Preparation Of Monoclonal Antibodies Against Transmissible Gastroenteritis Virus N Protein And Identification Of Antigenic Epitopes

Porcine transmissible gastroenteritis virus (TGEV), which is belonged to the familyCoronaviridae，is a kind of high contact digestive infectious disease. This enteropathogenicvirus causes acute and fatal diarrhea in piglets under2weeks of age. This disease began fromAmerica since1933, and now it has been widely distributed in the Northern Hemisphere,especially those temperate-frigid region.TGEV is sensitive to the heat, and can preserved long time in low temperature, thevirulence has no obviously change under the condition of liquid nitrogen. The genome of RNAencodes three major structural proteins: nucleoprotein(N), membrane(M) and spike(S) proteins.The phosphorylated N protein-is the most abundantly expressed structural protein and highlyconserved among all TGEV isolates; therefore, it can be used as a suitable target forserodiagnosis of the disease.In this study, we amplified the N protein gene by reverse transcription-polymerase chainreaction(RT-PCR), then insert the gene into the prokaryotic expression vectors pGEX-6P-1,named pGEX-6P-N. After sequencing, the confirmied plasmid was transformed into BL21(DE3)and induced by Isopropy1β-D-1-thiogalactopyranoside(IPTG). SDS-PAGE analysis showed thatthe protein was successfully expressed in supernatant. The fusion protein was purified by themethod of GST and the result showed that it was purified as expected. Western blot analysisrevealed that the recombinant protein could react with anti-TGEV serum.To prepare the MAbs against N protein of TGEV, the purified recombinant protein wereimmunized to BALB/c mice. Standard method was carried out for cell fusion, six hybridomacells were obtained and named2G10，3D7，4D12，5A8，5E8，5G7,respectively. The obtainedMAbs could react with the nature TGEV by western blot and immunofluorescence assay (IFA)analysis.In order to identify the core amino acid, three truncated parts(N1, N2, N3) as designedwere expressed successfully with pGEX-6P-1. Western blot analysis with sis MAbs revealedthat2G10,3D7,4D12,5A8could react with N3;5E8，5G7could react with N2. According tothe amino acids of N protein, a series of peptides segments were synthesized, two liner antigenicepitopes were established by indirect ELISA (246VTRFYGARSSSA257;189SVEQAVLAALKKLG202).MAbs against N protein were obtained in this study, and two antigenic epitopes were identified, which were in conformity with relative literature. It will increase the understanding ofantigenic structure and establishment the diagnose methods of N protein.