布氏菌病双价DNA疫苗免疫保护效果的研究

Construction of Bivalent DNA Vaccine and Evaluation of Its Immunoprotection Efficacy

【目的】构建布氏杆菌pcDNA3.1(+)-L7/L12-BCSP31双价DNA疫苗,观察其免疫保护效果。【方法】分别克隆L7/L12、BCSP31基因,构建pcDNA3.1(+)-L7/L12-BCSP31双价DNA疫苗。转染COS-7细胞,免疫细胞化学检测目的蛋白的表达。DNA疫苗免疫Balb/c小鼠,观察体液免疫和细胞免疫效果。布氏杆菌强毒株腹腔攻毒,观察双价DNA疫苗的免疫保护效果。【结果】构建的pcDNA3.1(+)-L7/L12-BCSP31双价DNA疫苗在COS-7细胞有目的蛋白的表达。双价DNA疫苗免疫小鼠,ELISA检测双价DNA疫苗产生了高水平的特异性IgG抗体,且抗体亚型以IgG2a为主。FCM检测双价DNA疫苗的CD4+/CD8+比值明显下降。ELISPOT检测到疫苗免疫后小鼠分泌IFN-γ的T细胞增多。攻毒实验证明双价DNA疫苗能够产生有效的保护效果;部分数据及攻毒结果表明双价DNA疫苗的效果优于单价。【结论】研制的pcDNA3.1(+)-L7/L12-BCSP31双价DNA疫苗免疫Balb/c小鼠能产生有效的免疫保护效果,且双价DNA疫苗效果优于单价。

[Objective] To Construct bivalent DNA vaccine encoding L7/L12,BCSP31 and to evaluate its immunogenicity and protective efficacy. [Methods]Brucella abortus L7/L12 （ ribosome protein ） and BCSP31 （ outer membrane lipoprotein ） gene were cloned into the eukaryotic expressing plasmid pcDNA3. 1 （ ＋ ） to get the recombinant plasmid infection in the spleens of vaccinated mice challenged with B. abortus A544. [Results] The bivalent DNA vaccine could be expressed in COS-7 cells. The typical T-helper 1-dominated immune response was determined by immunoglobulin G isotype analysis, FCM and ELISPOT. The high titer specific an- tibody against L7/L12-BCSP3, protein was detected by ELISA. Immunization with pcDNA3. 1 （ ＋ ） L7/L12- BCSP31 could reduce the bacterial burden relative to those in the control group. Some data showed that the bivalent DNA vaccine was better than the monovalent DNA vaccine. [Conclusion]pcDNA3. 1 （ ＋ ）-L7/L12-BC SP31 is effective for the production of cell-mediated responses in mice and a basis for the future studies of vaccination against brucellosis. The combination of the two gene is better than the only one which we describe be fore . The present work may be useful to the development of Brucella DNA vaccine.