副猪嗜血杆菌ompP2基因在猪肺泡巨噬细胞中的表达

为了进一步研究副猪嗜血杆菌(HPS)ompP2基因的功能,根据已发表的副猪嗜血杆菌核苷酸序列,设计并合成1对引物,PCR扩增HPS LZ株外膜蛋白ompP2基因,获得大小为1 158bp的核苷酸片段,该片段纯化后克隆至pEASY-Blunt载体,转化后鉴定,再与真核表达质粒pCI-neo经过酶切和连接反应,转化感受态大肠杆菌Trans-T1,双酶切鉴定,筛选阳性重组质粒并测序。通过脂质体法将pCI-neoompP2转染猪肺泡巨噬细胞,通过RT-PCR和Western blot方法检测重组质粒是否表达。结果显示,成功构建了含ompP2基因的副猪嗜血杆菌真核表达载体pCI-neo-ompP2,提取转染该质粒的猪肺泡巨噬细胞的mRNA,进行RT-PCR扩增,在1 000bp~2 000bp之间可见1条明显的条带。Western blot发现在42ku处出现目的条带。试验结果为后期研究副猪嗜血杆菌感染猪肺泡巨噬细胞的机理奠定了基础。

Expression of Haemophilus parasuis ompP2 Gene in Porcine Alveolar Macrophages

In order to study the function of Haemophilus parasuis (HPS)ompP2 gene,a pair of primers were designed and synthesized according to nucleotide sequence of HPS ompP2 gene.The ompP2 gene of HPS LZ was amplified strain.The amplified ompP2 gene was cloned into pEASY-Blunt vector.The ompP2 gene was sequenced and cloned into the eukaryotic expression vector pCI-neo.The recombinant plasmid was used to transform competent Trans-T1 and the positive clones were identified by restriction enzyme di-gestion.Thus,the recombinant pCI-neo-ompP2 was transfected to porcine alveolar macrophages (PAM). The expression of pCI-neo-ompP2 was detected by RT-PCR and Western blot.The result showed that a 1 158 bp ompP2 gene fragment was amplified from the HPS DNA.The eukaryotic expression vector pCI-neo-ompP2 containing ompP2 gene was constructed successfully.The 1 000-2 000 bp DNA was amplified from the mRNA extracted from the PAM transfected with pCI-neo-ompP2 by RT-PCR and the 42 ku pro-tein was detected by Western blot.This study provided an experiment fundation for studying the mecha-nism which HPS infects PAM.