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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。
OBJECTIVE: To express ScFv, a novel human anti-rabies glycoprotein scFv, using Escherichia coli and verify its activity. Methods: ScFv gene was obtained by gene fusion. The recombinant plasmid pET-22b (+) - ScFv was constructed and transformed into E. coli BL21 (DE3). The recombinant plasmid was induced by IPTG. Results: Western blotting showed that the target protein was expressed correctly, and the expressed product was in the form of inclusion body. The purified protein was purified by Ni-NTA column and refolded in vitro to obtain the ScFv protein with purity of 90%. The results of ELISA showed that the binding stability of ScFv in PBS and human serum was improved. Flow cytometry confirmed that the target protein could bind to rabies virus. The neutralization titer of ScFv was 40 U / mg. Conclusion: The prokaryotic expression system was successfully used to express human anti-GPRV ScFv and had certain neutralizing activity.