应用噬菌体展示技术筛选兔出血症病毒抗原模拟表位

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (8): 1281-1286.

应用噬菌体展示技术筛选兔出血症病毒抗原模拟表位

杨廷亚^1,2，王芳¹*，姜平²，胡波¹，范志宇¹，魏后军¹，薛家宾¹

1. 江苏省农业科学院兽医研究所，农业部兽用生物制品工程技术重点实验室，国家兽用生物制品工程技术研究中心，南京210014； 2. 南京农业大学动物医学院，南京 210095

收稿日期:2012-02-08 修回日期:1900-01-01 出版日期:2012-08-24 发布日期:2012-08-24
通讯作者: 王芳

Screening of Antigen Mimotopes of Rabbit Hemorrhagic Disease Virus by Phage Display Technology

YANG Tingya^{1, 2}, WANG Fang ¹*, JIANG Ping², HU Bo¹, FAN Zhiyu¹, WEI Houjun ¹, XUE Jiabin¹

1. Key Laboratory of Veterinary Biologicals Engineering and Technology of Ministry of Agriculture, National Center for Engineering Research of Veterinary Bioproducts, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China；2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2012-02-08 Revised:1900-01-01 Online:2012-08-24 Published:2012-08-24
Contact: WANG Fang

摘要/Abstract

摘要： 以抗兔出血症病毒（RHDV）的单克隆抗体A3c作为靶物质，应用噬菌体展示技术筛选RHDV抗原表位。将纯化的单抗A3c包被固相载体，经3轮亲和筛选后，挑取25株噬菌体单克隆并扩增，用ELISA测定后，提取阳性克隆单链DNA并测序，用阳性噬菌体克隆免疫小鼠制备高免血清，检测筛选抗原表位的免疫原性。结果表明：3轮亲和筛选后，特异性噬菌体克隆得到了有效富集，25株噬菌体单克隆中有19株为阳性克隆；测序结果表明，获得了与抗原高度同源的序列GTDDMDPGTTAA，即抗原的模拟表位，其中，氨基酸基序DXXDP为表位中的核心氨基酸；制备的小鼠高免血清与抗原具有较好的反应性，阳性噬菌体克隆与兔RHDV高免血清也具有较好的反应性。因此，该表位具有良好的免疫原性和反应原性。该研究为RHDV抗原表位的研究和新型疫苗的探索积累了资料。

关键词: 兔出血症病毒, 单克隆抗体, 噬菌体展示技术, 模拟表位

Abstract: The monoclonal antibody (McAb) A3c against rabbit hemorrhagic disease virus had been used as solidphase selective molecule to screen the epitope of RHDV by phage display technology. McAb A3c was coated on the solidphase and then three rounds of biopanning were carried out; 25 phages were selected and amplified to be identified by ELISA, and the positives were sequenced; the serum of mice immunized three times was prepared to determine the immunogenicity of the epitope. The results showed that the specific phages were enriched effectively after three rounds of biopanning; 19 of 25 phages were positive. The sequence analysis of the positive clones showed that highly homogenous sequence (GTDDMDPGTTAA) comparing to antigen was got, which is the antigen mimotopes of RHDV. The sequence DXXDP was the core amino acids in the epitope. In addition, the serum of mice reacted with antigen well and so did positive phages with hyperimmune serum of rabbit against RHDV, which indicated that the epotipe had good immunogenicity and reactionogenicity. This study provide theoretical basis for studying the epitope of RHDV and new vaccines.

Key words: rabbit hemorrhagic disease virus, monoclonal antibody, phage display technology, mimotopes

杨廷亚;王芳;姜平;胡波;范志宇;魏后军;薛家宾. 应用噬菌体展示技术筛选兔出血症病毒抗原模拟表位[J]. 畜牧兽医学报, 2012, 43(8): 1281-1286.

YANG Tingya;;WANG Fang;JIANG Ping;HU Bo;FAN Zhiyu;WEI Houjun;XUE Jiabin . Screening of Antigen Mimotopes of Rabbit Hemorrhagic Disease Virus by Phage Display Technology[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(8): 1281-1286.

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