• 中国精品科技期刊
  • 中国高校百佳科技期刊
  • 中国中文核心期刊
  • 中国科学引文数据库核心期刊
Advanced Search
Cloning,over-expression and in vitro activity of aminoglycoside phosphotransferase from clinical MRSA isolates[J]. Journal of China Pharmaceutical University, 2010, 41(1): 81-85.
Citation: Cloning,over-expression and in vitro activity of aminoglycoside phosphotransferase from clinical MRSA isolates[J]. Journal of China Pharmaceutical University, 2010, 41(1): 81-85.

Cloning,over-expression and in vitro activity of aminoglycoside phosphotransferase from clinical MRSA isolates

More Information
  • Aim: To clone and over-express the gene encoding aminoglycoside (AG) phosphotransferase (APH) from clinical MRSA isolates in E.coli and to develop an assay method for the recombinant APH. Methods: The susceptibility of clinical MRSA isolates to AGs was tested by disk diffusion.A nucleic acid sequence encoding APH was amplified from the genomic DNA of an isolate and ligated to expression vector pET-28a,and then transformed into E.coli BL21 (DE3).After purification of the recombinant protein by affinity chromatography,the phosphorylation activity of the enzyme was determined by ESI-MS and disk diffusion. Results: All 6 clinical MRSA isolates were unsusceptible to AGs.After cloning and expression,the recombinant APH was purified to 90%. The in vitro activity assay indicated that the recombinant protein could inactivate kanamycin B in the assay mixture within 2 h. Conclusion: The recombinant APH showed excellent enzymatic activity.The assay method was simple and convenient,which may provide the basis of developing a screening model for APH inhibitors.
  • Related Articles

    [1]MA Wenxuan, HAN Yuhong, LIN Ang, ZHAO Weijun. A comparative analysis of vaccine immunity induced by heterologous booster with Ad5-nCoV via different routes of administration[J]. Journal of China Pharmaceutical University, 2024, 55(1): 137-146. DOI: 10.11665/j.issn.1000-5048.2024011701
    [2]HU Wanwan, DING Xuefeng, CAI Yanfei, CHEN Yun, DUAN Zuoying, JIN Jian, LI Huazhong. Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome[J]. Journal of China Pharmaceutical University, 2021, 52(4): 487-495. DOI: 10.11665/j.issn.1000-5048.20210412
    [3]TONG Ruinian, WU Xuri, CHEN Yijun. Application of TAR cloning in microbial secondary metabolites[J]. Journal of China Pharmaceutical University, 2018, 49(2): 129-135. DOI: 10.11665/j.issn.1000-5048.20180201
    [4]WANG Yuanxi, CHEN Junsheng, SHAO Lei, LI Ji′an, LIN Huimin, CHEN Daijie. Heterologous expression and in vitro activity of vancomycin glycosyltransferase GtfE[J]. Journal of China Pharmaceutical University, 2013, 44(3): 272-276. DOI: 10.11665/j.issn.1000-5048.20130317
    [5]SHI Wei, SUN Li, LIN Sen-sen, YUAN Sheng-tao, ZHANG Lu-yong. Construction of eukaryotic expression plasmid for human TF full-length,TF truncated and characterization of their biological function[J]. Journal of China Pharmaceutical University, 2009, 40(5): 465-470.
    [6]Pichia Expression of Human VEGF165[J]. Journal of China Pharmaceutical University, 2003, (6): 96-100.
    [7]Construction of a Novel Fusion Expression Vector[J]. Journal of China Pharmaceutical University, 2000, (2): 65-69.
    [8]The High Expression of hGRF Gene[J]. Journal of China Pharmaceutical University, 1999, (6): 68-72.
    [9]Molecular Cloning and Expression of Tryptophanase Gene of Escherichia coli[J]. Journal of China Pharmaceutical University, 1999, (2): 61-64.
    [10]The Construction of an E.coli Strain with Expression of Fumarate Hydratase[J]. Journal of China Pharmaceutical University, 1994, (3): 159-162.

Catalog

    Article views (5433) PDF downloads (3966) Cited by()

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return