Preparation Of Monoclonal Antibody To Transmissible Gastroenteritis Virus N Protein And Its Initial Application

Transmissible gastroenteritis（TGE）is an intestinal digestive system disease caused by Transmissible gastroenteritis virus（TGEV）.Pigs of all ages are susceptible,and sick pigs mainly show clinical symptoms such as vomiting,watery diarrhea and dehydration.The mortality of piglets within 2 weeks of age is as high as 70%～100%.In recent decades,TGE has broken out in pig herds,which has caused seriously endangers the pig industry.TGEV is a member of the coronavirus genus of the family Coronaviridae,and the viral genome is approximately 28.5kb.The nucleocapsid（N）protein coding gene is highly conserved.N protein is the only phosphorylated structural protein in TGEV and one of the most abundant viral structural proteins in infected cells.Pigs infected with TGEV early can produce high levels of antibodies against N protein,so TGEV N protein can be used as an ideal target antigen for early detection and diagnosis of large-scale outbreaks in pig herds.In addition,a large number of studies have shown that coronavirus N protein plays a key role in regulating viral replication and changing host cell progression.However,the function of TGEV N protein is in initial stage.In this article,the study used E.coli to successfully express TGEV N protein,and used purified N protein to immune BALB/C mice.The monoclonal antibody was prepared and identified the recognized epitope,and then the antibody was used in the subcellular localization of N protein.The main study contents and results are as follows:1.The cloning and prokaryotic expression of the TGEV N geneThe TGEV N gene fragment was obtained by double digestion of the HA-N plasmid and was ligated into the p ET-28a vector to obtain the prokaryotic expression recombinant plasmid,designated p ET-N.The recombinant plasmids were expressed by IPTG induction,and the expression of the TGEV N protein was indicated by SDS-PAGE.The induced protein was purified using cutting gel purification.SDS-PAGE showed that the N protein with higher purity was obtained and could be used for subsequent studies.2.Preparation of monoclonal antibody against TGEV N proteinBALB/C mice were immunized with purified protein,reinforced by three subcutaneous injections and three intraperitoneal injections,and obtained with mouse monoclonal antibody（Monoclonal Antibody,Mc Ab）,named 2F10.Western-Blot showed that the prepared Mc Ab did not react with TGEV Nsp14a prokaryotic expression protein and PK-15 cells uninfected with TGEV,and specifically recognized only TEGV N prokaryotic expression protein and N protein produced by TGV PK-15 with TGEV infection.The results showed that 2F10 Mc Ab has significant specificity.Subsequently,monoclonal antibodies were prepared by inducing mouse ascites.ELISA showed that the prepared murine Mc Ab serum antibody titer was 100×2¹¹and the antibody subclass was Ig G 2a.The results showed that the 2F10 Mc Ab had a higher potency and better specificity,providing favorable conditions for subsequent identification of epitopes.3.Identification of the recognition epitopes of monoclonal antibodies against N proteinIn this study,we identified the antigen epitope of TGEV N protein by stepwise truncating the full-length TGEV N gene sequence,and transfected the constructed truncated plasmid into PK-15 cells for recognition using 2F10 Mc Ab.The results of indirect Immunofluorescence assay（IFA）indicated a linear epitope recognized by 2F10Mc Ab of ²⁵⁵SSANFGDTDLV²⁶⁵.Identification of precise epitopes can provide conditions for studies of antigen structure and function,and lay the foundation for subsequent screening of dominant epitopes and elucidation of immune mechanisms.4.The initial application of monoclonal antibodies to the N proteinIn this study,we explored the initial application of 2F10 Mc Ab and found that the N protein produced after TGEV infection in PK-15 cells could be detected by IFA and was proportional to the infectious dose of TGEV.Subsequently,the subcellular localization of N protein was observed by 2F10 Mc Ab,and it was found that TGEV N protein had obvious nuclear entry and exit phenomenon,and N protein was mainly localized in the cell nuclelus at the beginning of infection.To explore the precise nuclear localization sequence,the predicted nucleus localization sequence（NLS）was identified by truncating plasmids.It lays the foundation for the study of the structure and biological function of TGEV N protein,and provides new minds and new methods for the prevention and control of diseases caused by TGE and other coronaviruses.In conclusion,a murine Mc Ab for TGEV N protein was successfully prepared,named 2F10;the TGEV N protein epitope identified by 2F10 Mc Ab was identified;Western-Blot showed that 2F10 Mc Ab had significant specificity;IFA demonstrated that2F10 Mc Ab can be used to detect the N protein produced after TGEV-infected cells;and2F10 Mc Ab was used to explore the localization point of TGEV N protein to the nucleus and the predicted NLS was identified.The preparation and application of TGEV N protein Mc Ab in this study can lay the foundation for the further analysis of the function of the protein,the understanding of the pathogenic mechanism of the virus and the establishment of early diagnosis methods.