泡桐内生枯草芽孢杆菌JDB-1草菌素的抑菌活性及其spaS基因的克隆

Antimicrobial Activity of Subtilin from Bacillus Subtilis JDB-1 and Cloning spaS Gene

用硫酸铵沉淀法从泡桐内生枯草芽孢杆菌JDB-1发酵液中粗提枯草菌素,采用纸片扩散法和琼脂糖扩散法分别检测枯草菌素对细菌和真菌的抑菌活性,采用PCR从菌株JDB-1基因组DNA中扩增出枯草菌素基因spaS,并克隆到pMD18-T载体,测定spaS基因的核苷酸序列.结果表明硫酸铵粗提的枯草菌素对大肠杆菌K12、恶臭假单胞杆菌AS1.1003、赤霉菌JSD-2、赤霉菌JSD-7和白色念珠菌ATCC10123等细菌和和真菌具有较强的抑制活性.DNA测序结果显示spaS基因大小为171bp,与已报道不同菌株spaS基因核苷酸序列的同源性为95%.

The bacterial subtilin was purified from the fermentation broth of Bacillus subtilis JDB-1 by using the ammonium sulfate precipitation,and its antimicrobial activity against five tested microorganisms was investigated by using the agar diffusion test or the paper disc test.The spaS gene encoding subtilin was amplified from genomic DNA of Bacillus subtilis JDB-1 by PCR,and the PCR products cloned into the pMD18-T vector and subsequently introduced the competent cells of E.coli DH5α.The recombinant plasmid pMD18-spaS was identified by PCR and then sequenced.The results showed that the purified subtilin displayed antimicrobial activities against E.coli K12,Pseudomonas putida AS1.1003,Gibberella moniliformis JSD-8 and Candida albicans ATCC10123,respectively.The sequence analysis showed that the coding region spaS gene of Bacillus subtilis JDB-1 was 171bp in length,and the nucleotide sequence similarity of the spaS genes between the JDB-1 strain and the previously reported other bacterial strains was 95%.