【摘要】 根据GenBank中公布的犬α-干扰素(CaIFN-α)基因核酸序列,去掉信号肽后设计并合成1对引物,引入EcoRⅠ和HindⅢ酶切位点。以提取的犬肝脏DNA为模板,利用PCR技术扩增CaIFN-α基因,并分别克隆至表达载体pBV220、pET-32a(His标签)中,转化大肠杆菌BL21(DE3)原核表达系统。重组融合蛋白经SDS-PAGE及Western blotting鉴定。纯化目的蛋白,并采用MDCK/VSV微量细胞病变抑制法检测其抗病毒活性。试验结果表明,与pBV220载体连接的目的基因表达的蛋白活性较低,而与pET-32a(His标签)连接的目的基因表达的蛋白活性较高,达2.56×106 U/mL。通过分析不同载体对IFN-α的表达情况,为生产高活性的IFN奠定基础。更多还原

【Abstract】 According to the sequence of canine interferon-α(CaIFN-α)gene published in GenBank,primers were designed,synthesized and introduced EcoRⅠand HindⅢ restriction sites.Using canine liver DNA as a template,CaIFN-α gene was amplified by PCR,then the gene was cloned into pBV220 and pET-32a(+)expression vector,and transformed into the BL21(DE3)expression system.Recombinant fusion protein was analysed by SDS-PAGE and Western blotting.Protein was purified and the antiviral activity was detected by MDCK/VSV cytopathic inhibition assay.The results showed that pBV220-α-IFN recombinant protein has a low activity,but the activity of pET32a-α-IFN recombinant protein was up to 2.56×106 U/mL.This study laid a foundation for production of highly active IFN.更多还原