本文關(guān)鍵詞：剛地弓形蟲peroxiredoxin基因的克隆、表達(dá)、純化及免疫保護(hù)性研究　出處：《山西醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 剛地弓形蟲 Peroxiredoxin 克隆 原核表達(dá) 蛋白純化 免疫原性 滴鼻免疫 免疫保護(hù)

【摘要】： 實(shí)驗(yàn)?zāi)康?對(duì)剛地弓形蟲peroxiredoxin(TgPrx)基因進(jìn)行克隆、表達(dá)、純化,分析其免疫原性,觀察TgPrx免疫小鼠誘導(dǎo)的黏膜及系統(tǒng)免疫應(yīng)答及其抗弓形蟲感染的能力,探討TgPrx作為弓形蟲疫苗候選抗原的可能性。 實(shí)驗(yàn)方法 第一部分:構(gòu)建重組質(zhì)粒pET30a/TgPrx,并在大腸桿菌中高效表達(dá)可溶性蛋白。收集、純化RH株弓形蟲速殖子,提取總RNA;設(shè)計(jì)合成引物并引入EcoR I和Xho I酶切位點(diǎn),RT-PCR擴(kuò)增編碼TgPrx的基因片段克隆到原核質(zhì)粒pET30a中,經(jīng)雙酶切、PCR及測序鑒定陽性克隆;在大腸桿菌BL21/DE3中用IPTG誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)SDS-PAGE進(jìn)行鑒定。 第二部分:對(duì)原核系統(tǒng)表達(dá)的rTgPrx進(jìn)行純化、免疫原性分析并制備抗血清。大量表達(dá)rTgPrx可溶性蛋白,以Ni-NTA層析法純化蛋白,用兔抗弓形蟲血清做Westen blotting分析其免疫原性;以純化的TgPrx皮下注射免疫Wistar大鼠,ELISA測定血清中抗體滴度;純化的重組rTgPrx蛋白加入不同濃度的蛋白酶抑制劑——苯甲基磺酰氟(Phenylmethane sulfonyl fluoride,PMSF),觀察該蛋白的降解情況;采用免疫組化方法,對(duì)Prx做初步定位。 第三部分:觀察不同劑量rTgPrx滴鼻免疫小鼠誘導(dǎo)的黏膜和系統(tǒng)免疫應(yīng)答及抗弓形蟲感染作用。BALB/c小鼠75只隨機(jī)分為5組,免疫組分別用10μg、20μg、30μg、40μg rTgPrx/只滴鼻免疫小鼠3次,各間隔2周,rTgPrx溶于20μL PBS中,對(duì)照組用等量PBS滴鼻。末次免疫后第14 d,用1×104個(gè)速殖子/只灌胃攻擊全部小鼠,觀察小鼠健康狀況。攻擊后第30 d,眼靜脈叢采血,頸椎脫臼處死小鼠,檢測腸液IgA和血清IgG,計(jì)數(shù)肝、腦組織內(nèi)弓形蟲速殖子,分離并計(jì)數(shù)腸上皮內(nèi)淋巴細(xì)胞(intestinal intraepithelial lymphocytes, IEL)及脾淋巴細(xì)胞。 實(shí)驗(yàn)結(jié)果 從弓形蟲RH株cDNA中擴(kuò)增出591 bp的TgPrx基因片段,并成功構(gòu)建重組質(zhì)粒pET30a/TgPrx;SDS-PAGE結(jié)果表明,目的基因在大腸桿菌BL21/DE3中高效表達(dá),相對(duì)分子量(Mr)約32kDa,比預(yù)期值大約7kDa。 Western blotting顯示純化后的rTgPrx能被兔抗弓形蟲免疫血清識(shí)別;免疫大鼠后可誘導(dǎo)產(chǎn)生高滴度的特異性抗體,該蛋白在-20℃保存2周時(shí)開始降解,反復(fù)凍融可加速其降解;免疫組化結(jié)果顯示大鼠抗TgPrx血清能特異性識(shí)別速殖子中的Prx,Prx分布部位呈棕色反應(yīng)。 攻擊后第7～14 d,小鼠出現(xiàn)豎毛、倦怠、活動(dòng)減少、飲水及采食減少等異常表現(xiàn),10μg組和對(duì)照組小鼠死亡4只,20μg、30μg組死亡6只,40μg組死亡9只。40μg組肝、腦組織內(nèi)蟲荷(速殖子數(shù))顯著低于10μg、20μg、30μg組和對(duì)照組(P0.05),40μg組血清IgG、IEL及脾淋巴細(xì)胞數(shù)量均高于對(duì)照組(P0.05),腸液IgA無差異。 結(jié)論 成功構(gòu)建重組質(zhì)粒pET30a/TgPrx并在大腸桿菌中高效表達(dá)可溶性蛋白。原核系統(tǒng)表達(dá)的rTgPrx具有免疫原性。40μg組rTgPrx滴鼻免疫更有效誘導(dǎo)了黏膜和系統(tǒng)免疫應(yīng)答,并有效抵抗弓形蟲感染。
[Abstract]:Experimental purpose The TgPrx gene of Toxoplasma gondii was cloned, expressed, purified, and its immunogenicity was analyzed. To observe the mucosal and systemic immune response induced by TgPrx and its ability to resist Toxoplasma gondii infection, and to explore the possibility of TgPrx as a vaccine candidate antigen for Toxoplasma gondii (Toxoplasma gondii). Experimental method Part one: construct recombinant plasmid pET30a / TgPrx and express soluble protein in Escherichia coli, collect, purify Toxoplasma gondii Tachyzoites of RH strain, extract total RNAs; Primers were designed and synthesized, and EcoR I and Xho I restriction sites were used to amplify the gene fragment encoding TgPrx by RT-PCR. The fragment was cloned into prokaryotic plasmid pET30a and digested by double enzyme. The positive clones were identified by PCR and sequencing. The expression was induced by IPTG in Escherichia coli BL21/DE3, and the expressed product was identified by SDS-PAGE. The second part: the rTgPrx expressed in prokaryotic system was purified, the immunogenicity was analyzed and antiserum was prepared. The soluble protein of rTgPrx was expressed in large quantities, and the protein was purified by Ni-NTA chromatography. The immunogenicity of Toxoplasma gondii was determined by Westen blotting. The antibody titers in serum were determined by subcutaneous injection of purified TgPrx into Wistar rats by Elisa. The purified recombinant rTgPrx protein was added with Phenylmethane sulfonyl fluoride, a protease inhibitor with different concentrations. PMSF was used to observe the degradation of the protein. Immunohistochemical method was used to locate Prx. Part three: 75 mice were randomly divided into 5 groups to observe the mucosal and systemic immune response induced by nasal immunization with different doses of rTgPrx and the anti-Toxoplasma gondii infection. The mice in the immunized group were immunized with 10 渭 g rTgPrx40 渭 g rTgPrx40 渭 g rTgPrx1 for 3 times, and the rTgPrx was dissolved in 20 渭 L PBS at intervals of 2 weeks. The control group was given the same dose of PBS nasal drip. On the 14th day after the last immunization, all the mice were challenged with 1 脳 104 tachyzoites per gavage only to observe the health status of the mice. 30 days after the attack, blood was collected from the ocular venous plexus. The mice were killed by dislocation of cervical vertebrae, IgA in intestinal fluid and serum were detected, and Toxoplasma gondii Tachyzoites in liver and brain were counted. Intestinal intraepithelial lymphocytes (IELs) and splenic lymphocytes were isolated and counted. Experimental results The TgPrx gene fragment of 591bp was amplified from cDNA of Toxoplasma gondii RH strain and the recombinant plasmid pET30a / TgPrx was constructed successfully. SDS-PAGE results showed that the target gene was highly expressed in Escherichia coli BL21/DE3 with a relative molecular weight of about 32 kDa, which was about 7 kDa than the expected value. Western blotting showed that purified rTgPrx could be recognized by rabbit anti-Toxoplasma immunity serum. High titer specific antibody was induced after immunizing rats. The protein began to degrade at -20 鈩，

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