摘要
P65蛋白是猪肺炎支原体的主要免疫优势蛋白,与其他支原体无交叉反应,常作为猪肺炎支原体检测的靶蛋白。本研究通过基因合成获得P65基因中含有3个稀有密码子的前922bp,利用PCR从Mhp168株扩增获得P65基因后段,然后通过重叠延伸PCR(SOE-PCR)将前后段连接后,克隆入pET-32a(+)中,获得重组质粒pET-32a(+)/mhp P65,经IPTG诱导获得87ku的重组融合蛋白,Western blot分析表明其与Mhp血清呈强阳性反应。将该重组蛋白免疫小鼠获得了高滴度的特异性抗体,表明该蛋白具有良好的免疫原性。本研究成功表达了可溶性的Mhp P65融合蛋白,其具有较好的免疫原性,这为猪肺炎支原体P65蛋白的功能研究以及诊断学方法的建立和亚单位疫苗的研制提供了帮助。
Protein P65,the immunodominance protein of Mycoplasma hyopneumoniae(Mhp),and shows no cross reaction with other Mycoplasmas,is usually used as the target protein or gene for Mhp detection.In this study,the front 922bp fragment containing three rare codons in P65gene was obtained by gene synthesis,and the other fragment of P65gene was amplified by PCR from Mhp 168strain,then the mutated P65gene was obtained through the Splicing by overhang extension(SOE-PCR)with the surplus segment connection,finally the recombinant plasmid pET-32a(+)/P65was constructed by inserting the mutated P65gene into expression vector pET-32a(+).Under IPTG induction,E.coli BL21(DE3)/pET-32a(+)/P65expressed the recombinant P65protein with 87kD.The result of Western blot analysis showed that the recombinant protein has satisfactory immunoreactivity.High level of antibody was obtained in BALB/c mice after inoculating the recombinant P65protein,which indicated the recombinant P65protein has a good immunogenicity.We had successfully obtained the recombinant P65protein of Mhp by expressing in the E.coli,it provide the support for Mhp detection and subunit vaccine,and also offers the help for the further study of the pathogenic mechanism of this disease.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2014年第2期262-267,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
江苏省农业科技自主创新资金[CX(13)3066]
关键词
猪肺炎支原体
P65基因
原核表达
免疫原性
Mycoplasma hyopneumoniae(Mhp)
P65gene
prokaryotic expression
immunogenicity
