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Non-structural protein 1 from Japanese encephalitis virus expressed in E. coli retains its molecular weight and immunogenicity.
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2019-11-29 , DOI: 10.1016/j.pep.2019.105548
Jae-Won Choi 1 , Hyo-Ji Eom 2 , Hak Yong Kim 1
Affiliation  

Japanese encephalitis virus (JEV) is a member of the Flavivirus genus and has recently attracted attention as a high-risk pathogen in the Asia-Pacific region, with up to 30% mortality in the afflicted patients. Recent outbreaks of flavivirus-associated infections around the world have put the focus on non-structural protein 1 (NS1) as a candidate for diagnostic and vaccine researches on flaviviruses. Although the JEV NS1 protein has been expressed in eukaryotic cells, attempts to express JEV NS1 in E. coli are on due to advantages such as rapid growth, easy manipulation, low cost, and high yield. However, the challenges of low yield and poor solubility of the proteins expressed in E. coli remain to be overcome. Herein, we reported successful expression of the JEV NS1 protein in E. coli Rosetta(DE3) strain. We standardized the temperature, induction time, as well as the concentration of the inducer for optimizing the expression of JEV NS1 in E. coli. Further, we successfully obtained soluble JEV NS1 from inclusion bodies by partial refolding during elution and gradual refolding during dialysis. Furthermore, the JEV NS1 protein was found to retain its molecular weight and was able to induce an immune response in the mouse. Western blot and indirect enzyme-linked immunosorbent assay were performed using the blood of the immunized mouse and purified JEV NS1 in this study. Hence, JEV NS1 expressed in and isolated from E. coli Rosetta(DE3) strain holds potential for application in vaccine development and diagnostic studies to combat Japanese encephalitis outbreaks in the future.

中文翻译:

在大肠杆菌中表达的日本脑炎病毒的非结构蛋白1保留了其分子量和免疫原性。

日本脑炎病毒(JEV)是黄病毒属的成员,最近作为亚太地区的高危病原体引起了人们的关注,患病患者的死亡率高达30%。全球范围内与黄病毒相关感染的近期爆发已将非结构蛋白1(NS1)用作黄病毒诊断和疫苗研究的候选对象。尽管JEV NS1蛋白已经在真核细胞中表达,但是由于诸如快速生长,易于操作,低成本和高产量的优点,尝试在大肠杆菌中表达JEV NS1。然而,在大肠杆菌中表达的蛋白质的低产量和差的溶解性的挑战仍然有待克服。在本文中,我们报道了JEV NS1蛋白在大肠杆菌Rosetta(DE3)菌株中的成功表达。我们标准化了温度,诱导时间,以及用于优化JEV NS1在大肠杆菌中表达的诱导剂浓度。此外,通过洗脱过程中的部分重折叠和透析过程中的逐步重折叠,我们成功地从包涵体中获得了可溶性JEV NS1。此外,发现JEV NS1蛋白保留其分子量,并能够在小鼠中诱导免疫反应。在这项研究中,使用免疫小鼠的血液和纯化的JEV NS1进行了蛋白质印迹和间接酶联免疫吸附测定。因此,在大肠杆菌Rosetta(DE3)菌株中表达并从中分离的JEV NS1有望在疫苗开发和诊断研究中应用,以对抗未来的日本脑炎暴发。此外,通过洗脱过程中的部分重折叠和透析过程中的逐步重折叠,我们成功地从包涵体中获得了可溶性JEV NS1。此外,发现JEV NS1蛋白保留其分子量,并能够在小鼠中诱导免疫反应。在这项研究中,使用免疫小鼠的血液和纯化的JEV NS1进行了蛋白质印迹和间接酶联免疫吸附测定。因此,在大肠杆菌Rosetta(DE3)菌株中表达并从中分离的JEV NS1有望在疫苗开发和诊断研究中应用,以对抗未来的日本脑炎暴发。此外,通过洗脱过程中的部分重折叠和透析过程中的逐步重折叠,我们成功地从包涵体中获得了可溶性JEV NS1。此外,发现JEV NS1蛋白保留其分子量,并能够在小鼠中诱导免疫反应。在这项研究中,使用免疫小鼠的血液和纯化的JEV NS1进行了蛋白质印迹和间接酶联免疫吸附测定。因此,在大肠杆菌Rosetta(DE3)菌株中表达并从中分离的JEV NS1有望在疫苗开发和诊断研究中应用,以对抗未来的日本脑炎暴发。JEV NS1蛋白被发现保留其分子量,并能够在小鼠中诱导免疫反应。在这项研究中,使用免疫小鼠的血液和纯化的JEV NS1进行了蛋白质印迹和间接酶联免疫吸附测定。因此,在大肠杆菌Rosetta(DE3)菌株中表达并从中分离的JEV NS1有望在疫苗开发和诊断研究中应用,以对抗未来的日本脑炎暴发。JEV NS1蛋白被发现保留其分子量,并能够在小鼠中诱导免疫反应。在这项研究中,使用免疫小鼠的血液和纯化的JEV NS1进行了蛋白质印迹和间接酶联免疫吸附测定。因此,在大肠杆菌Rosetta(DE3)菌株中表达并从中分离的JEV NS1有望在疫苗开发和诊断研究中应用,以对抗未来的日本脑炎暴发。
更新日期:2019-11-30
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