日本血吸虫重组烯醇化酶研究

发布时间：2018-04-19 23:24

  本文选题：日本血吸虫 + 烯醇化酶蛋白　； 参考：《福建农林大学》2009年硕士论文

【摘要】： 日本血吸虫病是我国一种危害严重的人畜共患寄生虫病。由于治疗药物吡喹酮不能解决重复感染难题,而且有可能诱导产生抗药性,因此有必要研制高效、安全的抗血吸虫病疫苗和新治疗药物。血吸虫同其它寄生虫一样,通过新陈代谢在宿主中进行物质和能量交换。目前一般认为,血吸虫的能量主要是通过糖酵解过程获得的。由烯醇化酶参与糖酵解途径,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,所以本文对这个基因进行了克隆、表达、定位及生物学功能的初步研究。 1在本实验室双向电泳研究基础上,根据NCBI中GenBank登录号L23324设计特异引物克隆获得编码日本血吸虫烯醇化酶SjEno的编码基因。SjENO基因的ORF含1305bp,编码434个氨基酸,理论分子量47250.37。构建了pET28a(+)-SjEno重组质粒转入宿主菌BL21(DH5α),并于大肠杆菌中成功表达并纯化。 2经Western blotting显示表达产物能被日本血吸虫成虫抗原免疫兔血清所识别,具有良好的抗原性。应用该重组蛋白免疫BALB/c小鼠,获得了24.28%的肝组织减卵率和21.45%的粪便减卵率,显示该重组蛋白可诱导部分的免疫保护效果。rSjEno重组蛋白免疫组小鼠在3次免疫后产生了高滴度的特异性IgG抗体。 3利用免疫荧光技术观察SjEno在日本血吸虫28d及42d成虫中的表达定位。免疫荧光观察表明,在血吸虫28d及42d成虫的体表均可见荧光信号,在虫体内部和消化道肠管中也可见荧光信号。在血吸虫头、中、尾部的荧光信号没有明显差异。研究结果表明,日本血吸虫SjEno蛋白可以在虫体的体被表膜部位表达。 4检测日本血吸虫SjEno重组蛋白的酶学活性,用连续监测法,利用紫外分光光度计检测在240nm时磷酸烯醇丙酮酸酯吸光度的增加量(正向反应2-PGA→PEP)或减少量(反向反应PEP→2-PGA)。试验结果显示2-PGA→PEP中所测得的活性为35.81±2.02U(mg protein)~(-1);反向反应PEP→2-PGA测得的活性为15.87±0.966U(mg protein)-1。不管使用何种底物rSjEno酶最佳活性pH值范围为6.5-7。在检测浓度范围内KCL、LiCL、NaCL、MgCL_2、CaCL_2在检测浓度范围内对rSjEno酶活性有一定的抑制作用。ZnCL2在以2-PGA为底物的rSjEno对酶活性均有抑制作用。而ZnCL_2的浓度为2.5mM-7.5mM则对以PEP为底物的rSjEno酶有很强的激活作用。温度值范围从15℃到45℃对rSjEno酶活性的影响不大。
[Abstract]:Schistosomiasis japonicum is a serious zoonotic parasitic disease in China. Since praziquantel can not solve the problem of repeated infection and may induce drug resistance, it is necessary to develop an efficient and safe anti-schistosomiasis vaccine and new therapeutic drugs. Like other parasites, Schistosoma exchanges matter and energy in the host through metabolism. At present, it is generally believed that the energy of Schistosoma japonicum is mainly obtained by glycolysis. Enolase involved in glycolysis pathway is a key pathway of cell energy metabolism, and plays an important role in the growth and development of animals. Therefore, the cloning, expression, localization and biological function of this gene have been studied in this paper. 1 on the basis of two-dimensional electrophoresis in our laboratory, according to GenBank accession number L23324 in NCBI, a specific primer clone was designed to obtain the ORF encoding Schistosoma japonicum enolase SjEno. The ORF encoding SjENO gene contained 1305 BP, encoding 434 amino acids, and the theoretical molecular weight was 47250.37. The recombinant plasmid pET28a( pET28a- SjEno) was transformed into the host strain BL21(DH5 伪, and was successfully expressed and purified in E. coli. 2 Western blotting showed that the expressed product could be recognized by the sera of rabbits immunized with Schistosoma japonicum adult worm antigen and had good antigenicity. The recombinant protein was used to immunize BALB/c mice and obtained 24.28% liver oocyte reduction rate and 21.45% fecal oocyte reduction rate. The results showed that the recombinant protein could induce partial immune protection. The mice immunized with rSjEno recombinant protein produced high titer of specific IgG antibody after three times of immunization. 3Immunofluorescence technique was used to observe the expression of SjEno in adult Schistosoma japonicum for 28 days and 42 days. Immunofluorescence observation showed that fluorescent signals could be seen on the body surface of adult Schistosoma japonicum 28 days and 42 days, and also in the inner body and intestinal tract of digestive tract. In the head of schistosomiasis, there is no significant difference in fluorescence signals in the tail of Schistosoma japonicum. The results showed that the SjEno protein of Schistosoma japonicum could be expressed in the membrane of the body. (4) the enzymatic activity of Schistosoma japonicum SjEno recombinant protein was detected by continuous monitoring and UV spectrophotometer was used to detect the increase or decrease of phosphoenolpyruvate absorbance (PEP + 2-PGA) in 240nm. The results showed that the activity of 2-PGA PEP was 35.81 卤2.02U(mg protein ~ (-1) and that of reverse reaction PEP 2-PGA was 15.87 卤0.966U(mg protein ~ (-1). The optimal pH range of rSjEno enzyme activity was 6.5-7 regardless of the substrate used. In the range of detection concentration, rSjEno enzyme activity was inhibited by KCL-LiCL-NaCL-NaCL-2CaCL2 in the range of detection concentration. ZnCL2 inhibited the activity of rSjEno in rSjEno with 2-PGA as substrate. However, the concentration of ZnCL_2 was 2.5mM-7.5mM, which could activate the rSjEno enzyme with PEP as substrate. The temperature range from 15 鈩，

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