作　　者： ; (王伟佳）;

机构地区： 中山大学

出　　处： 《中国病毒病杂志》 2021年第2期128-134,共7页

摘　　要： 目的通过原核表达系统表达可溶性塔城病毒1(Tacheng tick virus 1,TcTV-1)重组融合核蛋白(nucleoprotein, NP)并纯化获得纯度高和均一度好的目的蛋白。方法利用聚合酶链式反应(polymerase chain reaction, PCR)从经密码子优化的重组质粒中扩增TcTV-1核蛋白的编码基因,构建带有小分子泛素样修饰蛋白(small ubiquitin-like modifier, SUMO)融合标签的重组质粒pET-21a-TcTV-1-NP,转化入表达菌株BL21(DE3),异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside, IPTG)诱导高表达量菌株。采用镍离子金属鳌合亲和层析介质(Ni-NTA)亲和层析、离子交换层析和凝胶过滤层析提纯目的蛋白。结果经PCR扩增的目的基因TcTV-1核蛋白和质粒载体pET-21a进行重组质粒构建,电泳结果显示,在约6 000 bp处可见条带,与重组质粒pET-21a-TcTV-1-NP大小相一致;重组质粒转入大肠埃希菌BL21(DE3),在16℃,0.2 mmol/L IPTG诱导下表达出大量可溶性蛋白,收集并重悬菌体后,经低温高压和超声破碎后,细菌上清液经Ni-NTA亲和层析纯化,在250 mmol/L咪唑洗脱液中获得目的蛋白;去除融合标签后,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-polyacrylamide gel electrophoresis, SDS-PAGE)结果显示在相对分子质量为50 000附近处出现明显条带,与目的蛋白相对分子质量为48 800相一致;目的蛋白通过阳离子交换层析,得到1个蛋白洗脱峰;经凝胶过滤层析再次纯化,收集相应的蛋白洗脱峰,经SDS-PAGE结果显示目的蛋白大小正确,纯度较高。结论 TcTV-1重组融合核蛋白在大肠埃希菌内以可溶性形式成功表达,为进一步研究TcTV-1核蛋白的作用机制和相应血清学检测方法的开发提供了重要的基础。 Objective To express and purify the recombinant fusion nucleoprotein NP of Tacheng tick virus 1. Methods Recombinant expression plasmid of pET-21 a-TcTV-1-NP was constructed.The plasmid was transformed into Escherichia coli BL21(DE3) strain, and the strain was induced by IPTG.The expressed protein was purified using Ni-NTA affinity chromatography, ion exchange chromatography and gel filtration chromatography. Results The recombinant TcTV-1 NP was constructed into pET-21 a vector with SUMO tag.Electrophoresis gel showed visible bands at about 6 000 bp which is consistent with the size of recombinant plasmid pET-21 a-TcTV-1-NP.The recombinant plasmid was transformed into Escherichia coli BL21(DE3), and a large amount of soluble protein was expressed under the addition of 0.2 mmol/L IPTG at 16 ℃.After purification by Ni-NTA affinity chromatography, SDS-PAGE indicated that the target protein was in the fragment containing 250 mmol/L imidazole.After removing the fusion tag, SDS-PAGE showed that an obvious band appeared at the molecular weight of 50 000, which is consistent with the target protein of 48 800.After removing the fusion tag, a protein elution peak was obtained by cation exchange chromatography and gel filtration chromatography, the corresponding protein elution peaks were collected.SDS-PAGE identified the high purity of the target protein. Conclusions TcTV-1 NP recombinant fusion protein is successfully expressed in Escherichia coli as soluble form.

关 键 词： 布尼亚病毒 塔城病毒 核蛋白 蛋白纯化

领　　域： [医药卫生—病原生物学] [医药卫生—基础医学]