刚地弓形虫抗缓殖子期抗原1单克隆抗体的制备与应用

Preparation and application of monoclonal antibody against Toxoplasma gondii bradyzoite antigen 1

目的 制备弓形虫抗缓殖子期抗原1 (BAG1)的单克隆抗体,探究其在弓形虫缓殖子鉴定上的应用。方法分别收集碱性培养基(pH 8.2)诱导5 d后的弓形虫ME49虫株(体外诱导)和慢性感染后2个月的小鼠脑组织匀浆中的ME49虫株(体内形成)。提取体外诱导的弓形虫ME49虫株缓殖子总RNA, RT-PCR扩增bag1开放读码框片段,亚克隆至原核表达载体pET-28a (+),转化至BL21 (DE3)大肠埃希菌,用1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达BAG1,采用Ni-NTA柱纯化重组蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测重组蛋白BAG1表达情况。取6只BALB/c小鼠,皮下注射纯化的重组BAG1蛋白(20μg/鼠) 3次后(首次免疫用弗氏完全佐剂,后两次免疫用等量弗氏不完全佐剂,每隔2周免疫1次),选择血清抗体效价高的小鼠,取脾组织分离脾细胞,与SP2/0骨髓瘤细胞融合后,用纯化的BAG1蛋白经间接ELISA筛选阳性杂交瘤细胞,经多次亚克隆筛选稳定分泌抗BAG1单克隆抗体的杂交瘤细胞株,收集培养上清,间接ELISA法检测抗体效价和抗体亚型。取BALB/c小鼠4只,液体石蜡腹腔注射(0.5 ml/鼠) 1周后,腹腔注射单克隆杂交瘤细胞106个/鼠,2周后收集腹水,亲和色谱法纯化抗BAG1的单克隆抗体。制备体外诱导和体内形成的弓形虫裂解蛋白,以纯化后的抗BAG1单克隆抗体为一抗,蛋白质免疫印迹(Western blotting)检测ME49虫株中BAG1蛋白的表达情况,间接免疫荧光试验(IFA)检测ME49虫株缓殖子情况。结果RT-PCR扩增获得bag1的开放读码框片段,长690 bp,编码229个氨基酸。重组质粒pET-28a (+)-bag1经菌落PCR和测序后鉴定正确。SDS-PAGE结果显示,重组BAG1蛋白相对分子质量(Mr)约为27 000,以可溶性蛋白和包涵体形式表达。间接ELISA检测结果显示,纯化的BAG1蛋白免疫小鼠3次后,血清抗体平均效价可达1∶102 400。融合筛选获得4株阳性单克隆杂交瘤细胞株,分别为mAb1H03、 mAb4B04、 mAb5H07、 mAb8B12,以mAb4B04单克隆抗体效价最高,达1∶25 600,该抗体亚型为IgG1型。Western blotting结果显示,mAb4B04单克隆抗体可识别体外诱导和体内形成的ME49虫株BAG1蛋白。IFA结果显示,mAb4B04单克隆抗体可检测到体外诱导和体内形成的ME49虫株缓殖子。结论 制备了抗弓形虫BAG1的单克隆抗体,其中效价最高的mAb4B04单克隆抗体能特异性识别弓形虫ME49虫株的BAG1蛋白和缓殖子。

Objective To prepare monoclonal antibodies against Toxoplasma gondii bradyzoite antigen 1(BAG1and explore its application in identification of T. gondii bradyzoite. Methods The bradyzoites of T. gondii ME49strain(in vitro induced) after induction in alkaline medium(pH 8.2) for 5 days and the ME49 strain obtained from mouse brain tissue homogenate(in vivo formed) 2 months after chronic infection were collected. Total RNA was extracted from the in vitro induced T. gondii ME49 bradyzoite and amplified by RT-PCR for the bag1 open reading frame segment, which was subcloned into the prokaryotic expression vector pET-28a(+) and transformed into BL21(DE3) Escherichia coli. The recombinant E. coli was induced by 1 mmol/L IPTG to express BAG1 protein, which was purified by Ni-NTA column, and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Six BALB/c mice were immunized subcutaneously with 20 μg purified BAG1 protein three times(Freunds complete adjuvant was used for the primary immunization, and the same amount of Freunds incomplete adjuvant was used for two boosting at an interval of two weeks). The mice with a high antibody titer were selected to collect spleen tissues for separating splenocytes, which were fused with SP2/0 myelomacells. The positive hybridoma cells were screened by indirect ELISA using purified BAG1 protein, and then underwent multiple subcloning screening for the hybridoma cell line presenting stable secretion of monoclonal antibody against BAG1. The supenant of the hybridoma cell line was collected, of which the antibody titer and sub-typing were determined by indirect ELISA. Four BALB/c mice were injected intraperitoneally with 0.5 ml liquid paraffin for one week, further intraperitoneal injection was delivered with 106monoclonal hybridoma cells/mouse. The mice ascites was collected and the monoclonal antibody against BAG1 was purified by affinity chromatography. Lysed protein was prepared from the in vitro induced and in vivo formed ME49 toxoplasma, of which the BAG1 expression was examined by Western blotting using the purified anti-BAG1 monoclonal antibody as the first antibody, and the bradyzoite status was checked by indirect immunofluorescence assay(IFA). Results The open reading frame fragment of bag1 was obtained by RT-PCR, which was 690 bp, encoding 229 amino acids. The recombinant plasmid pET-28a(+)-bag1 was identified by PCR and subsequent sequencing. SDS-PAGE results showed that the relative molecular weight( Mr) of the recombinant BAG1 protein was about 27 000, which was expressed in the form of soluble protein and inclusion body. The results of indirect ELISA showed that the average titer of serum antibody could reach 1 ∶ 102 400 after immunizing mice with purified BAG1 protein for 3 times. Four positive monoclonal hybridoma cell lines were obtained by fusion screening,which were mAb1H03, mAb4B04, mAb5H07, and mAb8B12, respectively. mAb4B04 clone had the highest titer of1 ∶ 25 600, and the antibody subtype was IgG1. Western blotting showed that the m Ab4B04 antibody could detect the BAG1 protein of ME49 strain induced by the alkaline medium in vitro and the BAG1 protein of ME49 strain from mouse brain homogenate. IFA results showed that the mAb4B04 antibody could successfully detect ME49 bradyzoites induced by alkaline medium in vitro and in the BAG1 protein of ME49 strain in the mouse brain homogenate. Conclusion Monoclonal antibodies against T. gondii BAG1 were produced, among them the m Ab4B04antibody with the highest titer can specifically recognize BAG1 protein and bradyzoite of T. gondii ME49 strain.