重组人IFNα-2b融合表达及纯化工艺研究  

Study on the fusion expression and purification technology of recombinant human interferon alpha-2b

作　　者：赵鑫[1,2] 张国利[2] 付玉和 李泽鸿[1] 张培培[2] 李爽[1] 田园[2] 刘雨玲 吴广谋[2] 岳玉环[2] 

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]军事医学科学院军事兽医研究所,长春130122

出　　处：《黑龙江畜牧兽医》2016年第12期38-41,共4页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了利用多分子伴侣表达载体在大肠埃希氏菌中融合表达人干扰素（IFNα）-2b，并进行IFNα-2b纯化工艺研究，试验采用人工设计并合成Smt3-IFN（x-2b克隆至自行构建的P32-TrxA载体中，将重组表达质粒P32-TrxA—Smt3-IFNα-2b转化至大肠埃希氏菌（E．coil）BL21（DE3），经IPTG诱导表达、SDS—PAGE分析，在此基础上利用金属螯合层析、SUMO蛋白酶酶切、阴离子交换层析以及凝胶层析建立了IFNot-2b的纯化工艺。结果表明：重组质粒经酶切鉴定及测序证明构建正确；融合蛋白分子质量约为40ku，主要以可溶形式表达，表达量占菌体总蛋白的30％以上；纯化的IFNα-2b分子质量约为17ku，蛋白纯度达到95％以上，利用细胞病变抑制法测得干扰素的效价为1．0×10^8IU／mg。说明成功在大肠埃希氏菌中表达并经过简易工艺纯化了重组蛋白IFNα-2b。To utilize a multi - molecular chaperone expression vector to express the fusion protein of interferon alpha - 2b （ IFN α - 2b） in Escherichia coli and study the purification technology of the IFN α -2b protein, Smt3 -IFNα -2b was artificially designed and synthesized in the test. The Smt3 - IFNα - 2b was cloned into a self - constructed vector P32 - TrxA to build a recombinant expression plasmid P32 - TrxA - Smt3 - IFNα - 2b, and then the recombinant expression plasmid was transformed into Escherichia coli BL21 （ DE3 ）. After inducing with IPTG, the fusion protein was analyzed by SDS - PAGE. On this basis, the purification technology of the IFN α - 2b protein was constructed using metal chelate chromatography, small ubiquitin -like modifier （SUMO） protease digestion, anion exchange chromatography and gel chromatography. The results showed that the recombinant plasmid was constructed correctly, which was proved by restriction enzyme digestion and sequencing; the fusion protein had a molecular mass of about 40 ku, and it was expressed in soluble form, while the expression level of fusion protein accounted for more than 30% of total bacterial proteins; the purified IFNα -2b had a molecular mass of about 17 ku and the purity production was above 95%, and the IFN titer determined by cytopathic effect inhibition assay was 1.0 × 10^8 IU/mg. The results indicate that the recombinant IFNα -2b is successfully expressed in Escherichia coli, and was purified through simple purification process.

关 键 词：IFNΑ-2B 表达 纯化 P300-TrxA—SUMO载体 层析 

分 类 号：TQ929.1[轻工技术与工程—发酵工程]