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Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification

Received: 11 December 2018     Published: 12 December 2018
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Abstract

Mycobacterium tuberculosis is a pathogen of tuberculosis and has relatively high epideictic and mortality among human. It is the second most life-threatening infectious disease second only to AIDS,so it is very important for the prevention and control of tuberculosis. In our experiment, RV1987 and TB7.7 recombinant plasmids of mycobacterium tuberculosis were constructed. The specific protein RV1987 and TB7.7 of mycobacterium tuberculosis were prokaryotic expressed, purified and identified in Escherichia coli. Our experiment lay a sound foundation for the subsequent serological diagnosis of tuberculosis as to obtain the high-purity target protein. Methods: use the genome of H37Rv as template to amplify the target gene and cloned it into prokaryotic expression vector, then transformed it into E. coli C43 (DE3) strain. After that, go through induced, expressed and purified in order to obtain recombinant protein.Results: the recombinant plasmid of RV1987 protein and TB7.7 protein was successfully constructed and expressed to obtain purified products, which were purified by affinity chromatography with a purity greater than 90%. The concentrations of RV1987 and TB7.7 proteins were 413 microns /mL and 1693 microns /mL. Rv1987 protein was expressed in inclusion body. TB7.7 protein was expressed in both supernatant and inclusion body, and the expression in supernatant was slightly higher than In inclusion body. Western Blot showed that all the above proteins could react with the positive serum of tuberculosis and had good reactivity. Conclusion: Escherichia coli is the most common system for gene engineering to express heterogenous proteins and widely used by researchers because of its relatively high expression efficiency. The experimental results showed that we obtained by prokaryotic expression and purification of two specific protein has good response to the original, in the subsequent experiments is obtained by testing several protein specificity and sensitivity, we can on the basis of the existing stimulus antigen screen is suitable for our country crowd IGRAs stimulus antigen, as rapid auxiliary diagnosis of tuberculosis (TB), thus improve the serum antibody detection of TB case detection and reduce the misdiagnosis rate.

Published in Science Discovery (Volume 6, Issue 6)
DOI 10.11648/j.sd.20180606.34
Page(s) 529-534
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2018. Published by Science Publishing Group

Keywords

Mycobacteria, Rv1987, TB7.7, Protein Expression, Purification, WB Identification

References
[1] 张文宏,李忠明.全球结核病控制六十年规划的成果,现状和展望[J].中华微生物学和免疫学杂志,2013,33(1):47-55。
[2] World Health Organization. Global tuberculosis report 2017.Geneva: World Health Organization, 2017: 23. http://www.who.int/tb/publications/global report/gtbr2017_ main_text.pdf
[3] Alcaide F, Galí N, Domínguez J, et al. Usefulness of a New Mycobacteriophage-Basd Technique for Rapid Diagnosis of Pulmonary Tuberculosis[J]. Journal of Clinical Microbiology, 2003,41(7):2867.
[4] Wallis R, Doherty T, P, Vahedi M, et al. Biomarkers for tuberculosis disease activity, cure, and relapse[J]. Lancet Infectious Diseases, 2010,10(2):70-71.
[5] Clinical infectious diseases : an official publication of the Infectious Diseases Society of America: Published by The University of Chicago Press; 1992.
[6] 李文彬,万康林.结核分枝杆菌RD区蛋白研究进展[J].中国人兽共患病学报,2018,34(04):362-371。
[7] Sha S. Shi X. Deng G, et al. Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice[[J]. Microbiol Res, 2017,197: 74-80. DOI: 10.1016/j. mieres.2017.01.004.
[8] Yanzhi Lu,Jian Kang,Huanhuan Ning,Lifei Wang,Yanhui Xu,Ying Xue,Zhikai Xu,Xingan Wu,Yinlan Bai. Immunological characteristics of Mycobacterium tuberculosis subunit vaccines immunized through different routes [J]. Microbial Pathogenesis,2018.
[9] Renshaw PS, Lightbody KL, Veverka V, et al. Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6 [J]. Embo Journal, 2005, 24( 14):2491-2498.
[10] 林楠,余琴,刘英杰,et al. Ag85A、Ag85B、16kDa和38kDa用于结核病血清学诊断的评价[J].中国卫生检验杂志,201618):2585-2586。
[11] Osadaoka M, Tateishi Y, Hirayama Y, et al. Antigen 85A and mycobacterial DNA-binding protein are targets of immunoglobulin G in individuals with pasttuberculosis[J]. Microbiology & Immunology, 2013,57(1):30.
[12] 董恩军,张灵霞,张翠英.结核分枝杆菌Ag85A和Ag85B蛋白在结核病诊断的价值[J].中国热带医学,2011,11(3):279-280。
[13] Bai Y, Xue Y, Gao H, et al.Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mousemodel[J]. Protein Expression & Purification, 2008, 59(2):189-196.
[14] 余琴,林楠,张爱洁,徐伟,刘英杰,万康林.Rv2031c、38kDa及融合蛋白CFP10-ESAT6用于结核病血清学诊断的评价[J].中国病原生物学杂志,2017,12(05):397-401。
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  • APA Style

    Xiangru Li, Jifu Ma, Ying Xu, Lijie Chen, Jinmeng Song, et al. (2018). Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification. Science Discovery, 6(6), 529-534. https://doi.org/10.11648/j.sd.20180606.34

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    ACS Style

    Xiangru Li; Jifu Ma; Ying Xu; Lijie Chen; Jinmeng Song, et al. Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification. Sci. Discov. 2018, 6(6), 529-534. doi: 10.11648/j.sd.20180606.34

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    AMA Style

    Xiangru Li, Jifu Ma, Ying Xu, Lijie Chen, Jinmeng Song, et al. Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification. Sci Discov. 2018;6(6):529-534. doi: 10.11648/j.sd.20180606.34

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  • @article{10.11648/j.sd.20180606.34,
      author = {Xiangru Li and Jifu Ma and Ying Xu and Lijie Chen and Jinmeng Song and Feng Shi},
      title = {Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification},
      journal = {Science Discovery},
      volume = {6},
      number = {6},
      pages = {529-534},
      doi = {10.11648/j.sd.20180606.34},
      url = {https://doi.org/10.11648/j.sd.20180606.34},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.sd.20180606.34},
      abstract = {Mycobacterium tuberculosis is a pathogen of tuberculosis and has relatively high epideictic and mortality among human. It is the second most life-threatening infectious disease second only to AIDS,so it is very important for the prevention and control of tuberculosis. In our experiment, RV1987 and TB7.7 recombinant plasmids of mycobacterium tuberculosis were constructed. The specific protein RV1987 and TB7.7 of mycobacterium tuberculosis were prokaryotic expressed, purified and identified in Escherichia coli. Our experiment lay a sound foundation for the subsequent serological diagnosis of tuberculosis as to obtain the high-purity target protein. Methods: use the genome of H37Rv as template to amplify the target gene and cloned it into prokaryotic expression vector, then transformed it into E. coli C43 (DE3) strain. After that, go through induced, expressed and purified in order to obtain recombinant protein.Results: the recombinant plasmid of RV1987 protein and TB7.7 protein was successfully constructed and expressed to obtain purified products, which were purified by affinity chromatography with a purity greater than 90%. The concentrations of RV1987 and TB7.7 proteins were 413 microns /mL and 1693 microns /mL. Rv1987 protein was expressed in inclusion body. TB7.7 protein was expressed in both supernatant and inclusion body, and the expression in supernatant was slightly higher than In inclusion body. Western Blot showed that all the above proteins could react with the positive serum of tuberculosis and had good reactivity. Conclusion: Escherichia coli is the most common system for gene engineering to express heterogenous proteins and widely used by researchers because of its relatively high expression efficiency. The experimental results showed that we obtained by prokaryotic expression and purification of two specific protein has good response to the original, in the subsequent experiments is obtained by testing several protein specificity and sensitivity, we can on the basis of the existing stimulus antigen screen is suitable for our country crowd IGRAs stimulus antigen, as rapid auxiliary diagnosis of tuberculosis (TB), thus improve the serum antibody detection of TB case detection and reduce the misdiagnosis rate.},
     year = {2018}
    }
    

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  • TY  - JOUR
    T1  - Mycobacterium Tuberculosis Rv1987, TB7.7 Protein's Prokaryotic Expression, Purification and Identification
    AU  - Xiangru Li
    AU  - Jifu Ma
    AU  - Ying Xu
    AU  - Lijie Chen
    AU  - Jinmeng Song
    AU  - Feng Shi
    Y1  - 2018/12/12
    PY  - 2018
    N1  - https://doi.org/10.11648/j.sd.20180606.34
    DO  - 10.11648/j.sd.20180606.34
    T2  - Science Discovery
    JF  - Science Discovery
    JO  - Science Discovery
    SP  - 529
    EP  - 534
    PB  - Science Publishing Group
    SN  - 2331-0650
    UR  - https://doi.org/10.11648/j.sd.20180606.34
    AB  - Mycobacterium tuberculosis is a pathogen of tuberculosis and has relatively high epideictic and mortality among human. It is the second most life-threatening infectious disease second only to AIDS,so it is very important for the prevention and control of tuberculosis. In our experiment, RV1987 and TB7.7 recombinant plasmids of mycobacterium tuberculosis were constructed. The specific protein RV1987 and TB7.7 of mycobacterium tuberculosis were prokaryotic expressed, purified and identified in Escherichia coli. Our experiment lay a sound foundation for the subsequent serological diagnosis of tuberculosis as to obtain the high-purity target protein. Methods: use the genome of H37Rv as template to amplify the target gene and cloned it into prokaryotic expression vector, then transformed it into E. coli C43 (DE3) strain. After that, go through induced, expressed and purified in order to obtain recombinant protein.Results: the recombinant plasmid of RV1987 protein and TB7.7 protein was successfully constructed and expressed to obtain purified products, which were purified by affinity chromatography with a purity greater than 90%. The concentrations of RV1987 and TB7.7 proteins were 413 microns /mL and 1693 microns /mL. Rv1987 protein was expressed in inclusion body. TB7.7 protein was expressed in both supernatant and inclusion body, and the expression in supernatant was slightly higher than In inclusion body. Western Blot showed that all the above proteins could react with the positive serum of tuberculosis and had good reactivity. Conclusion: Escherichia coli is the most common system for gene engineering to express heterogenous proteins and widely used by researchers because of its relatively high expression efficiency. The experimental results showed that we obtained by prokaryotic expression and purification of two specific protein has good response to the original, in the subsequent experiments is obtained by testing several protein specificity and sensitivity, we can on the basis of the existing stimulus antigen screen is suitable for our country crowd IGRAs stimulus antigen, as rapid auxiliary diagnosis of tuberculosis (TB), thus improve the serum antibody detection of TB case detection and reduce the misdiagnosis rate.
    VL  - 6
    IS  - 6
    ER  - 

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Author Information
  • College of Life Science, Shihezi University, Shihezi, China

  • College of Life Science, Shihezi University, Shihezi, China

  • College of Life Science, Shihezi University, Shihezi, China

  • College of Life Science, Shihezi University, Shihezi, China

  • College of Life Science, Shihezi University, Shihezi, China

  • College of Life Science, Shihezi University, Shihezi, China

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