抗人红细胞单链抗体与猪瘟E2蛋白双功能融合蛋白的构建及生物学活性检测

Preparation and bioactivity of anti-human red blood cell ScFv and CSFV E2 bifunctional fusion protein

摘要:

为构建具有凝集性、免疫反应性的双功能融合蛋白，本研究采用重叠延伸PCR方法将2E8ScFv (抗人红细胞H抗原单链抗体基因) 和mE2 (猪瘟病毒E2蛋白主要抗原编码区基因) 拼接成融合基因2E8mE2，并插入原核表达载体pET-DsbA，将重组表达质粒pET-DsbA-2E8mE2转化入大肠杆菌Escherichia coli BL21 (DE3) PlysS中进行IPTG 诱导表达，表达的融合蛋白经SDS-PAGE和Western blotting分析鉴定，结果表明：2E8mE2融合基因在大肠杆菌中获得了表达，表达产物以包涵体形式存在，分子量约为65 kDa，与预期的大小一致。分别采用亲和层析法和谷胱甘肽再氧化法对融合蛋白进行纯化和复性，红细胞凝集试验证实：2E8mE2融合蛋白复性效果良好，既能够与人红细胞结合，又能够与猪瘟病毒抗体反应，具有双功能特性。

Abstract:

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.