Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

抗SARS-CoV-2刺突糖蛋白受体结合域单链抗体的筛选及鉴定

目的 构建抗严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-Co V-2)刺突糖蛋白(spike protein,S)受体结合域(receptor-binding domain,RBD)的单链抗体(single-chain fragment variable,scFv)噬菌体展示文库,筛选特异性scFv,并进行功能鉴定。方法 提取RBD蛋白免疫小鼠的脾细胞m RNA,逆转录为c DNA,以其为模板扩增scFv的重链可变区(hight chain fragment of variable,VH)和轻链可变区(light chain fragment of variable,VL)基因,经过重叠延伸PCR扩增(splicing overlap extension PCR,SOE-PCR)组装为scFv基因片段。将scFv基因片段插入噬菌体载体,构建scFv噬菌体展示文库。经4轮生物淘洗,筛选出与RBD结合能力较强的scFv基因,进行重组表达、纯化和生物活性鉴定。结果 构建的scFv噬菌体文库滴度为6.0×10^(11)pfu/m L。经4轮生物淘选后,共筛选出4株与RBD结合较强的scFv,分别为scFv11、scFv12、scFv25、scFv28。scFv相对分子质量约为27 000,多以包涵体形式存在,纯化后可与HRP标记的鼠抗His单克隆抗体发生特异性结合,浓度达2.4 mg/m L,纯度约90%。4株scFv均可与RBD重组蛋白发生特异性结合;除scFv28外,其他3株scFv均可与野生型和多突变株S蛋白结合;与RBD均存在剂量依赖性相互作用,亲和力动态拟合解离常数(K_(D))分别为8.9、5.92、10.67、2.36 nmol/L,稳态拟合解离常数(K_(D))分别为5.3、6.5、8.7、5.8 nmol/L;scFv11、scFv12和scFv25可同时识别3个独立的RBD多肽,包括RBD2(S^(334~353))、RBD9(S^(439~458))和RBD13(S^(499~518));在线服务器SWISS-MODEL对scFv进行同源建模的模型质量良好,可用于分子对接;scFv11及人肺泡上皮细胞表面血管紧张素转换酶2(human angiotensin converting enzyme 2,ACE2)与RBD相互作用的界面仅部分重合,scFv12和scFv25与RBD的相互作用界面和ACE2与RBD相互作用界面存在较大差异。结论 本研究通过构建抗SARS-Co V-2 RBD的scFv噬菌体文库,筛选并制备了可与SARS-Co V-2 RBD特异结合的scFv,为进一步抗SARS-Co V-2药物和检测试剂的研发提供了实验依据。