FMDV整联蛋白受体b1亚基的克隆及其配体黏附区多抗的制备

Cloning of Foot-and-Mouth Disease Virus Integrin Receptor b1 Subunit and Antibody Production to Its Ligand-binding Domain

摘要:

采用RT-PCR技术从牛气管组织扩增出2400 bp的b1基因, 回收纯化连入PGEM-T载体, 测序。用Expasy软件对b1基因的抗原性进行分析, 选取胞外区334~861 bp的配体结合区与6×His融合, 在大肠杆菌中大规模诱导表达, 并经Ni2+亲和柱层析纯化。通过SDS-PAGE鉴定后, 应用纯化蛋白免疫新西兰家兔, 获得效价在1:12 800以上的多抗, Western blotting鉴定表明此抗体可特异性的与表达的融合蛋白作用。

Abstract:

We produced β1 gene which is about 2400 bp by reverse transcription polymerase chain reaction (RT-PCR) from bovine trachea, reclaimed and purified, then cloned the amplified fragment to pGEM-T easy vector, confirmed by sequencing. The immuno-dominant epitope of b1 gene was chosen by computer analysis and then syncretized ligand-binding domain from 346 bp to 843 bp of ecytoplasm with six histidine, expressed LBD protein massly in E. coli BL21 (DE3), and identified by SDS-PAGE. The fusion protein was purified with Ni-NTA affinity chromatography and immunized New Zealand rabbits preparing of its polyclonal antibody, the specific antibody titer was above 1:12 800 detected by indirect ELISA, the result of Western blot showed that this antibody could be recognized by LBD fusion protein.