Prokaryotic Expression Of N Protein Of Porcine Detalcoronavirus,Preparation Of Polyclonal Antibody And Establishment Of Serological Diagnosis Method

Porcine deltacoronavirus(PDCoV)is a newly discovered coronavirus.It mainly infects piglets and causes severe enteritis accompanied by diarrhea and vomiting and other symptoms.The symptoms are symptomatically indistinguishable from those that caused by Porcine transmissible gastroenteritis virus(TGEV)and Porcine epidemic diarrhea virus(PEDV).In clinic the co-infection of PDCoV and TGEV or PEDV is common,which causes huge economic losses to the swine industry.Up to now,there are no commercial reagents and vaccines that can be used for PDCoV diagnosis and prevention.Therefore,the research on diagnosis and vaccine of PDCoV is imminent.The N protein of Coronavirus is a kind of phosphorylated multifunctional structural protein,which is the most conserved structural protein in the four structural proteins of coronavirus and is one of the most structural proteins of coronavirus in infected cells.These characteristics can be used to establish a rapid,efficient and accurate serological diagnostic methods.In this test,the prokaryotic expression and purification N protein of PDCoV was carried out,and New Zealand white rabbits were immunized for obtaining the polyclonal antiserum of N protein.The indirect ELISA method for detecting PDCoV antibody was established by using N protein as coated antigen.Specific studies are as follows:1.Prokaryotic expression and purification of N protein of PDCoVAccording to the analysis of PDCoV N gene sequence of HKU15-44 strain in Genbank,a pair of primers targeting the N gene of PDCoV were designed.Recombinant plasmid PMD18-T-PDCoV-N was used as template to amplify N gene fragment by PCR method.The fragment was cloned into prokaryotic expression vector p ET-28 a and then constructed recombinant expression plasmid p ET-28a-PDCoV-N.The recombinant plasmid was identified by double enzyme digestion and sequencing,and then transformed into E.coli BL21.Through the optimization of IPTG concentration and induction time,We successfully obtained the size of about 42.9k Da of recombinant N protein.Most of the recombinant protein was expressed in soluble protein and purified by nickel column purification kit.Western Blot confirmed that the recombinant N protein had good immunogenicity.The recombinant N protein can be used as the preparation of rabbit polyclonal serum and antigen of indirect ELISA method.2.Preparation and application of polyclonal serum against N protein of PDCoVThe purified recombinant N protein was emulsified with Freund's adjuvant and inoculated New Zealand white rabbits three times,we obtained rabbit polyclonal serum against recombinant N protein of PDCoV.Western Blot was used to detect the specific reaction between the polyclonal serum and recombinant N protein of prokaryotic expression,and the titer was 1:12000.At the same time,the whole PDCoV virus was used as antigen,the titer of the polyclonal serum was 1:12800 determined by indirect ELISA method,which showed that the recombinant N protein had good immunogenicity.Infected ST cells with PDCoV,We used rabbit polyclonal serum(1:50,1:100,1:200,1:400,1:800,1:1000,1:2000,1:3000,1:4000,1:5000 dilution)as the first antibody to indirect immunofluorescence test,the results showed that the polyclonal antibody could identify PDCoV in ST cells,and the optimal dilution ratio of the first antibody was 1:200,which proved that the prepared polyclonal serum can be used in the indirect immunofluorescence assay for the detection of PDCoV antigen..3.Establishment and preliminary application of indirect ELISA method for PDCoV N proteinWith recombinant N protein as coating antigen,PDCoV standard positive serum as first antibody,rabbit Anti-pig Ig G as second antibody and Continuous optimization of reaction conditions,the indirect ELISA method for detecting PDCoV antibody was established.Briefly,Recombinant N protein was coated at concentration of 2?g/m L and coated overnight at 4 ?;blocked with 1% BSA for 2h at 37?;100?L serum was 80 fold diluted and added to each well and incubated for 1h at 37?;100?L 6000 fold diluted rabbit anti-swine Ig G was added to each well for another reaction time of 1h at 37?;100?L chromogen was added to each well for 15 min at 37?.The indirect ELISA method was applied to detect porcine serum,the mean value of 40 OD450 values lower sera plus 3 times of standard deviation(X+3SD)was the critical value of the positive and negative serum,which determined the critical value of serum of PDCoV was 0.272.Specificity test showed that the method had good specificity and no cross reaction with other viruses(Porcine epidemic diarrhea virus,Porcine transmissible gastroenteritis virus Classical swine fever virus,Pseudorabies virus,Porcine reproductive and respiratory syndrome virus,Porcine circovirus).There is no more than 5% variation coefficient of intro-batch and in the inter-batch tests,which shows that the established indirect ELISA method has good repeatability and stability.The indirect ELISA method was used to detect 165 serum samples from pig farms in Henan,89 positive samples of PDCoV antibody were detected and the positive rate was 53.93%.10 pig sera with OD450 below or slightly higher than 0.272 were choosed for indirect immunofluorescence test,the sera with OD450 below 0.272 were negative for PDCoV antibody and the sera with higher than 0.272 positive for PDCoV antibody,the results showed that the established ELISA method had high accuracy.In conclusion,the indirect ELISA method established in this study can be used for the detection of PDCoV antibody in pig serum,which provides a feasible technical means for serological investigation of PDCoV infection in swine.