High Expression Of CSFV E2 Antigen In E.coli And The Preparation Of The Expressed Protein

In order to gain high expression in E.coli the full CSFV E2 gene was modified by replacing its six rare codons with E.coli biased ones and then cloned into expressing vector pET-28(a). The resulted expression plasmid, referred as pETE2(SW) was used in transformation of E.coli strain BL21(DE3)pLysS and eventual E2 expression in the form of inclusion body at the level high up to 28% of total bacterial protein was gained after induction with IPIG.For the sake of preparing genetic engineered antigen of CSFV above E2 expressed as insoluble form of inclusion body was prepared by sonication of the bacterial cells and then subjected to denaturation in 8 mole/L urea. Meanwhile shrunk E2, referred as mE2 which is 1/3 part at N terminal of intact E2 containing the major antigenic region and was engineered in previous study was also prepared in the same way. Immoble metal affinity chromatography (IMAC) and QAE-Sephadex A50 ion-exchange chromatography were compared for the efficiency of purifying E2 protein after denaturation. The result showed that QAE-Sephadex A50 ion-exchange was better in purity and recovery of final product than that of IMAC. Afterward the optimized protocol was established for renaturation of ion-exchange purified E2 product and denatured mE2 applying glutathione reoxidation resulting in a reasonable renaturation efficiency of 70% for E2 and 19%-39% for mE2. The renaturated product was further concentrated by precipitation using ammonium sulphate or PEG20000 and the soluble E2 and mE2 proteins were prepared. In order to obtain higher purity the renatured protein (E2 and mE2) were further purified by IMAC and immunoaffinity chromatography, and the purity of mE2 and E2 proteins eventually reached 97% and 90% respectively. Compared with proteins in denaturation, the reactivitypanel of CSFV specific monoclonal and polyclonal antibodies.In addition E2 gene was cloned into pEZZ18 protein A gene fusion vector resulting in the construction of the recombinant plasmid E2/PEZZ18. After transformation E.coli HB101 could produce soluble E2 protein as evidenced by Western bloting.