利用重叠延伸PCR构建红色荧光蛋白布鲁氏菌表达载体

doi:10.16431/j.cnki.1671-7236.2018.09.006

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (9): 2394-2400.doi: 10.16431/j.cnki.1671-7236.2018.09.006

利用重叠延伸PCR构建红色荧光蛋白布鲁氏菌表达载体

罗成, 齐江矫, 王元元, 高剑峰

石河子大学生命科学学院, 石河子 832003

收稿日期:2018-01-02 出版日期:2018-09-20 发布日期:2018-09-26
通讯作者: 高剑峰 E-mail:jianfengg@shzu.edu.cn
作者简介:罗成(1991-),男,新疆伊犁人,硕士生,研究方向:动物基因工程,E-mail:275960344@qq.com
基金资助:
国家重点基础研究发展计划（973计划）（2010CB530200）

Construction of Red Fluorescent Protein Expression Vector of Brucella Using Overlapping Extension PCR Method

LUO Cheng, QI Jiangjiao, WANG Yuanyuan, GAO Jianfeng

College of Life Science, Shihezi University, Shihezi 832003, China

Received:2018-01-02 Online:2018-09-20 Published:2018-09-26

摘要/Abstract

摘要：

为了构建能够在布鲁氏杆菌宿主细胞和宿主个体中稳定表达红色荧光蛋白的载体，试验通过PCR分别获得核糖体结合位点-红色荧光蛋白（RBS-Red）与布鲁氏菌特异DNA（BDNA）片段，利用重叠延伸PCR获得RBS-Red-BDNA重叠片段；将重叠片段插入pLB载体，利用Sma Ⅰ与Sac Ⅱ对重组pLB载体和pMC-221质粒进行双酶切后连接目的片段，连接产物转化大肠杆菌DH5α感受态细胞；提取的质粒电转布鲁氏菌16M菌株感受态细胞，将电转后的16M-pMC-Red菌株涂布于含有氯霉素抗性的布鲁氏菌培养基，倒置荧光显微镜下观察菌株颜色；16M-pMC-Red菌株侵染小鼠巨噬细胞，制成细胞爬片，激光共聚焦荧光显微镜下观察小鼠巨噬细胞，鉴定红色荧光蛋白表达情况。结果显示，利用重叠延伸PCR技术成功改造了红色荧光蛋白布鲁氏菌双启动子表达载体；将改造获得的质粒电转进布鲁氏杆菌16M菌株，菌株荧光鉴定能够稳定表达红色荧光蛋白；电转成功的16M-pMC-Red菌株侵染小鼠巨噬细胞后，可以稳定表达红色荧光蛋白。试验构建的发红光质粒pMC-Red可以与发绿光质粒pMC-221联用，为不同种株布鲁氏菌之间的联合研究奠定了基础，也为布鲁氏菌病检测提供了一个以荧光检测为指标的新策略。

关键词: 布鲁氏杆菌; 红色荧光蛋白; 质粒改造; 侵染

Abstract:

To construct red fluorescent protein expression vector of Brucella host cells and individuals,RBS-Red and BDNA (Brucella DNA) fragments were obtained by PCR,and the RBS-Red-BDNA overlapping fragments were obtained by overlapping extension PCR method in this experiment.The overlapped fragments were inserted into pLB vector,the recombinant pLB vector and pMC-221 plasmid were digested by Sma Ⅰ and Sac Ⅱ double enzyme,the ligation fragments were transformed into E.coli DH5α,the extract plasmid was electroporated into Brucella 16M competent cell,16M-pMC-Red strain was coated with Brucella culture medium containing chloramphenicol resistance,and observed the strain color under inverted fluorescence microscope.The 16M-pMC-Red strain was used to infect the mouse macrophages and make the cell crawling slices,observation of mouse macrophages under laser confocal fluorescence microscopy and identification of red fluorescent expression.The results showed that the double promoter expression vector of red fluorescent protein in Brucella was successfully transformed by overlapping extension PCR method,the transformed plasmid was electroporated into Brucella strain 16M,and the strain could stably express the red fluorescent protein.The 16M-pMC-Red strain in mouse macrophages could stably express the red fluorescent protein.The red light-producing plasmid pMC-Red could be used together with the green light-emitting plasmid pMC-221,which laid a foundation for the research of different strains of Brucella,and also provided a new strategy using fluorescence detection as an indicator for the detection of brucellosis.

Key words: Brucella; red fluorescent protein; plasmid transformation; infection

中图分类号:

S852.61⁺4

罗成, 齐江矫, 王元元, 高剑峰. 利用重叠延伸PCR构建红色荧光蛋白布鲁氏菌表达载体[J]. 《中国畜牧兽医》, 2018, 45(9): 2394-2400.

LUO Cheng, QI Jiangjiao, WANG Yuanyuan, GAO Jianfeng. Construction of Red Fluorescent Protein Expression Vector of Brucella Using Overlapping Extension PCR Method[J]. , 2018, 45(9): 2394-2400.

参考文献

[1] AYSEGUL U K,GOKHAN M,EMINE A.Clinical presentations and diagnosis of brucellosis[J].Recent Patents on Anti-Infective Drug Discovery,2013,8(1):34-41.
[2] 丁家波,冯忠武.动物布鲁氏菌病疫苗应用现状及研究进展[J].生命科学,2013,25(1):91-99.DING J B,FENG Z W.Current application of brucellosis vaccines and its research advances[J].Chinese Bulletin of Life Sciences,2013,25(1):91-99.(in Chinese)
[3] FENG Y,PENG X,JIANG H,et al.Rough Brucella strain RM57 is attenuated and confers protection against Brucella melitensis[J].Microbial Pathogenesis,2017,107:270-275.
[4] DILEK Y,CIFTDOGAN,SELDA A.Unrecognized pediatric and adult family members of children with acute brucellosis[J]. Brazilian Journal of Infectious Diseases,2017,21(5):520-524.
[5] 高彦辉,赵丽军,孙殿军,等.布鲁氏菌病防治基础研究现状与展望[J].中国科学(生命科学),2014,44(6):628-635.GAO Y H,ZHAO L J,SUN D J,et al.Status and perspective of basic research of prevention and control of brucellosis[J].Chinese Science(Life Science),2014,44(6):628-635.(in Chinese)
[6] MORIYON I,GRILLO M J,MONREAL D,et al.Rough vaccines in animal brucellosis:Structural and genetic basis and present status[J].Veterinary Research,2004,35(1):1-38.
[7] FRENTIN D,FAUCONNIER A,KOHLER S,et al.The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infec-tion[J].Cellular Microbiology, 2005,7(5):687-698.
[8] YANG X,SKYBERG J A,CAO L,et al.Progress in Brucella vaccine development[J].Frontiers in Biology,2013,80(1):60-77.
[9] ZHAN Y,YANG J,CHEERS C.Cytokine response of T-cell subsets from Brucella abortus-infected mice to soluble Brucella proteins[J].Infection and Immunity,1993,61(7):2841-2847.
[10] GROSS A,TERRAZA A,OUAHRANIBETTACHE S,et al.In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells[J].Infection and Immunity,2000,68(1):342-351.
[11] BARKER L P,BRORKS D M,SMALL P L.The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein[J].Molecular Microbiology,1998,29(5):1167-1177.
[12] KOHLER S S,OUAHRANI B M,LAYSSAC J,et al.Constitutive and inducible expression of green fluorescent protein in Brucella suis[J].Infection and Immunity,1999,67(12):6695-6697.
[13] ESKRA L,CANAVESSI A,CAREY M,et al.Brucella abortus genes identified following constitutive growth and macrophage infection[J].Infection and Immunity,2001,69(12):7736-7742.
[14] 胡梦薇,刘朋涛,严国,等.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[J].中国畜牧兽医,2016,43(8):1944-1950.HU M W,LIU P T,YAN G,et al.The fluorescent characterization and analysis of two Brucella attenuated vaccine infecting mouse macrophagocyte[J].China Animal Husbandry & Veterinary Medicine,2016,43(8):1944-1950.(in Chinese)
[15] DE M F,DI G A,PETRINI A,et al.Correlation between animal and human brucellosis in Italy during the period 1997-2002[J].Clinical Microbiology and Infection,2005,11(8):632-636.
[16] 赵凤菊.布鲁氏菌病诊断方法的研究进展[J].中国畜牧兽医,2011,38(8):193-196.ZHAO F J.Research progress of diagnosis of brucellosis[J].China Animal Husbandry & Veterinary Medicine,2011,38(8):193-196.(in Chinese)
[17] GAMAL W,MELZER E,CHRISTIPH W,et al.Comprehensive identification of immunodominant proteins of Brucella abortus and Brucella melitensis using antibodies in the sera from naturally infected hosts[J]. Molecular Sciences,2016,17(5):659-678.
[18] 王显军,李兰玉,赵鸿雁,等.建立双抗原夹心酶免疫试验对人畜布鲁氏菌抗体检测的研究[J].中国人兽共患病学报,2007,23(3):262-265.WANG X J,LI L Y,ZHAO H Y,et al.Study on the detection of Brucella antibody in human and animal by the establishment of double antigen sandwich enzyme immunoassay[J].Chinese Journal of Zoonoses,2007,23(3):262-265.(in Chinese)
[19] KIM J W,LEE Y J, HAN M Y,et al.Evaluation of immunochromatographic assay for serodiagnosis of Brucella canis[J].Journal of Veterinary Medical Science,2007,69(11):1103-1107.
[20] 朱明东,洪林娣.胶体金免疫层析法快速诊断牛布鲁氏菌病的研究[J].中国人兽共患病学报,2008,24(8):755-757.ZHU M D,HONG L D.A new method of rapid diagnosis for brucellosis by colloidal gold-immunochromatographic assay[J].Chinese Journal of Zoonoses,2008,24(8):755-757.(in Chinese)
[21] OLSEN S C.Immune responses of elk to initial and booster vaccinations with Brucella abortus strain RB51 or 19[J].Clinical and Vaccine Immunology,2006,13(10):1098-1103.
[22] AXEL C,ISABELLE J,MARIA J G,et al.Development and evaluation as vaccines in mice of Brucella melitensis Rev.1 single and double deletion mutants of the BP26 and Omp31 genes coding for antigens of diagnostic significance in ovine brucellosis[J].Vaccine,2004,22(21):2827-2835.

利用重叠延伸PCR构建红色荧光蛋白布鲁氏菌表达载体

Construction of Red Fluorescent Protein Expression Vector of Brucella Using Overlapping Extension PCR Method

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价