黑虎虾Crustin的克隆表达纯化及抑菌活性测定  

Cloning,Expression,Purification and Antimicrobial Activity of Crustin From Penaeus Monodon

作　　者：徐栋梁[1] 朱超亚[1] 彭永鹤 夏立新[1] 刘志刚[1] 

机构地区：[1]深圳大学医学院过敏反应与免疫学研究所,广东深圳518060 [2]深圳市圣西马生物技术有限公司,广东深圳518057

出　　处：《南昌大学学报（理科版）》2010年第6期598-602,共5页Journal of Nanchang University(Natural Science)

基　　金：国家863计划(2006AA100308);深圳大学科研创新团队基金(200904)

摘　　要：从黑虎虾血淋巴细胞中提取总RNA,经RT-PCR扩增编码Crustin成熟肽的cDNA序列,将其克隆至pMD18-T载体进行扩增,然后克隆到pET-32a(+)表达载体中,转化至E.coliOrigamiTM(DE3)中表达Crustin抗菌蛋白,后通过Ni2+亲和层析柱纯化获得Crustin蛋白,采用滤纸片扩散法检测该蛋白的抑菌活性。表达出的Crus-tin蛋白分子量约14.7 kDa,滤纸片扩散法显示Crustin蛋白对绿色木霉具有抑制活性。Hemolyph was collected from Penaeus monodon.The total RNA harvested from the hemocyte sample was used to amplify the sequence encoding an open reading frame for mature peptide of Crustin by RT-PCR.The sequence was cloned into pMD18-T vector and subjected to DNA sequence analysis.The DNA of Crustin was isolated from pMD18-T/Crustin and was cloned into pET-32a（＋） expression vector to allow expression of Crustin as a fusion protein in E.coli OrigamiTM（DE3）.The fusion protein could be purified effectively by His-Band resin chelating chromatography.The antimicrobial activities of Crustin was studied by agar diffusion test.15% SDS-PAGE revealed the molecular mass of Crustin is 14.7 kDa.The antimicrobial activity revealed that Crustin exhibited antifungal activity towards Trichoderma viride.

关 键 词：黑虎虾 抗菌肽 克隆 纯化 抑菌活性 

分 类 号：Q786[生物学—分子生物学]