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| 猪TLR3胞外区片段重组载体的构建、表达和多克隆抗体的制备 | |
| 引用本文： | 管齐赛,房永祥,贾怀杰,何小兵,周涛,杨孝朴,景志忠.猪TLR3胞外区片段重组载体的构建、表达和多克隆抗体的制备[J].甘肃农业大学学报,2012,47(3):1-6,12. |
| 作者姓名： | 管齐赛 房永祥 贾怀杰 何小兵 周涛 杨孝朴 景志忠 |
| 作者单位： | 1. 甘肃农业大学动物医学院,甘肃兰州730050;中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点实验室,甘肃兰州730046 2. 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点实验室,甘肃兰州730046 3. 甘肃农业大学动物医学院,甘肃兰州,730050 |
| 基金项目： | 国家自然科学基金，转基因生物新品种培育重大专项 |
| 摘 要： | 为制备猪TLR3(poTLR3)胞外区蛋白的多克隆抗体,从poTLR3克隆载体扩增胞外区基因序列连接到pGEX-4T-1载体上,转化大肠杆菌BL21(DE3),在IPTG的诱导下,表达GST-poTLR3融合蛋白,将表达的融合蛋白纯化,免疫家兔制备抗体,并以间接ELISA法测定抗体效价,利用Western-blot和细胞免疫荧光验证抗体与真核表达蛋结合的白特异性.结果表明:制备的多克隆抗体的效价可达1∶128 000,poTLR3胞外区蛋白在大肠杆菌中得到高效表达,并且制备的多克隆抗体特异性好、滴度高. |
| 关 键 词： | 猪TLR3 胞外区 原核表达 多克隆抗体 |
Prokaryotic expression of poTLR3 extracellular domain and preparation of polyclonal antibody |
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| GUAN Qi-sai , FANG Yong-xiang , JIA Huai-jie , HE Xiao-bing , ZHOU Tao , YANG Xiao-pu , JING Zhi-zhong.Prokaryotic expression of poTLR3 extracellular domain and preparation of polyclonal antibody[J].Journal of Gansu Agricultural University,2012,47(3):1-6,12. | |
| Authors: | GUAN Qi-sai FANG Yong-xiang JIA Huai-jie HE Xiao-bing ZHOU Tao YANG Xiao-pu JING Zhi-zhong |
| Institution: | 1,2(1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;2.State Key Laboratory of Veterinary Etiological Biology,Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Lanzhou Veterinary Research Institute,CAAS,Lanzhou 730046,China) |
| Abstract: | In order to preparation polyclonal antibody of porcine toll-like receptor 3(poTLR3) extracellular domain proteins,the extracellular domain of poTLR3 was amplified from the constructed plasmid pGEM-Teasy-poTLR3 and cloned into the prokaryotic expression vector pGEX-4T-1,the recombinant plasmid pGEX-4T-poTLR3 was transformed into BL21 to express fusion protein GST-poTLR3 under the induction of IPTG.Moreover,the GST-poTLR3 fusion proteins were purified,and the antiserum was made by the immunized rabbits.Furthermore,the titer of the polyclonal antibody was tested by the indirect ELISA,and specificity of the antibody was identified by western blot and immunofluorescence.The results indicated that the titer of the polyclonal antibody was up to 1∶128 000,and the antibody combined specifically with the expressed protein of poTLR3 in eukaryotic cell.Therefore,it was showed that the poTLR3 extracellular domain protein was highly expressed in BL21,and the antiserum with high titers and specificities was prepared,and which provides foundation for further investigation in poTLR3 function. |
| Keywords: | poTLR3 extracellular domain prokaryotic expression polyclonal antibody |
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