弓形虫翻译控制肿瘤蛋白的原核表达及促进组胺释放活性的研究
发布时间：2019-01-01 08:23
【摘要】：弓形虫(Toxoplasma gondii)主要寄生在细胞内,感染弓形虫的地区是呈全球式分布的,但不同的区域感染弓形虫的概率存在差异。当速殖子快速生长时,Th1型细胞免疫应答被快速诱导,从而产生炎性细胞因子。组胺是一个重要的炎性反应调节因子,是由翻译控制肿瘤蛋白(Translationally controlled tumor protein,TCTP)刺激嗜碱性粒细胞和肥大细胞所产生,从而起到诱发变态反应和过敏性炎症反应的作用。TCTP具有高度的保守性,但是有关弓形虫TCTP蛋白的功能和特性,目前为止并未见到文献报道,因此,对该蛋白组胺释放因子样活性和介导宿主炎性反应作用的研究将有助于揭示弓形虫的感染机制,对弓形虫病的防治以及对抑制感染该病的传播是非常重要的。为研究弓形虫感染后产生炎性反应的分子机制,本研究应用延伸PCR方法扩增弓形虫翻译控制肿瘤蛋白(TCTP)基因和GST基因,并通过重叠延伸PCR将TCTP和GST基因融合后,构建了 pET-30a-TCTP-GST原核表达的载体;将此载体进行转化试验,同时利用SDS-PAGE和Western-blot的试验方法对表达产物进行分析,之后利用Native-PAGE鉴定TgTCTP多聚体存在形式,并应用弗氏佐剂制备TgTCTP蛋白亚单位疫苗,接种小鼠制备抗蛋白的血清;利用间接免疫荧光检测方法(IFAT)鉴定TgTCTP的定位情况;同时利用组胺释放活性的竞争性ELISA的检测方法鉴定重组TgTCTP蛋白(rTgTCTP)对小鼠腹腔细胞释放组胺的活性的作用。结果表明,经PCR反应分别获得的弓形虫TCTP和GST基因大小为510 bp和690 bp,同时融合的TCTP和GST基因长度为1200 bp,构建了pET-30a-TCTP-GST的重组质粒;同时利用SDS-PAGE的试验检测到该重组质粒表达蛋白的分子量是47 ku,同时发现该蛋白具备良好的反应原性;检测到抗TgTCTP的小鼠血清多克隆抗体可以识别出19ku弓形虫虫体天然蛋白;TgTCTP能够分泌到胞浆内;重组TgTCTP蛋白(rTgTCTP)促进小鼠腹腔细胞释放组胺的活性。综上试验结果表明,通过检测发现TgTCTP能够定位于弓形虫的胞浆内,是一种分泌性蛋白,具有分泌到虫体胞核外侧的功能,同时利用组胺释放检测试剂盒发现TgTCTP蛋白对组胺释放的作用,结果显示,TgTCTP蛋白具有促进组胺释放的作用。综上所诉,本研究为进一步验证TgTCTP介导宿主炎性反应的分子机制奠定了基础。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is mainly parasitic in the cells, and the area infected with Toxoplasma gondii is global distribution, but the probability of Toxoplasma gondii infection varies in different regions. When the tachyzoites grow rapidly, the Th1 type cellular immune response is induced quickly, thus producing inflammatory cytokines. Histamine is an important inflammatory response regulatory factor, produced by translation control tumor protein (Translationally controlled tumor protein,TCTP, which stimulates basophil and mast cells. TCTP is highly conserved, but the function and characteristics of Toxoplasma gondii TCTP protein have not been reported so far. The study of histamine releasing factor like activity and mediating the host inflammatory response will be helpful to reveal the infection mechanism of Toxoplasma gondii, and it is very important to prevent and control Toxoplasma gondii and to inhibit the transmission of the infection of Toxoplasma gondii. In order to study the molecular mechanism of inflammatory response after Toxoplasma gondii infection, Toxoplasma gondii (Toxoplasma gondii) translation control (TCTP) gene and GST gene were amplified by extended PCR, and TCTP and GST genes were fused by overlapping extension PCR. The prokaryotic expression vector of pET-30a-TCTP-GST was constructed. The expression product was analyzed by SDS-PAGE and Western-blot, then the form of TgTCTP polymer was identified by Native-PAGE, and the TgTCTP protein subunit vaccine was prepared by Freund's adjuvant. Mice were inoculated to prepare anti-protein serum. The localization of TgTCTP was identified by indirect immunofluorescence assay (IFAT) and the effect of recombinant TgTCTP protein (rTgTCTP) on the release of histamine from mouse peritoneal cells was evaluated by the competitive ELISA method of histamine releasing activity. The results showed that the length of TCTP and GST genes of Toxoplasma gondii TCTP and GST were 510 bp and 690 bp, respectively. The length of TCTP and GST genes was 1200 bp, and the recombinant plasmids of pET-30a-TCTP-GST were constructed. At the same time, the molecular weight of the expressed protein was 47 ku, by SDS-PAGE test. The polyclonal antibody against TgTCTP could recognize the natural protein of Toxoplasma gondii 19ku; TgTCTP could be secreted into the cytoplasm; and the recombinant TgTCTP protein (rTgTCTP) could promote the release of histamine from mouse peritoneal cells. The results show that TgTCTP can locate in the cytoplasm of Toxoplasma gondii and is a secretory protein with the function of secreting to the outer side of the nucleus of the body of Toxoplasma gondii (Toxoplasma gondii). The effect of TgTCTP protein on histamine release was also found by using histamine release assay kit. The results showed that TgTCTP protein could promote histamine release. In summary, this study laid a foundation for further verification of the molecular mechanism of host inflammatory response mediated by TgTCTP.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7
，
本文编号：2397271
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is mainly parasitic in the cells, and the area infected with Toxoplasma gondii is global distribution, but the probability of Toxoplasma gondii infection varies in different regions. When the tachyzoites grow rapidly, the Th1 type cellular immune response is induced quickly, thus producing inflammatory cytokines. Histamine is an important inflammatory response regulatory factor, produced by translation control tumor protein (Translationally controlled tumor protein,TCTP, which stimulates basophil and mast cells. TCTP is highly conserved, but the function and characteristics of Toxoplasma gondii TCTP protein have not been reported so far. The study of histamine releasing factor like activity and mediating the host inflammatory response will be helpful to reveal the infection mechanism of Toxoplasma gondii, and it is very important to prevent and control Toxoplasma gondii and to inhibit the transmission of the infection of Toxoplasma gondii. In order to study the molecular mechanism of inflammatory response after Toxoplasma gondii infection, Toxoplasma gondii (Toxoplasma gondii) translation control (TCTP) gene and GST gene were amplified by extended PCR, and TCTP and GST genes were fused by overlapping extension PCR. The prokaryotic expression vector of pET-30a-TCTP-GST was constructed. The expression product was analyzed by SDS-PAGE and Western-blot, then the form of TgTCTP polymer was identified by Native-PAGE, and the TgTCTP protein subunit vaccine was prepared by Freund's adjuvant. Mice were inoculated to prepare anti-protein serum. The localization of TgTCTP was identified by indirect immunofluorescence assay (IFAT) and the effect of recombinant TgTCTP protein (rTgTCTP) on the release of histamine from mouse peritoneal cells was evaluated by the competitive ELISA method of histamine releasing activity. The results showed that the length of TCTP and GST genes of Toxoplasma gondii TCTP and GST were 510 bp and 690 bp, respectively. The length of TCTP and GST genes was 1200 bp, and the recombinant plasmids of pET-30a-TCTP-GST were constructed. At the same time, the molecular weight of the expressed protein was 47 ku, by SDS-PAGE test. The polyclonal antibody against TgTCTP could recognize the natural protein of Toxoplasma gondii 19ku; TgTCTP could be secreted into the cytoplasm; and the recombinant TgTCTP protein (rTgTCTP) could promote the release of histamine from mouse peritoneal cells. The results show that TgTCTP can locate in the cytoplasm of Toxoplasma gondii and is a secretory protein with the function of secreting to the outer side of the nucleus of the body of Toxoplasma gondii (Toxoplasma gondii). The effect of TgTCTP protein on histamine release was also found by using histamine release assay kit. The results showed that TgTCTP protein could promote histamine release. In summary, this study laid a foundation for further verification of the molecular mechanism of host inflammatory response mediated by TgTCTP.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7
，
本文编号：2397271
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