猪肺炎支原体168弱毒株P46-P65融合蛋白的免疫原性  

Immunogenicity of P46-P65 fusion protein of avirulent Mycoplasma hyopneumoniae strain 168 in mice

作　　者：彭丽萍[1] 刘思远[1] 朱晓萍[1] 陈珍[1] 姚昌茹 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》2017年第4期626-631,共6页Chinese Journal of Veterinary Science

基　　金：河北省科技支撑计划资助项目(13226603D)

摘　　要：根据猪肺炎支原体168(Mycoplasma hyopneumoniae 168,Mhp168)弱毒株p46和p65基因序列设计特异性引物,进行PCR扩增并构建表达载体pET-28a-p46,pET-28a-p65以及pET-28a-p46-p65。以表达载体pET-28a-p46和pET-28a-p46-p65为模板,将其中p46基因3个TGA密码子突变为TGG。原核表达并纯化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。分别选取抗体效价较高的小鼠进行攻毒试验。经间接ELISA试验证明单蛋白P46、P65以及融合蛋白P46-P65的血清效价分别为:1∶1 280000,1∶128 000和1∶2 560 000;攻毒试验证明,P46、P65单蛋白和P46-P65融合蛋白疫苗的保护率分别可达82%,78%和98%。融合蛋白P46-P65具有良好的免疫原性,并且要优于单蛋白P46和P65的免疫原性。The experiment researched the immunogenicity of the P46-P65 fusion protein of avirulent Mycoplasrna hyopneurnoniae strain 168. Specific primers were designed according to the gene se quence of p46 and p65 in avirulent Mycoplasma hyopneumoniae strain 168. The genes were ob- tained by PCR amplification to construct the prokaryotic expression vector pET-28a-p46, pET-28a- p65 and pET-28a-p46-p65. The three TGA in p46 gene were point mutated to TGG with the templates of pET-28a-p46 and pET-28a-p46-p65. 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 immune sorbent assay （ELISA）. The mice with high antibody titers were selected to conduct toxic substances test. Results of indirect ELISA indicated that the serum titers of P46,P65 single protein and fusion protein were 1 ： 1 280 000,1 ： 128 000 and 1 ： 2 560 000, respectively; conteraeting toxic substances test demonstrate that the protection rate of P46 and P65 single protein and fusion protein vaccine were up to 82%, 78% and 98%, respectively. The immunogenicity of the fusion protein P46-P65 was better than that of single proteins P46 and P65.

关 键 词：猪肺炎支原体 P46基因 P65基因 融合蛋白 

分 类 号：S858.28[农业科学—临床兽医学] S852.62[农业科学—兽医学]