【摘要】 目的:克隆树鼩γ干扰素(IFNγ)基因,在大肠杆菌中高效表达纯化并鉴定其免疫原性。方法:提取树鼩肺组织总RNA,经RT-PCR扩增出树鼩IFNγ基因,再克隆到原核表达载体p ET30a(+),构建重组表达质粒p ET-30a(+)-IFNγ,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达树鼩IFNγ;经金属螯合柱复性及纯化,Western印迹和ELISA检测其免疫原性,并检测不同组织中IFNγ的分布情况。结果:构建了重组表达质粒p ET-30a(+)-IFNγ,重组蛋白在30℃、1 mmol/L IPTG诱导4 h获得较高表达量,镍柱复性纯化后得到较高纯度的树鼩IFNγ。Western印迹显示IFNγ可与兔抗人IFNγ单克隆抗体特异性结合,应用Western印迹和ELISA分别检测树鼩IFNγ的免疫原性和抗体滴度,在树鼩的鼻、心、肝、肺、肾、气管中检测到IFNγ。结论:在大肠杆菌中高效表达了重组树鼩IFNγ,复性纯化后具有良好的免疫原性。更多还原

【Abstract】 Objective: To clone and express tree shrew interferon-γ(IFNγ) gene in E.coli, and purify the expressed product and identify its immunogenicity. Methods: The total RNA was extracted from tree shrews. The IFNγ gene of tree shrew was amplified by RT-PCR and cloned into prokaryotic expression vector p ET30 a(+) to construct recombinant expression plasmid p ET-30 a(+)-IFNγ. Transformation of E.coli BL21(DE3) competent cell,IPTG induced expression of tree shrew IFNγ. After refolded and purified by metal chelating column, the immunogenicity was detected by Western blot and ELISA, and the distribution of IFNγ in different tissues was detected. Results: Recombinant expression plasmid p ET-30 a(+)-IFNγ was constructed. The recombinant protein was highly expressed at 30℃ for 1 mmol/L IPTG for 4 h. The high purity tree shrew IFNγ was obtained after refolded and purified by nickel column. Western blot showed that IFNγ binds specifically to rabbit anti-human IFNγ monoclonal antibodies. The immunogenicity and antibody titer of IFNγ were detected by Western blot and ELISA. IFNγ was detected in the heart, liver, spleen, lung, kidney and trachea of tree shrew. Conclusion: The recombinant IFNγwas highly expressed in E.coli and had good immunogenicity after refolding and purification.更多还原