Cloning,Expression,Purification and Antimicrobial Activity of CrusSp from Scylla paramamosain
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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.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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Keywords:
- Scylla paramamosain /
- Antimicrobial peptide /
- Cloning /
- Purification /
- Antimicrobial activity
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