A型口蹄疫病毒1D蛋白在大肠埃希菌中的可溶性表达、纯化及电子显微镜检测

发布时间：2018-07-08 19:35

本文选题：A型口蹄疫病毒 + D蛋白　；参考：《解剖学报》2017年06期

【摘要】：目的本实验旨在表达出高可溶性的A型口蹄疫病毒1D蛋白,并通过电子显微镜检测,以期望形成纳米样颗粒。方法根据A型口蹄疫病毒核酸序列,得到FMDV A/GDMM/CHA/2013株的1D蛋白基因,并进行截短和优化,共132个氨基酸;同时,从结肠弯曲杆菌(Campylobacter coli)中分离得到135个氨基酸的铁蛋白(Fn)基因片段,将A型口蹄疫病毒1D蛋白与铁蛋白串联,设计并合成了口蹄疫病毒1D蛋白-铁蛋白片段,命名为CcFnt166AS。构建了CcFnt166AS融合Grifin、GST、MBP、Sumo、Thioredoxin、γ-crystallin、ArsC、PpiB、CeHSP17等9种不同可溶性标签的表达重组载体。分别转化至大肠埃希菌BL21(DE3)中,异丙基硫代半乳糖苷(IPTG)诱导,SDSPAGE电泳对融合蛋白的可溶性表达进行检测,筛选出高可溶性表达的CcFnt166AS融合蛋白。重组蛋白通过NiNTA Agarose亲和纯化,进行电子显微镜检测。结果成功构建9种CcFnt166AS表达载体;9个标签中,MBP与CcFnt166AS蛋白相融合的可溶表达效果最好,并获得了高纯度的MBP-CcFnt166AS重组蛋白质;电子显微镜结果显示,MBP-CcFnt166AS形成了纳米样颗粒。结论本实验建立了稳定获得CcFnt166AS重组蛋白质的方法。
[Abstract]:Objective to express a high soluble protein of foot-and-mouth disease virus (FMDV) 1D and to form nanoparticles by electron microscopy. Methods according to the nucleic acid sequence of FMDV type A foot-and-mouth disease virus, the 1D protein gene of FMDV A / GDMMP / che / 2013 strain was obtained and truncated and optimized for a total of 132 amino acids, and 135 amino acid ferritin (FN) gene fragments were isolated from Campylobacter coli. The 1D protein-ferritin fragment of foot-and-mouth disease virus (FMDV) was designed and synthesized by linking 1D protein with ferritin, named CcFnt166AS. CcFnt166AS fusion recombinant vector containing nine different soluble tags, such as Grifing GST-MBP, Sumo Thioredoxin, 纬 -crystallin Ars PpiBU CeHSP17, was constructed. After transformed into Escherichia coli BL21 (DE3), isopropyl thiogalactoside (IPTG) induced SDSPAGE electrophoresis was used to detect the soluble expression of the fusion protein, and a highly soluble CcFnt166AS fusion protein was selected. The recombinant protein was purified by NiNTA Agarose affinity and detected by electron microscopy. Results 9 CcFnt166AS expression vectors were successfully constructed, and the fusion of MBP with CcFnt166AS protein was the best, and the recombinant protein of MBP-CcFnt166AS with high purity was obtained, and the results of electron microscopy showed that MBP-CcFnt166AS formed nanoparticles. Conclusion A stable method for obtaining CcFnt166AS recombinant protein was established.
【作者单位】：河南农业大学农业部动物生化与营养重点开放实验室;
【基金】：农业部948重点计划(2011-G35) 国家转基因重大专项(2014ZX0801015B) 河南省高等学校重点科研项目(17A230013)
【分类号】：S852.65

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