摘要
利用RT-PCR技术从果蝇总RNA中克隆出富甘氨酸果蝇抗菌肽基因,将该基因插入原核表达载体pET-32a(+)的Nde I和Xho I酶切位点之间,然后用重组质粒转化大肠杆菌BL21(DE3)利用乳糖操纵子的葡萄糖效应IPTG诱导表达,经15%SDS-PAGE、Western blot分析,在大约8 kD处出现了含6×His标签的抗菌肽蛋白。再以Ni-N TA纯化树脂进行亲和层析、脱盐、冻干,成功制备了富甘氨酸果蝇抗菌肽蛋白。
The gene of antibacterial peptide was amplified from Drosophila total P, NA using RT-PCR,and then inserted into Nde I and Xho I enzyme-cutting sites of pET-32a plasmid. The recombinant vectors were transformed into the competent cell E. coli BL 21. By using the glucose effect or catabolite,the antibacterial peptide was expressed in E. coil BL21 （DE3） by the induction of IPTG. By the analysis of 15% SDS-PAGE and Western blot,the antibacterial peptide was found at 8 kD. After the expressed product was purified by affinity binding chromatography with Ni-NTA, salt-out and freeze-dried procedure,the product was obtained.
出处
《生物技术通报》
CAS
CSCD
北大核心
2009年第3期88-90,共3页
Biotechnology Bulletin
基金
国家自然科学基金(30370647)
关键词
果蝇
富甘氨酸抗菌肽
直接表达
纯化
葡萄糖效应
Drosophila Glysine-rich peptides Direct expression Purification Glucose effect
