猪传染性胸膜肺炎放线杆菌毒素Ⅲ A基因的克隆及其在巴斯德毕赤酵母中的分泌表达

Cloning and expression of Actinobacillus pleuropneumoniae Apx Ⅲ A gene in Pachia pastoris

应用PCR方法扩增猪传染性胸膜肺炎放线杆菌(Actinabacillus pleuropneumoniae,App)编码毒素A结构蛋白的完整基因,连接到载体pMD18-T中,经Not和Snab双酶切后连接到用同样酶切的表达载体pPIC9k上,转化GS115酵母感受态细胞,经甲醇诱导表达了分泌到胞外的Apx A蛋白。SDS-PAGE分析显示,表达的蛋白约为110 000,与预期相符。Dot-blot和ELISA检测结果表明,表达蛋白具有良好的抗原性和特异性。为进一步研究Apx A蛋白的结构和功能,研制App重组诊断抗原或亚单位疫苗奠定了基础。

The full length of gene encoding Apx Ⅲ A of Actinobacillus pleuropneumoniae （App） was amplified by polymerse chain reaction （PCR） and cloned into vector pMD18-T. Then the extracted plasmid DNA was digested with Snab I and Not 1, followed by subsequent ligation into the eykaryotic expression vector, pPIC9k, to con- struct an expression plasmid p9k-Apx Ⅲ A which was transformed into GS115 cells using LiCI method. Apx Ⅲ A protein was expressed by adding methanol and secreted into culture medium. SDS-PAGE analysis demonstrated the presence of expressed product with the size of approximately 110 000. Dot-blotting indicated that recombinant protein had a good antigenicity. The Apx Ⅲ A-ELISA was developed to detect the App antibody using the purified fusion protein. No cross reaction was found when other antisera were tested by this technique. It is suitable for the clinical diagnosis of actinobacillus pleuropneumoniae infection.