弓形蟲翻譯控制腫瘤蛋白的原核表達及促進組胺釋放活性的研究
發(fā)布時間：2019-01-01 08:23
【摘要】：弓形蟲(Toxoplasma gondii)主要寄生在細胞內(nèi),感染弓形蟲的地區(qū)是呈全球式分布的,但不同的區(qū)域感染弓形蟲的概率存在差異。當速殖子快速生長時,Th1型細胞免疫應答被快速誘導,從而產(chǎn)生炎性細胞因子。組胺是一個重要的炎性反應調(diào)節(jié)因子,是由翻譯控制腫瘤蛋白(Translationally controlled tumor protein,TCTP)刺激嗜堿性粒細胞和肥大細胞所產(chǎn)生,從而起到誘發(fā)變態(tài)反應和過敏性炎癥反應的作用。TCTP具有高度的保守性,但是有關弓形蟲TCTP蛋白的功能和特性,目前為止并未見到文獻報道,因此,對該蛋白組胺釋放因子樣活性和介導宿主炎性反應作用的研究將有助于揭示弓形蟲的感染機制,對弓形蟲病的防治以及對抑制感染該病的傳播是非常重要的。為研究弓形蟲感染后產(chǎn)生炎性反應的分子機制,本研究應用延伸PCR方法擴增弓形蟲翻譯控制腫瘤蛋白(TCTP)基因和GST基因,并通過重疊延伸PCR將TCTP和GST基因融合后,構(gòu)建了 pET-30a-TCTP-GST原核表達的載體;將此載體進行轉(zhuǎn)化試驗,同時利用SDS-PAGE和Western-blot的試驗方法對表達產(chǎn)物進行分析,之后利用Native-PAGE鑒定TgTCTP多聚體存在形式,并應用弗氏佐劑制備TgTCTP蛋白亞單位疫苗,接種小鼠制備抗蛋白的血清;利用間接免疫熒光檢測方法(IFAT)鑒定TgTCTP的定位情況;同時利用組胺釋放活性的競爭性ELISA的檢測方法鑒定重組TgTCTP蛋白(rTgTCTP)對小鼠腹腔細胞釋放組胺的活性的作用。結(jié)果表明,經(jīng)PCR反應分別獲得的弓形蟲TCTP和GST基因大小為510 bp和690 bp,同時融合的TCTP和GST基因長度為1200 bp,構(gòu)建了pET-30a-TCTP-GST的重組質(zhì)粒;同時利用SDS-PAGE的試驗檢測到該重組質(zhì)粒表達蛋白的分子量是47 ku,同時發(fā)現(xiàn)該蛋白具備良好的反應原性;檢測到抗TgTCTP的小鼠血清多克隆抗體可以識別出19ku弓形蟲蟲體天然蛋白;TgTCTP能夠分泌到胞漿內(nèi);重組TgTCTP蛋白(rTgTCTP)促進小鼠腹腔細胞釋放組胺的活性。綜上試驗結(jié)果表明,通過檢測發(fā)現(xiàn)TgTCTP能夠定位于弓形蟲的胞漿內(nèi),是一種分泌性蛋白,具有分泌到蟲體胞核外側(cè)的功能,同時利用組胺釋放檢測試劑盒發(fā)現(xiàn)TgTCTP蛋白對組胺釋放的作用,結(jié)果顯示,TgTCTP蛋白具有促進組胺釋放的作用。綜上所訴,本研究為進一步驗證TgTCTP介導宿主炎性反應的分子機制奠定了基礎。
[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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.7
，
本文編號：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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.7
，
本文編號：2397271
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