人源抗HBsAg scFv与重组复合干扰素融合蛋白的高效表达及活性鉴定

目的：制备人源抗乙型肝炎病毒表面抗原(HBsAg)单链抗体(scFv)与重组复合干扰素(consensus interferon，cIFN)的融合蛋白，并鉴定其活性。方法：把人源抗HBsAg scFv-cIFN融合蛋白的表达载体pRA-cIFNscFv转化到大肠杆菌菌株BL21之后大量表达抗HBsAg scFv-cIFN融合蛋白。SDS-PAGE和Western blotting鉴定表达蛋白，经纯化并变性复性包涵体蛋白后用ELISA、流式细胞仪、MTS非放射性活细胞比色定量法及HBsAg血凝抑制试验等进行融合蛋白的抗HBsAg抗体活性和干扰素活性测定。结果：SDS-PAGE和Western blotting结果显示，抗HBsAg scFv-cIFN融合蛋白在BL21大肠杆菌中大量表达，表达量超过10mg/L培养基；ELISA和流式细胞仪结果显示，所表达的目的蛋白具有抗HBsAg和IFNα的免疫活性，且特异性良好；MTS和HBsAg血凝抑制试验结果显示，融合蛋白具有明显的PLC/PRF/5细胞增殖抑制活性和HBsAg分泌抑制活性，表明所获得的抗HBsAg scFv-cIFN融合蛋白具有抗HBV病毒活性和HBV感染细胞增殖抑制活性。结论：用基因工程方法成功制备了人源抗HBsAg scFv-cIFN融合蛋白，此融合蛋白具有抗HBsAg抗体活性和IFNα活性，有待深入探讨此融合蛋白的应用价值。

Expression and functional analysis of fusion protein for the human anti-HBsAg scFv antibody fragment-consensus interferon

Objective:Express a human anti-HBsAg single chain antibody fragment(scFv)-consensus interferon (cIFN) fusion protein by bacterial expression system and analyse the function of the fusion protein.Methods:Human anti-HBsAg single chain monoclonal antibody cDNA encoding the variable regions of immunoglobulin from PBMC of Hepatitis B patient. Consensus interferon gene was produced by overlap PCR.A plasmid for production of cIFN-scFV fusion protein was constructed, then the expression vector pRA cIFN-scFV transformed with the E.coli strain BL21(DE3). The gene product was analysed SDS-PAGE and Western blotting, then was solubilized by guanidine hydyochloride, refolded by conventional dilution method, and purified using metal-chelating chromatography. The immune and functional analysis of the resulting fusion protein have been studied by ELISA,FACS(Flow cytometry),MTS assay and hemaglutination inhibition test.Results:The authors isolated and characterized the human anti-HBsAg single chain antibody fragment(scFv)-consensus interferon (cIFN) fusion protein. The resulting human anti-HBsAg scFv-cIFN fusion protein was bound to react with HBsAg and cIFN, this react show that highly specific and bioactivity.Conclusion:A human anti-HBsAg single chain antibody fragment(scFv)-consensus interferon (cIFN) fusion protein was produced by bacterial expression system in this study. This genetically engineered human anti-HBsAg scFv-cIFN fusion protein promises to be an important reagent for hepatitis B immunotherapy.