摘要
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 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 BI21 （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.
出处
《军事医学科学院院刊》
CSCD
北大核心
2007年第6期505-508,共4页
Bulletin of the Academy of Military Medical Sciences
关键词
单链抗体
狂犬病毒
活性
原核表达
single-chain variable fragments
rabies virus
activity
prokaryotic expression
