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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。
According to the sequence of p46 and p65 of attenuated Mycoplasma hyopneumoniae 168 (Mhp168), specific primers were designed and amplified by PCR. The expression vectors pET-28a-p46, pET-28a-p65 and pET-28a-p46- p65. Three TGA codons of the p46 gene were mutated to TGG using the expression vectors pET-28a-p46 and pET-28a-p46-p65 as templates. Prokaryotic expression and purification of protein and adjuvant in a volume ratio of 1: 1 emulsified mice were immunized by ELISA (enzyme-linked immunosorbent assay) antibody titers in mice serum. The mice with higher antibody titers were selected for the challenge test. The serum titer of single protein P46, P65 and fusion protein P46-P65 was 1: 1 280000,1:128 000 and 1: 2 560 000 respectively by indirect ELISA test. The challenge test proved that the single protein of P46, P65 and P46-P65 fusion protein vaccine protection rates of up to 82%, 78% and 98%. The fusion protein P46-P65 has good immunogenicity and is superior to the immunogenicity of the single proteins P46 and P65.