resources and environmental pollution.The effective diagnosis of animal diseases can greatly reduce the abuse of antibiotics.Procalcitonin as a protein of the body has high sensitivity and specificity,which is clinically a differential diagnosis index for serious bacterial infection diseases and viral infectious diseases.Therefore,this study takes canine PCT protein as the research object and explore the clinical applicability of canine PCT protein through the prokaryotic expression,protein purification,the establishment of indirect ELISA detection method and the application analysis of recombinant protein.First,the canine pET-30a-PCT expression vector was constructed and validated.Double restriction enzyme digestion and sequencing showed that the canine PCT protein gene was successfully cloned into pET-30 a.The results of SDS-PAGE showed that the recombinant plasmid induced by IPTG successfully expressed a protein with a molecular mass of 14.9 ku,which was consistent with the expected results.Second,recombinant protein purification and validation were made.The factors affecting protein expression were optimized.The SDS-PAGE results showed that induction temperature 28 ° C,IPTG concentration 1 mmol/L,induction 9 h,recombinant protein soluble expression is more.The recombinant protein was verified by using the His-tag monoclonal antibody as a primary antibody.The results of Western blot showed that the recombinant protein reacted specifically with the His-tagged antibody.Subsequently,the recombinant protein is purified and the mouse is immunized to prepare a polyclonal antibody.The results of SDS-PAGE,Western blot and indirect EIISA results showed that the purified protein had no miscellaneous bands and had good reactogenicity.The multi-antiserum titer reached 1:51200.Finally,the application of recombinant protein was analyzed.The serum of healthy dogs and inflammation dogs were detected by indirect ELISA.The results of ELISA showed thatPCT was successfully detected in the serum of inflammation dogs and positively correlated with the degree of inflammation.To sum up,this study provides a theoretical basis for further research on biological function of canine PCT protein,preparation of monoclonal antibody and development of immunocolloidal gold test paper through construction of prokaryotic expression vector of canine pET-30a-PCT,expression of recombinant protein,establishment of indirect ELISA method and analysis of its applicability.