He Fang-Ping, Lin Qing-Shan, Li Shao-Wei, Wei Min-Xi, Chen Zhen-Qin, Luo Wen-Xin, Chen Yi-Xin, Zhang Jun, Xia Ning-Shao
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, Xiamen 361005, China.
Bing Du Xue Bao. 2011 May;27(3):202-6.
In our previous study, a panel of 52 broadly cross-reactive H5-specific monoclonal antibodies (MAbs) were generated and characterized. The 13D4, one of these MAbs, has been demonstrated to protect mice against lethal challenge by 4 strains of H5N1 avian influenza virus representing the currently prevailing genetic populations, clades 1, 2.1, 2.2, and 2.3. Here, we further cloned the gene of the 13D4 MAb and constructed a single-chain variable fragment. Then, the 13D4 single-chain antibody (scFv) was expressed in secretory maner in Pichia pastoris. The supernatant of the culture was concentrated and subjected to ammonium sulfate precipitation. The purity of the 13D4 scFv was around 90% in SDS-PAGE following ion-exchange chromatography. We further investigated its binding property using hemagglutination inhibition (HI) test and blocking ELISA. The results indicated that the 13D4 scFv shared the same binding sites and comparable HI titer with the prototype murine 13D4 Mab. In conclusion, an anti-H5 single-chain wide-spectrum neutralizing antibody is prepared successfully in yeast system.
在我们之前的研究中，制备并鉴定了一组52种具有广泛交叉反应性的H5特异性单克隆抗体（MAb）。其中一种单克隆抗体13D4已被证明能保护小鼠免受代表当前流行基因群体（进化枝1、2.1、2.2和2.3）的4株H5N1禽流感病毒的致死性攻击。在此，我们进一步克隆了13D4单克隆抗体的基因并构建了单链可变片段。然后，13D4单链抗体（scFv）在毕赤酵母中以分泌方式表达。培养上清液经浓缩后进行硫酸铵沉淀。离子交换色谱后，13D4 scFv在SDS-PAGE中的纯度约为90%。我们进一步使用血凝抑制（HI）试验和阻断ELISA研究了其结合特性。结果表明，13D4 scFv与原型鼠源13D4单克隆抗体具有相同的结合位点和相当的HI效价。总之，在酵母系统中成功制备了一种抗H5单链广谱中和抗体。
