新型重组人源抗狂犬病毒糖蛋白单链抗体的制备及活性分析

目的：利用大肠杆菌表达新型人源抗狂犬病毒糖蛋白单链抗体ScFv,并验证其活性。方法：采用基因融合获得ScFv基因,构建重组表达质粒pET-22b（＋）-ScFv,转化大肠杆菌BL21（DE3）,经IPTG诱导获得高效表达。结果：Western印迹显示目的蛋白表达正确,表达产物以包涵体形式存在,经Ni-NTA柱纯化和体外复性,获得纯度达90%的ScFv蛋白。ELISA结果显示在PBS及人血清中ScFv的结合稳定性有所提高,流式细胞术证明目的蛋白能靶向结合狂犬病毒,通过中和效价测定实验测得ScFv的中和效价为40 U/mg。结论：成功利用原核表达系统实现了对人源抗GPRV ScFv的表达,并且具有一定的中和活性。

Preparation and bioassay of novel human anti-GPRV ScFv in E. coli

Objective:To clone and express the human anti-glycoprotein of rabies virus(GPRV) ScFv in E. coli and identify the activity of the protein for further investigation. Methods:The full-length gene of ScFv was generated by SOE and the recombinant expression vector pET-22b ( )-ScFv was constructed. The pET-22b( )-ScFv gene was cloned into E. coli BL21(DE3) , induced by IPTG to express target protein. Results:The protein was identified by Western blot. ScdsFv was expressed as inclusion body. About 90% purity of ScFv was obtained following renafuring and purifying via Ni-NTA. Flow cytometry(FCM) test showed that ScFv can combine with target GPRV, and the RFFIT showed that the neutralizing titer of ScFv was 40 U/mg. Conclusion; ScFv could be successfully expressed in the prokaryotic expression system, and the neutralizing activity was positive.