环形泰勒虫Tams1单个B细胞抗体的制备及鉴定

Preparation and identification of single B cell antibody against Tams1 ofTheileria annulata

本研究旨在利用单个B细胞抗体技术制备环形泰勒虫Tams1蛋白特异性单克隆抗体并对其反应性进行鉴定。构建重组质粒pET-30a-Tams1,并进行蛋白原核表达与纯化。纯化的Tams1蛋白免疫健康黄牛,第4次免疫后结合流式分选技术,从外周血单个核细胞获得分泌抗Tams1特异性单克隆抗体的B淋巴细胞(Ig M-/CD21+/抗原+B淋巴细胞)。经单细胞裂解及反转录后,通过巢式PCR扩增获得Ig G天然配对的轻/重链可变区域序列,并克隆至包含Ig G轻/重链恒定区域的真核表达载体。配对质粒共转染HEK-293T细胞后,收集细胞培养上清,Western-blot结果显示所得8株抗体均能与Tams1蛋白结合。同时IFA结果证明8株抗体可与裂殖子表面抗原Tams1特异性结合。上述结果表明,本研究成功获得8株Tams1特异性单克隆抗体,为后续该蛋白的功能研究及单个B细胞抗体制备技术平台的建立奠定了基础。

The purpose of this study was to prepare specific monoclonal antibodies against Tams1 protein of Theileria annulata by single B cell antibody technology and to identify its reactivity.The pET-30a-Tams1 recombinant plasmid was constructed,and the protein was expressed and purified.The purified Tams1 protein was used to immunize healthy cattle.After the fourth immunization,B lymphocytes secreting anti-Tams1-specific monoclonal antibodies(IgM-/CD21+/antigen+B lymphocytes)were obtained from peripheral blood mononuclear cells by flow sorting.After single cell lysis and reverse transcription,the light/heavy chain variable region sequence of IgG natural pairing was obtained by nested PCR amplification and cloned into the eukaryotic expression vector containing the constant region of IgG light/heavy chain.After co-transfection of HEK-293 T cells with paired plasmids,the cell culture supernatant was collected.Western-blot results indicated that all the eight antibodies could bind to Tams1 protein.At the same time,IFA results showed that the eight antibodies could specifically bind to the merozoite surface antigen Tams1.Above results suggested that 8 strains of Tams1 specific monoclonal antibodies were successfully obtained in this study,which laid a foundation for the subsequent function study of the protein and the establishment of a single B cell antibody preparation technology platform.