畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (6): 981-988.doi: 10.11843/j.issn.0366-6964.2015.06.014

• 预防兽医 • 上一篇    下一篇

犬瘟热病毒囊膜糖蛋白在杆状病毒-昆虫细胞系统中的表达及鉴定

虞一聪1,2,冯娜2,3*,闫飞虎2,盖微微2,王铁成2,王化磊2,3,郑学星2,3,赵永坤2,3,黄耕2,杨松涛2,高玉伟2,夏咸柱2,3,4*   

  1. (1.吉林农业大学动物科学技术学院,长春 130118;2.军事医学科学院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,长春 130122;3.中国农业科学院长春兽医研究所,长春 130122;4.江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州 225009)
  • 收稿日期:2014-09-28 出版日期:2015-06-23 发布日期:2015-06-23
  • 通讯作者: 夏咸柱,E-mail:xiaxzh@cae.cn;冯娜,E-mail:fengna0308@126.com
  • 作者简介:虞一聪(1989-),男,浙江慈溪人,硕士生,主要从事动物分子病毒学研究,E-mail:jordanyuyicong@163.com
  • 基金资助:

    国家公益性行业(农业)科研专项(201303042)

Expression and Identification of Glycoprotein of Canine Distemper Virus in the Baculovirus-Insect Cell System

YU Yi-cong1,2,FENG Na2,3* ,YAN Fei-hu2,GAI Wei-wei2,WANG Tie-cheng2,WANG Hua-lei2,3,ZHENG Xue-xing2,3,ZHAO Yong-kun2,3,HUANG Geng2,YANG Song-tao2,GAO Yu-wei2,XIA Xian-zhu2,3,4*   

  1. (1.College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;2.Key Laboratory of Zoonosis Prevention and Control of Jilin Province,Military Veterinary Institute of the Academy of Military Medical Sciences,Changchun 130122,China;3.Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China;4.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis,Yangzhou 225009,China)
  • Received:2014-09-28 Online:2015-06-23 Published:2015-06-23

摘要:

为表达具有天然构象的犬瘟热病毒(CDV)囊膜糖蛋白融合蛋白(F)和血凝素蛋白(H),本研究扩增小熊猫源CDV驯化致弱株LP的FH基因,克隆至pFastBacTM1载体中,测序验证后转化至DH10BacTM感受态细胞,同源重组获得穿梭质粒rBacmid-F、rBacmid-H,将其分别转染Sf9细胞获得重组杆状病毒rpFB-F、rpFB-H,并将表达的重组融合蛋白(rF)和血凝素蛋白(rH)进行IFA和Western blot鉴定。以犬抗CDV高免血清对重组杆状病毒感染细胞进行IFA鉴定,在感染细胞的细胞膜上可见特异性荧光反应;以鼠抗F、H蛋白的主要抗原表位区多克隆抗体对重组杆状病毒感染细胞进行Western blot检测,可见相对分子质量为63和68 ku左右的条带,分别为重组融合蛋白(rF)和血凝素蛋白(rH),大小与预期相符。两种囊膜糖蛋白在杆状病毒-昆虫细胞系统中均成功表达,且具有良好的反应原性。本研究为CDV病毒样颗粒疫苗的开发等工作奠定了基础。

Abstract:

The objectives of this study were to express the native structure of fusion protein (F) and hemagglutinin glycoprotein (H) of canine distemper virus (CDV).The F and H gene of lesser panda attenuated strain were amplified by PCR and cloned into pFastBacTM1 vector.The recombinant plasmids were sequenced,and then were transformed into competent DH10BacTM E.coli cells to construct shuttle plasmids,rBacmid F and rBacmid H,by homologous recombination.The recombinant plasmids were then transfected into Sf9 cells to construct recombinant baculoviruses and the expression products of rF and rH were identified with IFA and Western blot.The expression of rF and rH in insect cells infected with recombinant baculoviruses were identified by IFA with dog anti-CDV hyperimmune serum and were detected by Western blot with mouse polyclonal antibody against F and H major epitopes of CDV.The molecular weight of expressed F and H protein were identified as 63 and 68 kD,which were consistent with the expected value.The results show that both envelope glycoproteins are successfully expressed in baculovirus-insect cell expression system and have a good immunoreactivity.Our study laid the foundation for the development of the CDV virus-like particle vaccine.

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