黑腹果蝇抗真菌肽基因Drs和Drs-lC在原核中的表达及抗菌活性的初步测定

对黑腹果蝇(Drosophila m elanogaster)抗真菌肽基因Drs和它的同系物基因Drs-lC在pET-21d载体中与T7.Tag标签序列进行了融合表达,并对融合表达产物进行Xa因子切割前后的抗菌活性测定。首先通过长引物PCR获得5′端带有Xa因子识别切割序列的Drs和Drs-lC,分别克隆至pET-21d表达载体上,然后转化E.coliO rigam i(DE3),筛选得到阳性克隆。以IPTG诱导目的基因与载体上的T7.Tag标签序列融合表达,将表达产物进行初步纯化后,以Xa因子进行切割处理,然后测定处理前后样品的抗菌活性。结果显示与T7.Tag标签序列融合表达的D rs、D rs-lC样品和经Xa因子切割后的样品对供试真菌黄色镰刀菌(Fusarium culm orum)、尖孢镰孢菌(Fusarium oxs-porum)和粗糙脉孢菌(Neuropora crassa)均有明显的抑制作用。

Expression of the Antifungal Peptide Genes, Drs and Drs-lC From Drosophila melanogaster in E.coli and the Activity Detection of Their Expression Products

The antifungal peptide genes,Drs and Drs-lC from Drosophila melanogaster were cloned into the pET-21d vector and fusion expressed with T7·Tag in E.coli.The antifungal activity of expression product was detected.Firstly,the target genes,Drs and Drs-lC with a cleavage site of Xa factor at 5′ end were obtained by PCR with long primers,and then were cloned into the pET-21d vector.The recombinant vectors,pET-21d-xa-Drs and pET-21d-xa-lC were transformed into the host cells of E.coli Origami(DE3).Drs,Drs-lC fused with T7·Tag were induced to express by IPTG.The expression products were purified and digested by Xa factor.The result showed that the fore-and-aft samples by digestion of Xa factor had obvious antifungal activity to the tested fungi,Fusarium culmorum,Fusarium oxsporum and Neuropora crassa.