含抗菌肽基因的天然质粒在枯草杆菌中的表达

Expression of Natural Plasmid Harboring an Antimicrobial Peptide Gene in Bacillus subtilis

侧孢短芽孢杆菌抗菌肽具有高效、广谱、稳定的抗菌活性,该抗菌活性与其内源质粒密切相关。为研究侧孢短芽孢杆菌含抗菌肽基因质粒异源表达的可行性,本研究中将侧孢短芽孢杆菌S62^-9的天然质粒电转枯草芽孢杆菌Wb800受体细胞,筛选阳性转化子并对其表达产物性质进行研究。得到了产生抗菌活性产物菌株Z4,发酵6 h后开始产生抗菌物质,12~20 h产量达到最大,达620.96 AU/m L。Tricine^-十二烷基硫酸钠^-聚丙烯酰胺凝胶电泳看到S62^-9质粒在枯草芽孢杆菌中得到了成功的表达,反相高效液相色谱分析Z4表达产物含有与侧孢短芽孢杆菌抗菌肽相同物质。该物质抑菌谱广,稳定性好:对于G^+、G^-和真菌均有抑菌活性,而且对于G^+菌株的抑菌效果优于G^-菌株和真菌,121℃处理20 min后的效价保留率约为90%,100℃处理60 min后仍有81%的活性保留;p H 2~12的范围内效价保留率基本不发生变化;对蛋白酶较为敏感,胰蛋白酶和蛋白酶K处理后抗菌肽的效价保留率为60%左右,其他3种蛋白酶处理后也有一定程度的降低。本研究获得了抗菌肽的异源表达菌株Z4,为进一步工业生产抗菌肽提供支持,而且为该抗菌肽遗传基因定位于质粒上提供了证明。

The antimicrobial peptide produced by Brevibacillus laterosporus shows efficient,broad-spectrum,and stable antimicrobial activities,which are closely associated with the endogenous plasmid in B.laterosporus S62-9.In order to study the feasibility of heterologous expression of B.laterosporus plasmid with antimicrobial peptide genes,B.laterosporus S62-9plasmid was transferred into Bacillus subtilis Wb800by electroporation.The positive transformants were screened and the expression products were characterized.The results showed that strain Z4showed an antimicrobial activity after fermentation for6h,reaching the highest titer(620.96AU/mL)during12-20h.Tricine-SDS-PAGE indicated that B.laterosporus plasmid was successfully expressed in B.subtilis.Reversed-phase high performance liquid chromatography(RP-HPLC)analysis showed that the expression products of strain Z4and B.laterosporus antimicrobial peptide contained the same substances,which showed broad-spectrum antimicrobial activities with high stability.Both of them displayed strong antimicrobial activities against Gram-positive bacteria,Gram-negative bacteria and fungi,especially against Gram-positive bacteria.About90%and81%of the titer was retained after20min at121℃and after60min at100℃,respectively.The antimicrobial activity showed a slight change in the pH range of2-12.However,the antimicrobial peptide was more sensitive to proteases and the titer was reduced by about40%after treatment with trypsin or proteinase K,and was also reduced after treatment with three other proteases.The establishment of strain Z4may lay a foundation for the industrial production of antimicrobial peptide in the future,and provide evidence for positioning the B.laterosporus antimicrobial peptide gene on plasmid.