High-throughput Analysis Of Protein-protein Interaction By High Sensitivity Flow Cytometry Combined With GFP Reporter

Protein interaction,involoved in the whole process of life activity,is the foundation of any living body structure and life activity.Study on the interaction of proteins is of great significance for the analysis of protein function,interaction network construction,drug development and other issues to be solved.Generally,protein interaction is studied by immunoprecipitation,surface plasmon resonance,and fluorescence resonance energy transfer.However,these methods often require cumbersome steps such as extraction and purification of proteins,and the need for in vitro experiments,which can not reflect the true situation of the proteins in the normal physiological state.In recent years,with the development of biotechnology,yeast two-hybrid system has been developed as a molecular biology method.to study the protein-protein interaction in vivo.In the method,the genes of the interaction protein fragments are constructed into the corresponding plasmids so as to be expressed in the cell body.Thus,the protein-protein interaction would stimulate the transcription of the reporter gene,and then be determined by analyzing the reporter gene expression product.However,this method still has shortcomings such as long test period and high false positive rate.To solve these problems,a bacterial adenylate cyclase-based two hybrid(BACTH)system was developed to shorten the experimental period,reduce the false positive rate,and avoid the protein to be located in the nucleus.However,the existing detection methods,can not quantitative by detect protein interactions.In addition,these methods are based on cell population analysis that can not distinguish the heterogeneity between cells.The high sensitivity flow cytometry(HSFCM)can rapidly,multi-parameter and quantitatively analyze the cells at the single particle level.Combined with green fluorescent protein(GFP)reporter factor excludes the need for cell membrane treatment,as well as antibody incubation and other cumbersome steps,and has a stable fluorescence,no toxicity,versatility and versatility and can be directly detected,this thesis developed of a new BACTH system based method for detecting protein interaction at the level of single bacteria.The first chapter is the literature review.The main significance of protein interaction and the main research methods are introduced,and the yeast two-hybrid system and BACTH system are introduced emphatically.It is clear that the BACTH system shows advantages for study of protein interactions.At the end of this chapter,the ultra-high sensitivity flow detection device and its application in biochemical analysis are introduced.The second chapter is the construction of tandem expression vector.In order to introduce the plasmid pTW2 containing the reporter gene gfp into the BACTH systerrn,a pair of interacting protein genes were constructed on the same plasmid.Protein pairs related to the stability of the outer membrane of bacteria,pal and TolB,and TolB's mutant TolB ?22-25 and TolB ?22-23 were taken as experimental models.The pal was constructed into plasmid pKT25-His-tolB,pKT25-His-tolB ?22-25and pKT25-His-tolB?22-33.The constructed plasmid was then transferred to the reporter E.coli BTH101 competent cells and the expression of the interacting protein pair was judged by observing the blue spot and measuring the ?-galactosidase activity.In the third chapter,the plasmid pKT25-zip-lac-T18-zip was used as the research object,which was 'co-transfected with plasmid pTW2 into the report bacterial E.coli BTH101 competent cells.The expression of GFP was then stimulated by Zip-Zip interaction.The high sensitivity of HSFCM allows us to analyze bacteria fluorescence at the single cell level.By introducing multiple copies of the plasmid pTW2,increasing the copy number of the lacZ promoter to buffer the background cAMP signal and other endogenous physiological factors of interference,GFP can be quantified in liquid culture,and the detection sensitivity is very high,which greatly improves the accuracy of the experiment and greatly reduces the false positive rate.The fourth chapter presents the application of the method for the analysis of protein interactions with different interaction levels.Single plasmid type tandem expression vector pKT25-His-tolB-lac-T18-pal and pKT25-His-tolB ?22-33-lac-T18-pal was constructed to study the relationship between different colonies in the same system.Both the fluorescence and side scattering were detected to distinguish the different intensity of the interaction protein pairs.The fifth chapter summarizes the research work and achievements of this thesis,and prospects for further research in the future.