副猪嗜血杆菌Neu基因的克隆表达及表达产物的免疫原性

Cloning and expression of neuraminidase gene of Haemophilus parasuis and immunogenicity of its expressed product

为研究副猪嗜血杆菌(HPS)基因工程疫苗,以HPS5型毒株为模板,通过PCR扩增获得副猪嗜血杆菌神经氨酸酶(Neu)全基因,将其克隆至pMD18-T载体并对其进行测定和分析。结果表明,Neu全基因含一个2187bp的开放阅读框,编码一含729个氨基酸的蛋白,与Neu基因参考序列(登录号:NZ-ABKM01000006)的同源性为99.0%。以pMD-Neu基因为模板,重新设计引物,扩增得到HPSNeu蛋白的部分编码区,将其克隆至原核表达载体pET-28a(+)中,构建得到重组原核表达质粒pET-Neu。将pET-Neu在大肠杆菌Rosetta(DE3)中进行优化表达,经SDS-PAGE分析,重组蛋白Neu主要以包涵体形式表达,分子质量为52ku;Western-blot分析表明,重组蛋白Neu能与阳性血清发生特异性抗原反应。将纯化重组蛋白Neu试验性注射免疫昆明小鼠,攻毒试验结果显示,氢氧化铝佐剂组的保护性较低,仅有10%;而弗氏佐剂组具有较高的保护性,保护率高达60%。

The full-length Neu 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 amplified Neu gene was successfully cloned into pMD18-T vector and the complete open reading frame with an ATG initiation codon comprised 2187 nucleotides and encoded a protein of 805 amino acids.It shared 99.0% identity with that of the reference sequence.The HPS Neu gene fragment was amplified by PCR from pMD-Neu,and cloned into the prokaryotic expression vector pET-28a（＋） to obtain the recombinant prokaryotic expression vector pET-Neu.The pET-Neu was transformed into Escherichia coli Rosetta（DE3） and expressed optimally.The recombinant protein Neu of approximately 52ku was detected by SDS-PAGE and existed mainly in the form of inclusion bodies.Western-blotting showed that the recombinant protein Neu could specifically react with the antibodies in positive sera well.Kunming mice were immunized with the purified recombinant protein Neu,and challenged with HPS post-immunization.The results showed that the Al（OH）3 group got a poor protective（10%）,but the Freund＇s had a higher protection（60%）.