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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。
In order to fuse and express human interferon (IFNα) -2b in Escherichia coli using polymolecular chaperone expression vector and study IFNα-2b purification process, Smt3-IFNα-2b clone was artificially designed and synthesized to construct self-constructed P32-TrxA vector, the recombinant expression plasmid P32-TrxA-Smt3-IFNα-2b was transformed into E. coli BL21 (DE3), induced by IPTG and analyzed by SDS-PAGE. The purification of IFNα-2b was established by metal chelation chromatography, SUMO protease digestion, anion exchange chromatography and gel chromatography. The results showed that the recombinant plasmid was proved to be correct by restriction enzyme digestion and sequencing. The fusion protein was about 40 ku in molecular weight and mainly expressed in soluble form, accounting for more than 30% of the total bacterial protein. The molecular mass of purified IFNα-2b About 17 ku, the purity of the protein reached more than 95%, and the titers of interferon measured by cytopathic effect inhibition method were 1.0 × 10 ~ 8 IU / mg. It was successfully expressed in Escherichia coli and the recombinant protein IFNα-2b was purified by a simple process.