Preparation and biological function research on polyclonal anti-body against avian infectious bronchitis virus nsp4 protein

【Objective】This paper screened out the amino acid region with good antigenicity of non-structural protein4(nsp4) of avian infectious bronchitis virus(IBV) and prepared polyclonal antibody, so as to provide a material basis for further research on the function of nsp4 protein in the process of IBV replication and lay a foundation for the research and development of new diagnostic kits and vaccines for IBV.【Method】In this study, nsp4 protein of IBV Beaudette strain was prokaryotically expressed. Rabbit and chicken polyclonal antibodies were prepared by immunizing Japanese white rabbit and healthy negative chicken with the fusion protein nsp4 as immunogen. And the biological function of the prepared polyclonal antibodies were investigated by indirect enzyme linked immunosorbent assay(ELISA), Western blotting and indirect immunofluorescence assay(IFA).【Result】The nsp4 protein prokaryotic expression vector pCZN-1-HIS-nsp4 and nsp4 protein eukaryotic expression vector p VAX1-nsp4-HA were successfully constructed by selecting amino acids at positions 408-514 of nsp4 protein as prokaryotic expression sequences. The prokaryotic expression of fusion protein nsp4 was 13.6 k D, which was consistent with its predicted size, and the fusion protein nsp4 was able to react with positive whole virus serum against IBV GX-YL5 and M41 strains. The serum titers of prepared rabbit and chicken polyclonal antibodies were 1∶128000 and 1∶6400 respectively. Additionally, both polyclonal antibodies could react with the fusion protein nsp4 and IBV whole virus. Moreover, they could react with nsp4 protein expressed in chicken embryonic kidney(CEK) cells infected with IBV GX-YL5, Beaudette and M41 strains, and could react with nsp4 protein expressed in the transfected or IBV infected African green monkey kidney cells(Vero) Beaudette strain. The results of real-time fluorescence quantitative PCR showed that the viral loads of Vero cells increased gradually after transfection of nsp4 protein eukaryotic expression vector p VAX1-nsp4-HA. In other words, in vitro overexpressed nsp4 protein promoted IBV replication.【Conclusion】Two polyclonal antibodies against nsp4 of IBV strain with high titer, good reactivity and strong specificity are successfully prepared, which can be used as tools for the study of protein characteristics during cell localization and expression phase analysis. In addition, they can be used to analyze the action mechanism of nsp4 protein in the proliferation process of IBV, and also provide reference for the study of other coronavirus nsp4 protein.