抗大豆胰蛋白酶抑制剂单链抗体表达与纯化方法优化  

Improvement of expression and purification method of anti-Bowman-Birk inhibitor(BBI) ScFv

作　　者：刘玉婷[1] 王琼[1] 谢亮[1] 程小玲[1] 彭静[1] 屈品均 辛化伟[1] 

机构地区：[1]武汉科技大学生物医学研究院,湖北武汉430070

出　　处：《生物技术》2016年第3期287-292,共6页Biotechnology

基　　金：国家自然科学基金项目("端粒与端粒酶联系的分子调控机制及其在抗肿瘤中的潜在应用";No.31371385)

摘　　要：[目的]利用大肠杆菌毒素蛋白VTB的五聚体化结构域（VT1B）及梭菌纤维素酶的纤维素结合结构域（CBD）,异源表达其与抗大豆胰蛋白酶抑制剂单链抗体（anti-BBI Sc Fv）的融合蛋白,对该单链抗体表达与纯化方法进行优化。[方法]构建CBD-VT1B-anti-BBI Sc Fv融合蛋白大肠杆菌表达系统;应用Ni-NTA树脂与微晶纤维素两步亲和层析法提纯单链抗体融合蛋白。[结果]单链抗体融合蛋白在大肠杆菌中得到大量表达,通过两步纯化法获得了高纯度的融合蛋白,纯度比Ni-NTA树脂一步纯化法提高约12~34%;五聚体融合蛋白与纤维素的结合能力及蛋白纯化效率比单体蛋白提高约1倍。[结论]通过增加纤维素标签和五聚体化结构域,抗大豆胰蛋白酶抑制剂五聚体化单链抗体融合蛋白的纯化效果得到提高。[ Objective] The N-terminus pentamerization domain of the E. coli verotoxin B （VT1 B） and cellulose binding do- main（CBD） of the Clostridium cellulase was used to express the fusion protein of anti-Bowman-Birk inhibitor （anti-BBI） single chain fragment variant （ScFv） heterologously for improvement of its expression and purification method. [ Methods] The ScFv against the soy bean Bowman-Birk inhibitor （anti-BBI） was fused to VT1B domain,and the fusion construct was cloned into a prokaryotic expression vector in-frame with the cellulose-binding domain. The expressed fusion protein was purified through Ni- NTA and cellulose chromatography. [ Results ] The ScFv fusion proteins was highly expressed in bacteria, and a relatively puri- fied product was obtained with the two-step tandem purification method. Compared to the single step purification with Ni-NTA resin,the tandem purification method led to an increase of fusion protein purity by approximately 12 -34%. Pentamerization of the fusion protein led to an increase if its cellulose-binding capacity, and therefore, protein purification efficiency by approxi- mately 2 times. [ Conclusion ] By adding the VT1B domain and CBD domain, the purification of anti-Bowman-Birk inhibitor （BBI） ScFv was improved.

关 键 词：抗大豆胰蛋白酶抑制剂单链抗体(anti-BBI ScFv) 五聚体化结构域(VT1B) 纤维素结合结构域(CBD) 

分 类 号：Q789[生物学—分子生物学]