抗HBsAg×抗CD3 diabody-cIFN原核表达及活性鉴定  

Construction and prokaryotic expression of a fusion protein for the human anti-HBsAg×anti-CD3 diabody-consensus interferon

作　　者：刘顺爱[1] 郭晶晶[1] 余康康[1] 刘志英[1] 冯鑫[1] 徐道振[1] 

机构地区：[1]北京地坛医院传染病研究所

出　　处：《免疫学杂志》2008年第4期404-407,共4页Immunological Journal

基　　金：北京市优秀人才培养资助;北京市卫生局科研基金;北京市自然科学基金(7042028)

摘　　要：目的构建人源抗乙型肝炎病毒表面抗原(HBsAg)×人源化抗CD3双特异双链抗体(diabody)与重组复合干扰素(consensus Interferon,cIFN)的融合蛋白(抗HBsAg×抗CD3 diabody-cIFN,简称S×3Db-cIFN)的表达载体,在原核表达系统中进行表达,并鉴定表达蛋白的活性。方法对相关的模板重组载体进行限制性内切酶消化和T4连接酶的连接,构建目的重组载体pRA-cIFNSHOL和pRA-OHSL,经测序鉴定后分别转化到大肠杆菌BL21(DE3)中诱导表达,用SDS-PAGE和Western blotting进行表达鉴定,用GuHCl阶段透析法重新折叠获得目的蛋白S×3Db-cIFN。用ELISA和流式细胞术进行S×3Db-cIFN的抗HBsAg抗体活性和抗CD3抗体活性。结果成功构建了表达载体pRA-cIFNSHOL和pRA-OHSL,并实现了大肠杆菌中的高效表达和表达后的重新折叠,获得了目的蛋白S×3Db-cIFN,蛋白复性率为24%。用ELISA和流式细胞术初步鉴定融合蛋白的活性,结果显示S×3Db-cIFN具有明显的抗HBsAg抗体活性和抗CD3抗体活性。结论用基因工程方法成功制备了人源(化)抗HBsAg×抗CD3 diabody与cIFN的融合蛋白,此融合蛋白具有抗HBsAg抗体活性和抗CD3抗体活性,有可能成为有效的抗HBV免疫导向药物,有待进一步探讨其应用价值。Objective To construct a expression vector for a fusion protein of the human （humanized） anti-HBsAg × anti-CD3 diabody-consensus interferon （cIFN） and identify the fusion protein. Methods The human anti-HBsAg scFv, the humanized anti-CD3 scFv, and the consensus interferon gene were prepared from pRA-cIFNSHSL, pRA-OHOL, and pRA-OH5L which previously reported. The obtained genes were digested by restriction endonucleases, and then inserted to the expression plasmid pRA to produce recombinant vector pRA-cIFNSHOL and pRA-OHSL. Then transformed the expression vector to E. coli strain BL21 （DE3） and the expressed protein was detected by SDS-PAGE and Western blotting. The anti HBsAg x anti CD3 diabody-clFN fusion protein named S × 3Db-cIFN was refolded by stepwise dialysis method. The biological activity of S × 3Db-cIFN was studied by using ELISA and flow cytometry. Results The S ×3Db-cIFN fusion protein was expressed in E. coli strain BL21 （DE3） harboring the plasmid constructed for expression of anti HBsAg scFv, anti CD3 scFv, and consensus interferon. SDS-PAGE and Western blotting showed that recombinant proteins were present in inclusion bodies mainly. Refolding was achieved by stepwise dialysis method, while the biological activity of refolded protein was estimated by ELISA and flow cytometry. These results showed that S × 3Db- cIFN fusion protein possessed immunological activity against HBsAg and CD3 antibodies. Conclusion A human （humanized） anti-HBsAg × anti-CD3 diabody-cIFN is produced by bacterial expression system in this study The fusion protein promises to be an important reagent for hepatitis B immunotherapy.

关 键 词：HBSAG CD3 双链抗体 cIFN 融合蛋白 

分 类 号：R392[医药卫生—免疫学]