中国寄生虫学与寄生虫病杂志

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刚地弓形虫入侵宿主细胞过程中棒状体蛋白16的分泌与分布

姜诗晨,魏海霞,何成,邓胜群,夏菁,彭鸿娟*   

  1. 南方医科大学公共卫生与热带医学学院广东省热带病研究重点实验室,病原生物学系暨广东省普通高校新发传染病防治重点实验室,广州 510515
  • 出版日期:2016-06-30 发布日期:2016-10-28

Secretion and Distribution of Rhoptry Protein 16 during Toxoplasma gondii Invasion into Host Cells

JIANG Shi-chen, WEI Hai-xia, HE Cheng, DENG Sheng-qun, XIA Jing, PENG Hong-juan*   

  1. Key Laboratory of Prevention and Control for Emerging Infectious Diseases, Guangdong Higher Institutes/Department of Pathogen Biology, School of Public Health, Southern Medical University, Guangzhou 510515, China
  • Online:2016-06-30 Published:2016-10-28

摘要: 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cDNA为模板,PCR扩增Tgrop16基因,克隆至pET-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TgROP16经纯化后免疫新西兰大白兔,制备抗TgROP16多克隆抗体,用蛋白质印迹(Western blotting)和间接ELISA检测抗TgROP16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TgROP16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,TgROP16蛋白在宿主细胞中的分泌与分布。 结果 制备的抗TgROP16多克隆抗体,Western blotting分析结果显示,能与重组蛋白TgROP16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TgROP16蛋白表达量(TgROP16与其内参TgTubulin的灰度比值为0.373)较高于Pru株(TgROP16与其内参TgTubulin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,TgROP16蛋白定位于速殖子的顶端复合体,速殖子入侵HFFs细胞后,TgROP16蛋白显示在虫体外。 结论 制备的抗TgROP16多克隆抗体特异性和敏感性高;刚地弓形虫RH株速殖子的TgROP16蛋白表达量高于Pru株的;两株速殖子在入侵HFFs细胞前,TgROP16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。

关键词: 刚地弓形虫, 棒状体蛋白16, 多克隆抗体, 制备, 实时荧光定量PCR, 间接免疫荧光, 蛋白质 , 印迹

Abstract: Objective To examine the secretion and localization of Toxoplasma gondii Rhoptry protein 16 (ROP16) during invasion of different strains of T. gondii into host cells. Methods The Tgrop16 gene was amplified by PCR on the cDNA of T. gondii RH strain, subcloned into the plasmid pET-32a(+), and expressed in Escherichia coli BL21(DE3) under the induction of isopropyl β-D-1-thiogalactopyranoside. New Zealand rabbit was immuned with the expressed recombinant protein TgROP16 to produce polyclonal anti-TgROP16 antibody. The specificity and sensitivity of the polyclonal antibody were examined by Western blotting and indirect ELISA, respectively. The transcriptional and protein levels of Tgrop16 in T. gondii RH strain and Pru strain were determined by real-time PCR and Western blotting, respectively. The secretion and distribution of TgROP16 in human foreskin fibroblasts(HFFs) during the invasion by T. gondii RH strain and Pru strain were examined by indirect immunofluorescence assay(IFA). Results Western blotting showed a specific band at Mr of ~100 000, indicating that the specific rabbit-derived anti-TgROP16 polyclonal antibody was capable of recognizing TgROP16. Indirect ELISA revealed a titer of 1:25 600 for the antibody. The relative expression level of Tgrop16 in Pru strain[(7.786±0.206)] was 7 times than that in RH strain[(1.000±0.110)](P<0.05) as detected by real-time PCR, and TgROP16 protein level was higher in RH strain than in Pru strain. IFA showed that TgROP16 was localized on the apical complex of the unrecruited tachyzoite of T. gondii before invasion and was secreted out of the recruited tachyzoite after invasion. Conclusion The anti-TgROP16 polyclonal antibody has high specificity and sensitivity. The TgROP16 protein level is higher in the RH strain than in the Pru strain. For both strains, TgROP16 is localized on the apical complex of the unrecruited tachyzoite before invasion and secreted out of the recruited tachyzoite during invasion.

Key words: Toxoplasma gondii, Rhoptry protein 16, Polyclonal antibody, Preparation, Real-time PCR, Indirect immunofluorescence assay, Western blotting