摘要
为利用大肠杆菌表达系统高效表达抗菌肽Thanatin并避免其抗菌活性对宿主菌的影响,将抗菌肽Thanatin基因与家蚕核型多角体病毒多角体蛋白Polyhedrin基因融合克隆到表达载体pET28a(+)中,构建重组表达质粒pET28a(+)-Thanatin-polyhedrin,转化大肠杆菌BL21(DE3)中表达。SDS-PAGE检测显示经诱导表达的菌体中有与融合蛋白理论值(35 kD)相符的目的条带。凝胶扫描分析目的融合蛋白以包涵体的形式表达,且诱导后6 h融合蛋白的表达量最大,约占菌体总蛋白量的30%。纯化融合蛋白用盐酸羟胺切割后初步纯化获得具有抗菌活性的Thanatin蛋白。研究结果表明家蚕核型多角体病毒多角体蛋白能够作为抗菌肽Thanatin融合标签介导其高水平表达,有助于利用基因工程技术大规模生产抗菌肽Thanatin。
To obtain high efficiency expression of antimicrobial peptide Thanatin in E.coli and avoid its lethal effect on host cells,the Thanatin gene was fused to Polyhedrin gene of Bombyx mori nucleopolyhedrovirus and cloned into expression vector pET28a（＋） for construction of the recombinant expression plasmid pET28a（＋）-Thanatin-polyhedrin and then transformed into E.coli BL21（DE3） for expression.SDS-PAGE assay indicated that the induced bacterial expression products contained the target band correspondent to molecular weight of the fusion protein（35 kD）.Gel scanning analysis revealed that the target fusion protein was expressed as inclusion body and reached maximum expression level at 6 h after induction,accounting for approximately 30% of the total cellular proteins.The purified fusion protein was cleaved with hydroxylamine hydrochloride.After preliminary purification,Thanatin protein with antibacterial activity was obtained.These results demonstrated that the Polyhedrin of Bombyx mori nucleopolyhedrovirus can be used as a fusion label of antimicrobial peptide Thanatin and is capable of mediating high level expression of Thanatin,being favorable for large scale production of antimicrobial peptide Thanatin by means of genetic engineering.
出处
《蚕业科学》
CAS
CSCD
北大核心
2011年第6期1008-1013,共6页
ACTA SERICOLOGICA SINICA
基金
国家自然科学基金项目(No.10975002)
国家高技术研究发展计划"863"项目(No.2011AA100306-3)
安徽省国际科技合作项目(No.11030603028)
