日本血吸虫23KD膜蛋白的原核表达与纯化及双价膜锚定型和分泌型表达疫苗抗感染效果的比较研究
发布时间：2018-11-04 15:51
【摘要】：目的构建日本血吸虫23KD膜蛋白基因的原核表达质粒pET-_28a-sj23，将其转化大肠杆菌BL-21(DE3)，诱导其表达并纯化此蛋白，为制备亚单位疫苗和检测特异性抗体及细胞因子提供抗原。 方法采用RT-PcR的方法，以日本血吸虫成虫总RNA为模板，扩增得到sj23基因，并克隆至PMDl8-T载体中，经BamH I和EcoR I双酶切后定向克隆于原核表达质粒pET-28a，构建成pET-sj23质粒，并将此质粒转化大肠杆菌BL-21 (DE3)，使用IPTG诱导蛋白表达，sDs-PAGE观察其表达状态，镍柱亲和层忻纯化此蛋白，BcA法测定纯化蛋白浓度。 结果成功构建原核表达质粒pET-28a-sj23，并转化至大肠杆菌BL-21(DE3)，sDs-PAGE显示经IPTG诱导之后成功表达出约31.3kD左右的融合蛋白(其中N-端含有8 kD的原核多肽)，亲和层忻纯化重组蛋白，浓度为O.8 mg／ml。 结论成功制备了sj23抗原，为亚单位疫苗的研究及特异性抗体和细胞因子的检测打下了基础。 目的比较膜锚定型pIREs-sj14-Sj26表达疫苗和分泌型pIREs-sj14-Sj26，表达疫苗在血吸虫感染BALB／c小鼠体内的抗感染效果，初步探讨膜锚定与分泌型的疫苗的作用效果和可能机制。 方法大量提取并纯化膜锚定型pIREs-sj14-Sj26表达质粒、分泌型pIREs-sj14-Sj26，表达质粒以及空pIREs质粒，，并用此三种质粒肌肉注射免疫BALB／c小鼠，每组10只，并设置生理盐水组作为空白对照组。每隔2周加强免疫1次，共免疫3次。完成免疫后，每只小鼠经腹部感染40土1条尾蚴，尾蚴攻击感染45d后，各组小鼠经肠系膜静脉灌注法和压片法收集血吸虫成虫，并留取肝脏备用。分别计算每只小鼠中收集的血吸虫成虫数，计算每组小鼠的平均成虫数，按下式计算减虫率：减虫率=(对照组平均获成虫数．实验组平均获成虫数)／对照组平均检获成虫数×100%。称取鼠肝总重量，按常规方法用5%氢氧化钾消化肝组织，计算每克肝组织虫卵数(EPG)，按下式计算减卵率：减卵率=(对照组平均EPG-实验组平均EPG)／对照组平均EPG×100%。 结果膜锚定表达疫苗组减虫率和减卵率较分泌表达疫苗组有所增加，但无显著性差异(P0.05)，较空载体组和生理盐水组的差异有极显著性意义(P0.01)； 结论膜锚定型表达疫苗pIREs-sj14-Sj2e，组较分泌型表达疫苗可能能诱导小鼠产生更为有效的抗日本血吸虫感染的保护作用。
[Abstract]:Objective to construct the prokaryotic expression plasmid pET-_28a-sj23, of 23KD membrane protein gene of Schistosoma japonicum and transform it into Escherichia coli BL-21 (DE3) to induce its expression and purification. It provides antigen for the preparation of subunit vaccine and detection of specific antibodies and cytokines. Methods the sj23 gene was amplified from the total RNA of adult Schistosoma japonicum by RT-PcR and cloned into the PMDl8-T vector. BamH I and EcoR I were digested and cloned into the prokaryotic expression plasmid pET-28a,. The pET-sj23 plasmid was constructed and transformed into Escherichia coli BL-21 (DE3). The expression of the protein was induced by IPTG. The expression status of the protein was observed by sDs-PAGE. The protein was purified by nickel column affinity layer and the concentration of purified protein was determined by BcA method. Results the prokaryotic expression plasmid pET-28a-sj23, was successfully constructed and transformed into Escherichia coli BL-21 (DE3). SDs-PAGE showed that the fusion protein about 31.3kD was successfully expressed after IPTG induction. Purification of Recombinant protein by affinity layer with concentration of O.8 mg/ml. Conclusion sj23 antigen was successfully prepared, which laid a foundation for the study of subunit vaccine and the detection of specific antibodies and cytokines. Objective to compare the antiinfective effect of membrane anchored pIREs-sj14-Sj26 vaccine and secretory pIREs-sj14-Sj26, vaccine in BALB/c mice infected with Schistosoma japonicum, and to explore the effect and possible mechanism of membrane anchored vaccine and secretory vaccine. Methods A large number of membrane anchored pIREs-sj14-Sj26 expression plasmids, secretory pIREs-sj14-Sj26, expression plasmids and empty pIREs plasmids were extracted and purified. The three plasmids were injected intramuscularly into BALB/c mice with 10 mice in each group. The normal saline group was used as the blank control group. Immunization was done once every 2 weeks for 3 times. After immunization, each mouse was infected with 40 cercariae through abdomen. After 45 days of infection with cercariae, the adult Schistosoma japonicum was collected by mesenteric vein perfusion and tablet pressing in each group, and the liver was reserved. The number of adult Schistosoma japonicum collected from each mouse was calculated, and the average adult number of each group was calculated. The worm reduction rate was calculated as follows: worm reduction rate = (average adult number in the control group and adult number in the experimental group) / average seizure number of adult worms 脳 100% in the control group. The total weight of rat liver was taken out and the liver tissue was digested with 5% potassium hydroxide. The egg reduction rate (EPG),) per gram liver tissue was calculated by the following formula: egg reduction rate = (average EPG- average EPG in the control group) / average EPG 脳 100g in the control group. Results the worm reduction rate and egg reduction rate in the membrane anchored expression vaccine group were higher than those in the secretory expression vaccine group, but there was no significant difference (P0.05). There was a significant difference between empty vector group and saline group (P0.01). Conclusion compared with secretory pIREs-sj14-Sj2e, the membrane anchored expression vaccine pIREs-sj14-Sj2e, can induce more effective protective effect against schistosomiasis japonicum infection in mice.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
[Abstract]:Objective to construct the prokaryotic expression plasmid pET-_28a-sj23, of 23KD membrane protein gene of Schistosoma japonicum and transform it into Escherichia coli BL-21 (DE3) to induce its expression and purification. It provides antigen for the preparation of subunit vaccine and detection of specific antibodies and cytokines. Methods the sj23 gene was amplified from the total RNA of adult Schistosoma japonicum by RT-PcR and cloned into the PMDl8-T vector. BamH I and EcoR I were digested and cloned into the prokaryotic expression plasmid pET-28a,. The pET-sj23 plasmid was constructed and transformed into Escherichia coli BL-21 (DE3). The expression of the protein was induced by IPTG. The expression status of the protein was observed by sDs-PAGE. The protein was purified by nickel column affinity layer and the concentration of purified protein was determined by BcA method. Results the prokaryotic expression plasmid pET-28a-sj23, was successfully constructed and transformed into Escherichia coli BL-21 (DE3). SDs-PAGE showed that the fusion protein about 31.3kD was successfully expressed after IPTG induction. Purification of Recombinant protein by affinity layer with concentration of O.8 mg/ml. Conclusion sj23 antigen was successfully prepared, which laid a foundation for the study of subunit vaccine and the detection of specific antibodies and cytokines. Objective to compare the antiinfective effect of membrane anchored pIREs-sj14-Sj26 vaccine and secretory pIREs-sj14-Sj26, vaccine in BALB/c mice infected with Schistosoma japonicum, and to explore the effect and possible mechanism of membrane anchored vaccine and secretory vaccine. Methods A large number of membrane anchored pIREs-sj14-Sj26 expression plasmids, secretory pIREs-sj14-Sj26, expression plasmids and empty pIREs plasmids were extracted and purified. The three plasmids were injected intramuscularly into BALB/c mice with 10 mice in each group. The normal saline group was used as the blank control group. Immunization was done once every 2 weeks for 3 times. After immunization, each mouse was infected with 40 cercariae through abdomen. After 45 days of infection with cercariae, the adult Schistosoma japonicum was collected by mesenteric vein perfusion and tablet pressing in each group, and the liver was reserved. The number of adult Schistosoma japonicum collected from each mouse was calculated, and the average adult number of each group was calculated. The worm reduction rate was calculated as follows: worm reduction rate = (average adult number in the control group and adult number in the experimental group) / average seizure number of adult worms 脳 100% in the control group. The total weight of rat liver was taken out and the liver tissue was digested with 5% potassium hydroxide. The egg reduction rate (EPG),) per gram liver tissue was calculated by the following formula: egg reduction rate = (average EPG- average EPG in the control group) / average EPG 脳 100g in the control group. Results the worm reduction rate and egg reduction rate in the membrane anchored expression vaccine group were higher than those in the secretory expression vaccine group, but there was no significant difference (P0.05). There was a significant difference between empty vector group and saline group (P0.01). Conclusion compared with secretory pIREs-sj14-Sj2e, the membrane anchored expression vaccine pIREs-sj14-Sj2e, can induce more effective protective effect against schistosomiasis japonicum infection in mice.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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