gp10基因的原核表达及其联合异烟肼的体外抗结核菌活性

doi:10.13481/j.1671-587x.20150322

Abstract

Abstract:

Objective To express the mycobacteriophage D29 lysin gp10 in prokaryotic cells, and to investigate its antibacterial activities of alone and combined with isoniazid(INH)to Mycobacterium tuberculosis(MTB) in vitro. Methods The gp10 gene was amplified by PCR using the genome of mycobacteriophage D29 as a template and cloned into pET32a(+) vector. The constructed vector pET32a(+)-gp10 was identified by restriction enzyme digestion and nucleotide sequencing, and transformed to E.coli BL21(DE3) for expression. The minimal inhibitory concentration(MIC) of recombinant protein gp10 was detected by resazurin drugs combination microtiter assay (REDCA) for MTB standard strain H₃₇Rv and clinical isolate of multidrug-resistant MTB, the fractional inhibitory concentration index(FICI) was used to the antibacterial activity between INH and gp10 in MTB H₃₇Rv. Results The gp10 at a length of 1 479 bp was amplified by PCR, the recombinant plasmid pET32a(+)-gp10 was digested by EcoRⅠ and NotⅠ for 5 900 and 1 479 bp bands, gp10 sequence analysis was consistent with the sequence in GenBank, and it expressed a target protein with a relative molecular mass about 75 200. The MICs of recombinant protein gp10 were 256 mg·L^-1 against standard strain H₃₇Rv and clinical isolate of multidrug-resistant MTB. The FICI of combined INH and gp10 was 0.5 for MTB H₃₇Rv. Conclusion The recombinant protein gp10 is expressed successfully, and it shows good antibacterial activity against MTB H₃₇Rv and clinical isolate of multidrug-resistant MTB. Synergism in MTB H₃₇Rv is observed with gp10 combined with INH in vitro.

Key words: gp10 protein, isoniazid, Mycobacterium tuberculosis, antibacterial activity

CLC Number:

R378

PENG Wei, LI Youlun, FENG Yanmei, PENG Li. Prokaryotic expression of gp10 gene and antibacterial activity of gp10 gene combined with isoniazid to Mycobacterium tuberculosis in vitro[J].Journal of Jilin University Medicine Edition, 2015, 41(03): 548-552.

References

[1] O'Flaherty S, Ross RP, Coffey A. Bacteriophage and their lysins for elimination of infectious bacteria[J]. FEMS Microbiol Rev, 2009, 33(4):801-819.

[2] 杨文慧, 于龙, 胡凌飞, 等. 分枝杆菌噬菌体D29裂解酶基因的表达与鉴定[J]. 军事医学科学院院刊, 2009, 33(2):113-116.

[3] 熊玉霞, 杨致邦, 于拽拽, 等. 分枝杆菌噬菌体D29裂解酶gp10的原核表达及抗脓肿分枝杆菌活性[J]. 中国生物制品学杂志, 2011, 24(11):1269-1272.

[4] Bhusal Y, Shiohira CM, Yamane N. Determination of in vitro synergy when three antimicrobial agents are combined against Mycobacterium tuberculosis[J]. Int J Antimicrob Agents, 2005, 26(4):292-297.

[5] Campanerut PA, Ghiraldi LD, Spositto FL, et al. Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay[J]. J Antimicrob Chemother, 2011, 66(5):1044-1046.

[6] Martin A, Pasch F, Docx S, et al. Multicentre laboratory validation of the colorimetric redox indicator(CRI) assay for the rapid detection of extensively drug-resistant(XDR)Mycobacterium tuberculosis[J]. J Antimicrob Chemother, 2011, 66(4):827-833.

[7] Rodriguez-Cerrato V, Garcia P, Del Prado G, et al. In vitro actions of LytA, the major pneumococcal autolysin. With two bacteriophage lytic enzymes (Cpl-1 and Pal), cefotaxime and moxifloxacin against antibiotic -susceptible and -resistant Streptococcus pneumoniae strains [J]. J Antimicrob Chemother, 2007, 60(5):1159-1162.

[8] Drago L, Nicola1 L, Rodighiero V, et al. Comparative evaluation of synergy of combinations of β-lactams with fluoroquinolones or a macrolide in Streptococcus pneumoniae[J]. J Antimicrob Chemother, 2011, 66(4):845-849.

[9] Caleffi-Ferracioli KR, Maltempe FG, Siqueira VL, et al. Fast detection of drug interaction in Mycobacterium tuberculosis by a checkerboard resazurin method[J]. Tuberculosis, 2013, 93(6):660-663.

[10] Fenton M, Ross P, McAuliffe O, et al. Recombinant bacteriophage lysins as antibacterials[J]. Bioeng Bugs, 2010, 1(1):9-16.

[11] Lim JA, Shin H, Heu S, et al. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria[J]. J Microbiol Biotechnol, 2014, 24(6):803-811.

[12] Lai MJ, Lin NT, Hu A, et al. Antibacterial activity of Acinetobacter baumannii phage ΦAB2 endolysin(LysAB2) against both gram-positive and gram-negative bacteria[J]. Appl Microbiol Biotechnol, 2011, 90(2):529-539.

[13] Payne KM, Hatfull GF. Mycobacteriophage endolysins:diverse and modular enzymes with multiple catalytic activities[J]. PLoS One, 2012, 7(3):e34052.

[14] Lakshmanan M, Xavier AS. Bedaquiline—The first ATP synthase inhibitor against multidrug resistant tuberculosis[J]. J Young Pharm, 2013, 5(4):112-115.

[15] Morbidoni HR, Morbidoni HR. Recycling and refurbishing old antitubercular drugs:the encouraging case of inhibitors of mycolic acid biosynthesis[J]. Expert Rev Anti Infect Ther, 2013, 11(4):429-440.

[16] Schuch R, Lee HM, Schneider BC, et al. Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia[J]. J Infect Dis, 2014, 209 (9):1469-1478.

Prokaryotic expression of gp10 gene and antibacterial activity of gp10 gene combined with isoniazid to Mycobacterium tuberculosis in vitro

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 4

Metrics

Comments

Recommended 0

[1]	YIN Yutian, ZHANG Liang, ZHANG Yue, JIANG Lingling, WANG Yuhan, WANG Rui, ZHANG Peng, LI Bo. Analysis on multidrug-resistant situation of first-line anti-tuberculosis drugs in tuberculosis patients with positive sputum Mycobacterium tuberculosis culture [J]. Journal of Jilin University Medicine Edition, 2015, 41(05): 1080-1084.
[2]	LIN Tao, LI Minghe, HAO Ying, MAO Jixiong, BA Te, HAN Chengmin. Preparation of clindamycin-loaded PLGA microspheres and evaluation on its in vitro antibacterial activity [J]. Journal of Jilin University Medicine Edition, 2015, 41(01): 94-98.
[3]	. Bacteriostatic effect of extract of Ginkgo Ailoba leaves on Porphyromonas gingivalis in vitro [J]. Journal of Jilin University Medicine Edition, 2014, 40(05): 1018-1023.
[4]	QIN Hai-hong,JIA Lin-yu,YAN Jian-yi,LI De-wang,SUN Li-juan,WANG Hui-guo. In vitro and in vivo antibacterial activities of Impatiens Balsamina water extraction  [J]. Journal of Jilin University Medicine Edition, 2013, 39(1): 60-64.