Ⅱ型弓形虫肠道感染期分泌蛋白初步研究
发布时间:2018-12-15 07:43
【摘要】:弓形虫是一种专性的细胞内寄生虫,属于顶复门原虫,是重要的人兽共患寄生虫。弓形虫生活史复杂,需要中间宿主和终末宿主,中间宿主为包括人在内的多种温血脊椎动物,以及鸟类和鱼类;终末宿主为猫科动物。弓形虫感染对于免疫抑制或免疫缺陷的人群是致命的;孕妇妊娠期感染会造成流产、死胎或早产,母畜感染也会造成流产。当前,弓形虫病已经严重威胁人畜健康,对食品安全构成威胁,其中Ⅱ型弓形虫是导致人畜弓形虫病的主要基因型之一,但却缺乏有效的药物和疫苗来预防和治疗该病。了解弓形虫肠道感染期分泌蛋白、阐明其肠道入侵机制、寻找有效的抗原分子是控制弓形虫病的重要研究方向。为了解Ⅱ型弓形虫肠道感染期的蛋白分泌情况,采用新鲜繁殖的弓形虫卵囊经口感染昆明小鼠,感染后6 h,分离肠道分泌蛋白,进行质谱鉴定和生物信息学分析,试验重复3次。结果显示,3次试验共获得71个分泌相关蛋白,这些蛋白质主要与结合和催化相关,参与细胞过程和代谢过程,以细胞和细胞器成分为主。为了解弓形虫急性感染期基因表达差异,将小鼠经口感染Ⅱ型弓形虫包囊,10 d后取脑组织提取总RNA,进行转录组高通量测序。结果显示,与对照组相比,共检测到14 506个新转录本,其中12 247个属于已知编码蛋白基因的新的可变剪接亚型,315个属于新的蛋白编码基因的转录本,其余的1 944个属于长链非编码RNA。共发现454条差异表达基因,其中416条基因表达上调,38条基因表达下调。这些基因主要与细胞组成有关,参与分子结合、生物调节、细胞过程等。通过与弓形虫数据库进行比对,共获得了154个差异表达蛋白。为克隆、表达前述研究鉴定的肠道感染期分泌蛋白弓形虫丝/苏氨酸蛋白磷酸酶1(TgPP1)基因,为下一步阐明其特性和功能打下基础,以Ⅱ型弓形虫包囊cDNA为模板,利用PCR技术扩增丝/苏氨酸蛋白磷酸酶基因,连接到表达载体pET-30a(+)上进行原核表达,对纯化的重组蛋白进行Western blot试验,观察其免疫反应性。结果显示,成功地扩增了弓形虫丝/苏氨酸蛋白磷酸酶1(TgPP1)基因,其大小为921 bp。成功地表达了重组丝/苏氨酸蛋白磷酸酶1(rTgPP1),大小33 kDa,该蛋白能够被弓形虫感染的小鼠阳性血清所识别,表明重组蛋白具有良好的反应原性。综上所述,本研究成功地对Ⅱ型弓形虫入侵小鼠后的分泌蛋白组和转录组进行了分析,对鉴定的分泌蛋白丝/苏氨酸蛋白磷酸酶1基因进行了克隆和表达,对进一步了解弓形虫感染和致病机制、研发新型防控策略、预防和控制弓形虫病流行有重要意义。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii), an important zoonotic parasite, is a specific intracellular parasite. The life history of Toxoplasma gondii is complex, which requires intermediate and terminal hosts, which include a variety of warm-blooded vertebrates, including human beings, birds and fish, while the final hosts are cats. Toxoplasma gondii infection can be fatal in immunosuppressive or immunodeficient populations; infection during pregnancy can cause miscarriage stillbirth or premature birth and miscarriage in females. At present, Toxoplasma gondii has been a serious threat to human and animal health and food safety. Toxoplasma gondii type II is one of the major genotypes leading to Toxoplasma gondii in humans and animals, but it lacks effective drugs and vaccines to prevent and treat the disease. To understand the secretory proteins of Toxoplasma gondii intestinal infection, to elucidate the mechanism of intestinal invasion and to find effective antigen molecules are important research directions for controlling Toxoplasma gondii. In order to understand the protein secretion of Toxoplasma gondii during the period of intestinal infection, the newly propagated oocysts of Toxoplasma gondii were infected by mouth in Kunming mice. The secreted proteins were isolated 6 hours after infection and identified by mass spectrometry and bioinformatics analysis. The experiment was repeated three times. The results showed that 71 secretory proteins were obtained in three experiments. These proteins were mainly related to binding and catalysis, and involved in cell process and metabolism, mainly cell and organelle components. In order to understand the difference of gene expression in acute infection stage of Toxoplasma gondii, mice were infected with Toxoplasma gondii type 鈪,
本文编号:2380275
[Abstract]:Toxoplasma gondii (Toxoplasma gondii), an important zoonotic parasite, is a specific intracellular parasite. The life history of Toxoplasma gondii is complex, which requires intermediate and terminal hosts, which include a variety of warm-blooded vertebrates, including human beings, birds and fish, while the final hosts are cats. Toxoplasma gondii infection can be fatal in immunosuppressive or immunodeficient populations; infection during pregnancy can cause miscarriage stillbirth or premature birth and miscarriage in females. At present, Toxoplasma gondii has been a serious threat to human and animal health and food safety. Toxoplasma gondii type II is one of the major genotypes leading to Toxoplasma gondii in humans and animals, but it lacks effective drugs and vaccines to prevent and treat the disease. To understand the secretory proteins of Toxoplasma gondii intestinal infection, to elucidate the mechanism of intestinal invasion and to find effective antigen molecules are important research directions for controlling Toxoplasma gondii. In order to understand the protein secretion of Toxoplasma gondii during the period of intestinal infection, the newly propagated oocysts of Toxoplasma gondii were infected by mouth in Kunming mice. The secreted proteins were isolated 6 hours after infection and identified by mass spectrometry and bioinformatics analysis. The experiment was repeated three times. The results showed that 71 secretory proteins were obtained in three experiments. These proteins were mainly related to binding and catalysis, and involved in cell process and metabolism, mainly cell and organelle components. In order to understand the difference of gene expression in acute infection stage of Toxoplasma gondii, mice were infected with Toxoplasma gondii type 鈪,
本文编号:2380275
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