刚地弓形虫peroxiredoxin基因的克

发布时间：2018-01-14 19:36

  本文关键词：刚地弓形虫peroxiredoxin基因的克隆、表达、纯化及免疫保护性研究　出处：《山西医科大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： 刚地弓形虫 Peroxiredoxin 克隆 原核表达 蛋白纯化 免疫原性 滴鼻免疫 免疫保护

【摘要】： 实验目的 对刚地弓形虫peroxiredoxin(TgPrx)基因进行克隆、表达、纯化,分析其免疫原性,观察TgPrx免疫小鼠诱导的黏膜及系统免疫应答及其抗弓形虫感染的能力,探讨TgPrx作为弓形虫疫苗候选抗原的可能性。 实验方法 第一部分:构建重组质粒pET30a/TgPrx,并在大肠杆菌中高效表达可溶性蛋白。收集、纯化RH株弓形虫速殖子,提取总RNA;设计合成引物并引入EcoR I和Xho I酶切位点,RT-PCR扩增编码TgPrx的基因片段克隆到原核质粒pET30a中,经双酶切、PCR及测序鉴定阳性克隆;在大肠杆菌BL21/DE3中用IPTG诱导表达,表达产物经SDS-PAGE进行鉴定。 第二部分:对原核系统表达的rTgPrx进行纯化、免疫原性分析并制备抗血清。大量表达rTgPrx可溶性蛋白,以Ni-NTA层析法纯化蛋白,用兔抗弓形虫血清做Westen blotting分析其免疫原性;以纯化的TgPrx皮下注射免疫Wistar大鼠,ELISA测定血清中抗体滴度;纯化的重组rTgPrx蛋白加入不同浓度的蛋白酶抑制剂——苯甲基磺酰氟(Phenylmethane sulfonyl fluoride,PMSF),观察该蛋白的降解情况;采用免疫组化方法,对Prx做初步定位。 第三部分:观察不同剂量rTgPrx滴鼻免疫小鼠诱导的黏膜和系统免疫应答及抗弓形虫感染作用。BALB/c小鼠75只随机分为5组,免疫组分别用10μg、20μg、30μg、40μg rTgPrx/只滴鼻免疫小鼠3次,各间隔2周,rTgPrx溶于20μL PBS中,对照组用等量PBS滴鼻。末次免疫后第14 d,用1×104个速殖子/只灌胃攻击全部小鼠,观察小鼠健康状况。攻击后第30 d,眼静脉丛采血,颈椎脱臼处死小鼠,检测肠液IgA和血清IgG,计数肝、脑组织内弓形虫速殖子,分离并计数肠上皮内淋巴细胞(intestinal intraepithelial lymphocytes, IEL)及脾淋巴细胞。 实验结果 从弓形虫RH株cDNA中扩增出591 bp的TgPrx基因片段,并成功构建重组质粒pET30a/TgPrx;SDS-PAGE结果表明,目的基因在大肠杆菌BL21/DE3中高效表达,相对分子量(Mr)约32kDa,比预期值大约7kDa。 Western blotting显示纯化后的rTgPrx能被兔抗弓形虫免疫血清识别;免疫大鼠后可诱导产生高滴度的特异性抗体,该蛋白在-20℃保存2周时开始降解,反复冻融可加速其降解;免疫组化结果显示大鼠抗TgPrx血清能特异性识别速殖子中的Prx,Prx分布部位呈棕色反应。 攻击后第7～14 d,小鼠出现竖毛、倦怠、活动减少、饮水及采食减少等异常表现,10μg组和对照组小鼠死亡4只,20μg、30μg组死亡6只,40μg组死亡9只。40μg组肝、脑组织内虫荷(速殖子数)显著低于10μg、20μg、30μg组和对照组(P0.05),40μg组血清IgG、IEL及脾淋巴细胞数量均高于对照组(P0.05),肠液IgA无差异。 结论 成功构建重组质粒pET30a/TgPrx并在大肠杆菌中高效表达可溶性蛋白。原核系统表达的rTgPrx具有免疫原性。40μg组rTgPrx滴鼻免疫更有效诱导了黏膜和系统免疫应答,并有效抵抗弓形虫感染。
[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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