牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定

doi:10.16431/j.cnki.1671-7236.2017.07.025

Abstract

Abstract:

This study was aimed to construct a shuttle expression vector of Mycobacterium bovis(M.bovis) eis gene and identify its bioactivity in recombinant Mycobacterium smegmatis (M.smegmatis). M.bovis eis gene was cloned by PCR and the shuttle expression vector pMV261-Mbeis was constructed, then it was identified by double digestion and sequencing. The recombinant plasmid was transformed into M.smegmatis mc²155 by electroporation. The expression of M.bovis eis gene in M.smegmatis was detected by SDS-PAGE and Western blotting, and the amino acids sequence of the target protein was identified by mass spectrometry. The growth curve of recombinant M.smegmatis mc²155 containing pMV261-Mbeis was successfully constructed.The results showed that pMV261-Mbeis did not affect the growth of M. smegmatis in vitro. The results of SDS-PAGE and Western blotting confirmed that the M. bovis eis gene expressed the eis protein which was about 44 ku in M. smegmatis. Mass spectrometry proved that the protein was the eis protein of M. bovis.The expression vector pMV261-Mbeis was successfully constructed and the expressed recombinant protein was proved to be have biological activities in M. smegmatis, which laid a foundation for the further study of the function of eis protein in M. bovis.

Key words: eis gene; pMV261; Mycobacterium smegmatis

CLC Number:

S855.2

ZHANG Tong-ming, SONG Tian-qi, XIN Ting, ZHU Hong-fei, JIA Hong. Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis[J]. , 2017, 44(7): 2079-2085.

References

[1] Cousins D V. Mycobacterium bovis infection and control in domestic livestock[J]. Revue Scientifique Et Technique,2001,20(1):71-85.
[2] 程继忠,捕甫永穆. 外源基因在耻垢分枝杆菌中表达效率的研究[J]. 生物化学与生物物理进展,1997,3:249-253.
[3] Deretic V,Levine B. Autophagy,immunity,and microbial adaptations[J]. Cell Host Microbe,2009,5(6):527-549.
[4] Huang J,Brumell J H.Bacteria-autophagy interplay:A battle for survival[J]. Nature Reviews Microbiology,2014,12(2):101-114.
[5] 张海,师长宏,黄庆生,等. 自噬在结核分枝杆菌感染中的作用及相关基因的表达[J].细胞与分子免疫学杂志,2009,25(2):120-122.
[6] Pareja M E,Colombo M I. Autophagic clearance of bacterial pathogens:Molecular recognition of intracellular microorganisms[J]. Frontiers in Cellular & Infection Microbiology,2013,3(5):54.
[7] Jo E K,Shin D M,Choi A M. Autophagy:Cellular defense to excessive inflammation[J]. Microbes Infect,2012,14(2):119-125.
[8] Wang J,Huang C,Wu M,et al. MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG[J]. Journal of Infection,2015,70(4):415-426.
[9] Samuel L P,Song C H,Wei J,et al. Expression,production and release of the eis protein by Mycobacterium tuberculosis during infection of macrophages and its effect on cytokine secretion[J]. Microbiology,2007,153(2):529-540.
[10] 黄芳,刘家云,马越云,等. 结核分枝杆菌eis基因突变与氨基糖苷耐药的研究[J]. 现代生物医学进展,2011,11(7):1213-1215.
[11] He Z,Li S,Zhou X. Rapid identification and detection of intracellular survival testing of Mycobacterium smegmatis mc2155 that contains eis gene from Mycobacterium tuberculosis by flow cytometry[J]. Current Microbiology,2011,63(5):426-432.
[12] 易敏,周向东,李升锦,等. 结核分枝杆菌eis基因抑制人肺腺癌A549细胞自噬[J]. 基础医学与临床,2013,33(2):129-132.
[13] 易敏,李升锦,牟方红. 结核分枝杆菌eis基因对鼠巨噬细胞Raw264.7自噬影响的研究[J]. 中国微生态学杂志,2014,26(7):779-781.
[14] Wei J,Dahl J L,Moulder J W,et al. Identification of a Mycobacterium tuberculosis gene that enhances Mycobacterial survival in macrophages[J]. Journal of Bacteriology,2000,182(2):377-384.
[15] Kim K H,An D R,Sg J,et al. Mycobacterium tuberculosis eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7[J]. Proceedings of the National Academy of Sciences of the USA,2012,109(20):7729-7734.
[16] Shin D M,Jeon B Y,Lee H M,et al. Mycobacterium tuberculosis eis regulates autophagy,inflammation,and cell death through Redox-dependent signaling[J]. PLOS Pathogens,2010,6(12):417-422.
[17] Leistikow R L,Morton R A,Bartek I L,et al. The Mycobacterium tuberculosisdosr regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy[J]. Journal of Bacteriology,2009,192(6):1662-1670.
[18] Maurya V K,Singh K,Sinha S. Suppression of eis and expression of Wag31 and groes in Mycobacterium tuberculosis cytosol under anaerobic culture conditions[J]. Indian J Exp Biol,2014,52(8):773-780.
[19] Lella R K,Sharma C. Eis (enhanced intracellular survival) protein of Mycobacterium tuberculosis disturbs the cross regulation of T-cells[J]. Journal of Biological Chemistry,2007,282(26):18671-18675.
[20] Duan L,Yi M,Chen J,et al. Mycobacterium tuberculosis eis gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[J]. Biochemical & Biophysical Research Communications,2016,473(4):1229-1234.
[21] 何子纯,李升锦. 结核杆菌eis基因的克隆及其在耻垢分枝杆菌中的表达[J]. 中国生物工程杂志, 2010, 30(12):20-24.

Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 1

Recommended Articles

Metrics

Comments