Preparation And Immunological Analysis Of Monoclonal Antibodies Against Infectious Bronchitis Virus

Based on antigenic cross-reactivity and nucleotide sequence analysis,Coronaviruses(the order Nidovirales, the family Coronaviridae) are classified into three groups. Infectious Bronchitis Virus(IBV) is an important member ofγCoronaviruses.IBV infection can cause typical respiratory signs:gasping,coughing,sneezing,tracheal rales and nasal discharge.So far, more than 30 serotypes or variants of IBV have been identified worldwide,against which little or even no cross-protection existed,that's why the disease is hard to bring under control.Preparation of McAbs against IBV is a good way for quick detection of IBV.Firstly,IBV nucleocapsid(N) glycoprotein gene was amplified from plasmid template using polymerase chain reaction(PCR).Then,it was subcloned into vector pET-30a to construct recombinant plasmid pET-30a-N.The recombinant plasmid was transformed into E.coli BL21(DE3) and expressed by induction with IPTG.SDS-PAGE analysis demonstrated that there were two target proteins expressed and their sizes were 60kDa and 49 kDa respectively.After purified using Ni-NTA purification system,the HIS-tagged recombinant protein was blotted onto the nitrocellulose membrance,and then reacted with murine anti-IBV serum to determinate its antigenicity.The result showed the recombinant protein possessed a good antigenicity.Meanwhile recombinant M protein was expressed,purified and then stored at -70℃for use.Secondly,by fusing myeloma cells and the spleen cells of BALB/c mice immunized with IBV tl/CH/LDT3/03, and then detecting with the recomninant M and N protein respectively,two monoclonal antibodies(McAbs),designated as 3G11 and 4F10, were prepared.McAb 3G11 belonged to IgG1 subtype withκchain,while McAb 4F10 belonged to IgG2b subtype withκchain.Both ELISA and western blot showed that McAb 3G11 was directly against M protein and McAb 4G10 was directly against N protein.Both the two McAbs could react with the chosen IBV strains,indicating that the epitopes they were against might be highly conservative.In order to narrow the epitope zone of the two McAbs,truncated recombinant M protein: MP3-1(99-172aa),MP3-2(161-225aa) and truncated recombinant N protein:GST-N1(1-99aa), GST-N2(81-160aa), GST-N3(145-255aa), GST-N4(236-321aa), GST-N5(303-409aa) were expressed.Both western blot and ELISA showed that McAb 3G11 could react with HIS-M,MP3-1 and could not react with MP3-2,GST control.This indicated that the epitope zone of McAb 3G11 located at 99-172aa of M protein.While McAb 4F10 could react with GST-N3 and could not react with GST-N1,GST-N2,GST-N4,GST-N5 and GST control.This indicated that the epitope zone of McAb 4F10 located at 144-255aa of N protein.To conclude,this study is important to the foundation of new diagnostic methods and vaccine preparation.