O型口蹄疫病毒衣壳蛋白在大肠杆菌中的可溶性表达、纯化及电镜检测

Soluble expression,purification and electron microscopy of capsid protein from Otype foot and mouth disease virus in Escherichia coli

本试验旨在表达出高可溶性的O型口蹄疫病毒（FMDV）衣壳蛋白，并通过电镜检测，以期望形成纳米样颗粒。根据O型FMDV核酸序列，得到FMDVO／GER／Wup／82株的衣壳蛋白基因，并进行截短和优化，共133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（Ferritin）基因片段，将O型FMDV衣壳蛋白与铁蛋白串联，设计并合成了FMDV衣壳蛋白-铁蛋白基因片段，命名为SeFntl6599。构建了SeFnt16599融合Grifin、GST、MBP、Sumo、Thioredoxin、7-crystallin、ArsC、PpiB、CeHSP17等9种不同可溶性标签的表达重组载体。分别转化至大肠杆菌BL21（DE3）中，IPTG诱导，SDS-PAGE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SeFnt16599融合蛋白。重组蛋白通过Ni—NTA Agarose亲和纯化，进行电镜检测。结果表明，成功构建9种SeFnt16599表达载体；9个标签中，MBP与SeFnt16599蛋白相融合的可溶性表达效果最好，并获得了高纯度的MBP—SeFnt16599重组蛋白质；电镜结果显示，MBP—SeFnt16599形成了纳米样颗粒。本试验建立了稳定获得SeFnt16599重组蛋白质的试验方法，为FMDV衣壳蛋白新型结构疫苗的开发及后续研究奠定了基础。

The objective of this test is to express the high soluble capsid protein from foot-and- mouth disease virus type O in Escherichia coli ,and be detected by electron microscopy, expecting to form nanoparticles. Based on the nucleic acid sequences of the foot-and-mouth disease virus type O,got the capsid protein genes of FMDV O/GER/Wup/82 strain,based on optimization design of truncation were finally shortened to 133 aa. The 135 aa ferritin protion was isolated from Salmo- nella enterica and connected with the capsid protein of foot-and-mouth disease virus type O, then named SeFnt16599. We constructed prokaryotic expression vectors fused with nine different fusion tags （Grifin, GST, MBP, Sumo, Thioredoxin, 7-crystallin, ArsC, PpiB, CeHSP17 ） fragments, these recombinant plasmids were transformed into E. coli BL21, respectively （DE3） and induced by IPTG. SDS-PAGE analysis was used to identify the soluble expression of recombinant protein after ultrasonic decomposition. The fusion tag was selected based on promoting soluble expression of SeFnt16599 protein. Moreover, abundantly inducible expression recombinant protein was purified by Ni-NTA purification system, detected by electron microscopy. The results showed that we suc- Cessfully constructed the recombinant prokaryotic expression plasmid and got the high purity MBP-SeFnt16599 ;the result of electron microscopy showed that MBP-SeFnt16599 formed nano sized particles. These results suggested that we established a method to generate SeFnt16599 re protein,which will lay a foundation for the development and subsequent study of FM DV structure vaccine.