Epitopes Identification Of Nucleocapsid Protein Of Infectious Bronchitis Virus And Study On The ScFv Against IBV N Protein

Infectious bronchitis(IB),caused by infectious bronchitis virus(IBV),is an acute and highly contagious disease in chickens.IBV can infect all ages of chickens,which resultes in a decline in egg production and quality in layers and poor weight gain and reduction of the feedstuff repay in broilers,it also induces significant mortality from infection of respiratory and kidney in young chickens.In addition,the chicken infected by IBV are easily infected by the other pathogenic microbes,so IBV can aggravate the hazard to chicken through mixed infection,which caused enormous economy loss in global poultry industry.The virion has four structure proteins:the spike(S) protein,the small membrane(E) proteins,the membrane(M) protein,the nucleocapsid (N) protein.The N protein is a phosphorylated protein.The N protein directly binds viral genomic RNA and forms a helical ribonucleoprotein complex(RNP).N protein is also a major immunogenic protein,inducing the immunoresponse and especially the cellullar immunologic response.So N gene and N gene-encoded protein are hot spots on the study of IBV.IBV N protein is regard as a target protein in our study.In this study,utilizing an IBV strain tl/CH/LDT3/03 isolated from China stored in our lab to immunize the BALB/c mice for 4 times.The mouse spleen was harvested under sterilized condition for fusion with SP2/0 myeloma cells by PEG1450.Four mAbs were obtained using classical hybridoma technology.The results of indirect ELISA and western blotting showed that the four mAbs specifically reacted with the recombinant IBV N protein prepared in our study and natural N protein of IBV tl/CH/LDT3/03 strain.the four mAbs were all against the N protein of IBV tl/CH/LDT3/03 strain,designated 6H3,5E12,2C4 and 6F9.The number of chromosome of hybridomas 6H3,5E12,2C4 and 6F9 were 90,99,91 and 102 respectively.Immunoglobulin subtype analysis indicated that the heavy of the two mAbs 6H3 and 2C4 were IgG1,mAb 6F9 was IgG2b and mAb 5E12 was IgM.and light chains were allκ.Subsequently,the epitopes of these two mAbs were identified.Firstly N gene were divided into eight overlapping fragments and cloned into the downstream of GST-tag in the pGEX-6p-1 vector and transformed the Ecoli.BL21(DE3),the eight truncated recombinant proteins were obtained and designated GST-N1,GST-N2,GST-N3,GST-N4,GST-N5,GST-N1-1,GST-N1-2. GST-N1-3.According to the results of western blotting analysis,the antigenic epitope site was identified by mAb 6H3 located in 80-99 amino acid(aa),mAb 6F9 the antigenic epitope site was identified located in 64-82 aa.At the same time,different IBV isolates from China CK/CH/LDL/97Ⅰ,CK/CH/LHN/00Ⅰ,CK/CH/LSD/05Ⅰ,CK/CH/LSC/99Ⅰ,CK/CH/LHLJ/04Ⅴ, CK/CH/LHB/08Ⅰ,CK/CH/LAH/08Ⅱand the other four srains which showed different pathotype and tissue tropism were chosen to detect the reactivity of mAbs 6H3 and 6F9 by using western blotting analysis,The results indicated mAb 6H3 could recognize the natural nucleocapsid of all different IBV isolates except for CK/CH/LHB08Ⅰwhich indicated that the other 11 different IBV isolates shared one B cell linear epitope located in 80-99 aa.However,mAb 6F9 failed to react with the nucleocapsid of CK/CH/LAH08Ⅱand CK/CH/LHB08ⅠIBV isolates,but it can recognize the natural nucleocapsid of the others.Moreover,the mAb 6H3 were selected to constructed the single chain variable fragment (scFv) against IBV N protein.The mRNA of hybridoma cell 6H3 was extracted through the methods of TRIzol,A single chain variable fragment(scFv) was amplified by using splice-overlap extension polymerase chain reaction(SOE-PCR) through the flexible(Gly(4)Ser)(3) linker from the hybridoma cell line 6H3 which was against IBV nucleocapsid protein.Subsequently,the scFv gene was clone into the prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid.The recombinant plasmid was confirmed by sequencing and transformed into E.coli BL21(DE3),and then bacteria contained recombinant plasmid was induced by IPTG.The recombinant protein was purified using ProBond^TM Purification System and was refolded by urea dialyse.Then SDS-PAGE was used to detected the purification results of recombinant IBV N protein.The concentration of recombinant IBV N protein was measured by ultraviolet absorption and the purified IBV N protein was refolded under the 6～0mol/L urea gradient.After recombinant protein refolded,Sandwich enzyme-linked immunosorbent assay(ELISA) and western blotting were performed to detect the reactivity between the scFv and IBV.The results indicated that scFv could react specifically with IBV nucleocapsid protein.Moreover,the scFv could also reacted with the natural IBV nucleocapsid protein which was consistent with the parent mAb 6H3.The scFv gives an access to the development of the therapeutic biological agent and show its importance for research on the other coronavirus.