›› 2010, Vol. 37 ›› Issue (12): 68-73.

• 生物技术 • 上一篇    下一篇

鸭肝炎病毒鸡胚化弱毒MY株VP1基因克隆、原核表达及抗原性分析

向毅勇1,罗薇1,靳艳玲2,刘内生1,刘群1,龙虎1   

  1. (1.西南民族大学生命科学与技术学院,成都 610041; 2.中国科学院成都生物研究所,成都 610041)
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-12-20 发布日期:2010-12-20
  • 通讯作者: 罗薇

Cloning and Prokaryotic Expression of Chicken-embryo-adapted Attenuated Vaccine Strain MY of Duck Hepatitis Virus and Antigenicity of the Expressed Protein

XIANG Yi-yong1,LUO Wei1, JIN Yan-ling2,LIU Nei-sheng1,LIU Qun1,LONG Hu1   

  1. (1.College of Life Science and Biotechnology, Southwest University for Nationalities, Chengdu 610041, China;2.Chengdu Institute of Biology, Chinese Academy of Science, Chengdu 610041, China)
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-12-20 Published:2010-12-20
  • Contact: LUO Wei

摘要: 应用巢氏PCR(nested-PCR)扩增出1型鸭肝炎病毒鸡胚化弱毒MY株结构蛋白VP1基因片段,将VP1克隆到pMD18-T载体上,测序结果为714 bp,GenBank登录号:GU363950。对MY株VP1基因编码蛋白的主要抗原位点进行预测,aa208-aa222氨基酸区段表现很高的亲水性、抗原指数和表面可及性。VP1经EcoR Ⅰ和Xho Ⅰ双酶切后克隆至pET32a(+)原核表达载体,获得重组质粒pET-VP1,转入BL21 PLyss(DE3)细胞中,IPTG诱导后SDS-PAGE检测结果表明,在大肠杆菌中表达了1个相对分子质量为47 ku的融合蛋白。Western blotting分析结果表明,该重组蛋白可与鸭肝炎标准阳性血清发生特异性反应,具有良好的反应原性。

关键词: 鸭肝炎; 原核表达; SDS-PAGE; Western blotting

Abstract: he gene of structural protein VP1 was cloned from chicken-embryo-adapted attenuated vaccine strain MY of duck hepatitis type 1 virus by nested-PCR. Then, the VP1 gene was subcloned into the vector pMD18-T, and the gene fragment was 714 bp. In GenBank submitted for GU363950. The main antigen sites of VP1 gene from strain MY was predicted, and the segment containing 208-222 amino acid exhibited high hydrophilicity, antigenic index and surface probability. After being double digested by EcoR Ⅰand XhoⅠ, VP1 gene was subcloned into prokaryotic expression vector pET32a (+) to construct expression plasmid pET-VP1. The pET-VP1 was transformed into BL21 PLyss (DE3) and induced by IPTG. SDS-PAGE analyses showed that a fusion protein with relative molecular mass of 47 ku was expressed in Escherichia coli. Western blotting analyses showed that specific immune response could occur with this protein and duck hepatitis virus positive serum, and this protein had good reactionogenicity.

Key words: duck hepatitis; prokaryotic expression; SDS-PAGE; Western blotting

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