胸膜肺炎放线杆菌外膜脂蛋白的原核表达及免疫原性分析

以猪传染性胸膜肺炎放线杆菌(APP)血清1型SC-A株基因组DNA为模板,用PCR扩增外膜脂蛋白(OML)基因特异片段,并克隆于pMD18-T中,经酶切及核苷酸序列分析鉴定后,亚克隆于原核表达载体pET-32 a(+),成功构建了重组表达载体pET-mOML。以此转化大肠埃希氏菌BL21(DE3),IPTG诱导表达,SDS-PAGE鉴定,表达的融合蛋白(TRX-mOML)分子质量约为60 ku,表达产物主要以包涵体形式存在,采取非变性电泳方法对蛋白进行纯化,经ELISA检测,重组OML免疫小鼠可产生较高水平的抗OML抗体,这表明重组OML有较好的免疫原性。

Prokaryotic expression and immunogenicity analysis of outer membrane lipoprotein of Actinobacillus pleuropneumoniae

The gene fragment of outer membrane lipoprotein(OML) was amplified from genomic DNA of Actinobacillus pleuropneumoniae(APP) strain SC-A by PCR and cloned into pMD18-T vector.Plasmid DNA was extracted and digested with enzymes and sequenced to confirm its rightness.The OML gene was subcloned into the expression vector pET-32a(+),and the recombinant plasmid was named as pET-mOML.The plasmid pET-mOML was transformed into E.coli BL21 and expressed under the induction of 1.0 mmol/L IPTG.The results of SDS-PAGE showed a protein band with an approximate molecular weight of 60 ku.The fusion protein could react with mice serum against mOML,as confirmed by ELISA.In conclusion,the OML gene was highly expressed in the prokaryotic system and the expressed protein had a good immunogenicity.