猫干扰素α基因的克隆及其表达产物的抗病毒活性分析

Cloning,expression and antiviral activity of feline IFN-α

为制备重组猫IFN-α并检测其抗病毒活性,本研究采用水泡性口炎病毒(VSV)感染结合poly I:C刺激实验猫后,提取猫脾淋巴细胞总RNA并反转录为cDNA,以其为模板经RT-PCR方法扩增获得了567 bp的猫IFN-α基因,测序后进行生物信息学分析。结果显示,猫IFN-α有21个潜在磷酸化位点、1个N-糖基化位点和8个O-糖基化位点,二级结构以α-螺旋为主。将该基因经BamH I/Kpn I酶切处理后克隆至pCold-TF载体,构建重组表达质粒pCold-α,将其转化大肠杆菌BL21感受态细胞后获得了表达猫IFN-α的重组大肠杆菌pCold-α/BL21。重组菌经IPTG诱导后SDS-PAGE检测结果显示重组猫IFN-α蛋白获得高效表达,采用His标签蛋白纯化柱纯化后得到纯化蛋白浓度为340 mg/L。以VSV和猫冠状病毒(FCoV)为模式病毒,采用微量细胞病变抑制法检测了重组猫IFN-α的抗病毒效果,结果显示,其抗VSV活性为5.91×10^(5) IU/mg,抗FCoV活性可达6.25×10^(6) IU/mg,具有良好的抗病毒活性。本研究为猫干扰素的开发应用奠定了物质基础。

To prepare recombinant protein of feline IFN-α(feIFN-α)and evaluate its antiviral activity,the gene encoding the feIFN-α was amplified by RT-PCR with the length size of 567 base pairs from the spleen lymphocytes of cat after infection with VAS combined with poly I:C stimulation.The bioinformatics analysis showed that the feIFN-αhad twenty-one potential phosphorylation sites,one N-glycosylation site and eight O-glycosylation sites,and its secondary structure wereα-helix.Subsequently,the gene was digested with BamH I/Kpn I and subcloned into the pCold-TF vector to construct a recombinant E.coli pCold-α/BL21 expressing the feIFN-α.After induction by IPTG,fusion protein was highly expressed by using SDS-PAGE analysis,and 340 mg/L of target protein(21 ku)was obtained after protein purification with a histidine(His)affinity tags.The antiviral activity of the feIFN-α was evaluated by microcytopathic-inhibiting-assay in the cells infected with either VSV or FCoV.And the results showed that the antiviral activities of the feIFN-αagainst VSV and FCoV were 5.91×10_(5) IU/mg and 6.25×10^(6) IU/mg,respectively.The feIFN-αhad good antiviral activity,providing a reference for the further development of the feIFN-α.