关键词: 刚地弓形虫, 单链抗体, 绿色荧光蛋白, 融合表达

Abstract: Objective To construct, express and purify human scFv antibody （S1） against the recombinant SAG1 of Toxoplasma gondii and fused to green fluorescent protein （GFP）, and observe its binding capacity to tachyzoite of Toxoplasma gondii. Methods The GFP gene amplified from vector pEGFP-N1 was subcloned into procaryotic expression vector pET-26b（+）, then the S1 scFv antibody gene amplified from phagmid pIT-2-S1 was cloned into downstream of GFP gene. The recombinant plasmid pET-26b-GFPS1 proved by DNA sequencing was transformed into E.coli BL21, and induced for fusion expression of GFPS1 with IPTG, the green fluorescence of E.coli BL21 harboring plasmid pET-26b-GFPS1 was observed under the fluorescence microscope. The expressed GFPS1 was purified with Ni2+ chelating HiTrap HP column, and detected with SDS-PAGE. Toxoplasma tachyzoites were incubated with the recombinant GFPS1, and the binding bioactivity was observed under the fluorescence microscope. Results The fused gene of S1 and GFP was successfully cloned into procaryotic expression vector pET-26b proved by DNA sequencing. The green fluorescence of E.coli BL21 harboring plasmid pET-26b-GFPS1 was catched under the fluorescence microscope. The recombinant GFPS1 protein about Mr 53 000 was expressed in E.coli as inclusion body. The immunofluorescence detection verified that anti-rSAG1 scFv antibody S1 could specifically bind outer membrane of Toxoplasma tachyzoite. Conclusions The purified rGFPS1 shows a strong binding capacity to outer membrane of Toxoplasma tachyzoite using GFP as a labeling protein, and the constructed pET-26b-GFP can also be used for research on other targeting molecules.

Key words: Toxoplasma gondii, Single fold scFv antibody, Green fluorescent protein, Fusion expression