禽坦布苏病毒截短E蛋白单克隆抗体的制备及其抗原性分析

发布时间：2018-01-13 13:41

本文关键词：禽坦布苏病毒截短E蛋白单克隆抗体的制备及其抗原性分析　出处：《中国兽医科学》2016年07期 　论文类型：期刊论文

【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。
[Abstract]:In order to prepare monoclonal antibody against E protein truncated protein and analyze its antigenicity, the cDNA of Tambusuvirus YY1 isolate was used as template. The gene encoding truncated E protein was amplified by RT-PCR. It was successfully cloned into prokaryotic expression vector pET-28a (). The recombinant vector pET-28a (pET-28a) was transformed into E. coli BL21pLysS. The recombinant protein His-YY1E was highly expressed under the induction of IPTG. The protein can react with the serum of tambusuvirus infected duck and immunize mice with purified His-YY1E to prepare monoclonal antibody. After three rounds of screening and identification by limited dilution method, two monoclonal antibody cell lines 1C5 and 3B1111 secreting anti-E protein were obtained. The prepared monoclonal antibodies were associated with prokaryotic expression and E. The recombinant vector was constructed by transfection of truncated bodies in different regions of truncated protein E and pEGFP-C3. And detected by IFA. The truncated E antigen region recognized by monoclonal antibodies 1C5 and 3B11 was found to be between 35 amino acids (105 bases) at the C-terminal. The preparation of TBV subunit vaccine and the establishment of a rapid and specific detection method for TBV have laid a foundation.
【作者单位】：浙江大学农业部动物病毒学重点实验室;
【基金】：国家科技支撑计划项目(2015BAD12B01)
【分类号】：S852.65
【正文快照】： 坦布苏病毒(Tem busu virus,TM U V)是禽坦布苏病毒病的病原。2010年4月,在我国南方养鸭业比较密集的福建、江浙一带首先暴发了一种以蛋鸭产蛋急剧下降为主要临床特征、出血性卵巢炎为主要病理变化特征的传染病[1]。该病传播快、发病率高,给我国沿海地区养鸭业带来了严重的经

【相似文献】