布氏杆菌pCDNA3.1-L7L12核酸疫苗的构建及其免疫学评价

Construction of DNA vaccine carrying Brucella Abortus Ribosomal L7/L12 Gene and evaluation of its immune response in vaccinated mice

目的 获得布氏杆菌保护性抗原L7 L12重组蛋白及pCDNA3.1 L7 L12重组质粒 ,并比较其诱导特异性免疫应答的能力。方法 PCR扩增布氏杆菌核蛋白L7 L12基因分别构建至原核表达载体PET32a(+)和真核表达载体pCDNA3.1(+)中 ;pET32a L7 L12重组质粒转化BL2 1(DE3) ,所表达蛋白经SDS PAGE、免疫印迹分析、纯化后免疫小鼠 ;pCDNA3.1 L7 L12重组质粒配以GM CSF同时肌肉注射免疫小鼠 ,3次免疫后测定免疫功能进行免疫效果的评价。结果 ELISA、Westernblot检测到免疫鼠体内有特异性抗体产生 ,蛋白苗所诱导的抗体效价远远高于DNA疫苗 ;通过淋巴细胞增殖实验、细胞因子和CD分子测定表明DNA疫苗以诱发TH1 型免疫为主。结论 所构建的布氏杆菌DNA疫苗和蛋白苗均具有诱导特异性细胞和体液免疫应答的能力 ,DNA疫苗诱导产生的细胞免疫反应强于蛋白苗 ,可作为潜在的布氏菌新型疫苗 。

Objective To construct a recombinant plasmid encoding L7/L12 gene of brucella and compare immunogenicity with recombinant L7/L12 protein. Methods L7/L12 gene was cloned into the eurokaryotic expressing plasmid pCDNA3.1, then the recombinant plasmid was injected into Balb/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene.BL21(DE3) contained the PET32a-L7/L12 expression plasmid was induced by IPTG and the fusion protein was identified and then purified. Mice were immunized with this protein mixed with Freund's adjuvant. Results pCDNA3.1-L7/L12 recombinant expressing plasmid was successfully constructed.The L7/L12 protein was highly and stably expressed in E.coli. Animals immunized with the recombinant protein and pCDNA3.1-L7/L12 respectively both induced antigen specific antibody. Furthermore, only pCDNA3.1-L7/L12 induced a typical T-helper 1-dominated immune response, as determined by lymphocyte proliferation assay,cytokine and immunoglobulin G isotype analysis. Conclusion pCDNA3.1-L7/L12 can induce both humoral and cellular immune response. This vaccine could be used as a potential candidate vaccine for the control of brucellosis.