本文選題：日本血吸蟲 + 烯醇化酶蛋白　； 參考：《福建農(nóng)林大學(xué)》2009年碩士論文

【摘要】： 日本血吸蟲病是我國一種危害嚴重的人畜共患寄生蟲病。由于治療藥物吡喹酮不能解決重復(fù)感染難題,而且有可能誘導(dǎo)產(chǎn)生抗藥性,因此有必要研制高效、安全的抗血吸蟲病疫苗和新治療藥物。血吸蟲同其它寄生蟲一樣,通過新陳代謝在宿主中進行物質(zhì)和能量交換。目前一般認為,血吸蟲的能量主要是通過糖酵解過程獲得的。由烯醇化酶參與糖酵解途徑,是細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,所以本文對這個基因進行了克隆、表達、定位及生物學(xué)功能的初步研究。 1在本實驗室雙向電泳研究基礎(chǔ)上,根據(jù)NCBI中GenBank登錄號L23324設(shè)計特異引物克隆獲得編碼日本血吸蟲烯醇化酶SjEno的編碼基因。SjENO基因的ORF含1305bp,編碼434個氨基酸,理論分子量47250.37。構(gòu)建了pET28a(+)-SjEno重組質(zhì)粒轉(zhuǎn)入宿主菌BL21(DH5α),并于大腸桿菌中成功表達并純化。 2經(jīng)Western blotting顯示表達產(chǎn)物能被日本血吸蟲成蟲抗原免疫兔血清所識別,具有良好的抗原性。應(yīng)用該重組蛋白免疫BALB/c小鼠,獲得了24.28%的肝組織減卵率和21.45%的糞便減卵率,顯示該重組蛋白可誘導(dǎo)部分的免疫保護效果。rSjEno重組蛋白免疫組小鼠在3次免疫后產(chǎn)生了高滴度的特異性IgG抗體。 3利用免疫熒光技術(shù)觀察SjEno在日本血吸蟲28d及42d成蟲中的表達定位。免疫熒光觀察表明,在血吸蟲28d及42d成蟲的體表均可見熒光信號,在蟲體內(nèi)部和消化道腸管中也可見熒光信號。在血吸蟲頭、中、尾部的熒光信號沒有明顯差異。研究結(jié)果表明,日本血吸蟲SjEno蛋白可以在蟲體的體被表膜部位表達。 4檢測日本血吸蟲SjEno重組蛋白的酶學(xué)活性,用連續(xù)監(jiān)測法,利用紫外分光光度計檢測在240nm時磷酸烯醇丙酮酸酯吸光度的增加量(正向反應(yīng)2-PGA→PEP)或減少量(反向反應(yīng)PEP→2-PGA)。試驗結(jié)果顯示2-PGA→PEP中所測得的活性為35.81±2.02U(mg protein)~(-1);反向反應(yīng)PEP→2-PGA測得的活性為15.87±0.966U(mg protein)-1。不管使用何種底物rSjEno酶最佳活性pH值范圍為6.5-7。在檢測濃度范圍內(nèi)KCL、LiCL、NaCL、MgCL_2、CaCL_2在檢測濃度范圍內(nèi)對rSjEno酶活性有一定的抑制作用。ZnCL2在以2-PGA為底物的rSjEno對酶活性均有抑制作用。而ZnCL_2的濃度為2.5mM-7.5mM則對以PEP為底物的rSjEno酶有很強的激活作用。溫度值范圍從15℃到45℃對rSjEno酶活性的影響不大。
[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 偽, 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 鹵2.02U(mg protein ~ (-1) and that of reverse reaction PEP 2-PGA was 15.87 鹵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，

本文編號：1775210