Preparation Of Monoclonal Antibodies Against N Protein Of Porcine Deltacoronavirus And Primary Identification Of Antigenic Epitopes

The porcine deltacoronavirus(Porcine Deltacoronavirus,PDCo V)is a rising coronavirus,which can cause vomiting,diarrhea,dehydration and even death of piglets,causing great losses to the pig industry.The four structural proteins encoded by PDCo V are spike,nucleocapsid,membrane and envelope.Among them,N protein plays an important role in the process of viral RNA replication and production.It has a large amount of expression and strong conservation,often used as the diagnostic antigen for detection of virus antibodies by other coronaviruses.The molecular basis for the determination of antigen specificity is the epitope of the antigen.By studying it,it is beneficial to further design peptides,novel diagnostic reagents and novel vaccine molecules with immunological activity and neutralizing activity.In view of this,this study prepared the monoclonal antibodies against PDCo V N protein,screened and identified the antigenic epitopes,laying a foundation for the research of PDCo V diagnostic methods and the novel vaccines.In order to obtain PDCo V N gene recombinant protein,the prokaryotic expression vectors of p ET-32 a and p GEX-6p-1 of the N gene of PDCo V NH strain were constructed and transformed into E.coli BL21(DE3)competent cells,respectively.IPTG with a concentration of 1 m M induced the expression of the recombinant bacteria,and further purified and identified the expressed recombinant protein of PDCo V.The results showed that RT-PCR and the PDCo V NH virus RNA was used as the template,which was in accordance with the expected size,amplified the N gene of about 1 029 bp.The results of SDS-PAGE electrophoresis showed that PDCo V N gene was successfully expressed in E.coli BL21(DE3),and the molecular weight of the recombinant N protein expressed was 56 k Da and 64 k Da,which was in line with the expected size.Western blot results showed that the purified protein reacts specifically with PDCo V positive serum,indicating its good antigenicity.The expressed and purified His-PDCo V-N was used as an immunogen to immunize six~eight weeks old female BALB/c mice.17 hybridoma cell lines capable of stably secreting monoclonal antibodies against PDCo V N protein were prepared.The hybridoma cell lines were named 1G6,1F8,1E10,2G5,3A7,3F9,3E12,5D11,7B2,7E6,7G6,7F10,8E2,8D4,8E8,9G1,9G11.Identification of antibody subclasses showed that the antibody subclasses secreted by 3E12 and 9G11-cell lines were Ig G2 b type,the rest 15 were Ig G1 type,and the light chains were all ? chains.Western blot results showed that the prepared 17 monoclonal antibodies were immunoreactive with natural PDCo V N protein.In order to identify the epitopes recognized by the obtained monoclonal antibodies against PDCo V N protein,the N gene was truncated and Western blot was used to preliminarily confirm that the main epitope recognized by the monoclonal antibodies was aa51-115.In this study,17 strains of monoclonal antibodies that stably secrete anti-PDCo V N protein were obtained,and the major antigenic epitopes of PDCo V N protein were identified,for PDCo V N protein structure and function of research as well as diagnosis and vaccine PDCo V relevant prevention and control strategy design provides a material basis.