猪肺炎支原体168弱毒菌株P65-DnaKc融合蛋白的表达及免疫原性  

Expression and immunogenicity of fusion protein P65-DnaKc in attenuated strain of Mycoplasma hyopneumoniae 168

作　　者：刘思远[1] 刘东[1] 关婉怡[1] 鞠建松[1] 赵宝华[1] 

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024

出　　处：《中国兽医学报》2015年第9期1463-1467,共5页Chinese Journal of Veterinary Science

基　　金：河北省科技支撑计划资助项目(13226603D)

摘　　要：研究猪肺炎支原体168(Mycoplasma hyopneumoniae 168,Mhp168)弱毒株融合蛋白P65-DnaKc免疫原性。根据GenBank中Mhp168弱毒株p65和dnaKc基因的开放阅读框设计特异性引物,进行PCR扩增,构建原核表达载体pET-28α-P65,pET-28α-DnaKc以及pET-28α-P65-DnaKc,经原核表达并纯化获得的蛋白与佐剂以体积比1∶1乳化后免疫小鼠,通过酶联免疫吸附反应(ELISA)测定小鼠血清的抗体效价。选取抗体效价较高的小鼠进行攻毒试验,记录分析试验结果。经间接ELISA试验证明单蛋白P65、DnaKc以及融合蛋白P65-DnaKc均具有良好的免疫原性,血清效价分别为:1∶12 800、1∶51 200和1∶204 800,融合蛋白的血清抗体效价明显高于单蛋白;攻毒试验证明,P65-DnaKc重组蛋白疫苗的保护率可达90%。融合蛋白P65-DnaKc具有良好的优于P65和DnaKc单蛋白的免疫原性。The immunogenicity of fusion protein P65-DnaKc in attenuated strain of Mycoplasma hyopneumoniae 168 were studied. Specific primers were designed according to the open reading frame of p65 and dnaKc genes （p65,ID：16186824;dnaKc,ID：16186839） in Mhp168 attenuated strain from GenBank. The genes were obtained by PCR amplification,and prokaryotic expression vector pET-28a-P65, pET-28a-DnaKc and pET-28a-P65-DnaKc were constructed. The proteins were expressed in prokaryotes and the purified proteins were emulsified with adjuvant at a volume ratio of 1 ： 1 to immunize mice. The antibody titers of mouse serum were determined by enzyme- linked immunosorbent assay （ELISA）. The mice with high antibody titers were selected to con- duct virulence test,and the results were recorded and analyzed. Results of indirect ELISA indica- ted that both single protein P65,DnaKc and fusion protein P65-DnaKc had good immunogenicity, the serum titers of them were 1 ： 12 800,1 ： 51 200,and 1 ： 204 800,respectively. The antibody ti- ter of the fusion protein in the serum was significantly higher than that of single proteins. Viru- lence test demonstrated that the protection rate of recombinant P65-DnaKc protein vaccine was up to 90G. The immunogenicity of the fusion protein P65-DnaKc was better than that of single pro- teins P65 or DnaKc.

关 键 词：猪肺炎支原体 P65基因 dnaKc基因 融合蛋白 

分 类 号：S858.28[农业科学—临床兽医学]