长双歧杆菌NCC2705与宿主细胞Caco2相互作用的研究
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摘要
双歧杆菌被认为是人肠道内最重要的益生菌,是评价人健康状况的指标之一。它能够调节并维持肠道微生态平衡,调节机体免疫,拮抗致病菌,合成营养物质,调节血脂,防治肿瘤等,具有保健、防病、治病的三重功效,一直倍受关注。双歧杆菌的粘附是其定植和发挥生理作用的前提,近年来许多学者进行了双歧杆菌粘附机制的相关研究,但尚未有定论。
本研究采用比较蛋白质组学的研究方法,筛选出长双歧杆菌NCC2705与宿主细胞Caco2相互作用前后差异表达的蛋白,通过质谱鉴定和生物信息学分析,最后确定目标蛋白Enolase、Tuf作为本研究的对象。构建重组质粒并在大肠杆菌中进行了GST融合表达,利用GST pull down技术证明纯化蛋白Enolase、Tuf能够与真核细胞表达的Plg相互作用,证明表达的目标蛋白具有Plg受体的功能。最后通过竞争性抑制实验,证明Enolase和Tuf蛋白能抑制双歧杆菌的粘附。
本研究利用比较蛋白质组学筛选出34个长双歧杆菌NCC2705与宿主细胞Caco2相互作用的差异蛋白,为双歧杆菌粘附机制的研究提供了新的角度;从34个差异蛋白中筛选出两个细胞壁蛋白Enolase和Tuf作为研究对象,并证明是粘附相关蛋白,为进一步阐明双歧杆菌粘附机制奠定了基础。
Background
Bifidobacteria spp. Which is health-promoting groups represent one of the most important physiological microorganisms of human intestinal microbiota.It is one of the largest classes in the gut of human being.Bifidobacteria can regulate and equilibrate the balance of gut microbiota.Numerous health-promoting contributions of bifidobacteria in the human gastrointestinal tract may include improved digestion and absorption, immunostimulation,vitamin synthesis,antagonism of the growth of pathogens,regulation of the blood-fat and inhibition of the tumor.Bifidobacteria was always paid close attention by researcher since 1899.There are eleven species of them have relationships with human including Bifidobacterium longum.Among them Bifidobacterium longum NCC2705 which has a identify heredity background has been sequenced.Adeherence of bacteria is precondition of setting or producing the effect , yet no studies have so far definitely demonstrated the adhesion mechanism.Accepted the theory of adherence is adhesion---acceptor.Adhesion may be lipoteichoicacid、carbohydrate or protein , nevertheless acceptor is accpetde glycoprotein accordantly.
Objective
Use of the comparative proteomic to sieve the different protein between B. lougum NCC2705 whether interact with Caco2.Finally, we definite the interest proteins and then to authenticate their fuction.The purpose is illuminate the mechanism of B. lougum NCC2705 adhere to host intestinal epithelial cell.It is important to explain the mechanism of interaction with host. To accumulate the evidence of the interaction between B. lougum NCC2705 and human intestinal epithelial cell, and to enhance the adherence of the Bifidobacterium which is inexistent in human gut track.
Methods
Use of Immobilized pH gradient (IPG) strips at the first step of the 2-dimensional electrophoresis. Use of ImageMaster 2D Platinum 5.0 to analyze distinct proteins which expression amount on interaction with Caco2 than on contrast bacterium as higher as 3 times were termed as different one .The distinct proteins were appraised by matrix-assisted desorption ionization - time of flight (MALDI-TOF MS) and electrospray ionization tandem mass spectrometer (ESI–MS/MS).The result of 2-dimensional electrophoresis was authenticated by reverse transcript polymerase chain reaction (RT-PCR) .We choose the enlase and tuf as the interest proteins among the 34 distinct proteins,which are located on the bacterial cell wall and acceptors of Plg in other bacteria. We supposed that Enolase and Tuf very probably function as an adhesion promoter.The genes encoding Enolase and Tuf proteins were amplified by PCR . After dissected with restriction endonucleases the gene fragments were ligated into PGEX-4T-1 vector distincly. The transformed clones were sequenced and the right one named GST-Eno/ GST-Tuf.Use of the same way to construct the Flag-Plg. Flag-Plg was transfected into 293 cell line to test the expression.The purified Enolase and Tuf proteins were put into lysate which expressed the Flag-Plg.To test the interaction by western blot.To indentify the fuction of Enolase and Tuf,put the purified proteins into cell culture fluid along with B.lougum NCC2705.Then observed the condition of adherence by microscope,and counted the number of B. lougum NCC2705 that adhere to the Caco2 in twenty random visual field. The supernatant was detected the level of the cell factor LDH and TNF-a to solidify the hypothesis that Enolase and Tuf can competitive inhibit the adherence of B. lougum NCC2705.
Results
1. Get 34 distinct proteins which expression amount on interaction with Caco2 than on contrast bacterium as higher as 3 times were termed as different one by 2-dimensional electrophoresis,there are 17 up-regulation expressing proteins and 17 down-regulation expressing in the B. lougum NCC2705 which interaction with Caco2;
2. We found that the 34 distinct proteins correspond to 31 proteins species by mass spectra identification and analysis. Categories were taken from COGs,distinct proteins come from 9 category mainly;
3. Recombinant plasmid GST-Eno、GST-Tuf、Flag-Plg were constructed successfully;
4. Expressed the proteins GST、GST-Enolase、GST-Tuf in BL21 successfully,and get the purified proteins by affinity chromatography;
5. Flag-Plg expressed Plg in 293T cell line by Western blot testing;
6. Enolase and Tuf can interact with Plg in vitro by GST-pull down;
7. We tested adhesion after incubating B. lougum NCC2705 to Caco2 cell and B. lougum NCC2705 together with the purified proteins to Caco2.The bacterium numer of adherent to Caco2 which mix the purified proteins was notablely less than the other group, suggesting that Enolase and Tuf may promote the strain’s binding to epithelial cells.
8. Cytokine LDH and TNF-a which were stimulated by pathogenic bacteria were reduced by the incubation of Caco-2 cells with B. lougum NCC2705,and enhanced by incubation with Enolase or Tuf, to illustrate Enolase、Tuf can inhibit the adherence of B. lougum NCC2705
Conclusion
1. Use of the comparative proteomic to sieve the 34 proteins between B. lougum NCC2705 whether interact with Caco2 .This way is effective method to research the adherence of bacteria,and sieve proteins correlateing to adheren.
2. Enolase and Tuf are described to be diretly involved in the adhesion of B. lougum NCC2705 to Caco-2 cells.This study develop the direction of researching adherence that locus on bacteria interaction with human plasminogen (Plg) system in addition.
本研究采用比较蛋白质组学的研究方法,筛选出长双歧杆菌NCC2705与宿主细胞Caco2相互作用前后差异表达的蛋白,通过质谱鉴定和生物信息学分析,最后确定目标蛋白Enolase、Tuf作为本研究的对象。构建重组质粒并在大肠杆菌中进行了GST融合表达,利用GST pull down技术证明纯化蛋白Enolase、Tuf能够与真核细胞表达的Plg相互作用,证明表达的目标蛋白具有Plg受体的功能。最后通过竞争性抑制实验,证明Enolase和Tuf蛋白能抑制双歧杆菌的粘附。
本研究利用比较蛋白质组学筛选出34个长双歧杆菌NCC2705与宿主细胞Caco2相互作用的差异蛋白,为双歧杆菌粘附机制的研究提供了新的角度;从34个差异蛋白中筛选出两个细胞壁蛋白Enolase和Tuf作为研究对象,并证明是粘附相关蛋白,为进一步阐明双歧杆菌粘附机制奠定了基础。
Background
Bifidobacteria spp. Which is health-promoting groups represent one of the most important physiological microorganisms of human intestinal microbiota.It is one of the largest classes in the gut of human being.Bifidobacteria can regulate and equilibrate the balance of gut microbiota.Numerous health-promoting contributions of bifidobacteria in the human gastrointestinal tract may include improved digestion and absorption, immunostimulation,vitamin synthesis,antagonism of the growth of pathogens,regulation of the blood-fat and inhibition of the tumor.Bifidobacteria was always paid close attention by researcher since 1899.There are eleven species of them have relationships with human including Bifidobacterium longum.Among them Bifidobacterium longum NCC2705 which has a identify heredity background has been sequenced.Adeherence of bacteria is precondition of setting or producing the effect , yet no studies have so far definitely demonstrated the adhesion mechanism.Accepted the theory of adherence is adhesion---acceptor.Adhesion may be lipoteichoicacid、carbohydrate or protein , nevertheless acceptor is accpetde glycoprotein accordantly.
Objective
Use of the comparative proteomic to sieve the different protein between B. lougum NCC2705 whether interact with Caco2.Finally, we definite the interest proteins and then to authenticate their fuction.The purpose is illuminate the mechanism of B. lougum NCC2705 adhere to host intestinal epithelial cell.It is important to explain the mechanism of interaction with host. To accumulate the evidence of the interaction between B. lougum NCC2705 and human intestinal epithelial cell, and to enhance the adherence of the Bifidobacterium which is inexistent in human gut track.
Methods
Use of Immobilized pH gradient (IPG) strips at the first step of the 2-dimensional electrophoresis. Use of ImageMaster 2D Platinum 5.0 to analyze distinct proteins which expression amount on interaction with Caco2 than on contrast bacterium as higher as 3 times were termed as different one .The distinct proteins were appraised by matrix-assisted desorption ionization - time of flight (MALDI-TOF MS) and electrospray ionization tandem mass spectrometer (ESI–MS/MS).The result of 2-dimensional electrophoresis was authenticated by reverse transcript polymerase chain reaction (RT-PCR) .We choose the enlase and tuf as the interest proteins among the 34 distinct proteins,which are located on the bacterial cell wall and acceptors of Plg in other bacteria. We supposed that Enolase and Tuf very probably function as an adhesion promoter.The genes encoding Enolase and Tuf proteins were amplified by PCR . After dissected with restriction endonucleases the gene fragments were ligated into PGEX-4T-1 vector distincly. The transformed clones were sequenced and the right one named GST-Eno/ GST-Tuf.Use of the same way to construct the Flag-Plg. Flag-Plg was transfected into 293 cell line to test the expression.The purified Enolase and Tuf proteins were put into lysate which expressed the Flag-Plg.To test the interaction by western blot.To indentify the fuction of Enolase and Tuf,put the purified proteins into cell culture fluid along with B.lougum NCC2705.Then observed the condition of adherence by microscope,and counted the number of B. lougum NCC2705 that adhere to the Caco2 in twenty random visual field. The supernatant was detected the level of the cell factor LDH and TNF-a to solidify the hypothesis that Enolase and Tuf can competitive inhibit the adherence of B. lougum NCC2705.
Results
1. Get 34 distinct proteins which expression amount on interaction with Caco2 than on contrast bacterium as higher as 3 times were termed as different one by 2-dimensional electrophoresis,there are 17 up-regulation expressing proteins and 17 down-regulation expressing in the B. lougum NCC2705 which interaction with Caco2;
2. We found that the 34 distinct proteins correspond to 31 proteins species by mass spectra identification and analysis. Categories were taken from COGs,distinct proteins come from 9 category mainly;
3. Recombinant plasmid GST-Eno、GST-Tuf、Flag-Plg were constructed successfully;
4. Expressed the proteins GST、GST-Enolase、GST-Tuf in BL21 successfully,and get the purified proteins by affinity chromatography;
5. Flag-Plg expressed Plg in 293T cell line by Western blot testing;
6. Enolase and Tuf can interact with Plg in vitro by GST-pull down;
7. We tested adhesion after incubating B. lougum NCC2705 to Caco2 cell and B. lougum NCC2705 together with the purified proteins to Caco2.The bacterium numer of adherent to Caco2 which mix the purified proteins was notablely less than the other group, suggesting that Enolase and Tuf may promote the strain’s binding to epithelial cells.
8. Cytokine LDH and TNF-a which were stimulated by pathogenic bacteria were reduced by the incubation of Caco-2 cells with B. lougum NCC2705,and enhanced by incubation with Enolase or Tuf, to illustrate Enolase、Tuf can inhibit the adherence of B. lougum NCC2705
Conclusion
1. Use of the comparative proteomic to sieve the 34 proteins between B. lougum NCC2705 whether interact with Caco2 .This way is effective method to research the adherence of bacteria,and sieve proteins correlateing to adheren.
2. Enolase and Tuf are described to be diretly involved in the adhesion of B. lougum NCC2705 to Caco-2 cells.This study develop the direction of researching adherence that locus on bacteria interaction with human plasminogen (Plg) system in addition.
引文
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