| Infectious bronchitis(IB)is an acute,highly contagious disease caused by the Infectious bronchitis virus(IBV).IBV mainly results in the death of chicks,the decrease of feed conversion ratio in broilers and decrease of laying hens.The virus is easy to mutate and recombine with other IBV,thus resulting in the existence of new genotypes,which make it difficult to control.IB is mainly controlled by vaccination,but it still occurs in immunized chickens due to poor cross protection of vaccine.There is few standard antigen and antibody references for evaluation of the vaccine in China.In this study,the IBV-M41 and IBV-CK/CH/2014/FJ14 were amplified as the reference antigens,and polyclonal antibody were prepared by inoculation 14-day-old chickens with the virus.The antigens and antibodies prepared above are used as reference antigens and antibodies after a series of calibration.In this study,IBV-CK/CH/2014/FJ14 strain of S protein was expressed through the eukaryotic vector expression system,and 3 strains of monoclonal antibodies were obtained by screening with the expressed protein,which could provide corresponding biomaterials for the prevention and control,diagnosis of the disease.1.Preparation of reference antigen and antibody of chicken infectious bronchitis virusIn this study,IBV-M41 strain and IBV-CK/CH/2014/FJ14 strain were proliferated by inoculation into the allantoic cavity of SPF chicken embryo.The virus titer of the collected viruses was determined.The results showed that the EID50 value of IBV-M41 strain and IBV-CK/CH/2014/FJ14 strain was 105.8EID50/mL The TOC-ID50 value of IBV-M41 strain was 105.25TOC-ID50/mL,and the TCID50 value was 104.5TCID50/mL.The TOC-ID50 value of the IBV-CK/CH/2014/FJ14 strain is 105.5TOC-ID50/mL,and the TCID50 value is 104.75 TCID50/mL.PCR results showed that two antigen were not contaminated by Avian leukosis virus(ALV),Goose parvoviruses(GPV),Infectious bursal disease virus(IBDV),Infectious laryngotracheitis virus(ILTV),Marek’s disease virus(MDV),Reticuloendotheliosis virus(REV),and Chicken infectious anemia virus(CIAV).The results of sterility test and mycoplasma test after lyophilization indicated that both bacteria and mycoplasma were negative.The results of physical properties,loading difference coefficient,the homogeneity,stability and long-term preservation indicated the antigen were all qualified,that is,the prepared antigen can be used as candidate reference for IBV antigens.To obtain the sera anginst IBV-M41 and IBV-CK/CH/2014/FJ14 strains,14-day-old SPF chickens were inoculated with these viruses,respectively.Boosting immunization was made very7 days for three time.Serum was collected after 28 days of the third immunization.The serum to IBV-M41 strain was titrated Our results showed that the titers were 1:800 in IFA,1:1722 in the neutralization,1:1600 in ELISA,1:27 in HI,respectively.The titers of the sera to IBV-CK/CH/2014/FJ14 strain 1:800 in IFA,1:1488 in neutralization,and 1:1600 in ELISA respectively.These serum could not reacted with other virus,such as Reticuloendotheliosis virus(REV),Marek’s disease virus(MDV),Avian leukemia virus(ALV),Avian influenza virus(AIV).The results of sterility test and mycoplasma test after lyophilization showed negative The physical properties,loading difference,homogeneity,stability and long-term preservation for these sera were analyzed.The results indicated all sera arequalifiedand could be used as candidate reference antibody.2.Expression of chicken infectious bronchitis virus S protein in eukaryotic expression systemIn this study,the extracellular domain of the S gene of the IBV-CK/CH/2014/FJ14 strain was amplified by PCR,and cloned into the pFast-Bac-HTA vector for restriction enzyme digestion and sequencing identification.After the identification,it was transposed into E.coli DH10BacTM competent cells.In the cells,a recombinant bacmid rBacmid-FJ14-S was constructed,and then transfected into Sf9 cells mediated by liposomes to obtain recombinant virus rBac-FJ14-S.IFA results showed that the recombinant virus could react with the serum to IBV.Western blot analysis found a band near 140 kDa,which was consistent with the expected size,which indicated that the IBV S protein extracellular domain was successfully expressed.At the same time,the constructed pCAGGS-FJ14S1-IgGFc recombinant vector was transfected into 293T cells.IFA showed that the recombinant protein can specifically react with the chicken serum against IBV.Protein FJ14S1-IgGFc waspurified by Proten G column The results of SDSA-PAGE and Western blot analysis showed that the size of the purified protein was in line with expectations and the S1 protein was correctly expressed.3.Preparation of monoclonal antibody against S protein of chicken infectious bronchitis virusIn this study,BALB/C mice aged 6-8 weeks were immunized with IBV-CK/CH/2014/FJ14 allantoic fluid virus,which was condensed by hypercentrifugation.The mice were immunized every 7 days,and blood samples were collected from tail vein 7 days after immunization for three times.The titer of the serum was 1:800 in IF,A.Hybrodoma cells fused between Sp2/0 and the spleen cells immunized with the IBV were screened with 293T cells transfected with the recombinant pCAGGS-FJ14S1-IgGFc in IFA.Three monoclonal antibodies were obtained,which were named as Mab-IBV-S1-1H9,Mab-IBV-S1-5D3 and Mab-IBV-Sl-5G11,respectively.Western blot analysis showed that all the three Mab strains could react well with purified recombinant protein FJ14-S1-IgG. |
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