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

Xiangru Li; Jifu Ma; Ying Xu; Lijie Chen; Jinmeng Song; Feng Shi

doi:doi:10.11648/j.sd.20180606.34

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

Xiangru Li, Jifu Ma, Ying Xu, Lijie Chen, Jinmeng Song, Feng Shi

Published in Science Discovery (Volume 6, Issue 6)

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

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[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

Xiangru Li

College of Life Science, Shihezi University, Shihezi, China
Jifu Ma

College of Life Science, Shihezi University, Shihezi, China
Ying Xu

College of Life Science, Shihezi University, Shihezi, China
Lijie Chen

College of Life Science, Shihezi University, Shihezi, China
Jinmeng Song

College of Life Science, Shihezi University, Shihezi, China
Feng Shi

College of Life Science, Shihezi University, Shihezi, China

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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}
}

Copy | Download

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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