Journal of Jilin University Medicine Edition ›› 2015, Vol. 41 ›› Issue (03): 548-552.doi: 10.13481/j.1671-587x.20150322

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

PENG Wei, LI Youlun, FENG Yanmei, PENG Li   

  1. Department of Respiratory Medicine, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
  • Received:2014-11-09 Published:2015-08-01

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