关键词: 刚地弓形虫, 缓殖子期抗原1, 克隆, 表达, 单克隆抗体

Abstract:

Objective To prepare monoclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (BAG1) and explore its application in identification of T. gondii bradyzoite. Methods The bradyzoites of T. gondii ME49 strain (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 10⁶ monoclonal 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 (M_r) 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 of 1 ∶ 25 600, and the antibody subtype was IgG1. Western blotting showed that the mAb4B04 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 mAb4B04 antibody with the highest titer can specifically recognize BAG1 protein and bradyzoite of T. gondii ME49 strain.

Key words: Toxoplasma gondii, Bradyzoite antigen 1, Clone, Expression, Monoclonal antibody