Expression And Purification Of Hepatitis B Virus X Proteins Of Genotype B And C In Drosophila Expression System

Objective To express and obtain genotype B and C X Proteins of hepatitis B virus with high purity in Drosophila Expression System.Methords X genes of genotype B and C HBV were amplified seperately by means of PCR(Xb and Xc)and cloned into drosophila expression vectors pMT/BiP/V5/HisC in AgeI and BglII sites. The recombinant plasmids were co-transfected into S2 cells with the selection vector pCoBlast by Cellfectine. The drug resistant multiclonal cell lines were performed after blasticidin screening with the concerntration of 25μg/ml. Cells were cultured in large scale and the target proteins were expressed by the induction of CuSO4。Western-Blot analysis was used to identify the correctness of the target product and determine the optimal CuSO4 concentration and inducing interval. After expression in large scale, genotype B and C HBx with high purity were obtained by two different methods of purification.Results 1. The recombinant plasmids pMT/BiP-Xb-HisC and pMT/BiP-Xc-HisC , which harboring the genotype B and C HBx respectively, were constructed successfully. 2. The cell lines expressing HBx fusion proteins were performed. the optimal interval for CuSO4 induction was 72 h and the suitable inducing concentration of CuSO₄ was between 500μM and 1mM. 3. The HBx fusion proteins were obtained with the purity of 84.59% or 94.62% depending on different purifying methods used.Conclusion Genotype B and C HBx were sussessfully expressed in Drosophila expression system. The HBx fusion proteins with high purity were obtained, and it will be helpful for the further study on the structural and functional differences between genotype B and C HBx.