生物技术通报 ›› 2016, Vol. 32 ›› Issue (5): 234-239.doi: 10.13560/j.cnki.biotech.bull.1985.2016.05.031

• 研究报告 • 上一篇    下一篇

小反刍兽疫病毒F基因缺失体的克隆、表达及鉴定

邓瑞雪1, 蒙学莲2, 曾巧英1, 才学鹏2   

  1. 1.甘肃农业大学动物医学院,兰州 730070;
    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,兰州 730046
  • 收稿日期:2015-12-22 出版日期:2016-05-25 发布日期:2016-05-27
  • 作者简介:邓瑞雪,男,硕士,研究方向:预防兽医学;E-mail:drxd1@163.com ;才学鹏,男,研究员,研究方向:家畜寄生虫的研究;
  • 基金资助:
    国家自然科学基金项目(31300142),兰州市科技计划项目(2013-4-40)

Cloning,Expression and Identification of F Gene Deletants from Peste Des Petits Ruminants Virus

DENG Rui-xue1,MENG Xue-lian2,ZENG Qiao-ying1,CAI Xue-peng2   

  1. 1. College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070;
    2. State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences,Lanzhou 730046
  • Received:2015-12-22 Published:2016-05-25 Online:2016-05-27

摘要: 旨在研究小反刍兽疫病毒(PPRV)囊膜与宿主细胞的融合机制,构建F基因缺失体,并且在原核细胞内表达。鉴于F基因中包含的两段保守序列HR1和HR2在融合过程中具有关键性作用,分别构建pET30a-HR1和pET30a-HR2原核表达载体,将测序正确的重组质粒转化到大肠杆菌BL21(DE3)细胞内进行IPTG诱导表达,在最优表达条件下大量表达,并利用镍琼脂糖凝胶纯化蛋白。结果显示,获得的重组蛋白与预期大小一致且以可溶性方式表达,纯化的蛋白纯度大于90%;重组蛋白与抗His标签抗体和抗PPRV Nigeria 75/1株阳性血清均能发生反应,证明重组蛋白具有反应原性。

关键词: F基因缺失体, 原核表达, 小反刍兽疫病毒

Abstract: The F gene deletants were constructed and expressed in prokaryotic cell in order to clarify the fusion mechanism between the envelope of peste des petits ruminants virus(PPRV)and the host cells. Considering the key roles in the fusion process,HR1 and HR2,the two conserved fragments of F gene,were cloned into prokaryotic expression vector pET-30a,respectively. The recombinant plasmid pET30a-HR1 and pET30a-HR2 with the confirmed correct sequences were transformed into Escherichia coli BL21(DE3)and expressed by the induction of IPTG. The fusion proteins were largely expressed under the optimal condition and purified using the nickel agarose gel. The results showed that the relative molecular masses of the fusion proteins expressed in form of soluble protein were consistent with the expectation,and the purities of the expressed proteins were over 90%. The fusion proteins reacted with both anti-His tag antibody and anti-PPRV Nigeria75/1 positive serum,indicating that the fusion protein had reactionogenicity.

Key words: F gene deletant, prokaryotic expression, peste des petits ruminants virus