关键词: 刚地弓形虫, 棒状体蛋白2, 多克隆抗体, 免疫荧光定位

Abstract:  【Abstract】 Objective  To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization.  Methods  The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21（DE3） and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 μg purified recombinant ROP2 mixing with the same volume of Freund′s adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method.  Results  The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1 ∶ 102 400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane.  Conclusion  The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.

Key words: Toxoplasma gondii, ROP2, Polyclonal antibody, Immunofluorescence localization