小鼠干扰素epsilon在大肠杆菌中的可溶性表达

发布时间：2018-03-20 02:19

本文选题：干扰素　切入点：分子伴侣　出处：《免疫学杂志》2017年03期 　论文类型：期刊论文

【摘要】：目的构建小鼠干扰素epsilon蛋白的原核表达质粒,诱导表达和纯化后获得稳定的可溶性重组蛋白。方法将目的基因克隆到表达载体p ET28a-SUMO,与能同时表达3个分子伴侣gro ES、gro EL和tig的质粒p G-tf2一起转化至大肠杆菌表达菌株BL21(DE3),用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。通过Ni-NTA亲和层析柱、Desalting柱、Q-HP柱及Superdex75分子筛层析经行纯化后,用Ulp1酶切,并对目标蛋白进行质谱分析。对目标蛋白抑制肿瘤细胞增长活性、刺激细胞产生抗病毒蛋白的活性进行鉴定。结果成功构建了重组质粒p ET28a-SUMO-IFN-ε和分子伴侣共表达,获得较多可溶性蛋白。目标蛋白酶切后仍可溶,SDS-PAGE分析显示重组IFN-ε蛋白表观相对分子质量为22 000。质谱分析证实目标蛋白的序列和小鼠IFN-ε的序列吻合。小鼠IFN-ε能够抑制肿瘤细胞的生长并能诱导细胞产生抗病毒蛋白Mx A。结论小鼠IFN-ε蛋白可以和分子伴侣一起在原核系统中以可溶形式表达,并证明此蛋白具有生物学活性。
[Abstract]:Objective to construct the prokaryotic expression plasmid of mouse interferon epsilon protein. After induction, expression and purification, a stable soluble recombinant protein was obtained. Methods the target gene was cloned into the expression vector pET28a-SUMO.The recombinant plasmid pG-tf2, which could simultaneously express three molecular chaperones, gro Espgro El and tig, was transformed into Escherichia coli. Strain BL21DDE3 was induced by isopropyl- 尾 -D- thiogalactoside (IPTG) and purified by Ni-NTA affinity chromatography with desalting column Q-HP column and Superdex75 molecular sieve chromatography. The target protein was digested by Ulp1 and analyzed by mass spectrometry. The inhibition activity of target protein on tumor cell growth and the activity of stimulating cell to produce antiviral protein were identified. Results the recombinant plasmid pET28a-SUMO-IFN- 蔚 was successfully constructed and co-expressed with molecular chaperone. More soluble proteins were obtained. SDS-PAGE analysis showed that the apparent molecular weight of recombinant IFN- 蔚 protein was 22 000. Mass spectrometry confirmed that the sequence of target protein was consistent with that of mouse IFN- 蔚. Mouse IFN- 蔚 could be inhibited. Conclusion the mouse IFN- 蔚 protein can be expressed in a soluble form in the prokaryotic system together with the molecular chaperone, and can induce the cell to produce the antiviral protein MxA.Conclusion the mouse IFN- 蔚 protein can be expressed in the prokaryotic system together with the molecular chaperone. It is proved that the protein has biological activity.
【作者单位】：第三军医大学西南医院全军泌尿外科研究所;大坪医院妇产科;第三军医大学西南医院全军免疫学研究所;
【基金】：国家自然科学基金(81270845)
【分类号】：R3411

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