拟穴青蟹CrusSp的克隆表达纯化及抑菌活性测定 |
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引用本文: | 徐栋梁,李洁颖,彭永鹤,夏立新,刘志刚.拟穴青蟹CrusSp的克隆表达纯化及抑菌活性测定[J].华南师范大学学报(自然科学版),2011,0(1). |
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作者姓名: | 徐栋梁 李洁颖 彭永鹤 夏立新 刘志刚 |
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作者单位: | 1.1.深圳大学医学院 |
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基金项目: | 国家八六三计划:No.2006AA100308,深圳大学科研创新团队基金:No.200904. |
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摘 要: | 从拟穴青蟹血淋巴细胞提取总RNA,经RT-PCR扩增编码CrusSp成熟肽的cDNA序列,将其克隆至pMD18-T载体进行扩增,然后克隆至pET-32a(+)表达载体中,转化至E.coli OrigamiTM(DE3)中表达CrusSp蛋白,通过Ni2+亲和层析柱纯化获得CrusSp蛋白,表达出的CrusSp蛋白分子量约10.27 kDa,等电点为8.54,采用滤纸片扩散法检测该蛋白的抑菌活性.滤纸片扩散法显示CrusSp蛋白对绿色木霉具有抑制活性.
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关 键 词: | 拟穴青蟹 抗菌肽 克隆 纯化 抑菌活性 |
收稿时间: | 2010-09-15 |
Cloning,Expression,Purification and Antimicrobial Activity of CrusSp from Scylla paramamosain |
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Abstract: | Hemolyph was collected from Scylla paramamosain. The total RNA extracted from the hemocyte sample was used to amplify the sequence encoding an open reading frame of a mature peptide of CrusSp by RT-PCR. The sequence was cloned into pMD18-T vector and subjected to DNA sequence analysis. The DNA of CrusSp was isolated from pMD18-T/CrusSp and was cloned into pET-32a(+) expression vector to allow expression of CrusSp 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 CrusSp were studied by agar diffusion test. Analysis by 15% SDS-PAGE revealed that the molecular mass of CrusSp is 10.27 kDa. The antimicrobial activity revealed that CrusSp exhibited antifungal activity towards Trichoderma viride. |
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