旋毛虫Ts-Sp-7蛋白的表达纯化及其多克隆抗体的制备

Expression and purification of Trichinella spiralis Ts-Sp-7protein and preparation of its polyclonal antibody

为给旋毛虫病的诊断及疫苗研制提供备选抗原,对本实验室获得的旋毛虫Ts-Sp-7基因(GenBank登录号EU263332.1)进行PCR扩增,构建原核表达载体,进行表达纯化,Western-blot分析。制备多克隆抗体进行间接免疫荧光分析。通过PCR扩增出1372 bp的Ts-Sp-7基因。SDS-PAGE结果显示,该蛋白大小为46 ku左右,与理论分子质量一致。Western-blot结果显示,该目的蛋白与10000条/头感染剂量不同感染时间的猪阳性血清均呈现特异性条带。制备的多克隆抗体效价为1∶350000。Ts-Sp-7多克隆抗体的间接免疫荧光鉴定结果显示,Ts-Sp-7蛋白在旋毛虫肌幼虫期存在于虫体表面。

To afford alternative antigenic materials for the diagnosis and vaccine development of trichinellosis,PCR amplification of Trichinella spiralis Ts-Sp-7 gene(GenBank:EU263332.1)was performed and the prokaryotic expression vector was constructed,expressed,purified and identified by Western-blot.The polyclonal antibody was prepared,and analyzed by indirect immunofluorescence assay.The Ts-Sp-7 gene of about 1372 bp was amplified by PCR.The SDS-PAGE result showed that the size of the protein was about 47 ku,which was consistent with the theoretical molecular weight.Western-blot results showed that the target protein showed specific bands with the serum samples from swine infected with 10000 T.spiralis at different days post infection(dpi).The polyclonal antibody made by using the recombinant protein has a titer of 1∶350000.Indirect immunofluorescence analysis using Ts-Sp-7 polyclonal antibody revealed that the protein was present on the surface of the worm in the muscle larvae.