| 摘要: |
| 【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。 |
| 关键词: 猪白细胞介素18 真核表达 生物学活性 猪流感灭活疫苗 |
| DOI： |
| 分类号: |
| 基金项目:国家 “十一五”科技支撑计划专项（2006BAD06A08） |
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| Construction of eukaryotic expression plasmid of porcine IL-18 gene and identification of bioactivity of its expressed protein |
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LIU Zeng wen1a 2 CHEN Kai1b MI Cai hong1a LI Qian1b
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| Abstract: |
| 【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 EcoRⅠand XhoⅠ,then inserted into eukaryotic expression vector pcDNA3.1(+) to generate an expression plasmid pIL-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 pIL-18 was constructed,and its expression protein had bioactivity. |
| Key words: porcine interleukin-18 eukaryotic expression bioactivity swine influenza vaccine |
