蛋白纳米疫苗VP6-Ferritin高效免疫的结构基础研究
摘要
蛋白和核酸即是生物体的组成部分，也是生理功能的重要执行者，而其功能的执行往往与其结构密切相关，因此通过解析蛋白质的结构将有助于增强我们对于其功能的认识，理解其参与生命过程的意义。轮状病毒是引起婴幼儿腹泻的病毒之一，本实验室之前的研究结果显示病毒VP6蛋白与Ferritin蛋白融合成的蛋白纳米疫苗VP6-Ferritin，灌胃小鼠后可产生较高的Ig A和Ig G抗体水平，攻毒试验显示蛋白纳米疫苗对试验小鼠具有显著的保护力。本研究拟解析蛋白纳米疫苗VP6-Ferritin结构，试验取得如下研究，现结果如下：bridegroom
1. 应用基因定向克隆技术成功构建了VP6-Ferritin的原核表达载体pET 28a-Hrv-VP6-Ferritin，对该载体进行原核表达条件优化显示在37 ℃条件下，0.05mmol/L的IPTG诱导6 h左右达到最大值，使用SDS-PAGE电泳在60 kDa-70 kDa之间有一条明显的特异性蛋白条带与预期符合。在包涵体组分和总蛋白组分检测到VP6-Ferritin蛋白的条带，说明VP6-Ferritin蛋白在细菌中主要以包涵体的形式表达。
2. 对VP6-Ferritin在变性条件下纯化，当洗脱缓冲液的比例提高至30 %时，获得了大量高纯度的VP6-Ferritin蛋白。通过梯度柱上复性对VP6-Ferritin进行重折叠，使用Superdex 200 pg对蛋白进行均一性检测，成功获
I
得了VP6-Ferritin活性蛋白。透射电子显微镜观察复性后的蛋白质样品，发现对照组重组蛋白Ferritin在体外自组装为均匀的球状中空纳米颗粒，且纳米颗粒外表光滑，而VP6-Ferritin蛋白则呈现为球状实心纳米颗粒，且纳米颗粒外表粗糙，二者粒径相似，约为12-20 nm。应用Expasy对VP6-Ferritin 的单体分子量进行预测，预测分子量为64.225 kDa，加上位于VP6-Ferritin N端的6 His标签和HRV-3c元件的2588.65 Da，所预测的理论分子量为66.81 kDa，而经HPLC-MS鉴定得VP6-Ferritin的精确单体蛋白分子量为66.911 kDa，而VP6-Ferritin的分子筛显示VP6-Ferritin复性后是一个高分子量多聚体蛋白。应用原子力显微镜和冷冻电镜下进一步对蛋白的形态与粒径进行了观察，显示相对于对照蛋白Ferritin，VP6-Ferritin失去了Ferritin 之前棱角分明的结构，但依旧显示出是一个20 nm左右球形纳米颗粒结构；动态光散射显示蛋白会随着pH或解聚或自组装，蛋白在生理条件下粒径表现为24 nm，虽然比Ferritin粒径更大，但VP6-Ferritin同样显出了较好自组装能力。nation
3. 对蛋白进行了结晶，使用超滤浓缩管对VP6-Ferritin蛋白进行浓缩，将其浓缩至最大浓度2 mg/mL，滴加到结晶母液之上，在晶体培养的第12天时，在PEG结晶条件下初步观察到一个规整的晶体结构。
结论：本研究成功表达了蛋白纳米疫苗VP6-Ferriitn，发现其为球形纳米颗粒，拥有着很好的自组装能力，可以在特定条件下解聚和自组装。本研究工作为研究蛋白纳米疫苗的作用机制奠定了基础。
30 conds to mars
爱在招生部关键词：纳米疫苗VP6-Ferritin 蛋白表达纯化结构分析
bug什么意思中文STRUCTURAL BASIS OF HIGHLYbetsy
vastIMMUNOGENIC RESPONSE OF
insurancepolicy
PROTEIN-BASED NANOV ACCINE
VP6-FERRITIN
ABSTRACT
Protein and nucleric acids are parts of components, and also are one of the important executors of physiological functions. Therefore, analyzing the structure of proteins will help to enhance our understanding of their functions and roles in the process of life. Rotavirus is one of cau diarrhea in infants. Previous studies in this laboratory showed viral VP6 protein and Ferritin protein could fu into a new protein nano-vaccine VP6-Ferritin, this protein vaccine could produce a higher levels of Ig A and Ig G antibody levels in mice after oral. The virus challenge test showed that protein nano-vaccine have a significant protective effect on experimental mice. This study intends to analyze the structure of VP6-Ferritin, here are the results:
1.The prokaryotic expression vector pET 28a-Hrv-VP6-Ferritin ofcorncob
VP6-Ferritin was successfully constructed by using gene clone technology, the optimization of prokaryotic expression conditions of this vector showed that protein expressions reached the maximum value after 6 h in 37 ℃ and 0.05mmol/L IPTG.And SDS-PAGE electrophoresis shows that as expected there was an obvious specific protein band between 60 kDa-70 kDa. VP6-Ferritin was detected in full protein and inclusion body, it means that the mainly expression forms of VP6-Ferritin is inclusion body.be happy
2. VP6-Ferritin was purified under denaturation condition. When the proportion of elution buffer was incread to 30%, a large amount of high-purity VP6-Ferritin protein was obtained. VP6-Ferritin was refolded in desalting column by gradient refolding. Superdex 200 pg was ud to detect the homogeneity of the protein, and VP6-Ferritin active protein was successfully obtained. TEM was ud to detected the refold protein, the results shows that control group protein Ferritin could lf-asmble into uniform spherical hollow nanoparticles in vitro, and nanoparticles had a smooth appearance, while VP6-Ferritin protein was spherical solid nanoparticles with rough appearance, the particles size of Ferritin and VP6-Ferritin are similar, about 12-20 nm. Expasy was ud to predict MW of VP6-Ferritin monomer, and the predicted MW was 64.225 kDa, 6 His tag and HRV-3c elemen
t at the N end of VP6-Ferritin was 2588.65 Da, the theoretical MW was 66.81 kDa,
while HPLC-MS shows that the MW of VP6-Ferritin was 66.911 kDa; SEC results shows that VP6-Ferritin is a high MW polymer protein after refold; AFM and Cryo-EM was ud to detected the morphology and particle size of the protein, it shows that although VP6-Ferritin lost the angular structure compared to the control group Ferritin, it still shows that it is a spherical nanoparticle structure abount 20 nm; DLS reveals that protein will depolymerize or lf-asmble with pH, the particle size of VP6-Ferritin under physiological conditions is 24 nm. Although it is larger than Ferritin, VP6-Ferritin also shows a great lf-asmbly ability.
3. The protein was crystallized. VP6-Ferritin protein was concentrated by using ultrafiltration concentration tube,the maximum concentration of VP6-Ferritin is 2 mg/mL, then it was added to the crystal stock solution. On the 12th day of crystal culture, a regular crystal structure was obrved under PEG condition.
Conclusion: In this study, the protein nano-vaccine VP6-Ferriitn was successfully expresd, and it was found to be spherical nanoparticles with good lf-asmbly ability, which can be depolymerized and lf-asmbled under specific conditions. This paper lays a foundation for the study of the mechanism of action of protein nano vaccine.
