Prokaryotic Expression of CSFV E0 Protein and Development of an Indirect ELISA for Detection of Antibody
Abstract: E0 protein of Classical swine fever virus(CSFV) is of discriminatory feature in serum diagnostic test for developing CSFV Marker vaccine. It is also expected to be useful for monitoring CSFV prevalence,evaluating immunization efficacy and establishing immunization procedure. To obtain a large amount of E0 protein in vitro ,the Hog cholera lapinised virus(HCLV) E0 gene was cloned into prokaryotic expression plasmids, pGEX4T、pET30 and pMAL-p2X respectively.After the recombinant expression plasmids were transformed into BL21、BL21(DE3)、TB1、BL21-codonplus(DE3)-RP respectively, the expression product were analyzed by investigating the IPTG induction concentration ,incubation temperature and time. The results revealed that the E0 protein could be expressed in the systems, namely pGEX4T/BL21-codonplus(DE3)-RP and pMAL-p2X/ BL21-codonplus(DE3)-RP as inclusion bodies accounted for 15% and 30% of whole bacterial protein. Two methods , B-per reagent and triton-urine, were applied to wash the inclusion body and both ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">access to the relatively good effect . After the protein refolding of denaturant inclusion body following dialysis , we got the pure recombinant GST2E0 protein by GST affinity columns. Using the purified protein as coating antigen , an indirect EL ISA is developed for detecting the anti2E antibody in t he CSFV serum by exploring the concent ration of coating antigen and dilution degree of serum , which will provide a good f undament for development of CSFV antibody surveillance kit .