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草业学报 ›› 2014, Vol. 23 ›› Issue (4): 138-145.DOI: 10.11686/cyxb20140417

• 研究论文 • 上一篇    下一篇

禽流感血凝素HA超表达载体的构建及高表达百脉根的获得

张伟伟1,2,袁蓓2,张占路3,赵杨敏3,徐秉良1*,吴燕民2*   

  1. 1.甘肃农业大学草业学院,甘肃兰州730070
    2.中国农业科学院生物技术研究所,北京100081
    3.深圳市农科集团有限公司,广东深圳518040
  • 收稿日期:2014-01-15 出版日期:2014-08-20 发布日期:2014-08-20
  • 通讯作者: E-mail:Wuym65@sina.com,xubl@gsau.edu.cn
  • 作者简介:张伟伟(1988-),女,山东潍坊人,在读硕士。E-mail:zhangweiwei1002132@163.com
  • 基金资助:
    国家重点基础研究发展计划“973”项目(2014CB138701)和国家自然科学基金资助项目(31372361)资助

Vector construction and over-expression of an avian influenza virus hemagglutinin (HA) in transgenic Lotus corniculatus

ZHANG Wei-wei1,2,YUAN Bei2,ZHANG Zhan-lu3,ZHAO Yang-min3,XU Bing-liang1,WU Yan-min2   

  1. 1.College of Grassland Science,Gansu Agricultural University,Lanzhou 730070,China;
    2.Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China;
    3.Shenzhen Nongke Group Corporation,Shenzhen 518040,China
  • Received:2014-01-15 Online:2014-08-20 Published:2014-08-20

摘要: 为提高外源抗原蛋白的表达水平,根据已得到的HA-LTB融合基因,通过添加双增强子CaMV35S启动子、增强子Ω序列、内质网滞留信号KDEL序列及加长poly(A)序列等表达调控元件,构建HA-LTB高效融合植物表达载体。利用农杆菌介导方法转化百脉根,获得转基因阳性植株21株,经PCR和RT-PCR检测表明HA-LTB融合基因已整合到百脉根基因组中。利用ELISA检测转基因植株,结果表明LTB免疫佐剂与HA抗原蛋白均具有免疫原性,LTB最高表达量达到0.086 μg/mg(蛋白粗提液),HA最高表达量达到0.25 μg/mg(蛋白粗提液)。通过植物表达载体的设计和构建,使禽流感血凝素在百脉根中的表达量得到了显著提高,为禽流感可饲疫苗的研制奠定了基础。

Abstract: A safe and efficient plant expression vector,HA-LTB,which added double 35S promoter,Ω enhancer,endoplasmic reticulum retention signal KDEL sequence and extended poly (A) sequence was constructed. Twenty one transgenic Lotus corniculatus plants were obtained by the Agrobacterium-mediated method and the results of PCR and RT-PCR showed that the HA-LTB gene had been obtained from the plant genome. ELISA results of the B subunit of Escherichia coli heat-labile enterotoxin (LTB) showed that LTB could be expressed in transgenic L. corniculatus and had immunogenicity. It was concluded that hemagglutinin (HA) which was fused with LTB had immune activity. The highest expressions of LTB and HA were 0.086 μg/mg (crude protein extracts) and 0.25 μg/mg (crude protein extracts) respectively which has approached advanced international standards. A high level exogenous antigen protein expression platform which was a basis for research on the H5N1 avian influenza vaccine was established by optimizing the expression vector.

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