重组猪胸膜肺炎放线杆菌NLPI蛋白诱导小鼠抗攻击感染的免疫保护力研究  

Immune Protection Research Induced by Actinobacillus pleuropneumoniae Recombinant NLPI Protein Against Challenge Infection in Mice(Mus musculus)

作　　者：杨婷婷[1] 黄复深[1,2] 张雨龙 张宇航[1] 李宏 Yang Ting-Ting;Huang Fu-Shen;Zhang Yu-Long;Zhang Yu-Hang;Li Hong(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Engineering Research Center of Veterinary Drug,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,长沙410128 [2]湖南农业大学湖南省兽药工程技术研究中心,长沙410128

出　　处：《农业生物技术学报》2018年第8期1392-1400,共9页Journal of Agricultural Biotechnology

基　　金：湖南省教育厅重点课题(No.16A101)

摘　　要：猪传染性胸膜肺炎(porcine contagious pleuropneumonia,PCP)是猪(Sus scrofa)的一种呼吸道疾病,影响世界养猪业,其病原菌为猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP),现有疫苗由于保护效果不甚理想,因此有必要寻找新的疫苗候选分子。本研究检测了猪APP重组NLPI蛋白(recombinant NLPI,rNLPI)的免疫保护作用。根据Gen Bank中已报道的APP nlpi基因序列设计引物对,然后以APP的基因组为模板PCR扩增nlpi基因,测序后进行生物信息学分析,发现该基因(Gen Bank No.:MH027649)由900 bp组成,编码1个含299个氨基酸的蛋白质,为一外膜脂蛋白,此外膜蛋白在多种APP血清型之间具有很高同源性。将nlpi基因亚克隆至p ET32a(+)载体,转化大肠杆菌(Escherichia coli)BL21(DE3),用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导表达APP rNLPI,并经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDSPAGE)和蛋白质印迹(Western blot,WB)确认。结果表明,在34 k D处有特异性条带,可被相应的阳性血清识别。动物保护实验采用BALB/c小鼠(Mus musculus)进行,酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)用于测定免疫球蛋G(immunoglobulin G,IgG)抗体滴度。结果表明,弗氏不完全佐剂+50μg rNLPI、弗氏不完全佐剂+30μg rNLPI、弗氏不完全佐剂+10μg rNLPI、30μg rNLPI免疫后,可诱导小鼠分别产生40%、20%、10%和20%的存活率;佐剂组和生理盐水对照组存活率为0。幸存小鼠慢慢恢复健康,并正常采食。可见,rNLPI皮下免疫可诱导小鼠产生免疫应答和较高的抗攻击感染的免疫保护力。该研究结果为进一步筛选猪传染性胸膜肺炎分子疫苗积累了基础资料。Porcine contagious pleuropneumonia （PCP） is a serious respiratory disease with great harm to pigs （Sus scrofa）, caused by Actinobacillus pleuropneumoniae （APP） infection. It is one of the major epidemic diseases in the world pig industry. Existing vaccines can not give full protection against App infection, so it is necessary to screening new vaccines. In this study, immune protection of recombinant NLPI （rNLPI）vaccination against App challenged infection was detected. Based on APP nlpi sequences in GenBank, the primers were designed. APP nlpi sequence was obtained by PCR from APP genome. Sequence analysis and function predication was made by bioinformatics software. The results revealed that the length of this gene （GenBank No.： MH027649） was 900 bp and its predicat protein was a 299 amino acid resides of an puptative outer membrane lipoprotein. Homology analysis showed that it shared high sequence similarity between different serotypes of APP. The APP nlpi was inserted into pET32a （＋）. Recombinant protein rNLPI was produced in Escherichia coli BL21 after induced by isopropyl β-D-1-thiogalactopyranoside （IPTG） and then determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. The 34 kD-protein was detected and showed good immunogenicity. The protective efficacy of rNLPI experiment was tested in 6-week-old specific-pathogen-free BALB/c mice （Mus musculus）. The serum immunoglobulin G （IgG） level was assayed by enzyme-linked immune sorbent assay （ELISA）. The results showed that 50 μg rNLPI＋Frund＇s incompleted adjuvant, 30 μg rNLPI＋ Frund＇s incompleted adjuvant and 10 μg rNLPI＋Frund＇s incompleted adjuvant and 30 μg rNLPI respectively induced 41%, 22%, 10% and 20% survival rate. No mice survived the challenge in adjuvant group and control group. The rest living mice of experiment groups slowly returned to normal health status. From above, rNLPI induced a partial immune protection of mice against App challenged

关 键 词：猪胸膜肺炎放线杆菌 重组NLPI蛋(rNLPI) 免疫保护效果 小鼠 

分 类 号：S852.61[农业科学—基础兽医学]