肺炎链球菌双组分系统WalK蛋白的截短表达及激酶活性分析
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摘要
目的对肺炎链球菌(Streptococcus pneumoniae,S.pn)双组分系统WalK蛋白进行截短表达及激酶活性分析。方法构建重组表达菌株,通过Ni-NTA亲和层析纯化蛋白,利用Kinase-Glo~化学发光试剂盒检测蛋白活性,并用ELISA和Western blot方法鉴定其抗原性和抗体特异性。结果成功获得高浓度、高纯度、有活性的截短WalK重组蛋白。ELISA与Western Blot检测结果均表明目的蛋白具有良好免疫原性及抗体特异性。结论成功获得高表达的S.pn WalK截短蛋白,且该蛋白具有较高的激酶活性、免疫原性及抗体特异性,本研究为后续以该蛋白为靶点的疫苗研发及抗菌药物筛选提供实验基础。
Objective To investigate the truncated expression,activity and identification of WalK protein of Streptococcus pneumoniae(S.pn).Methods:The recombinant strain was constructed and the protein was purified by NiNTA affinity chromatography.The activity of the protein was measured using the Kinase-Glo kit and the protein was identified by ELISA and Western blot.Results:Successful access to high concentration,high purity,active truncated protein.ELISA and Western Bloting test results show that the successful access to a good immunogenicity and antibody specificity of the target protein.Conclusion:The WalK truncated protein is successfully obtained by highly expressed,and its activity,immunogenicity and antibody specificity are not affected by truncation,which provide the experimental basis for the follow-up vaccine development and antimicrobial drug target research.
Objective To investigate the truncated expression,activity and identification of WalK protein of Streptococcus pneumoniae(S.pn).Methods:The recombinant strain was constructed and the protein was purified by NiNTA affinity chromatography.The activity of the protein was measured using the Kinase-Glo kit and the protein was identified by ELISA and Western blot.Results:Successful access to high concentration,high purity,active truncated protein.ELISA and Western Bloting test results show that the successful access to a good immunogenicity and antibody specificity of the target protein.Conclusion:The WalK truncated protein is successfully obtained by highly expressed,and its activity,immunogenicity and antibody specificity are not affected by truncation,which provide the experimental basis for the follow-up vaccine development and antimicrobial drug target research.
引文
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[7]刘华勇.抑制葡萄球菌组氨酸激酶YycG的噻唑烷酮类衍生物的抗菌活性及作用机制研究[D].复旦大学,2014.
[2]Reynolds CA,Finkelstein JA,Ray GT,et al.Attributable healthcare utilization and cost of pneumonia due to drug-resistant streptococcus pneumonia:a cost analysis[J].Antimicrob Resist Infect Control,2014,3(16):657-668.
[3]李南,王非,胥文春,等.肺炎链球菌双组份系统中的组氨酸激酶(YycG)的同源模建与分析[J].生物工程学报,2009,25(2):207-214.
[4]Schweizer I,Blattner S,Maurer P,et al.New Aspects of the Interplay between Penicillin Binding Proteins,murM,and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19AIsolates from Hungary[J].Antimicrob Agents Chemother,2017,61(7):414-417.
[5]Cockeran R,Herbert JA.Exposure of a 23Fserotype strain of Streptococcus pneumoniae to cigarette smoke condensate is associated with selective upregulation of genes encoding the two-component regulatory system11(TCS11)[J].Biomed Res Int,2014,47(3):47-51.
[6]Huang RZ,Zheng LK,Liu HY,et al.Thiazolidione derivatives targeting the histidine kinase YycG are effective against both planktonic and biofilm-associated Staphylococcus epidermidis[J].Acta Pharmacol Sin,2012,33(3):418-425.
[7]刘华勇.抑制葡萄球菌组氨酸激酶YycG的噻唑烷酮类衍生物的抗菌活性及作用机制研究[D].复旦大学,2014.