肺炎链球菌非经典分泌蛋白GAPDH的分泌机制及其与热休克蛋白DnaJ的相互作用研究
发布时间：2019-05-15 16:47
【摘要】：目的明确肺炎链球菌非经典分泌蛋白GAPDH的分泌是否由细菌裂解引起,并确定GAPDH分泌必需结构域。同时,验证该蛋白与热休克蛋白的DnaJ的相互作用,为深入研究GAPDH的分泌机制奠定基础。方法原核表达GAPDH重组蛋白并制备其多克隆抗体,利用该多克隆抗体通过Western Blot评价GAPDH在肺炎链球菌中的保守性。缺失肺炎链球菌主要自溶酶LytA使其丧失自溶能力,通过Western Blot比较野生菌和LytA缺陷菌GAPDH的分泌是否存在差异。同时以只在细胞内表达的CodY蛋白为参照,鉴定肺炎链球菌对数生长早期GAPDH的分泌是否伴有细菌裂解。利用Jpred3软件分析GAPDH二级结构和功能结构域,利用Swiss-Model软件对GAPDH进行同源模建,根据生物信息学结果进行截短表达设计。利用分子克隆技术将不同GAPDH截短表达基因片段克隆到异位表达载体pJW-v25质粒,经测序鉴定后将重组质粒转化肺炎链球菌D39,采用0.15mm Zn2+诱导表达含有GFP标签的GAPDH。分离亚组份,采用Western Blot检测截短表达的GAPDH在肺炎链球菌的定位,初步确定分泌必需结构域。采用结构替代和氨基酸突变两种方法进一步验证分泌必需结构域在GAPDH分泌中的作用,并确定参与GAPDH分泌的关键性氨基酸。将肺炎链球菌GAPDH分泌必需结构域和枯草芽孢杆菌168菌株的GAPDH同源序列进行替换,评价该分泌必需结构域是否具有普遍性。在肺炎链球菌中,将GAPDH的结构域Ⅰ进行直接连接或柔性结构连接后,与GFP标签蛋白融合表达,观察融合蛋白的分泌及表面定位情况,以确定结构域Ⅰ能否作为一个信号肽介导蛋白质的分泌。在肺炎链球菌中异位表达枯草芽孢杆菌168菌株GAPDH,观察其分泌情况,探讨GAPDH在肺炎链球菌和枯草芽孢杆菌中是否具有共同的分泌途径。采用大肠杆菌双杂交系统、直接结合实验、生物膜干涉技术验证GAPDH和热休克蛋白DnaJ的相互作用,并初步探讨DnaJ对GADPH分泌的影响。结果获得了纯度达90%的GAPDH重组蛋白,并制备了效价达107的抗GAPDH多克隆抗体。Western blot结果显示,在对数生长早期二型肺炎链球菌荚膜型D39和荚膜缺陷型R6细胞壁及培养上清中均检测到GAPDH的表达,但是并没有检测到CodY的表达。同时,主要自溶酶LytA缺失后,培养上清依旧检测到GAPDH的表达,与野生菌相比无明显差异。提示,肺炎链球菌GAPDH的分泌不是由于细菌自溶引起。用Jpred3软件分析发现,GAPDH具有两个结构域：结构域Ⅰ和Ⅱ。结构域Ⅰ由N末端的1-150aa和C末端α螺旋(317-335aa)构成。结构域Ⅱ由151-316aa构成。对GAPDH进行同源建模,也发现GAPDH N末端和C末端在空间上相互靠近构成结构域Ⅰ。根据以上生物信息学结果我们设计了GAPDH截短表达。Western blot结果显示,结构域Ⅰ的N末端1-30aa和(或)C末端α螺旋缺失后,GAPDH都不能定位于细菌表面及分泌到培养上清,提示结构域Ⅰ是GAPDH在细胞表面定为及分泌的必需结构域。氨基酸突变结果显示,N末端的氨基酸3VKVGIN9、18GGG20和C末端325RTLEYF330突变后GAPDH的表面定位和胞外分泌都缺失,提示这些氨基酸是GAPDH的表面定位和分泌的关键氨基酸。此外10F突变后,GAPDH在上清中消失,但仍出现于细胞表面,提示该突变只影响了GAPDH在上清的分泌,并不影响GAPDH在细胞表面的定位；而322QLV324突变后,GAPDH不能定位到细胞表面,但是可以分泌到培养上清,提示该突变只影响了GAPDH在细胞表面的定位,并不影响GAPDH在上清的分泌。单独的结构域Ⅰ与GFP融合后,其只表达于胞内,上清中没有检测到融合蛋白,柔性结构连接的结构域Ⅰ也不能分泌到胞外,提示单独的结构域Ⅰ不能介导蛋白质的分泌。枯草芽孢杆菌168菌株GAPDH在肺炎链球菌中能够表达,但在细胞表面和上清中均检测不到该蛋白,提示其在肺炎链球菌中不能定位到细胞表面及分泌到胞外,表明GAPDH在枯草芽孢杆菌和肺炎链球菌中的分泌途径存在差异。大肠杆菌双杂交、ELISA直接结合实验和BLI生物膜干涉技术结果显示GAPDH和热休克蛋白DnaJ具有直接的相互作用。结论肺炎链球菌GAPDH的分泌并不是细菌自身裂解引起的,结构域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关键氨基酸。肺炎链球菌和枯草芽孢杆菌中的GAPDH分泌途径存在差异。另外,GAPDH和热休克蛋白DnaJ具有直接的相互作用。这些结果为进一步研究肺炎链球菌非经典分泌蛋白GAPDH的分泌奠定了坚实的实验基础和理论基础。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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