弓形虫SAG1重组抗体制备及抗原表位鉴定

Preparation of Toxoplasma gondii SAG1recombinant antibody and identification of its epitope

重组抗体相较于多克隆抗体和杂交瘤单克隆抗体具有更好的稳定性和特异性,本研究以弓形虫表面抗原1(surface antigen 1,SAG1)为靶标制备了重组抗体。将弓形虫SAG1(45~198aa)的基因序列被克隆到pET-32a原核表达质粒,转化大肠杆菌BL21感受态细胞后获得弓形虫SAG1(45~198aa)重组蛋白。重组蛋白经镍柱纯化后通过皮下注射免疫BALB/c小鼠,取免疫后小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0融合,制备SAG1单克隆杂交瘤。通过兼并引物克隆杂交瘤抗体的重链和轻链序列得到重组抗体表达质粒,将重组抗体表达质粒转染HEK293T细胞获得重组抗体。通过pET-32a和pCMV-EGFP质粒表达SAG1多肽片段鉴定重组抗体识别的SAG1抗原表位。结果显示,免疫印迹和免疫荧光试验确定SAG1重组抗体的特异性,SAG1重组蛋白的抗原表位序列为SAG1(84~90aa)。本研究展示了一种简便的重组抗体制备过程,可为其他蛋白的抗体制备提供借鉴,制备的重组抗体具有详细抗原表位信息,有望成为弓形虫诊断的工具抗体。

Compared with polyclonal antibody and hybridoma monoclonal antibody,recombinant antibody is more popular for its stability and specificity.A recombinant antibody against Toxoplasma gondii(T.gondii)surface antigen 1(SAG1)was generated in the present study.SAG1(45-198aa)recombinant protein was prepared via prokaryotic expression system.BALB/c mice were immunized with purified recombinant protein by subcutaneous injection.Spleen cells of immunized mice were harvested and fused with mouse myeloma SP2/0cells to generate a hybridoma against SAG1.Heavy chain and light chain sequences of hybridoma were cloned into plasmid(Addgene ID:28217),recombinant antibodies were obtained from culture supernatant of HEK293Tcells transfected with the plasmid.The epitope of recombinant antibodies was verified by Western blot against SAG1polypeptides expressed via pET-32aand pCMV-EGFP.Results showed that the recombinant antibody worked well in Western blot and immunofluorescence experiments,and the epitope recognized by the recombinant antibody was defined as SAG1(84-90aa).This study provides a simple way to generate recombinant antibody,the recombinant antibody generated against SAG1(84-90aa)may be used for diagnosis of T.gondii infection in the future.