鸡白细胞介素2和白细胞介素18基因的原核及真核表达

Pronuclear and Eukaryotic Expression of Chicken Interleukin-2 and Interleukin-18 Genes

【目的】克隆并原核和真核表达无特定病原(SPF)鸡白细胞介素2(ChIL-2)和白细胞介素18(ChIL-18)基因,为后续开展ChIL-2和ChIL-18蛋白功能、生物学活性及联合应用的免疫佐剂研究打下基础。【方法】运用RT-PCR扩增ChIL-2和ChIL-18基因,并进行测序和序列比对分析,分别构建其重组原核表达质粒pET32a-ChIL-2和pET28a-ChIL-18及重组真核表达质粒pVAX1-ChIL-2和pVAX1-ChIL-18,并将重组原核表达质粒pET32a-ChIL-2和pET28a-ChIL-18分别转化大肠杆菌BL21,将重组真核表达质粒pVAX1-ChIL-2和pVAX1-ChIL-18分别转染非洲绿猴肾(Vero)细胞。【结果】重组原核表达质粒pET32a-ChIL-2和pET28a-ChIL-18经IPTG诱导表达,其表达产物经SDS-PAGE和Western blotting分析,结果表明融合蛋白ChIL-2和ChIL-18在大肠杆菌中获得高效表达,表达的蛋白分子量分别为27和23 kD;重组真核表达质粒pVAX1-ChIL-2和pVAX1-ChIL-18转染Vero细胞24 h后进行倒置荧光显微镜检测,结果显示融合蛋白ChIL-2和ChIL-18在Vero细胞中均有较高表达。【结论】克隆获得的ChIL-2和ChIL-18基因可在原核和真核细胞中高效表达。

【Objective】Cloning and prokaryotic and eukaryotic expression of specific pathogen-free(SPF) chicken interleukin 2(ChIL-2) and interleukin 18(ChIL-18) protein were to lay the foundation for future research on chicken IL-2 and IL-18 protein function, biological activity and combined application of immune adjuvants.【Method】The ChIL-2 and ChIL-18 genes were amplified by the reverse transcription polymerase chain reaction(RT-PCR) and genes sequence alignment and sequencing were performed. Prokaryotic recombinant expression plasmids pET32 a-ChIL-2,pET28 a-ChIL-18 and eukaryotic recombinant plasmids pVAX1-ChIL-2,pVAX1-ChIL-18 were constructed respectively. The recombinant prokaryotic expression plasmids pET32 a-CHIL-2 and pET28 a-CHIL-18 were transformed into Escherichiacoli BL21, and the eukaryotic expression plasmids pVAX1-ChIL-2,pVAX1-ChIL-18 were transfected into Vero cells, respectively.【Result】Prokaryotic recombinant plasmids pET32 a-ChIL-2 and pET28 a-ChIL-18 were induced by IPTG,and expression products were obtained respectively. SDS-PAGE and Western blotting analysis showed that ChIL-2 and ChIL-18 proteins were highly expressed in Escherichiacoli with molecular weights of 27 and 23 kDa. The eukaryotic recombinant plasmids pVAX1-ChIL-2,pVAX1-ChIL-18 were transfected into Vero cells for 24 hours. The results showed that the proteins of ChIL-2 and ChIL-18 were well expressed in Vero cells.【Conclusion】The chicken ChIL-2 and ChIL-18 genes were successfully cloned. ChIL-2 and ChIL-18 genes were highly expressed in both prokaryotic and eukaryotic cells.