《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (8): 1944-1950.doi: 10.16431/j.cnki.1671-7236.2016.08.003

• 生物技术 • 上一篇    下一篇

两种布鲁氏菌弱毒株侵染小鼠巨噬细胞过程的荧光表征及分析

胡梦薇, 刘朋涛, 严国, 高剑峰   

  1. 石河子大学生命科学学院, 石河子 832003
  • 收稿日期:2016-01-21 出版日期:2016-08-20 发布日期:2016-08-23
  • 通讯作者: 高建峰 E-mail:jianfengg@shzu.edu.cn
  • 作者简介:胡梦薇(1991-),女,新疆乌鲁木齐人,硕士生,研究方向:引进与杂交畜禽品种生物学特征研究,E-mail:15699219011@163.com
  • 基金资助:

    国家重点基础研究发展计划(973计划)(2010CB530200)

The Fluorescent Characterization and Analysis of Two Brucella Attenuated Vaccine Infecting Mouse Macrophagocyte

HU Meng-wei, LIU Peng-tao, YAN Guo, GAO Jian-feng   

  1. College of Life Science, Shihezi University, Shihezi 832003, China
  • Received:2016-01-21 Online:2016-08-20 Published:2016-08-23

摘要:

通过对绿色荧光蛋白(GFP)布鲁氏菌弱菌株M5(GFP-M5)和S19(GFP-S19)侵染小鼠巨噬细胞(RAW264.7)及对其与胞内溶酶体、内质网、高尔基体初次结合所用时间进行测定,探讨分析两种布鲁氏菌弱毒株侵染小鼠巨噬细胞过程的荧光表征。将GFP-M5和GFP-S19分不同时间段分别侵染RAW264.7,利用激光共聚焦和流式细胞仪观察和检测。结果显示,GFP-M5和GFP-S19均构建成功。布鲁氏菌M5和S19及GFP-M5和GFP-S19侵染RAW264.7后胞内生存能力无明显差异。GFP-M5和GFP-S19侵染30 min后均已进入小鼠巨噬细胞,2 h分别到达溶酶体、内质网和高尔基体。而两种弱毒株在1、2、3、4 h与各细胞器结合率相近。流式细胞仪检测结果显示,GFP-M5和GFP-S19侵染RAW264.7的GFP+细胞含量无显著差异(P>0.05)。结果提示,两种弱毒株在侵染进入宿主细胞的初期及侵袭能力并没有明显差异。

关键词: 布鲁氏菌; 绿色荧光蛋白(GFP); RAW264.7; 共聚焦显微镜; 流式细胞仪

Abstract:

The experiment was conducted to discuss the difference of binding time of green fluorescent protein B.melitensis M5 (GFP-M5) and B.abortus S19 (GFP-S19) infecting the mouse macrophagocyte (RAW264.7),lysosome,endoplasmic reticulum and golgi body in the initial stage and compare the binding rate of GFP-M5,GFP-S19 with organelle in different timeline,respectively,by confocal laser scanning microscope (CLSM) and flow cytometry.The result showed that GFP-M5 and GFP-S19 were successfully constructed.The intracellular survival ability of Brucella M5,Brucella S19,GFP-M5 and GFP-S19 were not obvisouly affected after infecting RAW264.7.GFP-M5 and GFP-S19 could enter the macrophagocyte in 30 mins,and in 2 h the Brucella could reach lysosome,endoplasmic reticulum and golgi body.In addition,the binding time for two attenuated vaccine did not show differences in 1,2,3 and 4 h.The content of GFP+ cell produced by RAW264.7 infected by GFP-M5 and GFP-S19 did not show significant differences (P>0.05).Therefore,the two strains did not have significant differences in the invasion ability in the initial stage of infecting host cell.

Key words: Brucella; green fluorescent protein; RAW264.7; confocal laser scanning microscope; flow cytometry

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