日本血吸虫磷酸甘油酸变位酶SjPGAM的研究

发布时间：2018-01-16 18:30

  本文关键词：日本血吸虫磷酸甘油酸变位酶SjPGAM的研究　出处：《中国农业科学院》2010年硕士论文　论文类型：学位论文

  更多相关文章： 日本血吸虫 磷酸甘油酸变位酶 重组表位疫苗 蒿甲醚 酶活性

【摘要】： 血吸虫病是由血吸虫感染引起的分布广泛、危害严重的人兽共患寄生虫病。研制开发有效的血吸虫病疫苗及新治疗药物是血吸虫防控的重大科技需求,寻找新的疫苗侯选分子和药物靶标一直受到寄生虫学者们的关注。由磷酸甘油酸变位酶(Phosphoglycerate mutas PGAM)基因家族产物与其它相关基因产物所构成的糖代谢途径,是细胞能量代谢的一个关键途径,对动物的生长发育起着重要的作用。本研究以日本血吸虫糖酵解途径关键酶SjPGAM为研究对象,开展了该基因的表达、重组抗原酶活性测定;重组表位疫苗构建、表达及动物免疫保护试验;抗血吸虫药物蒿甲醚对重组蛋白SjPGAM酶活性及对不同发育时期血吸虫虫体SjPGAM基因表达的影响等研究,为深入探讨SjPGAM的生物学功能,评估其作为疫苗候选分子和药靶的应用前景提供基础。 一、日本血吸虫磷酸甘油酸变位酶SjPGAM的克隆、表达及酶活性测定研究成功构建了pET28a-SjPGAM重组表达质粒,优化了表达条件,在大肠杆菌体系中获得可溶性、具有酶活性的重组抗原。酶动力学研究表明重组蛋白活性单位为0.438u/mg,米氏常数(Km值)为3.78mmol,Kcat(转换率)为5.26s-1,Kcat/Km为1.39 104M-1S-1。PH值为7时重组蛋白的酶活力最高;最适反应温度在37-40℃之间;EDTA对rSjPGAM活性具有明显的抑制作用;10mM金属阳离子Mn~(2+)、Ni~(2+)、Mg~(2+)、Zn~(2+)对SjPGAM酶活性均有激活作用,其中Ni~(2+)较为明显,能使酶活力恢复到40%。 二、SjPGAM重组表位疫苗的研究利用生物信息学技术对日本血吸虫糖酵解途径关键酶SjPGAM和SjEnol(烯醇化酶)的抗原表位进行在线分析,预测并筛选了SjPGAM和SjEnol富含MHCⅡ细胞结合表位且与宿主同源性较小的肽段,将对应编码的核苷酸序列进行拼接,构建重组表达质粒pET32a(+)-BSjPGAM-BSjEnol,并在E.coli BL21中成功表达。将纯化的重组蛋白免疫动物,结果显示,与空白组相比,pET32a-BSjPGAM-BSjEnol组获得39.7%(p0.05)的减虫率和64.9%(p0.05)的肝减卵率,比单一重组抗原pET28a-SjPGAM和pET28a-SjEnol诱导了更高的免疫保护作用。 三、抗血吸虫药物蒿甲醚对SjPGAM重组蛋白酶活性及不同发育阶段血吸虫虫体基因表达影响的研究试验发现抗血吸虫药物蒿甲醚对SjPGAM重组蛋白酶活性有明显抑制作用;对试验感染日本血吸虫的兔子,分别于感染后6d、9d、13d、17d、41d天肌肉注射蒿甲醚,一天后收集虫体,相对定量real-time PCR检测表明,蒿甲醚处理后对不同发育阶段血吸虫虫体SjPGAM基因表达均有不同程度的抑制作用。显示SjPGAM可能是蒿甲醚抗血吸虫作用的潜在药物靶点之一。
[Abstract]:Schistosomiasis is a serious zoonotic parasitic disease caused by schistosomiasis infection. The development of effective schistosomiasis vaccine and new therapeutic drugs is an important scientific and technological demand for prevention and control of schistosomiasis. The search for new vaccine candidate molecules and drug targets has been the focus of parasitologists. Phosphoglycerate mutas PGAM). The glycometabolism pathway between gene family products and other related gene products. It is a key pathway of cell energy metabolism, and plays an important role in the growth and development of animals. In this study, the key enzyme SjPGAM of the glycolytic pathway of Schistosoma japonicum was studied, and the expression of the gene was carried out. Detection of recombinant antigenase activity; Construction, expression and animal immune protection test of recombinant epitope vaccine; The effects of artemether on the activity of recombinant protein SjPGAM enzyme and on the expression of SjPGAM gene in Schistosoma japonicum at different developmental stages were studied. It provides a basis for further studying the biological function of SjPGAM and evaluating its application prospect as vaccine candidate and drug target. The main results are as follows: 1. The cloning, expression and enzyme activity determination of SjPGAM from Schistosoma japonicum phosphoglycerate translocation enzyme were studied. The recombinant expression plasmid of pET28a-SjPGAM was successfully constructed, and the expression conditions were optimized. A soluble recombinant antigen with enzyme activity was obtained in Escherichia coli system. The enzyme kinetic study showed that the recombinant protein activity unit was 0.438 渭 / mg. The conversion rate of Kcat (conversion rate) was 5.26s-1. The enzyme activity of the recombinant protein with Kcat/Km 1.39104M-1S-1.PH = 7:00 was the highest. The optimum reaction temperature is 37-40 鈩，

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