O型口蹄疫病毒VP1蛋白在大肠埃希菌中的可溶性表达、纯化及纳米样结构观察

doi:10.16098/j.issn.0529-1356.2018.01.020

摘要/Abstract

摘要：

目的本实验旨在高效表达可溶性的O型口蹄疫病毒VP1蛋白，并形成纳米样颗粒。方法根据O型口蹄疫病毒核酸序列，得到FMDV O/MYA/7/98株的VP1蛋白基因，并进行截短和优化，共73个氨基酸；同时，从肠道沙门菌(Salmonella enterica）中分离得到135个氨基酸铁蛋白(Fn)基因片段，将O型口蹄疫病毒VP1蛋白与铁蛋白串联，设计并合成了口蹄疫病毒VP1蛋白-铁蛋白基因片段，命名为SeFnt16798。构建了SeFnt16798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，SDS-PAGE电泳对融合蛋白的可溶性表达进行检测，筛选高效可溶性表达的SeFnt16798融合蛋白。重组蛋白通过Ni-NTA Agarose亲和纯化，进行电子显微镜检测。结果实验成功构建9个SeFnt16798表达载体；9个标签中，MBP与SeFnt16798蛋白相融合的可溶性表达效果最好，并获得了高纯度的MBP-SeFnt16798重组蛋白质；电子显微镜结果显示，MBP-SeFnt16798形成了纳米样颗粒。结论本实验建立了稳定获得SeFnt16798重组蛋白质的方法。

关键词: O型口蹄疫病毒, VP1蛋白, 铁蛋白, 融合标签；纳米颗粒, 电子显微镜, 大肠埃希菌

Abstract:

Objective To establish a method to express the soluble VP1 protein from foot-and-mouth disease virus type O in Escherichia coli, (E. coli) and to observe its nanoparticles. Methods VP1 gene from FMDV O/MYA/7/98 strain was obtained according to the sequence of O-type foot-and-mouth disease virus. Truncation and optimization were performed. The fusion tages were screened to obtain soluble expression of SeFnt16798 protein. The 73 aa ferritin protein was isolated from Salmonella enteric and linked with the VP1 protein, which was named SeFnt16798. Prokaryotic expression vectors were fused with nine different fusion tages(Grifin, GST, MBP, Sumo, Thioredoxin, γ-crystallin, ArsC, PpiB and CeHSP17)fragments were constructed. These recombinant plasmids were transformed into Escherichia coli BL21 (DE3) and induced by isopropyl β-D-thiogalactiside(IPTG). SDS-PAGE analysis was used to identify the soluble expression of recombinant protein after ultrasonic decomposition. The fusion tag that could significantly promote soluble expression of SeFnt16798 protein was selected. Abundantly inducible expression recombinant protein was purified by Ni-NTA purification system and detected by electron microscopy. Results Recombinant expression of SeFnt16798 was successfully constructed and had the high purity MBP-SeFnt16798. The result of electron microscopy showed that MBP-SeFnt16798 formed nano-sized particles. Conclusion We established a stable method to obtain recombinant protein of SeFnt16798, which may lay a foundation for the development and subsequent study of FMDV structure vaccine.

Key words: O-type foot and mouth disease virus, VP1 protein, Ferritin, Fusion tag, Nanoparticle, Electron rnicroscopy, Escherichia coli

郭玉堃明胜利郭婉莹杨国宇郭豫杰. O型口蹄疫病毒VP1蛋白在大肠埃希菌中的可溶性表达、纯化及纳米样结构观察[J]. 解剖学报, 2018, 49(1): 118-123.

GUO Yu-kun MING Sheng-li GUO Wan-ying YANG Guo-yu GUO Yu-jie. Soluble expression and purification of VP1 protein from O-type foot-and-mouth disease virus in Escherichia coli and its nanometer structure observation[J]. Acta Anatomica Sinica, 2018, 49(1): 118-123.

参考文献

［1］Mahy BW. Introduction and history of foot-and-mouth disease virus［J］. Curr Top Microbiol Immunol, 2005,288:1-8.

［2］Domingo E, Baranowski E, Escarmís C, et al. Foot-and-mouth disease virus［J］. Comp Immunol Microbiol Infect Dise, 2002, 25(5-6):297308.

［3］Deng GM. Current situation of foot and mouth disease and prevention and control points ［J］. Veterinary Orientation, 2013,(8):9-11. (in Chinese)

邓光明. 口蹄疫的流行态势和防控要点［J］. 兽医导刊,2013,(8):9-11.

［4］Grubman MJ, Baxt B. Foot-and-mouth disease［J］. Clin Microbiol Rev, 2004, 17(2):465-493.

［5］Mason PW, Grubman MJ, Baxt B. Molecular basis of pathogenesis of FMDV［J］. Virus Res, 2003, 91(1):9-32.

［6］Nel AE, Mdler L, Velegol D, et al. Understanding biophysicochemical interactions at the nanobio interface［J］. Nat Mater, 2009, 8(7):543-557.

［7］Zhu Y, Cai X, Li J, et al. Synchrotron-based X-ray microscopic studies for bioeffects of nanomaterials［J］. Nanomedicine, 2014, 10(3):515-524.

［8］Zhu Y, Li W, Zhang Y, et al. Excessive sodium ions delivered into cells by nanodiamonds: implications for tumor therapy［J］. Small, 2012, 8(11):1771-1779.

［9］Li J, Zhu Y, Li W, et al. Nanodiamonds as intracellular transporters of chemotherapeutic drug［J］. Biomaterials, 2010, 31(32):8410-8418.

［10］Lynch I, Salvati A, Dawson KA. Protein-nanoparticle interactions: What does the cell see［J］? Nat Nanotechnol, 2009, 4(9):546-547.

［11］Ahmed H, Vasta GR. Unlike mammalian GRIFIN, the zebrafish homologue (DrGRIFIN) represents a functional carbohydrate-binding galectin［J］. Biochem Biophys Res Commun, 2008, 371(3):350-355.

［12］Huang XB, Kang QZh, Fu G, et al. Expression of soluble GST-NAP fusion protein and GST-Tag-cleavage on column ［J］. Journal of Zhengzhou University(Natural Science Edition), 2015, (4): 94-98. (in Chinese)

黄夏冰, 康巧珍, 傅国,等. GST-NAP融合蛋白可溶性表达及柱上切割GST标签［J］. 郑州大学学报(理学版), 2015(4):94-98.

［13］Su N, Wu JJ, Li Y, et al. Construction of MBP fusion heparinase Ⅲ efficient expression system in E.coli［J］. Journal of Chemical Industry and Engineering, 2014, 65(7): 2829-2842. (in Chinese)

苏楠, 吴敬君, 李晔,等. MBP融合肝素酶Ⅲ大肠杆菌高效表达体系构建［J］. 化工学报, 2014, 65(7):2829-2842.

［14］Tang YL, Mi ZL, Zhong HQ, et al. Expressing the soluble AtRD22 protein of Arabidopsis thaliana in Escherichia coli using SUMO technology ［J］. Journal of Shenzhen University(Science & Engineering), 2015, 32(6): 610-616. (in Chinese)

唐玉林, 米子岚, 钟活权,等. 利用SUMO技术表达可溶性的拟南芥AtRD22蛋白［J］. 深圳大学学报(理工版), 2015, 32(6):610-616.

［15］Yang XH, Liu HG, Liu X, et al. Thioredoxin and impaired spatial learning and memory in the rats exposed to intermittent hypoxia［J］. Chin Med J(Engl), 2012, 125(17):3074-3080.［16］Mohr BG, Dobson CM, Garman SC, et al. Electrostatic origin of in vitro aggregation of human γ-crystallin［J］. J Che Phys, 2013, 139(12):121914.

［17］El Sayed NAM, Zayed HH, Sharawy MI. ARSC: Augmented reality student cardan augmented reality solution for the education field［J］. Computers & Education, 2011, 56(4):1045-1061.

［18］Steelman SM, Jackson ND, Conant E. Ehlers-danlos syndrome in a quarter horse gelding: a case report of PPIB-independent hereditary equine regional dermal asthenia［J］. J Equine Vet Sci, 2014, 34(4):565-568.

［19］Ezemaduka AN, Yu J, Shi X, et al. A small heat shock protein enables escherichia coli to grow at a lethal temperature of 50℃ conceivably by maintaining cell envelope integrity［J］. J Bacteriol, 2014, 196(11):2004-2011.

［20］Gao P, Li ZhY, Liu JSh. Cloning of whole ORF coding genes of FMDV and construction of recombinant adenoviral vector［J］.Current Biotechnology, 2014, 4(2):107-112. (in Chinese)

高鹏, 李志勇, 柳纪省. FMDV全ORF编码基因的克隆及其腺病毒载体的构建［J］. 生物技术进展, 2014, 4(2):107-112.

［21］Liu JX, Chen ChF,Qiao J，et al. Construction and identification of recombinant vaccinia virus of O-type foot-and-mouth disease virus vp1 gene［J］.Heilongjiang Animal Science and Veterinary Medicine, 2011,(1):96-98. (in Chinese)

刘佳旭,陈创夫,乔军,等. O型口蹄疫病毒vp1基因重组痘苗病毒的构建与鉴定［J］. 黑龙江畜牧兽医,2011,(1):96-98.

［22］Xie YL, Ma Q, Li ZhY.Expression and identification of capsid protein of foot-and-mouth disease virus type O in insect cells［J］. Journal of Anhui Agricultural Sciences, 2015(8):95-97. (in Chinese)

解银丽, 马琪, 李志勇. O型口蹄疫病毒衣壳蛋白在昆虫细胞中的表达及其抗原性鉴定［J］. 安徽农业科学, 2015(8):95-97.

［23］Jiao Y, Gong JF, He XP, Expression of FMDV VP1 gene by pichia pastoris and the assessment of its immunogenicity ［J］. Progress in Veterinary Medicine, 2010, 31(2):73-77. (in Chinese)

焦颖, 龚劲峰, 何校澎,等. O型口蹄疫病毒VP1基因在毕赤酵母中的表达及免疫原性分析［J］. 动物医学进展, 2010, 31(2):73-77.

［24］Zhang YL, Guo YJ, Wang KY, et al. Enhanced immunogenicity of modified hepatitis B virus core particle fused with multiepitopes of foot-and-mouth disease virus.［J］. Scand J Immunol, 2007, 65(4):320-328.

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