Tan Lik Chern Melvin, Chua Anthony Jin Shun, Goh Li Shan Liza, Pua Shu Min, Cheong Yuen Kuen, Ng Mah Lee
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
Protein Expr Purif. 2010 Nov;74(1):129-37. doi: 10.1016/j.pep.2010.06.015. Epub 2010 Jul 1.
Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus.
节肢动物传播的黄病毒，如登革病毒（DENV）和西尼罗河病毒（WNV），对全球社区构成重大健康威胁。由于全球范围内DENV和WNV感染数量不断增加，开发有效的疫苗仍然是全球卫生的优先事项。由于黄病毒包膜结构域III（DIII）蛋白具有高度免疫原性，能够诱导针对野生型病毒的中和抗体，它既是一种潜在的蛋白亚单位疫苗候选物，也是一种合适的诊断试剂。在此，我们描述了使用金属亲和膜色谱法作为一种快速且改进的替代方法，用于从DENV血清型1-4以及WNV的纽约株、萨拉芬德株、温格勒株和库京株中纯化重组DIII（rDIII）抗原。确定了这些蛋白表达、溶解、复性和纯化的最佳条件。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和使用针对相应病毒产生的抗体进行的酶联免疫吸附测定（ELISA）对纯化的蛋白进行了确认。纯化的rDIII蛋白的生物学功能通过其在小鼠体内产生能够中和病毒的DIII特异性抗体的能力得到证实。
