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目的:克隆刚地弓形虫RH株速殖子棒状体蛋白(rhoptry protein,ROP)16基因,并构建ROP16基因的原核表达系统,检测和定位ROP16蛋白的表达。方法:以弓形虫RH株速殖子基因组DNA为模板,PCR扩增弓形虫ROP16基因,克隆至p ET-32a(+)载体,在大肠埃希菌Rosetta中诱导表达。经KCl染色切胶法纯化重组ROP16蛋白,并制备其兔多克隆抗体。采用蛋白质印迹法和间接免疫荧光法检测和定位ROP16在弓形虫速殖子内的表达。结果:成功表达并纯化重组ROP16蛋白,制备了其多克隆抗体。蛋白质印迹法检测出相对分子质量为74 000的特异性条带,间接免疫荧光实验显示ROP16分布于弓形虫速殖子胞质内。结论:经原核表达重组ROP16制备的多克隆抗体能检测和定位ROP16在弓形虫速殖子内的表达。
OBJECTIVE: To clone Rhoptry protein (ROP) 16 gene of Toxoplasma gondii RH strain and construct prokaryotic expression system of ROP16 gene to detect and locate ROP16 protein expression. Methods: Toxoplasma gondii RHOP tachyzoite genomic DNA was used as a template to amplify the Toxoplasma gondii ROP16 gene and cloned into p ET-32a (+) vector and induced in Escherichia coli Rosetta. Recombinant ROP16 protein was purified by KCl staining and polyclonal antibody was prepared. The expression of ROP16 in Toxoplasma gondii tachyzoites was detected and localized by Western blotting and indirect immunofluorescence. Results: The recombinant ROP16 protein was successfully expressed and purified and its polyclonal antibody was prepared. The specific band of 74 000 was detected by Western blotting. Indirect immunofluorescence assay showed that ROP16 was located in Toxoplasma gondii cytoplasm. CONCLUSION: The polyclonal antibody produced by prokaryotic recombinant ROP16 can detect and locate the expression of ROP16 in Toxoplasma gondii tachyzoites.