肺炎鏈球菌非經(jīng)典分泌蛋白GAPDH的分泌機(jī)制及其與熱休克蛋白DnaJ的相互作用研究
發(fā)布時(shí)間：2019-05-15 16:47
【摘要】：目的明確肺炎鏈球菌非經(jīng)典分泌蛋白GAPDH的分泌是否由細(xì)菌裂解引起,并確定GAPDH分泌必需結(jié)構(gòu)域。同時(shí),驗(yàn)證該蛋白與熱休克蛋白的DnaJ的相互作用,為深入研究GAPDH的分泌機(jī)制奠定基礎(chǔ)。方法原核表達(dá)GAPDH重組蛋白并制備其多克隆抗體,利用該多克隆抗體通過(guò)Western Blot評(píng)價(jià)GAPDH在肺炎鏈球菌中的保守性。缺失肺炎鏈球菌主要自溶酶LytA使其喪失自溶能力,通過(guò)Western Blot比較野生菌和LytA缺陷菌GAPDH的分泌是否存在差異。同時(shí)以只在細(xì)胞內(nèi)表達(dá)的CodY蛋白為參照,鑒定肺炎鏈球菌對(duì)數(shù)生長(zhǎng)早期GAPDH的分泌是否伴有細(xì)菌裂解。利用Jpred3軟件分析GAPDH二級(jí)結(jié)構(gòu)和功能結(jié)構(gòu)域,利用Swiss-Model軟件對(duì)GAPDH進(jìn)行同源模建,根據(jù)生物信息學(xué)結(jié)果進(jìn)行截短表達(dá)設(shè)計(jì)。利用分子克隆技術(shù)將不同GAPDH截短表達(dá)基因片段克隆到異位表達(dá)載體pJW-v25質(zhì)粒,經(jīng)測(cè)序鑒定后將重組質(zhì)粒轉(zhuǎn)化肺炎鏈球菌D39,采用0.15mm Zn2+誘導(dǎo)表達(dá)含有GFP標(biāo)簽的GAPDH。分離亞組份,采用Western Blot檢測(cè)截短表達(dá)的GAPDH在肺炎鏈球菌的定位,初步確定分泌必需結(jié)構(gòu)域。采用結(jié)構(gòu)替代和氨基酸突變兩種方法進(jìn)一步驗(yàn)證分泌必需結(jié)構(gòu)域在GAPDH分泌中的作用,并確定參與GAPDH分泌的關(guān)鍵性氨基酸。將肺炎鏈球菌GAPDH分泌必需結(jié)構(gòu)域和枯草芽孢桿菌168菌株的GAPDH同源序列進(jìn)行替換,評(píng)價(jià)該分泌必需結(jié)構(gòu)域是否具有普遍性。在肺炎鏈球菌中,將GAPDH的結(jié)構(gòu)域Ⅰ進(jìn)行直接連接或柔性結(jié)構(gòu)連接后,與GFP標(biāo)簽蛋白融合表達(dá),觀察融合蛋白的分泌及表面定位情況,以確定結(jié)構(gòu)域Ⅰ能否作為一個(gè)信號(hào)肽介導(dǎo)蛋白質(zhì)的分泌。在肺炎鏈球菌中異位表達(dá)枯草芽孢桿菌168菌株GAPDH,觀察其分泌情況,探討GAPDH在肺炎鏈球菌和枯草芽孢桿菌中是否具有共同的分泌途徑。采用大腸桿菌雙雜交系統(tǒng)、直接結(jié)合實(shí)驗(yàn)、生物膜干涉技術(shù)驗(yàn)證GAPDH和熱休克蛋白DnaJ的相互作用,并初步探討DnaJ對(duì)GADPH分泌的影響。結(jié)果獲得了純度達(dá)90%的GAPDH重組蛋白,并制備了效價(jià)達(dá)107的抗GAPDH多克隆抗體。Western blot結(jié)果顯示,在對(duì)數(shù)生長(zhǎng)早期二型肺炎鏈球菌莢膜型D39和莢膜缺陷型R6細(xì)胞壁及培養(yǎng)上清中均檢測(cè)到GAPDH的表達(dá),但是并沒(méi)有檢測(cè)到CodY的表達(dá)。同時(shí),主要自溶酶LytA缺失后,培養(yǎng)上清依舊檢測(cè)到GAPDH的表達(dá),與野生菌相比無(wú)明顯差異。提示,肺炎鏈球菌GAPDH的分泌不是由于細(xì)菌自溶引起。用Jpred3軟件分析發(fā)現(xiàn),GAPDH具有兩個(gè)結(jié)構(gòu)域：結(jié)構(gòu)域Ⅰ和Ⅱ。結(jié)構(gòu)域Ⅰ由N末端的1-150aa和C末端α螺旋(317-335aa)構(gòu)成。結(jié)構(gòu)域Ⅱ由151-316aa構(gòu)成。對(duì)GAPDH進(jìn)行同源建模,也發(fā)現(xiàn)GAPDH N末端和C末端在空間上相互靠近構(gòu)成結(jié)構(gòu)域Ⅰ。根據(jù)以上生物信息學(xué)結(jié)果我們?cè)O(shè)計(jì)了GAPDH截短表達(dá)。Western blot結(jié)果顯示,結(jié)構(gòu)域Ⅰ的N末端1-30aa和(或)C末端α螺旋缺失后,GAPDH都不能定位于細(xì)菌表面及分泌到培養(yǎng)上清,提示結(jié)構(gòu)域Ⅰ是GAPDH在細(xì)胞表面定為及分泌的必需結(jié)構(gòu)域。氨基酸突變結(jié)果顯示,N末端的氨基酸3VKVGIN9、18GGG20和C末端325RTLEYF330突變后GAPDH的表面定位和胞外分泌都缺失,提示這些氨基酸是GAPDH的表面定位和分泌的關(guān)鍵氨基酸。此外10F突變后,GAPDH在上清中消失,但仍出現(xiàn)于細(xì)胞表面,提示該突變只影響了GAPDH在上清的分泌,并不影響GAPDH在細(xì)胞表面的定位；而322QLV324突變后,GAPDH不能定位到細(xì)胞表面,但是可以分泌到培養(yǎng)上清,提示該突變只影響了GAPDH在細(xì)胞表面的定位,并不影響GAPDH在上清的分泌。單獨(dú)的結(jié)構(gòu)域Ⅰ與GFP融合后,其只表達(dá)于胞內(nèi),上清中沒(méi)有檢測(cè)到融合蛋白,柔性結(jié)構(gòu)連接的結(jié)構(gòu)域Ⅰ也不能分泌到胞外,提示單獨(dú)的結(jié)構(gòu)域Ⅰ不能介導(dǎo)蛋白質(zhì)的分泌�？莶菅挎邨U菌168菌株GAPDH在肺炎鏈球菌中能夠表達(dá),但在細(xì)胞表面和上清中均檢測(cè)不到該蛋白,提示其在肺炎鏈球菌中不能定位到細(xì)胞表面及分泌到胞外,表明GAPDH在枯草芽孢桿菌和肺炎鏈球菌中的分泌途徑存在差異。大腸桿菌雙雜交、ELISA直接結(jié)合實(shí)驗(yàn)和BLI生物膜干涉技術(shù)結(jié)果顯示GAPDH和熱休克蛋白DnaJ具有直接的相互作用。結(jié)論肺炎鏈球菌GAPDH的分泌并不是細(xì)菌自身裂解引起的,結(jié)構(gòu)域Ⅰ的是其表面定位及分泌所必需的,但不足以使蛋白分泌到胞外。N末端氨基酸3VKVGIN9、18GGG20和C末端325RTLEYF330是GAPDH的表面定位和分泌的關(guān)鍵氨基酸。肺炎鏈球菌和枯草芽孢桿菌中的GAPDH分泌途徑存在差異。另外,GAPDH和熱休克蛋白DnaJ具有直接的相互作用。這些結(jié)果為進(jìn)一步研究肺炎鏈球菌非經(jīng)典分泌蛋白GAPDH的分泌奠定了堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)和理論基礎(chǔ)。
[Abstract]:Objective To determine whether the secretion of non-classical secretory protein GAPDH of S. pneumoniae is caused by bacterial lysis, and to determine the necessary domain of GAPDH secretion. At the same time, the interaction between the protein and the DnaJ of the heat shock protein is verified, which lays the foundation for further study of the secretion mechanism of the GAPDH. Methods The recombinant protein of GAPDH was expressed and its polyclonal antibody was prepared. The conservative of GAPDH in Streptococcus pneumoniae was evaluated by Western Blot using the polyclonal antibody. The main autoplasmin, LytA, of S. pneumoniae was deleted and its self-dissolving ability was lost, and there was a difference in the secretion of GAPDH from the wild and Lydia-deficient bacteria by Western Blot. At the same time, it was determined whether the secretion of GAPDH in the logarithmic growth of S. pneumoniae was accompanied by bacterial lysis with reference to the CodY protein expressed in the cells. The secondary structure and functional domain of GAPDH were analyzed by means of Jpred3 software, and the homologous model of GAPDH was carried out by using the Swiss-Model software, and the truncated expression was designed according to the results of bioinformatics. The pJW-v25 plasmid was cloned into pJW-v25 plasmid by using a molecular cloning technique, and the recombinant plasmid was transformed to S. pneumoniae D39 by sequencing, and the GAPDH containing the GFP label was induced with 0.15 mm of Zn2 +. The subcomponent was isolated and the truncated expression of GAPDH was detected by Western Blot to determine the secretory essential domain. The role of the secretory essential domain in the secretion of GAPDH is further verified by the two methods of structural substitution and amino acid mutation, and the key amino acids involved in the secretion of GAPDH are determined. The GAPDH secretion necessary domain of S.pneumoniae and the GAPDH homologous sequence of the Bacillus subtilis 168 strain are replaced to evaluate whether the secretory required domain is universal. In the streptococcus pneumoniae, the domain I of the GAPDH is directly connected or the flexible structure is connected, and is fused and expressed with the GFP label protein to observe the secretion of the fusion protein and the surface positioning condition, so as to determine whether the domain I can mediate the secretion of the protein as a signal peptide. GAPDH of Bacillus subtilis 168 was heterotopic in S. pneumoniae, and its secretion was observed, and whether GAPDH had a common secretory pathway in S. pneumoniae and Bacillus subtilis was discussed. The interaction between the GAPDH and the heat shock protein DnaJ was verified by using the double-hybrid system of E. coli, and the effect of DnaJ on the secretion of GADPH was also discussed. As a result, the GAPDH recombinant protein with the purity of up to 90% was obtained, and the anti-GAPDH polyclonal antibody with the titer of 107 was prepared. The results of Western blot showed that the expression of GAPDH was detected in both the cell wall and the culture supernatant of both the S. pneumoniae membrane type D39 and the membrane defect type R6 in the early stage of logarithmic growth, but the expression of CodY was not detected. At the same time, the expression of GAPDH was still detected by the culture supernatant after the deletion of LytA, and there was no significant difference with the wild bacteria. It is suggested that the secretion of GAPDH of S. pneumoniae is not caused by the autolysis of bacteria. GAPDH has two domains: domains I and II, using Jpred3 software analysis. Domain I consists of N-terminal 1-150aa and C-terminal polar helix (317-335aa). Domain II is composed of 151-316aa. Homologous modeling of GAPDH was performed, and the N-terminal and C-terminal of the GAPDH were also found to be spatially close to each other to form the domain I. Based on the above bioinformatics results, we designed the GAPDH truncated expression. Western blot showed that the GAPDH could not be located on the surface of the bacteria and secreted into the culture supernatant after the N-terminal 1-30aa and/ or the C-terminal of the domain I was deleted, suggesting that the domain I was the required domain of the GAPDH on the surface of the cell and secreted. The amino acid mutation results showed that the surface location and extracellular secretion of the GAPDH after the mutation of the amino acid 3VKVGIN9, 18GGG 20 and the C-terminal 325RTLEYF330 at the N-terminal were deleted, suggesting that these amino acids were the key amino acids for the surface localization and secretion of the GAPDH. In addition, after the 10-F mutation, GAPDH disappeared in the supernatant, but appeared on the surface of the cell, suggesting that the mutation only affected the secretion of GAPDH on the supernatant, and did not affect the localization of the GAPDH on the cell surface; and after the 322-QLV324 mutation, the GAPDH could not be localized to the cell surface, but could be secreted into the culture supernatant, It is suggested that the mutation only affects the localization of GAPDH on the cell surface, and does not affect the secretion of GAPDH on the supernatant. After the single domain I was fused with GFP, it was only expressed in the cell, and the fusion protein was not detected in the supernatant. The domain I linked to the flexible structure could not be secreted to the extracellular domain, suggesting that the separate domain I could not mediate the secretion of the protein. The Bacillus subtilis 168 strain GAPDH can be expressed in the streptococcus pneumoniae, but the protein is not detected in both the cell surface and the supernatant, suggesting that it cannot be positioned on the surface of the cell and secreted into the extracellular domain in the streptococcus pneumoniae, It is shown that the secretory pathway of GAPDH in Bacillus subtilis and S. pneumoniae is different. The direct interaction of GAPDH and the heat shock protein DnaJ was shown by the direct binding experiment of E. coli and the BLI biofilm interference technique. Conclusion The secretion of the GAPDH of S. pneumoniae is not caused by the self-lysis of the bacteria, and the domain I is necessary for its surface localization and secretion, but not enough to secrete the protein to the extracellular domain. The N-terminal amino acids 3VKVGIN9, 18GGG20 and C-terminal 325RTLEYF330 are the key amino acids for the surface localization and secretion of GAPDH. There is a difference in the secretory pathway of the GAPDH in S. pneumoniae and Bacillus subtilis. In addition, the GAPDH and the heat shock protein DnaJ have a direct interaction. These results provide a solid experimental basis and a theoretical basis for further study of the secretion of non-classical secretory protein GAPDH of Streptococcus pneumoniae.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
[Abstract]:Objective To determine whether the secretion of non-classical secretory protein GAPDH of S. pneumoniae is caused by bacterial lysis, and to determine the necessary domain of GAPDH secretion. At the same time, the interaction between the protein and the DnaJ of the heat shock protein is verified, which lays the foundation for further study of the secretion mechanism of the GAPDH. Methods The recombinant protein of GAPDH was expressed and its polyclonal antibody was prepared. The conservative of GAPDH in Streptococcus pneumoniae was evaluated by Western Blot using the polyclonal antibody. The main autoplasmin, LytA, of S. pneumoniae was deleted and its self-dissolving ability was lost, and there was a difference in the secretion of GAPDH from the wild and Lydia-deficient bacteria by Western Blot. At the same time, it was determined whether the secretion of GAPDH in the logarithmic growth of S. pneumoniae was accompanied by bacterial lysis with reference to the CodY protein expressed in the cells. The secondary structure and functional domain of GAPDH were analyzed by means of Jpred3 software, and the homologous model of GAPDH was carried out by using the Swiss-Model software, and the truncated expression was designed according to the results of bioinformatics. The pJW-v25 plasmid was cloned into pJW-v25 plasmid by using a molecular cloning technique, and the recombinant plasmid was transformed to S. pneumoniae D39 by sequencing, and the GAPDH containing the GFP label was induced with 0.15 mm of Zn2 +. The subcomponent was isolated and the truncated expression of GAPDH was detected by Western Blot to determine the secretory essential domain. The role of the secretory essential domain in the secretion of GAPDH is further verified by the two methods of structural substitution and amino acid mutation, and the key amino acids involved in the secretion of GAPDH are determined. The GAPDH secretion necessary domain of S.pneumoniae and the GAPDH homologous sequence of the Bacillus subtilis 168 strain are replaced to evaluate whether the secretory required domain is universal. In the streptococcus pneumoniae, the domain I of the GAPDH is directly connected or the flexible structure is connected, and is fused and expressed with the GFP label protein to observe the secretion of the fusion protein and the surface positioning condition, so as to determine whether the domain I can mediate the secretion of the protein as a signal peptide. GAPDH of Bacillus subtilis 168 was heterotopic in S. pneumoniae, and its secretion was observed, and whether GAPDH had a common secretory pathway in S. pneumoniae and Bacillus subtilis was discussed. The interaction between the GAPDH and the heat shock protein DnaJ was verified by using the double-hybrid system of E. coli, and the effect of DnaJ on the secretion of GADPH was also discussed. As a result, the GAPDH recombinant protein with the purity of up to 90% was obtained, and the anti-GAPDH polyclonal antibody with the titer of 107 was prepared. The results of Western blot showed that the expression of GAPDH was detected in both the cell wall and the culture supernatant of both the S. pneumoniae membrane type D39 and the membrane defect type R6 in the early stage of logarithmic growth, but the expression of CodY was not detected. At the same time, the expression of GAPDH was still detected by the culture supernatant after the deletion of LytA, and there was no significant difference with the wild bacteria. It is suggested that the secretion of GAPDH of S. pneumoniae is not caused by the autolysis of bacteria. GAPDH has two domains: domains I and II, using Jpred3 software analysis. Domain I consists of N-terminal 1-150aa and C-terminal polar helix (317-335aa). Domain II is composed of 151-316aa. Homologous modeling of GAPDH was performed, and the N-terminal and C-terminal of the GAPDH were also found to be spatially close to each other to form the domain I. Based on the above bioinformatics results, we designed the GAPDH truncated expression. Western blot showed that the GAPDH could not be located on the surface of the bacteria and secreted into the culture supernatant after the N-terminal 1-30aa and/ or the C-terminal of the domain I was deleted, suggesting that the domain I was the required domain of the GAPDH on the surface of the cell and secreted. The amino acid mutation results showed that the surface location and extracellular secretion of the GAPDH after the mutation of the amino acid 3VKVGIN9, 18GGG 20 and the C-terminal 325RTLEYF330 at the N-terminal were deleted, suggesting that these amino acids were the key amino acids for the surface localization and secretion of the GAPDH. In addition, after the 10-F mutation, GAPDH disappeared in the supernatant, but appeared on the surface of the cell, suggesting that the mutation only affected the secretion of GAPDH on the supernatant, and did not affect the localization of the GAPDH on the cell surface; and after the 322-QLV324 mutation, the GAPDH could not be localized to the cell surface, but could be secreted into the culture supernatant, It is suggested that the mutation only affects the localization of GAPDH on the cell surface, and does not affect the secretion of GAPDH on the supernatant. After the single domain I was fused with GFP, it was only expressed in the cell, and the fusion protein was not detected in the supernatant. The domain I linked to the flexible structure could not be secreted to the extracellular domain, suggesting that the separate domain I could not mediate the secretion of the protein. The Bacillus subtilis 168 strain GAPDH can be expressed in the streptococcus pneumoniae, but the protein is not detected in both the cell surface and the supernatant, suggesting that it cannot be positioned on the surface of the cell and secreted into the extracellular domain in the streptococcus pneumoniae, It is shown that the secretory pathway of GAPDH in Bacillus subtilis and S. pneumoniae is different. The direct interaction of GAPDH and the heat shock protein DnaJ was shown by the direct binding experiment of E. coli and the BLI biofilm interference technique. Conclusion The secretion of the GAPDH of S. pneumoniae is not caused by the self-lysis of the bacteria, and the domain I is necessary for its surface localization and secretion, but not enough to secrete the protein to the extracellular domain. The N-terminal amino acids 3VKVGIN9, 18GGG20 and C-terminal 325RTLEYF330 are the key amino acids for the surface localization and secretion of GAPDH. There is a difference in the secretory pathway of the GAPDH in S. pneumoniae and Bacillus subtilis. In addition, the GAPDH and the heat shock protein DnaJ have a direct interaction. These results provide a solid experimental basis and a theoretical basis for further study of the secretion of non-classical secretory protein GAPDH of Streptococcus pneumoniae.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
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