Clone, Expression Of T3SS-associated Genes Of Pseudomonas Syringae And Protein Purification As Well As Preparation Of Polyclonal Antibodies

Type Ⅲ secretion system(T3SS) of Gram-negative bacteria is transmembrane channel formed by multicomponent protein complex.It can secret proteins into the extracellular environment or directly into the host cell,stimulating the signal transduction pathway of host cell,which can result in a series of cellular effects such as cytoskeletal rearrangement caused by allosteric actin,activation of transcriotion factors,ion channel stimulation,appeared apoptosis in some cells.Phosphatidylcholine(PC) is the major component of membrane phospholipids in eukaryote.Based on bacterial genome database,it is speculated that more than15%of the total number of bacteria may contain PC.And the vast majority of bacteria containing PC are associated with eukaryotic pathogens or symbionts. Synthesis of bacterial PC mainly include N-methylated and/or phosphatidyl choline synthase(PCS) pathways.Studies in vitro have found that membrane phospholipid composition has a very important impact on the tanslocation efficiency,topology,stability,transportion during secretion as assembled complex and sorting of membrane protein as well as the activity of the enzyme.During2010, we conducted the research of the role of phosphatidylcholine synthase gene (PCS) on the Pathogenicity of pathogenic bacteria, and after two years’ hard work, we successfully constructed the PCS-mutant of two model bacteria Pseudomonas sp.593and P. syringae pv. Syringae van Hall1336,and we found that the missing of PC caused the loss of the pathogenic capacity of P. syringae pv. Syringae van Hall1336, which is closely related to the loss of the function of Type Ⅲ secretion system. So after our establishing that PC regulates the function of Type Ⅲ secretion system, and in order to further explore its molecular mechanisms,we are to compare the differences on the main T3SS proteins between two modes’wildtype and mutant respectively,from transcription level,translation level and transportion level. So we try to clone and express all the T3SS-associated genes of P. syringae pv. syringae van Hall1336,and then purify them.inject those purified proteins into New Zealand rabbits for polyclonal antibodies.The current achievements are:1Based on the already reported genome information of P. syringae pv. Syringae B728a,we find out that its T3SS includes27ORFs.So we speculate the T3SS of P. syringae pv. syringae van Hall1336also have27genes.Except3genes previously cloned in our laboratory,we have cloned22genes from P. syringae pv. syringae van Hall1336,and we conducted the corresponding bioinformatic analysis for the above22genes.2Connect the above22genes with expression vector pET23a or pGEX-6p-1to form recombination plasmids,then thansform recombination plasmids into E. coli BL21(DE3) or E. coli Transta for gene expression and protein purification.There are16genes successfully expressed and10proteins successfully purified.3We successfully produce6polyclonal antibodies by injecting the purified proteins into New Zealand rabbits,which which will be used in the following Western Blot experiment.By comparing the hybridization signal differences on the cytoplasmic proteins、membrane proteins、extracellular proteins between the two modes’wildtype and mutant, respectively,we can speculate if PC regulate the function of T3SS by the way which PC influence the transportion of T3SS component proteins.