| 鸭瘟病毒gB蛋白N端抗原域的高效表达及其免疫特性初步研究 |
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| 引用本文： | 潘华奇,曹瑞兵,王楠,刘丽,刘磊,胡江春,陈溥言.鸭瘟病毒gB蛋白N端抗原域的高效表达及其免疫特性初步研究[J].农业生物技术学报,2008,16(5). |
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| 作者姓名： | 潘华奇 曹瑞兵 王楠 刘丽 刘磊 胡江春 陈溥言 |
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| 作者单位： | 中国科学院沈阳应用生态研究所 南京农业大学农业部动物疫病诊断与免疫重点开放实验室 甘肃农业大学动物医学院 |
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| 摘 要： | 在分析鸭瘟病毒gB蛋白抗原性的基础上，设计一对引物克隆gB蛋白N端抗原性较好的抗原域编码基因, 克隆至表达载体pET32a中, 构建了原核表达质粒pET-gB1。将pET-gB1转化至感受态E. coli BL21(DE3) 中, 经IPTG诱导和SDS-PAGE 分析, 可见约42.4kDa 的目的蛋白以包涵体形式表达。Western blotting 分析发现, 表达产物与抗鸭瘟的鼠阳性血清发生特异性反应。将包涵体溶解于8mol/L的尿素中, 利用His•Bind试剂盒获得纯化的蛋白, 将纯化的蛋白皮下注射免疫小鼠, 间接ELISA法测得抗体的效价, MTT 法检测免疫小鼠的T 淋巴细胞增殖反应能力。结果说明该融合蛋白能够诱导机体产生较强的体液免疫和细胞免疫。
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| 关 键 词： | 鸭瘟病毒 糖蛋白B 原核表达 抗原域 免疫原性 |
| 收稿时间： | 2007-12-5 |
| 修稿时间： | 2008-1-23 |
Prokaryotic expression of N-terminal antigenic domain of duck plague virus gB |
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| Abstract: | According to the antigenic analysis of duck plague virus (DPV) gB protein , one pair of primers were designed, with which the gene fragment coding the high antigenic domain of DPV N-terminal gB protein was amplified from the DPV genome. The segment was cloned into pET32a vector to obtain the recombinant pET-gB1 plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in very high level as inclusion body after induced with IPTG. The expressived product analyzed by SDS- PAGE and Western blotting. The result indicated that the fusion protein (pET-gB1) has specific antigenicity. The inclusion body was solubilized in urea (8mol/L) . The purified protein was obtained by use of His•Bind affinity chromatography. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein and normal saline. To evaluate the prophylaxtic efficacy of fusion protein , Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. |
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