Development And Application Of An Indirect ELISA For Detection Of Antibodies Against Infections PHEV Using Recombinant N Protein

Porcine hemagglutinating encephalomyelitis virus(PHEV)causes encephalomyelitis and belongs to the betacoronavirus family,or vomiting and wasting disease in suckling piglets.Their mortality rate is higher,up to 100%when piglets under 3 weeks old are infected with PHEV,.At present,there are some commonly used diagnostic methods for the disease:Real time-PCR,nest PCR,Electrical microscopic observation,VN and so on.These methods have played a main role in the production,but there are insufficient that the operation is complex and takes time,costs high and so on.It is unsuitable mathod for farmers in rural areas.In addition,the enzyme linked immune sorbent assay(ELISA)plate was coated with the purified PHEV inactivated,and an indirect ELISA was developed for rapid detection of antibodies against PHEV by optimizing reaction conditions.Although this method has the characteristics of simple operation,cheap,high sensitivity and specificity,but using purified PHEV as coating antigen to establish the diagnostic method of ELISA,which has the shortcomings such as preparing numerous antigen difficultly and spreading virus easily.Therefore,a easy,safe,efficient and accurate diagnostic technology established is urgently needed.Nucleocapsid protein(N)of coronavirus is highly conserved,which is a larger proportion of viral structural protein.High levels of antibodies against N protein can be detected in the early stage of infection.It is a diagnostic antigen and has important application value in the way of detecting virus infection and evaluation of vaccine immune effect.In order to establish an indirect ELISA method which is more simple in the process of preparation of the antigen and more secure during operation,Firstly,according to the PHEV N protein gene sequence published on GenBank,we designed and synthesized specific primers of N protein gene and which was amplified by PCR.Furthermore N prokaryotic expression recombinant plasmid(pET28a-N)was constructed by enzyme digestion,ligation and transformation;and then it was transformed into BL21(DE3)expression bacteria,and its expression induced by IPTG High purity protein was obtained by affinity chromate grapy purification.The target protein detected by SDS-PAGE,the results showed that N protein expressed mainly in the form of:inclusion bodies in E.Coli5 purified successfully and obtained high concentration protein which was a 55 kDa fusion protein.Western blot detection indicated that the recombinant protein could specifically react with antiserum against PHEV.The reaction was optimized using 2μg/mL N protein as coat antigen and 1:10000 dilution of HRP-labeled goat anti-procine IgG.Two hundred of serum samples collected from Tianjing were detected using N-ELISA method.The positive rate of serum sample was 83.5%,which showed 90,91%consistency with the results obtained by HEV-ELISA.Compared to porcine epidemic diarrhea virus(PEDV),classical swine fever virus(CSFV),porcine transmissible gastroenteritis virus(TGEV),pseudo rabies virus(PRV),Escherichia coli and so on,there had no cross-reactivity.Therefore,this method can be used for clinical detection of serum antibodies and epidemiological investigation of PHEV.The research results will provide important diagnosis technology for PHE.