本文關(guān)鍵詞：日本血吸蟲磷酸甘油酸變位酶SjPGAM的研究　出處：《中國農(nóng)業(yè)科學(xué)院》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 日本血吸蟲 磷酸甘油酸變位酶 重組表位疫苗 蒿甲醚 酶活性

【摘要】： 血吸蟲病是由血吸蟲感染引起的分布廣泛、危害嚴(yán)重的人獸共患寄生蟲病。研制開發(fā)有效的血吸蟲病疫苗及新治療藥物是血吸蟲防控的重大科技需求,尋找新的疫苗侯選分子和藥物靶標(biāo)一直受到寄生蟲學(xué)者們的關(guān)注。由磷酸甘油酸變位酶(Phosphoglycerate mutas PGAM)基因家族產(chǎn)物與其它相關(guān)基因產(chǎn)物所構(gòu)成的糖代謝途徑,是細(xì)胞能量代謝的一個關(guān)鍵途徑,對動物的生長發(fā)育起著重要的作用。本研究以日本血吸蟲糖酵解途徑關(guān)鍵酶SjPGAM為研究對象,開展了該基因的表達(dá)、重組抗原酶活性測定;重組表位疫苗構(gòu)建、表達(dá)及動物免疫保護試驗;抗血吸蟲藥物蒿甲醚對重組蛋白SjPGAM酶活性及對不同發(fā)育時期血吸蟲蟲體SjPGAM基因表達(dá)的影響等研究,為深入探討SjPGAM的生物學(xué)功能,評估其作為疫苗候選分子和藥靶的應(yīng)用前景提供基礎(chǔ)。 一、日本血吸蟲磷酸甘油酸變位酶SjPGAM的克隆、表達(dá)及酶活性測定研究成功構(gòu)建了pET28a-SjPGAM重組表達(dá)質(zhì)粒,優(yōu)化了表達(dá)條件,在大腸桿菌體系中獲得可溶性、具有酶活性的重組抗原。酶動力學(xué)研究表明重組蛋白活性單位為0.438u/mg,米氏常數(shù)(Km值)為3.78mmol,Kcat(轉(zhuǎn)換率)為5.26s-1,Kcat/Km為1.39 104M-1S-1。PH值為7時重組蛋白的酶活力最高;最適反應(yīng)溫度在37-40℃之間;EDTA對rSjPGAM活性具有明顯的抑制作用;10mM金屬陽離子Mn~(2+)、Ni~(2+)、Mg~(2+)、Zn~(2+)對SjPGAM酶活性均有激活作用,其中Ni~(2+)較為明顯,能使酶活力恢復(fù)到40%。 二、SjPGAM重組表位疫苗的研究利用生物信息學(xué)技術(shù)對日本血吸蟲糖酵解途徑關(guān)鍵酶SjPGAM和SjEnol(烯醇化酶)的抗原表位進行在線分析,預(yù)測并篩選了SjPGAM和SjEnol富含MHCⅡ細(xì)胞結(jié)合表位且與宿主同源性較小的肽段,將對應(yīng)編碼的核苷酸序列進行拼接,構(gòu)建重組表達(dá)質(zhì)粒pET32a(+)-BSjPGAM-BSjEnol,并在E.coli BL21中成功表達(dá)。將純化的重組蛋白免疫動物,結(jié)果顯示,與空白組相比,pET32a-BSjPGAM-BSjEnol組獲得39.7%(p0.05)的減蟲率和64.9%(p0.05)的肝減卵率,比單一重組抗原pET28a-SjPGAM和pET28a-SjEnol誘導(dǎo)了更高的免疫保護作用。 三、抗血吸蟲藥物蒿甲醚對SjPGAM重組蛋白酶活性及不同發(fā)育階段血吸蟲蟲體基因表達(dá)影響的研究試驗發(fā)現(xiàn)抗血吸蟲藥物蒿甲醚對SjPGAM重組蛋白酶活性有明顯抑制作用;對試驗感染日本血吸蟲的兔子,分別于感染后6d、9d、13d、17d、41d天肌肉注射蒿甲醚,一天后收集蟲體,相對定量real-time PCR檢測表明,蒿甲醚處理后對不同發(fā)育階段血吸蟲蟲體SjPGAM基因表達(dá)均有不同程度的抑制作用。顯示SjPGAM可能是蒿甲醚抗血吸蟲作用的潛在藥物靶點之一。
[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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