弓形虫棒状体蛋白ROP7的生物信息学分析、原核表达及组织学定位

发布时间：2018-04-11 00:14

  本文选题：弓形虫 + ROP7　； 参考：《吉林农业大学》2015年硕士论文

【摘要】：弓形虫病是一种呈世界范围流行的人兽共患寄生虫病。弓形虫病严重影响神经系统,眼部病变,新生儿全身性疾病,尤其是免疫系统薄弱的人。而免疫功能正常的成年人,感染通常无明显症状,一般为隐性感染。当机体免疫力受到损伤,弓形虫隐性感染被激活,引起广泛的组织损伤和严重的病变,也是艾滋病患者死亡的主要原因。弓形虫不但危害人类健康,也严重的影响着畜禽牧业的发展,造成严重的经济损失。目前关于弓形虫病尚无良好的药物以及可实际商业化生产应用的疫苗,故寻找有效预防弓形虫病的疫苗是当前研究的方向。弓形虫顶端复合体—棒状体,在入侵宿主细胞与机体感染起着重要作用,其分泌的棒状体蛋白ROP7与弓形虫入侵宿主细胞有关,其在纳虫空泡上的拓扑结构还未确定,抗原表位及对宿主是否具有免疫保护性也不清楚。(1)本研究通过生物信息学技术对弓形虫棒状体蛋白ROP7的理化性质、是否含有信号肽和跨膜结构域、可溶性、二级结构及抗原表位等进行了预测,结果发现弓形虫RH株棒状体蛋白ROP7基因全长为1728bp,编码575个氨基酸,N-端有一1-33位氨基酸的信号肽序列,12-34位氨基酸的跨膜结构域,具有多个翻译后修饰位点,多个抗原表位,这在理论上支持了弓形虫棒状体蛋白ROP7具有潜在的免疫源性,有成为候选疫苗基因的潜能。(2)根据生物信息软件的在线预测,将弓形虫RH株棒状体蛋白ROP7基因信号肽部位去掉,从余下的序列中截取561bp进行PCR扩增,将扩增的目的片段回收纯化后,克隆到pMD18-T载体,重组质粒pMD18-T-Tg2ROP7经NcoⅠ和NotⅠ双酶切后,插入到pET-28a原核表达载体中,重组质粒经测序鉴定正确的转入DE3感受态,经IPTG诱导,重组蛋白ROP7进行SDS-PAGE与Western blot分析,重组Tg2ROP7表达大小为22kDa。NI-NTA进行纯化,透析后用PEG 20000浓缩测定浓度为0.3mg/mL。(3)应用纯化的蛋白免疫小鼠,制备Tg2ROP7鼠源性多克隆抗体,ELISA检测其效价为1∶6400,并将其成功应用于弓形虫Tg2ROP7在弓形虫速殖子偏顶端的免疫荧光定位试验。本研究不但在理论上证明弓形虫棒状体蛋白ROP7具有抗原性,在技术上证明其作为弓形虫病核酸疫苗的可行性,同时还将生物信息学技术应用于弓形虫蛋白的预测,这都将为深入了解弓形虫入侵宿主细胞机理、弓形虫病疫苗的研发和弓形虫诊断试剂盒的研制奠定基础。
[Abstract]:Toxoplasmosis (Toxoplasma gondii) is a worldwide epidemic zoonotic parasitic disease.Toxoplasma gondii severely affects the nervous system, eye lesions, neonatal systemic diseases, especially in people with weak immune systems.In adults with normal immune function, the infection is usually asymptomatic and usually recessive.When body immunity is damaged, Toxoplasma gondii recessive infection is activated, causing extensive tissue damage and serious lesions, which is also the main cause of death in AIDS patients.Toxoplasma gondii not only harm human health, but also seriously affect the development of animal husbandry, causing serious economic losses.At present, there is no good medicine for toxoplasmosis and the vaccine can be commercialized, so it is the research direction to find a vaccine to prevent Toxoplasma gondii effectively.The rodlike body of Toxoplasma gondii apical complex plays an important role in invading host cell and body infection. The rodlike protein ROP7 secreted by Toxoplasma gondii is related to Toxoplasma gondii invading host cell, and its topological structure on the vacuole of Toxoplasma gondii has not been determined.In this study, the physicochemical properties of Toxoplasma gondii rodlike protein ROP7, whether it contains signal peptides and transmembrane domains, and the solubility of Toxoplasma gondii rodlike protein ROP7 by bioinformatics were also unclear.The secondary structure and antigen epitopes were predicted. The results showed that the ROP7 gene of RH strain of Toxoplasma gondii was 1728 BP, encoding 575 amino acids with a 1-33 amino acid signal peptide sequence and a 12-34 amino acid transmembrane domain.Having multiple post-translational modification sites, multiple antigenic epitopes, which theoretically supports the potential immunogenicity of Toxoplasma gondii coryloid protein ROP7, which has the potential to be a candidate vaccine gene, according to the online prediction of bioinformatics software.The signal peptide of ROP7 gene of RH strain of Toxoplasma gondii was removed, 561bp was intercepted from the remaining sequence for PCR amplification, the amplified target fragment was recovered and purified, then cloned into pMD18-T vector. The recombinant plasmid pMD18-T-Tg2ROP7 was digested by Nco 鈪，

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