猪IL-18基因真核表达质粒的构建及其表达产物的生物学活性

Construction of eukaryotic expression plasmid of porcine IL-18 gene and identification of bioactivity of its expressed protein

【目的】构建猪IL-18的重组质粒,检测其表达产物的生物学活性,为进一步研究猪IL-18基因的结构与功能奠定基础。【方法】通过PCR技术从含猪IL-18全基因的克隆质粒中扩增猪IL-18基因,定向克隆到真核表达载体pcDNA3.1(+)中,构建重组质粒pIL-18,在脂质体作用下转染猪肾PK15细胞。【结果】重组质粒pIL-18经酶切、测序鉴定,证实含有目的片段,且连接、构建正确。通过RT-PCR检测,证实了猪IL-18在PK15细胞中的表达;SDS-PAGE分析结果表明,表达产物是与猪IL-18相符的约22 ku的蛋白条带;Western blot证实,表达产物能与猪IL-18单克隆抗体发生特异性反应。pIL-18对H1亚型猪流感灭活疫苗免疫增强作用的研究表明,pIL-18能够提高猪流感灭活疫苗诱发的细胞免疫应答。【结论】成功地构建了猪IL-18的重组质粒pIL-18,并在PK15细胞中获得了瞬时表达,且具有一定的免疫原性。

[Objective] The research was to construct eukaryotic expression vector of porcine IL-18 and detect bioactivity of its expressed protein. [Method] Porcine IL-18 gene was amplified by PCR with recombinant plasmid pGEM IL-18 as a template. The PCR product was digested with EcoR Ⅰ and Xho Ⅰ,then inserted into eukaryotic expression vector pcDNA3. 1 （＋） to generate an expression plasmid plL-18 and transfected into the PK15 cells by lipofectamine 2 000. [Result] Porcine IL-18 mRNA expression was found in the PK15 cells. SDS-PAGE and Western blot indicated that porcine IL-18 gene had already inserted into PK15 cell chromosome. The results showed that recombinant protein was about 22 ku. The recombinant protein can react with porcine IL- 18 monoclonal antibody. Immunoenhancement effect of recombinant expression plasmid pIL-18 on swine influenza vaccine was observed by proliferation response of the T lymphocytes from spleen. It can obviously enhance the cell-mediated immune response. [Conclusion] The recombinant plasmid plL-18 was constructed,and its expression protein had bioactivity.