【摘要】 旨在克隆到猫ω干扰素和γ干扰素(FeIFN-ω和FeIFN-γ)基因的基础上,分析它们的一些生物学特性,并进行可溶性原核表达和简化纯化工艺,为后期作为抗病毒药物的开发奠定基础。根据GenBank登录的FeIFN-ω和FeIFN-γ基因序列,对其编码氨基酸的信号肽序列进行分析,设计引物用于克隆不包含信号肽的成熟蛋白基因序列,分别插入到原核表达载体pET28a-SUMO,转化入BL21感受态细胞进行表达,并通过不同的诱导时间、温度和控制IPTG浓度优化其表达条件,在纯化阶段摸索简易工艺。结果表明,FeIFN-ω基因编码区长591 bp,共编码196个氨基酸,信号肽序列为前23位氨基酸;FeIFN-γ基因编码区长504 bp,共编码167个氨基酸,信号肽序列为前20位氨基酸。将克隆到的FeIFN-ω和FeIFN-γ基因通过BamHⅠ和HindⅢ酶切位点带入到含SUMO标签的原核表达载体,构建成功的重组质粒分别命名为pET28a-SUMO-FeIFN-ω和pET28a-SUMO-FeIFN-γ。优化条件后表明SUMO-FeIFN-ω融合蛋白在IPTG终浓度为0.6 mmol/L时,16℃诱导6 h表达量最高;SUMO-FeIFN-γ融合蛋白在IPTG终浓度为0.8 mmol/L时,16℃诱导9 h表达量最高。最终表达出均以可溶性形式存在的两种融合蛋白,经镍柱纯化,用SUMO蛋白酶去除标签和经两次截留柱纯化获得不含标签的干扰素,分子质量约18 ku和17 ku,与预期大小相符,表明FeIFN-ω和FeIFN-γ蛋白成功得到表达和纯化。更多还原

【Abstract】 The purpose of this study was to clone the feline interferon ω and interfero γ(FeIFN-ω and FeIFN-γ) genes, analyze their biological characteristics in order to express soluble prokaryotes and simplify the purification process, which lay a foundation for the development of antiviral drugs in the future.According to the sequences of FeIFN-ω and FeIFN-γ gene in GenBank, the signal peptide sequence was analyzed, and primers were designed to clone the coding mature protein without signal peptide sequence.Then the obtained protein sequences were inserted into the prokaryotic expression vector pET28 a-SUMO,respectively.The vector was transformed into BL21 competent cells for expression, then the conditions of IPTG and temperature were optimized by different induction time, and the purification technology processing was simplified.The results showed that the coding region of FeIFN-ω gene is 591 bp, encoding 196 amino acids, and the first 23 amino acids were signal peptide sequences; the coding region of FeIFN-γ gene is 504 bp, encoding 167 amino acids, and the first 20 amino acids were signal peptide sequences.The cloned FeIFN-ω and FeIFN-γ genes were transferred into SUMO tagged prokaryotic expression vectors through the restriction sites of BamH Ⅰ and Hind Ⅲ,the recombinant plasmids were successfully constructed and named pET28 a-SUMO-FeIFN-ω and pET28 a-SUMO-FeIFN-γ,respectively.The optimized conditions showed that the expression of SUMO-FelFN ω fusion protein was the highest when the final concentration of IPTG was 0.6 mmol/L and incubated at 16 ℃ for 6 h; when the final concentration of IPTG was 0.8 mmol/L and incubated at 16 ℃ for 9 h, the expression of SUMO-FeIFN-γ fusion protein was the highest.Both expressed fusion proteins were existed in soluble form.The expressed proteins were purified by a nickel column, the SUMO protease was used to remove the tag and purified by two intercepting columns to obtain tag-free interferon.The molecular weights are about 18 ku and 17 ku, which are consistent with the expected size, indicating that FeIFN-ω and FeIFN-γ proteins were successfully expressed and purified.更多还原