家蚕拟抗微生物肽Gloverins基因(Bmglv)的原核表达及抗菌活性鉴定

Prokaryotic expression and activity detection of the gloverin-like genes (Bmglv) of Bombyx mori

家蚕Bombyx mori基因组全测序于2004年完成后,由家蚕基因组注释发现了35条拟抗微生物肽基因序列。本研究选取其中的5条Gloverins同系物(isoforms)基因(BmglvB,BmglvA2,BmglvA3,BmglvA5和BmglvA6),克隆至pET-21d载体,转化Escherichia coli RosettaTM(DE3)宿主菌进行表达;克隆表达的5个家蚕Gloverins同系物基因大部分以可溶形式表达,经Ni-NTA亲和层析纯化和Sephadex G-10脱盐处理后,采用平板孔穴抑菌法测定其抗菌活性。结果表明:注释发现的5条家蚕拟抗微生物肽Gloverins同系物基因,具有与在其他昆虫中已鉴定报道的Gloverin相似的抗革兰氏阴性细菌的功能,实验确认了它们就是家蚕Gloverins抗微生物肽基因。

Thirty-five members of the gene family in seven gene families have been discovered from silkworm genome by the bioinformatics analysis after draft map of silkworm genome had been completed in 2004, of which seven pieces belong to gloverin gene families. In this study, five silkworm gloverin-like genes, namely BmglvB, BmglvA2, BmglvA3, BmglvA5 and BmglvA6, were cloned and expressed in the pET-21d expression vector/Escherichia coli RosettaTM （DE3）. The expressed products of gloverin-like gene family were purified successfully by the Ni-NTA affinity chromatography and desalted by Sephadex G-10 chromatography under native conditions, respectively. By use of agarose diffusion assay, the antimicrobial activity of the gloverin-like genes was defined. The results showed that the five silkworm gloverin-like genes had the similar activity to Gram-negative bacterial as reported in other insects, and the five gloverin-like peptides were confiremed as new antimicrobial peptides.