登革2型病毒E蛋白结构域III的表达及多克隆抗体制备

登革病毒(Dengue virus,DENV)属于黄病毒科(Flaviviridae),黄病毒属(Flavivirus),为单股正链RNA病毒,有4个不同的血清型(DENV-1,2,3,4),主要通过埃及伊蚊(Aedes aegypti)和白纹伊蚊(Aedes albopictus)传播,可引起登革热、登革出血热、登革休克综合征等多种疾病1,2]。E蛋白是位于DENV表面的结构蛋白,由495个氨基酸组成,它既含有黄病毒亚群特异的和登革病毒血清型特异的抗原表位,又有与中和,血凝抑制作用有关的抗原表位,是病毒颗粒的主要包膜蛋白3]。Modis等研究表明,DENV-2型E蛋白以延伸的二聚体形式平铺在病毒表面,折叠成3个不…

Expression of Domain III of the DENV-2 E Protein and Preparation of Anti-His-D2EIII Polyclonal Antibody

The gene fragment coding for amino acids 281 to 395 of the E protein of DENV-2 (New Guinea C strain) was amplified by PCR, comprising Domain III (amino acids 295 to 395) of the E protein. The fragment was cloned into pMD18-T vector and subcloned to expression vector pET-28a and pMAL-c2X. The recombinant plasmid pET-28a-D2EIII was transformed into E.coli BL21(DE3) and the pMAL-c2X-D2EIII was transformed into E.coli TB1. The induced recombinant proteins were purified by His-tag and MBP-tag affinity chromatography, respectively. The purified protein His-D2EIII was used to immunize rabbit three times at two-week intervals, the immunized rabbit produced high titer anti-His-D2EIII polyclonal antibody. The result of western blot indicated that the expressed fusion protein could react with the polyclonal antibody against Domain III of E protein.