A Preliminary Study On Secretion Mechanism Of Chlamydia Trachomatis GlgA Protein And Screening Of Interaction Proteins

Objective : Searching for the molecular mechanisms regulating the expression and secretion of GlgA protein in Chlamydia trachomatis,the yeast two-hybrid system was used to screen out the molecules interacting with GlgA from the human cervical cancer epithelial cell cDNA library,providing a theoretical basis for further studying the molecular function of GlgA and clarifying the pathogenic mechanism of Chlamydia trachomatis.Methods:(1)Analyze the ORFs of the Ct D standard strain and design specific amplification primers for the chlamydia coding gene.The PCR amplification technique was used to obtain the target gene.The constructed PGEX-6P-1/GlgA prokaryotic expression recombinant plasmid was transformed into E.coli BL-21,and the recombinant GlgA protein was induced at a concentration of 0.4 mM IPTG.(2)The SignalP-4.1 software was amplified to predict GlgA protein N terminal signal peptide.C.trachomatis infected He La cells were treated individually with C16 compounds,C1 compounds,or both of them to observe the effect of blocking type II or type III secretion system on GlgA secretion.Novobiocin treatment,plaque-forming assay,as well as shuttle plasmid transformation technique were used to construct either plasmid-free or plasmid-compensate C.trachomatis strains,then the strains were further identified by detecting both the plasmid encoding gene and protein.Indirect immunofluorescence assay was carried out to evaluate the effects of the plasmid deletion on GlgA protein expression and secretion.(3)Yeast two-hybrid technology was used to screen the GlgA interaction protein: GlgA gene cDNA clone was constructed on the yeast two-hybrid bait vector,and the vector was subjected to PCR amplification,Sfi I digestion,and the resulting cloned fragment was digested with the enzyme.The pGBKT7 bait vector plasmid was ligated and the PCR primers were used to amplify the plasmid primers,and the recombinant bait plasmid pGBKT7-GlgA was digested and sequenced.The bait plasmids pGBKT7-GlgA and pGBKT7 were transformed into AH109 yeast competent cells,and the toxicity and autoactivation of the bait plasmid pGBKT7-GlgA were detected.Simultaneously screening human cervical cancer epithelial cell cDNA library and purifying enough plasmids to transform yeast cells.After fusion of the yeast containing the bait plasmid p GBKT7-GlgA with the human cervical cancer epithelial cell cDNA library,the hybrid products were separately plated on SD/-Positive clones were screened on Trp,SD/-leu,SD/-His,SD/-Ade solid culture plates.Extraction of yeast plasmids,analysis of positive clones(transfection of yeast plasmids into E.coli,extraction of positive cloning plasmids to the company for sequencing).Results:(1)The result of restriction enzyme digestion and sequencing showed that the gene sequence of GlgA is correct.The purified GlgA protein was analyzed by 12% SDS-PAGE electrophoresis and WB analysis.It was found that there was a clear protein band at the corresponding position,and the molecular weight of the protein was consistent with the expected result.(2)The full length GlgA is predicted to contain no putative signal peptide at its N terminus.Neither C16 or C1 compounds can inhibit GlgA secretion into the cytosol of chlamydia-infected cells.The plasmid-encoded gene pgp7,the plasmid-encoded protein Pgp3,the genome-encoded protein GlgA were all detected in the cultures infected with either wild type serovar D or CTD1-pGFP::SW2 transformant but not the plasmid-free CTD1 organisms.(3)The pGBKT7-GlgA decoy plasmid was successfully constructed and proved to be non-toxic,self-activating,and capable of expression in yeast.Two rounds of screening were performed after the bait plasmid was transferred to the library,and a total of 11 positive clones were obtained.Conclusion:Preliminary confirmed that the expression and secretion of GlgA protein is not dependent on bacteria type II or III secretion system,but closely related to the plasmid of C.trachomatis.GlgA cDNA clones were used as bait to screen human yeast two-hybrid libraries.After two rounds of library screening,a total of 11 initial positives were screened.BLAST analysis of DNA sequencing results showed that these11 positive clones encode 4 proteins respectively.,roughly divided into the following categories:(1)binding proteins(such as CAPG);(2)transcription factors(such as CXXC1);(3)apoptosis-related proteins(such as tumor suppressor PHB);(4)lipoproteins(such as APOA1BP),all of them have important biological functions.