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目的观察刚地弓形虫(Toxoplasma gondii)不同毒力株的棒状体蛋白16(ROP16)在入侵宿主细胞过程中的分泌及分布。方法以刚地弓形虫RH株速殖子c DNA为模板,PCR扩增Tgrop16基因,克隆至p ET-32a(+)载体,在大肠埃希菌(Escherichia coli)BL21(DE3)中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。重组蛋白Tg ROP16经纯化后免疫新西兰大白兔,制备抗Tg ROP16多克隆抗体,用蛋白质印迹(Western blotting)和间接ELISA检测抗Tg ROP16多克隆抗体的特异性和敏感性。用实时荧光定量PCR(real-time PCR)检测刚地弓形虫RH株和Pru株速殖子Tgrop16基因的转录水平,Western blotting分析RH株和Pru株Tg ROP16蛋白的表达量。用间接免疫荧光(IFA)检测弓形虫RH株和Pru株速殖子在入侵人包皮成纤维细胞(HFFs细胞)过程中,Tg ROP16蛋白在宿主细胞中的分泌与分布。结果制备的抗Tg ROP16多克隆抗体,Western blotting分析结果显示,能与重组蛋白Tg ROP16结合,在相对分子质量(Mr)约100 000处有一特异性条带;间接ELISA检测结果显示,抗体效价为1∶25 600。实时荧光定量PCR检测结果显示,Pru株Tgrop16基因表达量(7.786±0.206)是RH株(1.000±0.110)的7倍(P<0.05)。Western blotting分析结果显示,RH株中Tg ROP16蛋白表达量(Tg ROP16与其内参Tg Tubulin的灰度比值为0.373)较高于Pru株(Tg ROP16与其内参Tg Tubulin的灰度比值为0.232)。IFA检测结果显示,RH株和Pru株速殖子在入侵HFFs细胞前,Tg ROP16蛋白定位于速殖子的顶端复合体,速殖子入侵HFFs细胞后,Tg ROP16蛋白显示在虫体外。结论制备的抗Tg ROP16多克隆抗体特异性和敏感性高;刚地弓形虫RH株速殖子的Tg ROP16蛋白表达量高于Pru株的;两株速殖子在入侵HFFs细胞前,Tg ROP16蛋白分布于速殖子顶端复合体,在入侵宿主细胞过程中被分泌出虫体。
Objective To observe the secretion and distribution of ROP16 in different virulence strains of Toxoplasma gondii during its invasion into host cells. Methods The Tgrop16 gene was amplified by PCR from tachyzoites RH strain tachyzoites cT DNA clone and cloned into p ET-32a (+) vector. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) Ki-β-D-thiogalactoside (IPTG) induces expression. The recombinant protein Tg ROP16 was purified and immunized New Zealand white rabbits to prepare anti-Tg ROP16 polyclonal antibody. The specificity and sensitivity of anti-Tg ROP16 polyclonal antibody were detected by Western blotting and indirect ELISA. The transcription level of Tgrop16 gene in T.gondii RH strain and Pru strain tachyzoites was detected by real-time PCR, and the expression of Tg ROP16 protein in RH strain and Pru strain was analyzed by Western blotting. Indirect immunofluorescence (IFA) was used to detect the secretion and distribution of Tg ROP16 protein in host cells during invasion of human foreskin fibroblasts (HFFs) by Tachyzoites RH strains and Pru strains. Results The anti-Tg ROP16 polyclonal antibody was prepared. The result of Western blotting showed that it could bind with the recombinant protein Tg ROP16 and had a specific band at about 100,000 molecular weight (Mr). The indirect ELISA showed that the antibody titer 1:25 600. Real-time PCR results showed that the expression of Tgrop16 gene in Pru strain (7.786 ± 0.206) was seven-fold higher than that in RH strain (1.000 ± 0.110) (P <0.05). The result of Western blotting showed that the expression level of Tg ROP16 in RH strain was higher than that of Pru strain (the ratio of the grayscale of Tg ROP16 to its Tg Tubulin was 0.232). The results of IFA showed that Tg ROP16 protein was located in the tachyzoite apical complex before the tachyzoites of RH strain and Pru invaded HFFs cells. Tachyzoites invaded HFFs cells and the Tg ROP16 protein was displayed outside the insect cells. CONCLUSION: The anti-Tg ROP16 polyclonal antibody is highly specific and sensitive. The Tg ROP16 protein expression of tachyzoites in T. gondii RH strain is higher than that of Pru strain. Before the two tachyzoites invade HFFs cells, Tg ROP16 Proteins are distributed in the tachyzoite apical complex and are secreted by the host cells during their invasion.