猪IL-18基因的原核表达及重组蛋白的纯化

Prokaryotic expression of porcine interleukin-18 gene and purification of the expressed IL-18 protein

以pcDNA3.1-pIL-18为模板,采用PCR技术扩增到了猪白细胞介素18(IL-18)的成熟蛋白基因,通过KpnⅠ+SacⅠ双酶切及连接反应,构建了pET32c-pIL-18原核表达质粒。经过限制性内切酶分析、PCR鉴定及DNA序列测定证实,重组质粒中的基因片段连接正确。之后,重组质粒转化大肠杆菌BL21(DE3),于37℃、1.0 mmol/L IPTG条件下诱导表达。菌体裂解产物经SDS-PAGE分析,在分子质量约为33 ku处出现了预期的目的蛋白。用8 mol/L脲对表达产物变性,经Ni2+-NTA柱纯化,透析复性,得到了纯化的IL-18蛋白。Western-blot分析证实,纯化的重组IL-18蛋白具有反应活性。上述研究结果为重组IL-18的应用奠定了基础。

The mature protein gene of porcine interleukin-18 was amplified from recombinant plasmid pcDNA3.1-pIL-18 by PCR. The prokaryotic expression plasmid pET32c-pIL-18 was constructed through Kpn Ⅰ ＋ Sac Ⅰ digestion and ligation, and the recombination was identified by enzyme digestion, PCR amplification and DNA sequencing. The recombinant plasmid was transformed into Escherichia coli BL21 （DE3） competent cells, and expression of the protein was induced with IPTG at 37℃ overnight. SDS-PAGE analysis showed that the recombinant plasmid was highly expressed in E. coli, and molecular weight of the expressed protein was approximately 33 ku. After denaturation with 8 mol/L urea, the recombinant IL-18 protein which was purified with Ni^2＋-NTA His Bind Resin was refolded by dialysis against PBS and water. Western-blotting analysis confirmed that the purified protein had immunogenicity. These results provide the foundation for the practical application of the recombinant IL-18.