College of Life Science, Jilin Agricultural University, Changchun, China.
Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, China.
PLoS One. 2022 Mar 31;17(3):e0266220. doi: 10.1371/journal.pone.0266220. eCollection 2022.
We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. Further analysis demonstrated that TAT-HuScFv could inhibit the hemagglutination activity of the 300 TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.
我们基于 H5N1 禽流感病毒（A/猫鼬/上海/SH-1/2012）的 M1 蛋白开发了一种人类细胞内抗体，并对该抗体的特性进行了表征。使用 H5N1 病毒的 cDNA 作为模板，通过 RT-PCR 扩增 M1 蛋白序列，在细菌表达系统 BL21（DE3）中表达并纯化。通过使用 M1 作为抗原的噬菌体抗体文库筛选，获得了针对 M1 蛋白的人类高亲和力单链抗体（HuScFv）。通过融合 HIV 的 TAT 蛋白转导结构域（PTD）基因表达重组 TAT-HuScFv 蛋白，制备针对禽流感病毒的人类细胞内抗体。进一步分析表明，TAT-HuScFv 可以抑制 300 TCID50 H1N1 病毒的血凝活性，从而初步验证了该抗体的通用性。经过两轮 M1 蛋白分解，鉴定出 TAT-HuScFv 抗原结合位点为 239 位的丙氨酸（A）。综上所述，我们的数据描述了一种针对禽流感病毒保守序列具有高结合活性的重组抗体。这种细胞内重组抗体可阻断感染细胞内病毒的 M1 蛋白，从而抑制 H5N1 病毒的复制和繁殖。
