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| The Preparation of the Streptolysin O Active Recombinant Protein of Streptococcus pyogenes |
| YANG Si-yuan, PAN Jing-mei, WANG Shuo, DENG Kai-xuan, DENG Qiang, HUANG Xin-he, LI Xue-ru |
| School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China |
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Abstract The SMART algorithing (htpp://smart.emble-heideberg.de) was used for the structural analysis of Streptolysin O (Slo) in Streptococcus pyogenes. The location of protein domain shows that Slo contains a Thiol_cytolysin domain which is composed of 461 amino acids and a transmembrane domain in the amino terminus. A pET101-GENE protein expression system was used to construct genetically engineered bacteria which only express the Thiol_cytolysin domain fusion protein. The recombinant protein was purified using Ni-NTA Resins affinity chromatography. SDS-PAGE analysis of the purified recombinant protein showed that the relative molecular weight of protein is about 50kDa as same as we predicted by bioinformation. Hemolysis testing showed that the purified recombinant protein possessed the hemolytic activity in red blood cells. In addition, the polyclonal antibody against recombinant Slo was prepared in rats. The titre of antiserum was 1 ∶128 000 detected by ELISA. Analysis of Western blot with the polyclonal antibody showed that hemolysin was detected only in culture supermentant of Streptococcus pyogenes. This result showed that recombinant protein is a specific antigen in Streptococcus pyogenes.
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Received: 03 March 2016
Published: 25 June 2016
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| [1] Debra E B. Population biology of the human restricted pathogen, Streptococcus pyogenes infection. Genetics and Evolution, 2009,9(4):581-593.
[2] Li-ping Xu, Pei-feng Li. Invasive mechanisms underlying group A streptococcal pathogenesis through human plasminogen. Chemistry of Life,2010,30(3):391-396.
[3] Carapetis J R,Steer A C,Mulholl E K,et al.The global burden of group A streptococcal diseases. Lancet Infect Dis,2005,5(11):685-694.
[4] Kai-hu Yao. the classification and drug resistance status of group A streptococcus in domestic pediatric. Chinese Journal of Practical Pediatrics,2012,27 (4):262-264.
[5] Ming-huan Zheng,Zhen-quan Jiao, Li-jie Zhang, et al. Genetic analysis of Group A Streptococcus isolates recovered during acute Glomeru lonephritis outbreaks in Guizhou province of China.Journal of Clinical Microbiology,2009, 47(3):715-720.
[6] Yang Yong-hong, Shen Xu-zhuang. Recognizing the importance of the study in group A streptococcus and its related pediatric diseases. The Journal of Clinical Pediatrics,2006, 24(6):443-444.
[7] Maghnus O'Seaghdha. Streptolysin O and its Co-Toxin NAD-glycohydrolase protect Group A streptococcus from Xenophagic Killing. Plos Pathogens,2013,6(9):1-14.
[8] Cara C Mozola, A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by streptolysin O. Molecular Microbiology, 2014,94(3):675-687.
[9] Jia-yun Zhong, Tao Zhang. The expression and purification of recombinant streptolysin O. Scientific Chinese,2015,9 :67-69.
[10] Qi Zhang. Clinical research of blood anti-streptolysin O in patients with ankylosing spongylitis China & Foreign Medical Treatment, 2015,4:72-73.
[11] Sun Hua, Zhang Zi-de. Expression and purification and of human hydrogen peroxidase in E.coli and identification of its biological activity. Journal of Jinan University,2014,35(2):187-191.
[12] Limbago B, Penumalli V,Brian W, et al. Role of streptolysin O in a mouse model of invasive Group A streptococcal disease. Infetion and Immunity,2000,68(11):6384-6390.
[13] Yin Zhi-kui, Deng Zhi-jian. Expression of aldolase of toxoplasma gondii and preparation of the polyclonal antibodies against this protein. Chinese Journal of Cellular and Molecular Immunology, 2011,27(10):1114-1116.
[14] Hu Shan,Ma Chao,LI Xue-ru, et al. Correlation of enolase and the maturation of SpeB in Streptococcus pyogenes. Chemical Journal of Chinese Universities,2013,4(34):886-892.
[15] Chen Xi. Review on classification and representative species of hemolysin. Chinese Agricultural Science Bulletin,2008,24(8):16-22.
[16] Susanne C F,David B Ascher. Structural studies of Streptococcuspyogenes streptolysin O provide insights into the early steps of membrane penetration. Mol Biol,2014,426:785-792.
[17] Qin Yang. Expression and purification of Tir-cytoskeleton coupling protein from E.coli O157:H7 and preparation, application of its polyclonal antibodies. Chong Qing:Chongqing Medical University,2007,46.
[18] Sun Wen, Pan Xiu-zhen. Molecular cloning, hemolytic activity, and immunological characterization of suilysin from S.suis2. Journal of Henan University(Medical Science),2014,3(33):179-184.
[19] Hisashi K, Yutaka F. Expression of recombinant streptolysin O and specific antibody production. J Mol Microbiol Biotechnol, 2005, 10:64-68.
[20] Tang Xin-jie, Sheng Ling-ling, ZHANG Qun, et al. Effects of streptolysin O on increasing cell permeability Chinese Journal of Tissue Engineering Research, 2012, 16(46): 8689-8692.
[21] Horohov D W,Loynachan A T. The use of streptolysin O(SLO) as an adjunct therapy for Rhodococcus equi pneumonia in foals.Veterinary Microbiology, 2011,154:156-162. |
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