胸膜肺炎放线杆菌RTX毒素抗原优势决定簇的筛选和融合表达  

Screening and fusion expression of antigenic determinant regions of RTX toxins secreted by Actinobacillus pleuropneumoniae

作　　者：耿琰 蔡金双 伭婷 李玉峰[1] GENG Yan;CAI Jinshuang;XUAN Ting;LI Yufeng(College of Veterinary Medicine,Nanjing Agricultural University/Key Laboratory of Bacteriology,Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院/农业农村部细菌学重点实验室,江苏南京210095

出　　处：《畜牧与兽医》2023年第5期87-94,共8页Animal Husbandry & Veterinary Medicine

基　　金：“十四五”重点研发计划(2022YFD1800900)。

摘　　要：为了制备胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)亚单位疫苗和建立配套的诊断方法,本研究在对APP的RTX毒素(ApxⅠ~Ⅲ)生物学信息分析的基础上,以APP血清5型和8型的DNA为模板对ApxⅠ~Ⅲ分段扩增后进行截短表达,获得12个截短表达蛋白。利用Western blot对12个表达蛋白进行抗原性分析,确定ApxⅠ~Ⅲ的优势抗原决定簇分别为蛋白AⅠ2、AⅡ3和AⅢ2。以蛋白AⅡ3、AⅢ2、AⅠ2的顺序排列并在蛋白间加入GPGPG氨基酸序列,无缝克隆3个蛋白片段基因,通过原核表达获得融合蛋白A231。该蛋白可与临床APP阳性猪血清特异性结合,具有良好的免疫反应性。该研究的成功开展可为研制具有交叉保护力的亚单位疫苗及建立配套ELISA检测方法奠定基础。This study was to prepare a subunit vaccine for Actinobacillus pleuropneumoniae(APP)and establish a matching differential diagnostic method.Based on the analysis of the biological characteristics of Actinobacillus pleuropneumoniae-RTX-toxins(ApxⅠ-Ⅲ),the genomic DNAs from the APP strains of serotypes 5 and 8 were used as templates,and 12 truncated expression proteins were obtained by segmental expression of ApxⅠ-Ⅲ.The antigenicity analysis by Western blot confirmed that the dominant antigenic fragments of ApxⅠ-Ⅲ were proteins AⅠ2,AⅡ3 and AⅢ2.The proteins AⅡ3,AⅢ2,and AⅠ2 were tandemly linked with the GPGPG sequence.After seamless cloning of the gene sequences of the three protein fragments,the fusion protein A231 was obtained by prokaryotic expression.The protein was able to specifically react with the serum from APP clinically infected pigs,implying a potential immunogenicity of recombinant proteins.Our study laid a foundation for development of subunit vaccines with cross-protection and for establishment of a matching differential ELIAS method.

关 键 词：猪传染性胸膜肺炎放线杆菌 APX毒素 抗原决定区 免疫原性 

分 类 号：S852.6[农业科学—基础兽医学]