人源化抗Cry1B毒素蛋白单链抗体的原核表达及生物学活性测定

Eecretory expression and characterization of humanized anti-Cry1B scFv antibody in Escherichia coli

利用含有重组噬菌粒的噬菌体直接侵染大肠杆菌(Escherichia coli)HB2151,原核分泌表达了抗苏云金芽孢杆菌(Ba-cillus thuringiensis,Bt)Cry1B毒素蛋白的单链抗体(single chain-variable fragment,scFv),经纯化、鉴定抗原结合活性后,建立了Cry1B毒素蛋白的ELISA检测方法。方法以展示scFv抗体的重组噬菌体感染E.coli HB2151,经PCR和基因测序检测克隆scFv基因片段的完整性,用SDS-PAGE方法检测scFv在E.coli HB2151宿主菌中的表达水平,用ELISA测定法检测scFv的抗原结合活性,通过时间梯度优化获得可溶性表达蛋白的最佳培养时间。结果表明:经PCR、DNA电泳及基因测序等,均证实重组噬菌体对E.coli HB2151宿主菌侵染成功,SDS-PAGE电泳结果表明scFv抗体表达成功,纯化后的蛋白质量浓度为132μg.mL-1。以纯化的scFv蛋白为基础建立了对Cry1B毒素蛋白的间接竞争ELISA法,方法的抑制中质量浓度(IC50)为1.398μg.mL-1,最低检测限(IC10)为0.025 7μg.mL-1,线性检测范围为0.5～5.0μg.mL-1,scFv对Cry1C的交叉反应率为7.51%;与Cry1Ab、Cry1Ac的交叉反应率均小于0.1%;在培养温度30℃,1 mmol.L-1IPTG诱导条件下,scFv在E.coliHB2151宿主中的最佳诱导表达时间为12 h。本研究成功地将抗Cry1B毒素蛋白的scFv在E.coli HB2151中进行了可溶性表达,获得了具有抗原结合活性的scFv融合型抗体,为实际生产应用与试剂盒研发提供了基础。

In this study, single chain variable fragments （scFv）against Bacillus thuringiensis Cryl B were expressed by E. coli HB2151 in a soluble format, and based on that, a approach of in-directive sandwich ELISA was developed for the detection of Cryl B, and could be exploited in the transgenic crops testing with further study. Phage clones displaying scFv fragment against Cryl B were selected from Tomlinson J phage display library, after E. coli HB2151 cell infection, induction and expression, the scFv antibody presented in a soluble form was secretory expressed and purified, the antigen recognizing ability was confirmed by ELISA, and the integrity of anti-Cryl B scFv gene and its secretory expression in E. coli was identified by PCR, sequence analysis and SDS-PAGE. The results demonstrated that the selected clones had the antibody encoding genes inserted correctly, in the optimized culture condition of 30 ℃, 1 mmol. L-1 IFFG and culture 12 h, the soluble scFv antibody could be expressed successfully, and the concentration of purified scFv could reach to 132 μ.mL-1. An indirect competitive ELISA was developed based on the purified scFv antibody for Cryl B determination, the concentration of Cryl B ranged from 0.5 p.g-mL-1 to 5.0μg.mL-1, the IC50 and the detection limit（ IC10 ）of this approach reached to 1. 398 μg.mL-1 and 0.025 7 μg. mL-1 ,respectively, and the cross-reactivity for CrylC was 7.51% ,and for CrylAb,CrylAc was less than 0. 1%.