摘要
以pcDNA3.1-IL18为模板进行PCR扩增获得猪白细胞介素-18(IL-18)的成熟肽基因,以KpnⅠ、SacⅠ双酶切PCR产物作为供体,KpnⅠ、SacⅠ双酶切的pET41c和pET32c作为载体,连接载体供体,转化DH5α,经双酶切、PCR鉴定及测序筛选出阳性重组质粒,并命名为pET41c-IL18和pET32c-IL18。将pET41c-IL18和pET32c-IL18转化大肠埃希氏菌BL21(DE3),用1 mmol/L IPTG 37℃诱导表达。SDS-PAGE及W estern-blotting分析结果表明,所表达的重组蛋白主要以包涵体形式存在,pET32c-IL18重组蛋白分子质量约33 ku,与预期大小相符。将包涵体提取物用8 mol/L脲变性,经N i-NTA柱纯化,透析复性,得到了纯化的IL-18蛋白。
The mature protein gene of porcine interleukin-18 was amplified from recombinant plasmid pcDNA3.1- IL18 by PCR. After being digested by restriction endonucleases, the IL-18 mature protein gene was subcloned into the prokaryotic expression vector pET41c and pET32c. After restriction enzyme digestion, PCR identification and sequence analysis of the recombinant plasmid, the positive recombinant plasmid was selected and designated as pET41c-IL18 and pET32c-IL18. The pET41c-IL18 and pET32c-IL18 were transformed into Escherichia coli BL2, （DE3） , and then induced with IPTG at 37℃ overnight. SDS-PAGE and Western-blotting analysis Showed that the expressed recombinant IL-18 protein presented in inclusion bodies and was about 33 ku in molecular weight. Following denaturation with 8 mol/L urea, the recombinant IL-18 protein which was purified with Ni-NTA His Bind Resin was refolded by dialysis against PBS and water.
出处
《中国兽药杂志》
2007年第2期5-9,共5页
Chinese Journal of Veterinary Drug
