【摘要】 依据猫IFN-ω3和THY-α1的分子结构特征及大肠杆菌密码子的偏爱性设计引物,同时在IFN-ω3和THY-α1中间设计了1个(Gly-Gly-Gly-Gly-Ser)×3总共15肽的linker。采用搭桥PCR方法获得二者的融合基因。将该基因克隆至pBVIL载体中,构建了pBVIL-IFNω3-THYα1原核表达载体。将重组载体转化至Rosseta(DE3)细胞中,经过IPTG诱导表达及蛋白质分离纯化,获得了质量浓度为0.78 g/L的IFNω3-THYα1融合蛋白。经E玫瑰花环形成试验和细胞病变抑制试验,结果表明E玫瑰花环形成率平均为23.4%,融合蛋白的活力值为7.4%,细胞病变抑制试验同阳性对照组结果基本一致,融合蛋白中的IFNω3、THYα1均较好地保持各自的生物活性,为IFNω3-THYα1融合蛋白进一步的开发和应用奠定了基础。更多还原

【Abstract】 By analysising members of IFN family,the primers were designed based on IFN-ω3 and THY-α1 molecular structural characteristics and E.coli codon preference.Fifteen peptides linker containing(Gly-Gly-Gly-Gly-Ser)×3 was designed in the middle of IFN-ω3 and THY-α1.The fusion gene was obtained by the method of over-lapping PCR extension.The gene was cloned to prokaryotic expression vector pBVIL and constructed pBVIL-IFNω3-THYα1 prokaryotic expression system.The recombinant plasmids were transfected into Rosseta(DE3) cells and induced.there was a fusional protein with 0.78 g/L after separation and purification.The results carried on the E rosette assay and the virus inhibition on cell test were that E rosette formation rate was 23.4% and the fusion protein vigour value was 7.4% and cytopathic effect inhibition test basic coincides with positive control group.The IFNω3-THYα1 fusion protein was expressed after induction.We have detect the active of the pure protein.The result showed that the fusional protein has anti-virus activity of IFN-ω3 and it also has the activity of THY-α1.This study established the foundation for IFNω3-THYα1 fusion protein further development and application.更多还原