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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。
In order to prepare and analyze the antigenicity of the E protein truncated protein of tebufenozoviruses, the cDNA encoding the truncated E protein was amplified by RT-PCR using the cDNA of Tambuccino virus YY1 as a template , And successfully cloned into prokaryotic expression vector pET-28a (+). The recombinant vector pET-28a (+) - YY1E transformed E. coli BL21 (pLysS) highly expressed the recombinant protein His-YY1E under the induction of IPTG and reacted with the serum of duck infected with Tambusu virus. Monoclonal antibodies were prepared by immunizing mice with purified His-YY1E. After three rounds of screening and identification by limiting dilution, two McAbs 1C5 and 3B11 secreting anti-E protein were obtained. The prepared monoclonal antibody reacted with both prokaryotic and E protein in virus-infected cells. Further, the truncated E antigen region recognized by the monoclonal antibodies 1C5 and 3B11 was found to be 35 amino acids (105) at the C-terminal by transfecting truncated truncated E. coli pEGFP-C3 recombinant vectors with different regions of truncated protein E Base). This study lays the foundation for the further study of the function of Tambussu virus E protein, the development of subunit vaccine against Tambusu virus disease and the establishment of a rapid and specific Tambussu virus detection method.