【摘要】 为制备禽坦布苏病毒囊膜E蛋白截短蛋白的单克隆抗体并分析其抗原性,以坦布苏病毒YY1分离株的cDNA为模板,通过RT-PCR扩增编码截短E蛋白的基因,并将其成功克隆到原核表达载体pET-28a(+)中。重组载体pET-28a(+)-YY1E转化的大肠杆菌BL21(pLysS)在IPTG诱导下高效表达重组蛋白His-YY1E,该蛋白可与坦布苏病毒感染鸭的血清反应。用纯化的His-YY1E免疫小鼠制备单克隆抗体,采用有限稀释法经过3轮筛选和鉴定最终获得了2株分泌抗E蛋白的单克隆抗体细胞株1C5和3B11。制备的单克隆抗体与原核表达和病毒感染细胞中的E蛋白均能反应。进一步通过转染截短蛋白E不同区域截短体与pEGFP-C3构建的重组载体,并经IFA检测,发现单克隆抗体1C5和3B11识别的截短E抗原区域位于C端35个氨基酸(105个碱基)之间。本研究为进一步研究坦布苏病毒E蛋白的功能,研制坦布苏病毒病亚单位疫苗以及建立快速特异的坦布苏病毒检测方法奠定了基础。更多还原

【Abstract】 In order to obtain the gene which encodes truncated envelope protein E of Tembusu virus(TMUV), TMUV truncated E gene was amplified by RT-PCR using cDNA of TMUV YY1 isolate as template. The truncated E gene was then cloned into prokaryotic expression vector pET-28a(+) and the recombinant plasmid p ET-28a(+)-YY1 E was transformed into Escherichia coli BL21(p Lys S). The efficient expression of recombinant His-YY1 E was observed with IPTG induction, which showed positive reaction with serum from a TMUV infected duck. For preparation of monoclonal antibody(Mc Ab)against truncated E, BALB/c mice were immunized with purified recombinant His-YY1 E protein. After three times limited dilutions,two hybridoma cell strains(1C5 and 3B11) which secreted antibodies reacting with E were obtained. The McAbs could react with both recombinant protein by Western-blot analysis and native E protein expressed in TMUV-infected DF-1 cells and truncated-E transfected cells by IFA. Further IFA analysis of the reaction of McAbs with the truncated E transfected cells indicated that McAbs 1C5 and 3B11 recognized the antigenic domain located in 35 aa of the N terminal of the truncated E.This study providesa basis for further function studies of the TMUV E protein, for development of subunit vaccines against TMUV infection and establishment of rapid detection methods for TMUV.更多还原