【摘要】 发光杆菌能产生很多毒素杀死昆虫宿主.这些毒素中有一种叫Mcf致软因子,可以在大肠杆菌中表达并毒杀毛虫.通过同源性比对分析,在Mcf氨基酸序列中N端有一个BH3的区域,拥有BH3结构域的蛋白具有细胞凋亡的功能,推测保留N端BH3结构域的碳端截短Mcf毒素具有杀虫毒力.从发光杆菌属(Photorhabdus luminescens subsp.laumondii)中克隆了杀虫毒素基因mcf的5′端3960bp的核酸序列(包含BH3结构域),将该基因连接到E.coli表达载体pET28a上,并转入BL21(DE3).经IPTG诱导后,在SDS-PAGE上发现145kD的目的蛋白条带.利用亲和层析纯化得到纯毒素,分别对甜菜夜蛾一龄幼虫喂食生测和对甜菜夜蛾四龄幼虫(Spodoptera exigua)注射生测,显示该毒素具有喂食毒力和血腔毒力,证明含有BH3区域的碳端截短Mcf毒素有杀虫的生物活性.更多还原

【Abstract】 Photorhabdus bacteria produce a number of toxins to kill their insect hosts. The expression of Mcf is sufficient to allow Escherichia coli to persist within and kill caterpillars. Through searching for amino acids homology sequences in the protein Databases, BH3 domain was found in the N-terminal of Mcf toxin. It speculated the C-terminal truncated Mcf toxin with BH3 domain in the N-terminal might have the insecticidal activity because of the function of apoptosis of BH3 domain. The 5′-end of mcf insecticidal toxin gene from Photorhabdus luminescens subsp. Laumondii,which was 3 960 bp in size,was cloned into pET28a vector and transformed into BL21 (DE3). After induced by IPTG,a novel protein about 145 kDa in size was found on the SDS-PAGE gel. The expressed protein was subsequently purified by using affinity chromatography. The oral insecticidal activity of truncated Mcf toxin was tested against the first-instar larvae of Spodoptera exigua. Hemocoel insecticidal activity of truncated Mcf toxin was determined against mature larvae of Spodoptera exigua (fourth-instar). The present study demonstrated that the C-terminal truncated Mcf toxin had the insecticidal activity.更多还原