Studies on the Function of Recombinant Subunits A of A. Actinomycetemcomitans Cytolethal Distending Holotoxin
|School||Nanjing Medical University|
|Keywords||Actinobacillus actinomycetemcomitans The cytolethal expansion toxins A subunit Function|
Cell expansion of lethal toxin (cytolethal distending toxin, CDT) is a newly discovered a special bacterial toxins secreted by certain G-pathogens, can cause epithelial cells, fibroblasts and lymphocytes cell cycle G2 / M phase block. Actinobacillus actinomycetemcomitans (Aggregatibacter actinomycetemcomitans, Aa) is localized aggressive periodontitis suspicious pathogens, is also found to express CDT oral bacteria. The CDT CdtA, CdtB, and CdtC, CdtB has been shown is the toxic subunit clear function remains but CdtA and CdtC. [Objective] by cytotoxicity assay screening toxicity defects, missing mutation sites, a clear key amino acids to maintain the normal functioning of CdtA activity groups to explore the relationship between the the the Aa CdtA structure and function of. [Method] Aa ATCC 29522 genomic DNA as a template, using the PCR method to get wild-type cdtA, cdtB,, cdtC genes prokaryotic expression vector double digested cloned connection. The prebuilt wild type pET-15b-cdtA plasmid DNA as template, using site-directed mutagenesis techniques to obtain the mutant pET-15b-cdtAY105A, pET-15b-cdtAY181A, pET-15b-cdtAY125A. IPTG induction of protein expression by SDS-polyacrylamide gel electrophoresis and Western blot analysis and identification of protein expression. Ni-HisTrap HP prepacked columns of each of the recombinant protein in vitro purification, the purified recombinant CdtB vitro with supercoiled plasmid (pET-32a) DNA and incubated to observe the biological activity to determine whether each of the recombinant protein with biological activity. Wild-type and mutant CdtA with wild-type CdtB, respectively, equal amounts CdtC mixed in Reconstruction of the buffer solution, respectively, co-incubated with HeLa cells and CHO, MTT assay Hela cell proliferation inhibitory effect, colony formation number of cfu (colony-forming units) the number of living cells; inverted phase contrast microscope to observe the morphological changes of the expansion of the Hela cell lethal; flow cytometry analysis of Hela cell cycle arrest situation. [Results] sequencing, pET-15b-cdtA constructed wild-type prokaryotic expression vector pET-15b-cdtB, pET-15b-cdtC transfection E. coli each carry cdtA cdtB,, cdtC gene and have been included in the gene fragment with GenBank the Aa cdtA, cdtB, the cdtC gene sequence identity of up to 99%. The constructed mutant prokaryotic expression vector pET-15b-cdtAY105A, pET-15b-cdtAY181A, pET-15b-cdtAY125A mutation sites is entirely correct. The Western blot observed the with target protein of CdtA of His6-tag tag CdtB, CdtC, CdtAY105A CdtAY181A CdtAY125A. Separate wild type of CdtA of CdtB, and three mutant CdtAY105A, CdtAY181A CdtAY125A protein on cell growth had no inhibitory effect. Separate CdtC proteins and wild-type CDT holotoxin having a biological activity of the expansion of the cell lethality. Compared with the wild-type CDT holotoxin, the mutant holotoxin CdtAY105ABC the biological activity is significantly reduced, the mutant holotoxin CdtAY181ABC the biological activity is slightly enhanced, the mutant holotoxin CdtAY125ABC the biological activity of no significant change compared to. [Conclusion] This study has successfully obtained in vitro reconstitution of the wild-type CDT mutant CDT holotoxin preliminary screening the Aa CdtA two potential functional sites CdtAY105A CdtAY181A lay the foundation for further study the structure and function of the CdtA.