| 猪繁殖与呼吸综合征病毒ORF2基因的表达及其单克隆抗体的制备 |
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| 引用本文： | 刘金霞,周艳君,安同庆,仇华吉,王云峰,童光志.猪繁殖与呼吸综合征病毒ORF2基因的表达及其单克隆抗体的制备[J].中国预防兽医学报,2006,28(6):636-639,644. |
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| 作者姓名： | 刘金霞 周艳君 安同庆 仇华吉 王云峰 童光志 |
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| 作者单位： | 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,黑龙江,哈尔滨,150001 |
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| 基金项目： | 国家自然科学基金;国家重点基础研究发展计划(973计划) |
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| 摘 要： | 将猪繁殖与呼吸综合征病毒（PRRV）CH-1a株的GP2蛋白基因进行截短修饰后，克隆于pGEX6p-1载体中，转化大肠杆菌，并进行诱导表达。经SDS—PAGE分析发现，融合表达的蛋白rtGP2大小约40Ku，Western blot分析证实，表达的融合蛋白rtGP2能被PRRSV阳性血清所特异性识别。收获融合表达的rtGP2，免疫BALB／c小鼠，取脾细胞与SP2／0骨髓瘤细胞进行融合，分别以表达的融合蛋白rtGP2和GST蛋白作为包被抗原，通过间接ELISA方法对融合细胞的上清液进行检测，结果获得了1株能稳定分泌抗rtGP2蛋白抗体的杂交瘤细胞株，将其命名为26D8，利用PRRSV感染的Marc-145细胞进行间接免疫荧光检测结果发现，所获得的单克隆抗体能与PRRSV感染细胞产生特异性免疫荧光。亚型鉴定结果显示，该单克隆抗体为培G1型，其轻链均为κ链。本研究中融合表达的rtGP2蛋白及其单克隆抗体的获得，将为今后深入研究PRRSV GP2蛋白的功能与特性提供有益帮助。
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| 关 键 词： | 猪繁殖与呼吸综合征病毒 单克隆抗体 |
| 文章编号： | 1008-0589（2006）06-0636-04 |
| 收稿时间： | 2005-10-25 |
| 修稿时间： | 2005-10-25 |
Prokaryotic expression and development of monoclonal antibody against glycoprotein 2 (GP2) of PRRSV CH-1a strain |
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| LIU Jin-xia,ZHOU Yan-jun,AN Tong-qing,QIU Hua-ji,WANG Yun-feng,TONG Guang-zhi.Prokaryotic expression and development of monoclonal antibody against glycoprotein 2 (GP2) of PRRSV CH-1a strain[J].Chinese Journal of Preventive Veterinary Medicine,2006,28(6):636-639,644. |
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| Authors: | LIU Jin-xia ZHOU Yan-jun AN Tong-qing QIU Hua-ji WANG Yun-feng TONG Guang-zhi |
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| Institution: | National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China |
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| Abstract: | The ORF2 gene of PRRSV strain CH-la was truncated and cloned into a prokaryotic expression vector pGEX-6p-1, and a fusion-expressed protein rtGP2 of 40 Ku was obtained in E. coil The protein rtGP2 showed a strong reaction to the PRRSV-positive sera in Western-blotting. BALB/c mice were immunized intraperitoneally with purified rtGP2 protein. Murine myeloma cells were fused with the splenocytes of the immunized mice after the third immunization, An indirect ELISA coated with rtGP2 and GST was used to screen hybridomas for production of specific antibody in hybridoma culture fluid. One hybridomas clone which producedMAb steadily was obtained, named 26D8, The McAb showed strong reactivity in IFA on the PRRSV infected Marc-145 cells. The McAb is belonging to IgG1 isotype, and the light chain of the antibody is K chain, which was isotyped by ELISA using a commercial kit. The McAb against PRRSV GP2 protein could be used for further analysis of the structure and function of PRRSV. |
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| Keywords: | rtGP2 |
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