摘要
根据GenBank中登录的副猪嗜血杆菌(HPS)D15基因鸟枪序列(登录号:NZ_ABKM01000005)设计1对特异性引物,以HPS5型SP毒株DNA为模板,通过PCR方法扩增了D15基因。将其进行T-A克隆,构建pMD18D15质粒并进行序列测定和分析。结果表明,D15基因含有一个2418bp的开放阅读框,编码805个氨基酸,与D15基因参考序列的同源性为99.9%。该HPSD15基因的推导氨基酸序列具有典型Omp85蛋白家族的拓扑结构特征。以pMD18D15质粒为模板,用PCR方法扩增HPSD15基因,并将其克隆到pET-28a(+)中,构建pETD15原核表达质粒,将其转化大肠杆菌Rosetta(DE3)后,用IPTG诱导重组菌,并用SDS-PAGE和Western-blot检测诱导物。结果表明,pETD15重组表达质粒在大肠杆菌中实现了高效表达,融合蛋白的分子质量约为94ku,融合蛋白能被HPS阳性血清识别,提示该融合蛋白具有反应原性。昆明小鼠和豚鼠免疫试验结果表明,D15重组蛋白具有一定的诱导免疫保护反应的能力。
The full-length D15 gene was amplified from Haemophilus parasuis（HPS） serotype 5 SP strain by PCR with a pair of primers based on the whole genome shotgun sequence（Accession No.：NZ_ABKM01000005） in GenBank.Sequencing showed that the complete open reading frame（ORF） with an ATG initiation codon comprised 2 418 nucleotides and encoded a protein of 805 amino acids.It shared 99.9% identity with the reference sequence.The deduced amino acid from HPS D15 gene had the typical topology structure of Omp85 protein family.The D15 gene was successfully cloned into pMD18-T vector,and then subsequently sub-cloned into expression vector pET-28a（＋） and the recombinant plasmid was then transformed into competent Rosetta（DE3） bacteria.SDS-PAGE and Western-blot analyses revealed that the recombinant Rosetta（DE3） bacteria induced by IPTG were able to express the D15 protein with molecular weight of 94 ku.Kunming mice and guinea pigs were immunized with the recombinant D15 protein,the rusults showed that the protein could provide certain protective immunity against diseases caused by HPS.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第2期158-163,共6页
Chinese Veterinary Science
基金
湖南省科技重大专项(2007FJ1003)
湖南农业大学人才引进基金项目(04YJ03)
关键词
副猪嗜血杆菌
外膜蛋白
原核表达
免疫原性
Haemophilus parasuis
outer membrane protein
prokaryotic expression
immunogenicity
