【摘要】：目的構(gòu)建日本血吸蟲23KD膜蛋白基因的原核表達(dá)質(zhì)粒pET-_28a-sj23，將其轉(zhuǎn)化大腸桿菌BL-21(DE3)，誘導(dǎo)其表達(dá)并純化此蛋白，為制備亞單位疫苗和檢測特異性抗體及細(xì)胞因子提供抗原。 方法采用RT-PcR的方法，以日本血吸蟲成蟲總RNA為模板，擴(kuò)增得到sj23基因，并克隆至PMDl8-T載體中，經(jīng)BamH I和EcoR I雙酶切后定向克隆于原核表達(dá)質(zhì)粒pET-28a，構(gòu)建成pET-sj23質(zhì)粒，并將此質(zhì)粒轉(zhuǎn)化大腸桿菌BL-21 (DE3)，使用IPTG誘導(dǎo)蛋白表達(dá)，sDs-PAGE觀察其表達(dá)狀態(tài)，鎳柱親和層忻純化此蛋白，BcA法測定純化蛋白濃度。 結(jié)果成功構(gòu)建原核表達(dá)質(zhì)粒pET-28a-sj23，并轉(zhuǎn)化至大腸桿菌BL-21(DE3)，sDs-PAGE顯示經(jīng)IPTG誘導(dǎo)之后成功表達(dá)出約31.3kD左右的融合蛋白(其中N-端含有8 kD的原核多肽)，親和層忻純化重組蛋白，濃度為O.8 mg／ml。 結(jié)論成功制備了sj23抗原，為亞單位疫苗的研究及特異性抗體和細(xì)胞因子的檢測打下了基礎(chǔ)。 目的比較膜錨定型pIREs-sj14-Sj26表達(dá)疫苗和分泌型pIREs-sj14-Sj26，表達(dá)疫苗在血吸蟲感染BALB／c小鼠體內(nèi)的抗感染效果，初步探討膜錨定與分泌型的疫苗的作用效果和可能機(jī)制。 方法大量提取并純化膜錨定型pIREs-sj14-Sj26表達(dá)質(zhì)粒、分泌型pIREs-sj14-Sj26，表達(dá)質(zhì)粒以及空pIREs質(zhì)粒，，并用此三種質(zhì)粒肌肉注射免疫BALB／c小鼠，每組10只，并設(shè)置生理鹽水組作為空白對照組。每隔2周加強(qiáng)免疫1次，共免疫3次。完成免疫后，每只小鼠經(jīng)腹部感染40土1條尾蚴，尾蚴攻擊感染45d后，各組小鼠經(jīng)腸系膜靜脈灌注法和壓片法收集血吸蟲成蟲，并留取肝臟備用。分別計(jì)算每只小鼠中收集的血吸蟲成蟲數(shù)，計(jì)算每組小鼠的平均成蟲數(shù)，按下式計(jì)算減蟲率：減蟲率=(對照組平均獲成蟲數(shù)．實(shí)驗(yàn)組平均獲成蟲數(shù))／對照組平均檢獲成蟲數(shù)×100%。稱取鼠肝總重量，按常規(guī)方法用5%氫氧化鉀消化肝組織，計(jì)算每克肝組織蟲卵數(shù)(EPG)，按下式計(jì)算減卵率：減卵率=(對照組平均EPG-實(shí)驗(yàn)組平均EPG)／對照組平均EPG×100%。 結(jié)果膜錨定表達(dá)疫苗組減蟲率和減卵率較分泌表達(dá)疫苗組有所增加，但無顯著性差異(P0.05)，較空載體組和生理鹽水組的差異有極顯著性意義(P0.01)； 結(jié)論膜錨定型表達(dá)疫苗pIREs-sj14-Sj2e，組較分泌型表達(dá)疫苗可能能誘導(dǎo)小鼠產(chǎn)生更為有效的抗日本血吸蟲感染的保護(hù)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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本文編號：2310329