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

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Se Fnt16798。构建了Se Fnt16798融合Grifin、GST、MBP、Sumo、Thioredoxin、γ-crystallin、Ars C、Ppi B、Ce HSP17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Se Fnt16798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Se Fnt16798表达载体;9个标签中,MBP与Se Fnt16798蛋白相融合的可溶性表达效果最好,并获得了高纯度的MBP-Se Fnt16798重组蛋白质;电子显微镜结果显示,MBP-Se Fnt16798形成了纳米样颗粒。结论本实验建立了稳定获得Se Fnt16798重组蛋白质的方法。

Soluble expression and purification of VP1 protein from O-type foot-and-mouth disease virus in Escherichia coli and its nanometer structure observation

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.