畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (6): 1212-1221.doi: 10.11843/j.issn.0366-6964.2018.06.013

• 预防兽医 • 上一篇    下一篇

稳定表达SLAM受体的Vero和BHK21细胞系的建立及其对犬瘟热病毒分离效果的比较

朱璐1, 燕霞2, 代科1, 王正皓1, 赵玉佳1, 杨振1, 文心田1, 曹三杰1, 黄小波1, 伍锐1, 赵勤1, 文翼平1*   

  1. 1. 四川农业大学动物医学院猪病研究中心, 成都 611130;
    2. 成都大熊猫繁育研究基地, 成都 610081
  • 收稿日期:2017-10-16 出版日期:2018-06-23 发布日期:2018-06-23
  • 通讯作者: 文翼平,博士,E-mail:yueliang5189@163.com
  • 作者简介:朱璐(1992-),女,山西太原人,硕士生,主要从事动物传染病的研究工作,E-mail:happyzhulu@163.com
  • 基金资助:

    成都大熊猫繁育研究基金会资助项目(CPF:2015-05)

Establishment of Vero and BHK21 Cell Lines with a Stable Expression of Canine Distemper Virus Cell Receptor SLAM and Comparison of Their Effects on the Isolation of Canine Distemper Virus

ZHU Lu1, YAN Xia2, DAI Ke1, WANG Zheng-hao1, ZHAO Yu-jia1, YANG Zhen1, WEN Xin-tian1, CAO San-jie1, HUANG Xiao-bo1, WU Rui1, ZHAO Qin1, WEN Yi-ping1*   

  1. 1. Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;
    2. Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China
  • Received:2017-10-16 Online:2018-06-23 Published:2018-06-23

摘要:

旨在构建稳定表达SLAM受体的SLAM-Vero细胞系和SLAM-BHK21细胞系,并比较其对犬瘟热病毒(CDV)野毒株的敏感性,为CDV野毒株的快速分离与研究提供一种有效的工具。将真核表达载体Pcag-SLAM分别转染Vero细胞和BHK21细胞。经G418压力筛选结合有限稀释法筛选阳性克隆株,并通过RT-PCR、间接免疫荧光鉴定(IFA)和Western blot等方法对稳定表达SLAM受体的SLAM-Vero细胞系和SLAM-BHK21细胞系加以验证。应用这两种细胞系对3例临床犬瘟热病例的5种不同组织进行病毒分离,对分离得到的CDV进行RT-PCR及IFA鉴定。结果显示,经RT-PCR和Western blot验证,本研究成功构建出SLAM-Vero细胞系和SLAM-BHK21细胞系,SLAM-Vero细胞接种病毒12~24 h即可出现典型的CDV导致的细胞融合体CPE,利用SLAM-Vero细胞从3只CDV阳性犬的肺和脾中分离得到2株CDV,而SLAM-BHK21细胞、亲本Vero细胞及亲本BHK21细胞未能分离出病毒。研究表明,与SLAM-BHK21细胞相比,SLAM-Vero细胞对CDV的分离率较高,并能产生明显的CPE。在分离CDV野毒株时,选择肺和脾更容易在SLAM-Vero细胞系上分离出病毒。

Abstract:

The objective of this work was to establish a SLAM-Vero cell line and a SLAM-BHK21 cell line that stanbly expressing SLAM receptor, and then to compare their sensitivities to the canine distemper virus (CDV) wild strains, aiming at providing another tools for the rapid isolation of CDV for further study. The eukaryotic expression vector Pcag-SLAM, which contains the SLAM gene, was transfected into the Vero cells and BHK-21 cells, respectively. Then the cells which harbor the SLAM gene were subsequently screened with G418 combined with end point dilution, RT-PCR, immunofluorescence assay, and Western blot. The two cell lines were subsequently applied to isolate virus from 5 different tissues in 3 clinical cases. The isolated CDV was further identified by RT-PCR and Indirect Immunofluorescence assay. The results showed that the SLAM-Vero cells lines and SLAM-BHK21 cells lines were constructed successfully. SLAM-Vero inoculated virus for 12-24 h presented a typical CDV-induced cell fusion of CPE. By using SLAM-Vero cells, two strains of CDV were obtained from three CDV positive canine lungs and spleens, while SLAM-BHK21 cell lines, parental Vero cells and parental BHK21 cells failed to isolate the virus. The results indicated that CDV was more readily to replicate and proliferate in SLAM-Vero cells and easier to produce visible CPE than that in SLAM-BHK21 cells. Canine lungs and spleens are more ideal candidates for isolation of CDV when using the newly established SLAM-Vero cell lines in this report.

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