Prokaryotic Expression Of S Protein Fragment Of Cat Coronavirus And Establishment Of Indirect ELISA Antibody Detection Method

Feline coronavirus(FCoV)is a non segmented,single stranded positive strand RNA virus.The pathological types of FCoV are divided into two types:one is feline enteric coronavirus(FECV),which has a low mortality and is often ignored.The other is feline infectious peritonitis virus(FIPV),which has a high mortality rate,but there is no effective treatment in clinic.Although the pathogenesis,transmission law,prevention and treatment of FIP have been widely studied at home and abroad,FIP is still one of the feline infectious diseases with high clinical mortality.Due to the high mortality of FIP and the lack of targeted treatment,it is of great significance to establish a rapid,effective and accurate detection method for the early diagnosis and timely prevention of FCoV.Serological diagnosis technology has been applied to the diagnosis of many diseases.Among them,ELISA is a commonly used rapid detection method because of its short reaction time,fast and simple,no need for special equipment,and the reagent designed based on the principle of enzyme-linked immunosorbent assay has the characteristics of good stability,strong specificity and high sensitivity.Because there is no commercial FCoV indirect ELISA antibody detection kit in China.Therefore,in this study,bioinformatics technology was used to analyze the secondary structure,physicochemical properties,hydrophobicity,signal peptide cleavage sites,membrane penetrating helical structure,N-glycosylation sites and protein surface accessibility of FCoV S protein,so as to determine the intercepted amino acid region at position1127-1400 of S protein.Referring to the published FCoV S protein sequence in Gen Bank,a pair of specific primers were designed and synthesized to amplify the FCoV S protein fragment,and the purified FCoV S protein was combined with p EASY~?,The recombinant expression plasmid of FCoV S protein fragment was constructed.The identified recombinant expression plasmid was transformed into E.coli BL21 for induced expression.After the expression product was verified by SDS-PAGE,the target protein was purified.The S protein was identified and analyzed by SDS-PAGE and Western blot.The results showed that S protein was successfully expressed and purified,and the purified S protein had good biological activity.Using the purified S protein fragment as the coating antigen,an indirect ELISA method for detecting FCoV antibody was established by optimizing the reaction conditions such as the optimal coating concentration and incubation conditions,the optimal primary antibody concentration and incubation conditions,the optimal secondary antibody concentration and incubation conditions,and the optimal color development time.The established method was applied to epidemiological investigation and analysis of FCoV infection in cats in Fujian Province.The results are as follows:1、FCoV S protein was analyzed according to biological information,and1127-1400 amino acids were selected as the optimal expression region.2、Build peace successfully p EASY~?-S prokaryotic expression plasmid and optimized the protein induction expression conditions.FCoV S protein was successfully expressed and purified,and the recombinant protein had good reactivity with cat positive serum.3、Using the recombinant FCoV S protein expressed in this study,an indirect ELISA method for the detection of FCoV antibody was successfully established.The optimal reaction conditions were as follows:the optimal coating concentration of antigen was 4mg/ml,the optimal dilution ratio of primary antibody was 1:400,the optimal dilution ratio of secondary antibody was 1:20000,and the optimal color development time was 6 min.4、The indirect ELISA method of FCoV antibody established in this study has good sensitivity,specificity and repeatability.The positive coincidence rate between FCoV indirect ELISA and Western blot was95.1%.5、The indirect ELISA method of FCoV established in this study provides reference data for the prevalence of FCoV in Fujian through the detection of 172 feline serum samples collected clinically.The indirect ELISA method of FCoV established in this study can provide reference basis for the early diagnosis of FCoV,provide method basis for the epidemiological monitoring and comprehensive prevention and treatment of FCoV,and provide detection means for the antibody response of the subsequent vaccine prepared by FCoV.