人源抗狂犬病毒G蛋白单链抗体的表达和鉴定

Expression and Identification of Human Single Chain Antibody(ScFv) against Glycoprotein Antigen of Rabies Virus

目的对人源抗狂犬病毒G蛋白单链抗体A12进行可溶性表达及特异性和中和活性检测。方法表达人源单链抗体A12的菌株,经诱导后进行SDS-PAGE及Western blot检测,用流式细胞仪(FACS)检测表达狂犬病毒G蛋白的重组痘苗病毒感染Vero细胞的能力,用快速荧光灶抑制实验(RFFIT)检测表达产物的中和活性。结果Western blot显示表达产物与抗C-myc抗体在相对分子质量约30 000处出现强阳性表达带。FACS表明A12与表达狂犬病毒G蛋白的重组痘苗病毒感染的Vero细胞出现特异性反应。RFFIT表明,A12 ScFv在1∶27倍稀释时能完全中和病毒。结论已获得了A12可溶性表达,表达产物能与狂犬病毒G蛋白特异性结合,并对狂犬病毒具有一定的中和活性。

Objective To achieve the soluble expression of human A12 ScFv against glycoprotein antigen of rabies virus and determine the specificity and neutralizing capacity of expressed product. Methods The recombinant strain HB2151 containing A12 ScFv gene was induced by IPIG.The expressed product was identified by SDS-PAGE and Western blot.Determine the binding capacity of A12 ScFv to the Veto cells infected with recombinant vaccinia virus expressing the glycoprotein of rabies virus by FACS, and the neutralizing capacity of expressed product by RFFIT.Results SDS-PAGE showed an expression band with relative molecular weight of 30 000, Western blot showed reaction of the expressed product with antibody against C-myc. FACS showed specific reaction of A12 ScFv with the Veto cells infected with recombinant vaccinia virus expressing the glycoprotein of rabies virus. RFFTT proved that A12 ScFv at a dilution of 1：27 completely neutralized rabies virus. Conclusion The soluble expression of A12 ScFv was achieved, The expressed product could bind to the glycoprotein antigen of rabies virus and showed a certain neutralizing capacity to the virus.