Preparation and preliminary application of polyclonal antibodies of walleye dermal sarcoma virus(WDSV)accessory genes orfA and orfC

【Objective】 The study was done to prepare polyclonal antibodies of walleye dermal sarcoma virus(WDSV)accessory genes orfA and orfC and to analyze the orfA and orfC proteins expressed in yeast and mammalian cells.【Method】 In this research,the WDSV genes orfA and orfC were amplified from walleye dermal sarcoma virus complete genome and cloned into pET32a(+)to construct the prokaryotic expression vectors.Subsequently,Rosseta(DE3)competent cells were transformed with the constructed expression vectors,and the orfA and orfC recombinant proteins were prepared by induction with IPTG and purified by washing with 2 mol/L and 4 mol/L urea solution,successively.The purified proteins were used to immunize New Zealand rabbits using the method of hypodermic injection coupled with era vein booster immunization.The specificities of the polyclonal antibodies prepared were checked by Western blot analysis.Subsequently,the anti-orfA and anti-orfC rabbit serums were preliminarily applied to confirm the expression of orfA and orfC proteins in yeast and tumor cells.【Result】 The prokaryotic expression vectors of orfA and orfC were constructed and the expression was inducted successfully.The orfA and orfC recombinant fusion proteins were in the form of inclusion bodies.The method of hypodermic injection coupled with era vein booster immunization improved the antibody titers effectively.The expressions of orfA and orfC proteins in yeast and tumor cells were checked successfully with the anti-orfA and anti-orfC rabbit serums.【Conclusion】 The results showed that the polyclonal antibodies were prepared with high specificity and could be used in Western blot analysis for the expression of the target proteins in bacteria,yeast and mammalian cells.