【摘要】 昆虫抗菌肽具有广谱抗菌活性,克隆黄粉虫Tenebrio molitor抗菌肽基因,进行原核表达和活性检测,为昆虫抗菌肽推广应用奠定一定基础。根据GenBank公布的黄粉虫抗菌肽序列设计特异引物,以RT-PCR法从黄粉虫体内克隆了抗菌肽基因TmAMP3,将其亚克隆至pET-30a表达载体中,转化到大肠杆菌Escherichia coli BL21(DE3)中进行原核表达,SDS-PAGE检测融合蛋白的表达,同时检测生物活性。结果表明我们成功构建了重组质粒pET-30a-TmAMP3,大部分目的蛋白呈可溶性分泌表达。经Ni2+亲和层析,获得较纯融合蛋白HIS-TmAMP3,诱导表达的融合蛋白使BL21转化菌生长受到一定程度的抑制。融合蛋白在100℃煮沸10h、与pH2～12的缓冲液混匀后,依然保持稳定的抗菌活性,具有较强的稳定性。同时检测了融合蛋白对5种菌株的最小抑菌浓度(minimun inhibitory concentration,MIC)。本研究为具有稳定活性抗菌肽进行大规模生产发酵提供了理论基础。更多还原

【Abstract】 Insect antimicrobial peptides (AMPs) have wide antimicrobial spectrum. Cloning and prokaryotic expression of antimicrobial peptide TmAMP3 gene from Tenebrio molitor could provide a basis for further application of this antimicrobial peptide. TmAMP3 was amplified by RT-PCR using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank. The gene TmAMP3 was subcloned directionally into the prokaryotic expression vector pET-30a and transformed into Escherichia coli BL21. After IPTG introduction, fusion protein was detected in the supernatant of lysates by SDS-PAGE and purified with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices, and the fusion protein HIS-TmAMP3 was obtained. In the presence of IPTG, the growth of BL21 transformed by pET30a-TmAMP3 was inhibited. Under conditions of high temperature 100℃ for 10 h and strong acid or alkali, antibacterial activities of the fusion protein still existed. The minimum inhibitory concentration (MIC) of the fusion protein against five bacterial strains was also detected. The experiment offers a theoretical foundation to produce antimrobial peptides with stability on a large scale.更多还原