重组人IFNα-2b融合表达及纯化工艺研究

发布时间：2017-12-31 07:45

本文关键词：重组人IFNα-2b融合表达及纯化工艺研究　出处：《黑龙江畜牧兽医》2016年23期 　论文类型：期刊论文

更多相关文章： IFNα-b 表达纯化 P-TrxA-SUMO载体 层析

【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-IFNα-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IFNα-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40 ku,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17 ku,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。
[Abstract]:In order to use the expression of molecular chaperones vector in Escherichia coli fusion expression of human interferon alpha (IFN alpha) -2b, and purification of IFN alpha -2b test with artificial design and synthesis of Smt3-IFN alpha -2b was cloned into P32-TrxA vector to construct the recombinant expression plasmid P32-TrxA-Smt3-IFN, alpha -2b was transformed into Escherichia coli (E.coil) BL21 (DE3), the expression of SDS-PAGE induced by IPTG analysis, on the basis of the use of metal chelating chromatography, SUMO protease digestion, anion exchange chromatography and gel chromatography to establish the purification process of IFN alpha -2b. The results showed that the recombinant plasmid was verified by enzyme digestion and sequencing showed that the fusion protein was constructed correctly; molecular mass of about 40 Ku, mainly expressed in soluble form, the expression of the total protein of the bacteria accounted for more than 30%; the IFN alpha -2b molecular weight of the purified protein was about 17 Ku, the purity reached more than 95%, using cytopathic inhibition test The potency of interferon was 1 * 10~8IU/mg. indicating that the recombinant protein IFN alpha -2b. was successfully expressed in Escherichia coli and purified by a simple process.

【作者单位】：吉林农业大学生命科学学院;军事医学科学院军事兽医研究所;
【分类号】：R94;TQ920.1
【正文快照】： 人干扰素(IFN)α-2b是由白细胞产生的Ⅰ型干扰素[1],是一种抗肿瘤、抗病毒并且可以调节机体免疫功能的广谱类药物,其作用是在免疫系统发挥作用前使细胞形成抗病毒状态,并具有抑制病毒增殖、防止病毒扩增、使病毒失活的作用,同时通过诱导机体细胞产生多种细胞因子发挥协同抗病

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