Expression Of Classical Swine Fever Virus E0 Gene In The Prokaryotic And Eucaryotic Expression Systems

The glycoprotein EO of Classical swine fever virus(CSFV),besides being an envelope protein, possesses KNase activity,which is pertinent to viral persistent infection in the host. Since the important roles of EO protein in the viral infection .immunity and virus-host interaction.the homology of 21 CSFV strains was investigated by sequence analysis of EO genes in this study,which will provide some evidence for epidemiological study.In addition, the EO gene of hog cholera lapinized vaccine(HCLV) strain was expressed in the prokaryotic and eucaryotic systems ,and the recombinant proteins were preliminarily analyzed by immunological method.Firstly,the EO genes of CSFV-JL and CSFV-LN9912 strains were amplified by RT-PCR and nested PCR and then sequenced after cloned into T easy vector. Comparing the EO sequences with other strains EO by biosoftwares , DNAStar5.0 and Bioedit,the phylogenetic analyse revealed that all of the strains we have sequenced could be divided into groupl and groupII.Two amino acid streches of 15 CSFV strains EO,which form the RNase active site,and Histidine residues essential for RNase catalysis in both ones were highly conserved.The EO protein propertys of antigen epitope,hydrophobicity,isoelectric point were also predicted by bioinformatic method.Secondly, LN9912 EO gene was cloned into pGEX4T, pETSO and pMAL-p2X respectively, with strains BL21 and BL21 (DE3) codon plus, BL21(DE3) and BL21 (DE3) codon plus, TB1 and BL21 C DE3) codon plus as their corresponding expression hosts.The EO gene could be successfully expressed in the systems,namely pGEX4T/BL21 (DE3) codon plus and pMAL-p2X/BL21 (DE3) codon plus, by investigating the IPTG induction concentration .incubation temperature and time. The expression protein products as insoluble inclusion bodies accounted for 15% and 30% of whole bacterial protein. Two methods , B-per reagent only and triton-urine with sonication lysis of cells,were both access to the relatively good washing effect. After the protein refolding of denaturant inclusion body following dialysis,we got the pure recombinant GST-EO protein by GST affinity columns.Using the purified protein as coating antigen ,an indirect ELISA were developed for detecting the anti-EO antibody in the CSFV serum by exploring the concentration of coating antigen and dilution degree of serum.In order to get the soluble recombinant EO protein and inspect the protein expression status convinently,the EGFP and EO gene were ligated into baculovirus transfer vector .With the co-transfecting sf9 cells of baculovirus recombinant transfer vector and linearized viral DNA, and plaque purification in the posttransfection procedure,the pure recombinant baculovirus were harvested ,whichinfected the sf9 cells for amplifying to generate a P-l stock..In the meantime,the fluorescence microscopy detection indicated expressed EGFP protein to confirm the heterogenous protein expression of recombinant baculovirus.The PI-stock from a pure plaque was used to generate a high liter P-2 stock,which was determined in liter as 1.14 107pfu/ml by performing a plaque assay.When a volume of P-2 stock infected the sf9 cells with MOI 5-10 for expression,the strong fluorescence was obeserved on the day 3 of postinfection.Furthermore, the EGFP-EO gene was also cloned into pCI-dhfr,a CHO (dhfr") cell expression vector.Following the transfection and MTX selection ,some CHO cells presented green fluoresence,which will establish a good foundation for production of soluble EO glycoprotein and investigating its biological functions.