传染性喉气管炎病毒gD蛋白多克隆抗体制备与运用  

Preparation of polyclonal antibodies against gD protein of ILTV and application

作　　者：张晓彩[1] 赵妍[1] 张雪梅[1] 刘胜旺[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽呼吸道病创新团队,哈尔滨150001

出　　处：《东北农业大学学报》2016年第5期69-75,97,共8页Journal of Northeast Agricultural University

基　　金：农业部蛋鸡科技创新体系项目(CARS-41-K12)

摘　　要：鸡传染性喉气管炎（Infectious laryngotracheitis,ILT）是由传染性喉气管炎病毒（Infectious laryngotracheitis virus,ILTV）引起的一种急性接触性上部呼吸道病。ILTV表面糖蛋白g D能刺激机体产生良好体液和细胞免疫应答,编码该蛋白的g D基因是ILTV主要抗原性基因之一。以ILTV-LJS09株基因组为模板,PCR扩增g D基因主要抗原区域（54~344 aa）及g D基因,分别亚克隆至p GEX-6P-1和p CAGGS载体,构建原核表达载体p GEX-6P-1-g D163/1032和真核表达载体p CAGGS-g D。利用纯化ILTV、原核表达纯化获得重组蛋白r-g D和真核质粒p CAGGS-g D,对新西兰大白兔进行免疫与加强免疫,制备兔抗ILTV g D多克隆抗体。通过间接免疫荧光试验确定ILTV g D多克隆抗体和ILTV感染的鸡肝癌细胞表达蛋白发生反应,表明制备的ILTV g D多克隆抗体能识别ILTV天然抗原表位,最佳稀释度为11 000;用ILTV感染的LMH细胞培养物进行Western Blot分析,在40 ku左右出现一条特异性条带,与预期ILTV g D蛋白大小相符。激光共聚焦定位感染鸡肝癌细胞中ILTV g D蛋白,发现ILTV g D蛋白主要表达于感染细胞胞浆及融合细胞交界处。Infectious Laryngotracheitis（ILT） is a highly contagious respiratory system disease caused by Infectious Laryngotracheitis Virus（ILTV）. The ILTV g D gene was one of the main antigenic genes of ILTV, which can stimulate humoral immunity and cellular immune response in vivo. The major antigenic region of g D gene（54-344 aa） and the completed g D gene were amplified from ILTV-LJS09.The two fragments were subsequently inserted into p GEX- 6P-1 and p CAGGS, respectively. The two expression vector were named p GEX-6P-1-g D163/1032 and p CAGGS-g D. The purfied ILTV, recombinant protein r-g D, and p CAGGS-g D plasmid were used as antigen to prepare polyclonal antibody. The immunofluorescence assay showed that ILTV g D polyclonal antibody could react with ILTV infected LMH cell cultures and demonstrated that ILTV g D polyclonal antibody could identify natural antigen epitope of ILTV. The best dilution was 11 000 determined by immunofluorescence assay. Western Blot result revealed a specific band approximately 40 ku, which was consistent with ILTV g D. The result showed that the polyclonal antibody has specificity. Detecting the location of g D protein in ILTV infected LMH showed that g D protein expressed in cytoplasm and the junction of fused cells.

关 键 词：ILTV gD蛋白 原核表达与纯化 真核表达 多克隆抗体 细胞定位 

分 类 号：S852.659.1[农业科学—基础兽医学]