摘要
本实验主要对鲢鱼半胱氨酸蛋白酶抑制因子Cystatin进行原核表达和纯化鉴定,并研究重组Cystatin对铜绿假单胞菌的抑菌效果。首先用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导转入p ET-30-Cystatin的E.coli BL21(DE3)表达重组Cystatin蛋白,经Ni2+-NTA镍离子亲和层析对其进行纯化,该重组蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示单一条带,分子质量约20.6 k D,在TSK-GEL G2000SW凝胶过滤高效液相色谱上呈单一活性峰,纯度为94.27%,经标准曲线计算其分子质量为20.9 k D;其次,利用抑制剂滴定曲线法,以偶氮酪蛋白为底物,确定重组Cystatin对木瓜蛋白酶(45.375μmol)的一个抑制活性单位为0.718μg;最后通过滤纸片扩散法鉴定鲢鱼重组Cystatin对铜绿假单胞菌的抑菌效果,结果表明Cystatin的添加量为20、60、120、200 U/片时,抑菌圈直径分别为8、9、13、26 mm。鲢鱼重组Cystatin对铜绿假单胞菌的抑菌效果呈剂量依赖关系。
Prokaryotic expression, purification and identification of a cysteine proteinase inhibitor, cystatin, from Hypophthalmichthys molitrix were performed, and the antibacterial effect of the recombinant cystatin on Pseudomonas aeruginosa was investigated in this work. The cystatin was expressed by E. coli BL21(DE3) transformed with p ET-30-Cystatin with the induction of isopropyl-beta-D-galactose(IPTG), and purified by Ni2+-NTA affinity chromatography. The purified protein appeared as a single band on SDS-PAGE, corresponding to a molecular weight of approximately 20.6 k D. The same protein also appeared as a single active peak on the gel filtration HPLC of TSK-GEL G2000 SW with a purity of 94.27% and a relative molecular weight of 20.9 k D, as calibrated by standards. The inhibitory activity of the cystatin against papain(45.375 μmol) was 0.718 μg, which was determined by inhibitor titration method with Azocasein as the substrate. The antibacterial effect of the recombinant cystatin on Pseudomonas aeruginosa was tested by filter paper diffusion method, and it was found that the diameters of the inhibition zones were 8, 9, 13 and 26 mm with the addition of 20, 60, 120 and 200 units of the cystatin, respectively. This suggests that the antibacterial activity of the cystatin on Pseudomonas aeruginosa is dose-dependent.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2014年第21期133-137,共5页
Food Science
基金
国家自然科学基金青年科学基金项目(31101249)
关键词
半胱氨酸蛋白酶抑制剂
鲢鱼
原核表达
鉴定
铜绿假单胞菌
抑菌活性
cystatin
Hypophthalmichthys molitrix
prokaryotic expression
identification
Pseudomonas aeruginosa
antibacterial activity
