布鲁氏菌BP26蛋白细菌样颗粒疫苗的制备和免疫学评价  

Preparation and Immunological Evaluation of Bacterium-like Particle Vaccine Based on Brucella BP26 Outer Membrane Protein

作　　者：林海 涂飞 李楠[1,2] 孙洋 姜博文 纪雪 刘军 LIN Hai;TU Fei;LI Nan;SUN Yang;JIANG Bowen;JI Xue;LIU Jun(Changchun Veterinary Research Institute,Chinese Academy of Agriculture Sciences,Changchun 130122,China;Key Laboratory of Jilin Province for Zoonosis Prevention and Control,Changchun 130122,China)

机构地区：[1]中国农业科学院长春兽医研究所,吉林长春130122 [2]吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国兽医杂志》2024年第6期37-46,共10页Chinese Journal of Veterinary Medicine

基　　金：内蒙古自治区科技重大专项(2019ZD006)。

摘　　要：为了构建布鲁氏菌外膜蛋白BP26细菌样颗粒候选疫苗BLPs-BP26,并评价其免疫原性,本试验首先分别扩增羊种布鲁氏菌16M株编码BP26蛋白的整个基因序列和乳酸乳球菌MG1363株编码肽聚糖锚钩蛋白(PA)的整个基因序列,通过重叠延伸PCR技术将bp26基因与PA基因连接构建融合基因bp26-PA,将融合基因bp26-PA克隆至pET-28a原核表达载体并诱导表达融合蛋白BP26-PA。将表达正确的BP26-PA融合蛋白与细菌样颗粒(BLPs)结合构建BLPs-BP26,通过SDS-PAGE、Western blot、间接免疫荧光和冷冻超薄切片对BLPs-BP26进行鉴定。将BLPs-BP26免疫小鼠,通过酶联免疫吸附试验(ELISA)检测小鼠特异性IgG抗体水平,实时荧光定量反转录PCR(RT-qPCR)分析免疫小鼠脾脏组织中干扰素γ(IFN-γ)和白介素2(IL-2)的基因转录水平,ELISA方法检测免疫小鼠血清中IFN-γ和IL-2的蛋白表达水平。鉴定结果显示,BP26-PA蛋白成功展示在BLPs表面;免疫小鼠试验结果显示,小鼠血清特异性IgG抗体效价最高可达1∶1 024 000;与PBS组相比,BLPs-BP26组免疫小鼠脾脏组织中IFN-γ和IL-2基因的转录水平均极显著升高(P值分别为P<0.000 1和P<0.01),小鼠血清中IFN-γ的表达水平极显著升高(P<0.000 1)。结果表明,BLPs-BP26能刺激小鼠产生高水平的特异性IgG抗体,并可诱导小鼠产生细胞免疫应答。本试验为开发具有良好免疫原性且无安全隐患的布鲁氏菌新型疫苗提供了新的思路。In order to construct a Brucella outer membrane protein BP26 bacterium-like particles candidate vaccine(BLPs-BP26)and evaluate its immunogenicity,in this study,the entire gene sequence encoding BP26 protein of Brucella melitensis 16M strain and the entire gene sequence encoding protein anchor(PA)of Lactococcus lactis MG1363 strain were amplified separately.The bp 26 gene and the PA gene were linked by overlapping extension PCR technology to construct the fusion gene bp 26-PA.The fusion gene bp 26-PA was cloned into the pET-28a prokaryotic expression vector and induced to express the fusion protein BP26-PA.The expressed correct BP26-PA fusion protein was combined with bacterium-like particles(BLPs)to construct BLPs-BP26,which was identified by SDS-PAGE,Western blot,indirect immunofluorescence,and ultra-thin frozen section.BLPs-BP26 was used to immunize mice,and the levels of mouse-specific IgG antibodies were detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative reverse transcription PCR(RT-qPCR)was used to analyze the gene transcription levels of interferon-γ(IFN-γ)and interleukin-2(IL-2)in the spleen tissues of immunized mice.ELISA was used to detect the protein expression levels of IFN-γand IL-2 in mouse serum.The identification results showed that the BP26-PA protein was successfully displayed on the surface of BLPs.The results of mouse experiments showed that the titer of specific IgG antibodies in mouse serum could reach up to 1∶1024000.Compared with the PBS group,the transcription levels of IFN-γand IL-2 genes in the spleen tissues of mice immunized with BLPs-BP26 were significantly increased(P values were P<0.0001 and P<0.01,respectively),and the expression level of significantly increased ( P <0.000 1). The results indicate that BLPs-BP26 can stimulate mice to produce high levels of specific IgG antibodies and induce a cellular immune response in mice. This study provides a new idea for the development of a novel Brucella vaccine with good immunogenicity and no saf

关 键 词：表面展示技术 布鲁氏菌 BP26蛋白 细菌样颗粒疫苗 

分 类 号：S855.1[农业科学—临床兽医学]