Abstract：Objective: To express the His6CalR protein of Vibrio parahaemolyticus and characterize its DNAbinding activity,for further indepth study on the target genes and molecular regulatory mechanism. Methods: The entire coding region of calR in RIMD2210633 was amplified and then directionally cloned into the BamHⅠ and Hind Ⅲ sites of the vector pET28a. The recombinant plasmid pET28a was transformed into BL21λDE3 using heat shock. Overexpression of His6CalR in the LB media was induced by 1 mmol/L isopropylbDthiogalactoside (IPTG), and then purified under native conditions with a nickelresin column. The DNAbinding activity of His6CalR to the promoter DNA regions of exsC, exsD and VP1687 was analyzed using the electrophoretic mobility shift assay. Results: The His6CalR protein was expressed in supernatant at 37 ℃ and under 1 mmol/L IPTG induction. The purified His6CalR protein had the ability to bind to the upstream DNA promoter regions of exsC, exsD and VP1687. Conclusion: Active His6 CalR protein was successfully expressed and purificated, and can be used in the transcriptional regulatory mechanism of subsequent research in V. parahaemolyticus.
[1\]Park KS, Ono T, Rokuda M，et al. Functional characterization of two type Ⅲ secretion systems of Vibrio parahaemolyticus\[J\]. Infect Immun, 2004，72(11): 6659-6665.\[2\]Newton A,Kendall M, Vugia DJ, et al. Increasing rates of vibriosis in the United States, 1996-2010: review of surveillance data from 2 systems\[J\]. Clin Infect Dis, 2012，54( Suppl 5): S391-S395.\[3\]Hiyoshi H,Kodama T,Iida T, et al. Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice\[J\]. Infect Immun, 2010，78(4): 1772-1780.\[4\]Yu Y,Yang H,Li J, et al. Putative type Ⅵ secretion systems of Vibrio parahaemolyticus contribute to adhesion to cultured cell monolayers\[J\]. Arch Microbiol, 2012，194(10): 827-835.\[5\]Ono T,Park KS, Ueta M, et al. Identification of proteins secreted via Vibrio parahaemolyticus type Ⅲ secretion system 1\[J\]. Infect Immun, 2006, 74(2): 1032-1042.\[6\]Hertzberg KM,Gemmill R,Jones J,et al. Cloning of an EcoRⅠgenerated fragment of the leucine operon of Salmonella typhimurium\[J\]. Gene, 1980, 8(2): 135-152.\[7\]Maddocks SE, Oyston PC. Structure and function of the LysRtype transcriptional regulator (LTTR) family proteins\[J\]. Microbiology, 2008, 154(Pt 12): 3609-3623.\[8\]Dillon SC,Espinosa E,Hokamp K, et al. LeuO is a global regulator of gene expression in Salmonella enterica serovar Typhimurium\[J\]. Mol Microbiol, 2012, 85(6): 1072-1089.\[9\]GodePotratz CJ,Chodur DM, McCarter LL. Calcium and iron regulate swarming and type Ⅲ secretion in Vibrio parahaemolyticus\[J\]. J Bacteriol, 2010, 192(22): 6025-6038.\[10\]Whitaker WB,Parent MA,Boyd A, et al. The Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycininduced adult murine model\[J\]. Infect Immun, 2012, 80(5): 1834-1845.\[11\]张义全，高鹤，王丽，等.鼠疫菌HNS蛋白的表达与纯化及其DNA结合活性分析［J］.微生物学报, 2011, 51(5): 615-621.\[12\]Ante VM,Bina XR, Howard MF, et al. Vibrio cholerae leuO transcription is positively regulated by ToxR and contributes to bile resistance\[J\]. J Bacteriol, 2015, 197(22): 3499-3510.\[13\]Klauck E,Bohringer J, HenggeAronis R. The LysRlike regulator LeuO in Escherichia coli is involved in the translational regulation of rpoS by affecting the expression of the small regulatory DsrARNA\[J\]. Mol Microbiol, 1997, 25(3): 559-569.\[14\]Moorthy S, Watnick PI. Identification of novel stagespecific genetic requirements through whole genome transcription profiling of Vibrio cholerae biofilm development\[J\]. Mol Microbiol, 2005, 57(6): 1623-1635.
2017, Vol. 27 
