猪肺炎支原体P36基因在毕赤酵母中的表达

摘要

According to the sequence of P36 gene of Mycoplasma hyopneumoniae, a pair of primer was designed to amplify the whole P36 gene. Then the P36 gene was cloned into the pPICZα-A vector to construct the recombinant expression vector pPICZα-A-P36. The plasmid pPICZα-A-P36 was linearized with Sac Ⅰ digestion and was transformed into Pichia. pastoris X-33 by electroporation. The transformants were identified by PCR using 5' AOX1 and 3' AOX1 specific primers. SDS-PAGE and wetsern-blotting analysis showed that the P36 protein was successfully expressed with good reacting ability and secreted into the supernatant of medium when induced by methanol. The research provides the bases for establishing the diagnostic way of Mhp and preparing the engineering vaccine of Mhp.%根据GenBank中猪肺炎支原体P36基因序列设计1对特异性引物,通过PCR扩增出猪肺炎支原体特异性蛋白基冈P36,克隆入酵母表达载体pPICZα-A,构建分泌型重组酵母表达载体pPICZα-A-P36.PICZα-A-P36经Sac Ⅰ酶切线性化后电转化导入毕赤酵母菌株X-33.PCR鉴定为阳性的酵母转化子经甲醇诱导分泌P36蛋白于发酵卜清液中,通过SDS-PAGE电泳以及Western-blotting进行鉴定,结果表明,P36蛋白基因获得了分泌性表达,而且具有良好的反应原性,这为猪肺炎支原体免疫检测试剂盒和基因工程疫苗的研制奠定了基础.