猪白细胞介素18基因的克隆及真核表达重组质粒的构建

摘要

To obtain recombinant eukaryotic expression plasmid of porcine interleukin-18(IL-18),the whole gene of porcine IL-18 gene was amplified from porcine spleen,lung and lymph nodes by RT-PCR and cloned into eukaryotic expression vector pZJ-1.The recombinant expression plasmid pZJ-IL-18 were identified by enzyme digestion and sequencing analysis,and was transfected into 293T cells.The expression of IL-18 was detected by Real-time PCR and Western lblotting in both gene and protein levels.The results showed that the eukaryotic expression plasmid of porcine IL-18 was constructed and could express transiently in 293T cells.Western blotting result confirmed that porcine IL-18 polyclonal antibody could react specifically with approximately 17 ku expression products,and indicated that IL-18 could express correctly and be responsive.This study constructed the eukaryotic expression plasmid of porcine IL-18 gene which could express transiently in 293T cells,and laid the foundation for studying function of IL-18.%为获得表达猪白细胞介素18(interleukin-18,IL-18)的真核表达重组质粒,试验通过RT-PCR从猪脾脏、肺脏和淋巴结组织中扩增猪IL-18全基因并定向克隆到真核表达载体pZJ-1,测序分析和酶切鉴定正确后,转染至293T细胞中,并通过实时荧光定量PCR和Western blotting分别在基因和蛋白水平检测IL-18的表达.结果表明,试验成功构建了真核表达重组质粒pZJ-IL-18,且可以在293T细胞中表达IL-18基因.Western blotting试验证实,猪IL-18多克隆抗体能与约17 ku的表达产物发生特异性反应,表明IL-18能正确表达且具有反应原性.本试验构建了表达猪IL-18基因的真核表达质粒,并在293T细胞中瞬时表达,为进一步研究IL-18的功能奠定基础.