Cloning, expression and biological activity analysis of coagulase gene coa of Staphylococcus aureus
GUO Hai-yong1, ZHAO Xue2, ZHU Xiao-yu1, ZHAO Yan-tong1, LIN Yi-dan2, SHI Shuang1, LI Shuo1, JI Yin-duo3, SONG Qin-ye2
1. School of Life Science,Jilin Normal University,Siping 136000, China; 2. College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071000,China; 3. College of Veterinary Medicine, University of Minnesota, St Paul 55108,USA
Abstract：The object of this study is to express the coagulase(Coa)of Staphylococcus aureus (S. aureus) in vitro and evaluate its biological activity. According to the coa gene sequence of S. aureus in GenBank, a pair of primers was designed. DNA was extracted from S. aureus strain USA300 community-acquired methicillin resistant Staphylococcus aureus(CA-MRSA) 923, and used as a template for PCR to amplify the coa gene. The coa gene was cloned into pET-24b (+) expression vector to construct the recombinant expression plasmid pET-24b-coa. After double digestion and sequencing analysis, the recombinant plasmids were transformed into competent cell E.coli BL21(DE3) and the coa gene was expressed by IPTG induction. The expression product was identified by SDS-PAGE and Western blot analysis. The coagulation activity of rCoa was measured in human whole blood and rabbit plasma in vitro, and the immunogenicity of rCoa was evaluated through detecting specific antibodies against rCoa in serum of BALB/c mouse immunized with purified rCoa by ELISA and Western blot. The results showed that rCoa, about 74.8 ku in line with expectations, was expressed in E.coli BL21(DE3) in the form of an inclusion body. The purified rCoa could coagulate human blood and rabbit plasma and stimulate mouse to produce high titer specific antibodies against Coa in serum. These results lay a foundation for further study on the function of S. aureus coagulase and the development of anti-infective agents and vaccine candidates targeting Coa.
郭海勇, 赵雪, 朱小雨, 赵研彤, 林依丹, 施爽, 李铄, 计银铎, 宋勤叶. 金黄色葡萄球菌凝固酶coa基因的克隆、表达及其生物活性分析[J]. 中国人兽共患病学报, 2020, 36(9): 696-702.
GUO Hai-yong, ZHAO Xue, ZHU Xiao-yu, ZHAO Yan-tong, LIN Yi-dan, SHI Shuang, LI Shuo, JI Yin-duo, SONG Qin-ye. Cloning, expression and biological activity analysis of coagulase gene coa of Staphylococcus aureus. Chinese Journal of Zoonoses, 2020, 36(9): 696-702.
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2020, Vol. 36 
