【摘要】 采集南美白对虾血淋巴,从血细胞中提取总RNA,经RT-PCR扩增编码penaeidin-2成熟肽的开放阅读框cDNA序列,将其克隆至pGEM-T载体,进行序列测定,然后克隆至pET-32a(+)表达载体中,在E.coliOriga-mi B(DE3)pLysS中表达含6个His的重组对虾肽融合蛋白Pen-24,通过His Band resin Ni2+层析柱纯化获得Pen-24蛋白,采用纸片扩散法和液体生长抑制法检测该蛋白的抑菌活性.经SDS-PAGE分析,Pen-24蛋白的分子量为24.1×103.1μg对虾肽融合蛋白Pen-24与5 U的氨苄青霉素活性相当,对革兰氏阳性细菌、革兰氏阴性细菌和抗氨苄青霉素大肠杆菌均具有抑菌活性.更多还原

【Abstract】 Hemolymph was collected from P.vannamei.The total RNA harvested from the hemocyte sample was used to amplify the sequence encoding an open reading frame for mature peptide of penaeidin-2 by RT-PCR.The sequence was cloned into pGEM-T vector and subjected to DNA sequence analysis.The DNA of penaeidin-2 was isolated from pGEM-T/Pen and was cloned into pET-32a(+) expression vector to allow expression of Pen-24 as a fusion protein in E.coli Origami B(DE3)pLys~S.The fusion protein could be purified effectively by His-Band resin chelating chromatography.The antibacterial activities of Pen-24 was studied by scrip diffusion analysis and the liquid growth inhibition assays. SDS-PAGE revealed the molecular weight of Pen-24 is 24.1×10~3.The antibacterial activity revealed that Pen-24 displayed antibacterial activity against Gram-positive,Gram-negative bacteria and E.coli against ampicillin and 1 μg Pen-24 fusion protein had identical antibacterial activity with 5 U ampicillin.更多还原