拟穴青蟹CrusSp的克隆表达纯化及抑菌活性测定

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.

Cloning,Expression,Purification and Antimicrobial Activity of CrusSp from Scylla paramamosain

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.