Prokaryotic expression of E^rns protein of classical swine fever virus and preparation of its polyclonal antibody

【Objective】 The study aimed to prepare polyclonal antibody against the E^rns protein of the classical swine fe‐ ver virus（CSFV），which could provide theoretical basis for further exploring the structure and function of the E^rns protein，as well as for elucidating the pathogenic mechanisms of CSFV. 【Method】 Bioinformatics methods were used to predict the structure of the E^rns protein. The CSFV E^rns gene was cloned by PCR using the eukaryotic expression vector pCMVHA-E^rns as a template，and the prokaryotic expression vector pET-32a-E^rns was constructed. The pET-32a-E^rns vector was then transformed into Escherichia coli BL21（DE3）receptor cells，IPTG was added to induce the expression of E^rns protein，confirming the expression form of E^rns protein. The target protein was purified by Ni-NTA affinity chromatography to obtain high-purity E^rns protein. Mice were immunized using the back subcutaneous multiple injection method，and after 4 immunizations，blood was collected to isolate serum for the preparation of polyclonal antibodies against the E^rns protein， and their titer and specificity were assessed. 【Result】 The E^rns protein lacked signal peptide and transmembrane domains， making it a hydrophilic protein that contained multiple B-cell epitopes. The size of the E^rns gene fragment was 681 bp，and its recombinant protein was primarily expressed as inclusion bodies. The polyclonal antibodies against the E^rns protein had titers of 1∶64000 for the prokaryotic expressed recombinant protein and 1∶1000 for the eukaryotic expressed recombinant protein，and they could specifically recognize CSFV. 【Conclusion】 The prepared polyclonal antibodies against the E^rns protein possess high titer and strong specificity，making them suitable for detecting the expression levels of the E^rns protein in cells using ELISA and Western blotting. The preparation of E^rns protein polyclonal antibodies contributes to a better under‐ standing of the role of E^rns protein in CSFV infection and immune processes. This has significant implications for further research into the function of E^rns protein，the biological characteristics of CSFV，and the development of prevention and diagnostic methods for CSFV.