大熊猫白细胞介素-18的原核表达及免疫功能分析

Prokaryotic expression and immune function analysis of giant panda interleukin-18

以本实验室构建的含有GpIL-18编码区序列的质粒pMD-GpIL-18为模板,PCR扩增GpIL-18ORF后连接到pET-28a原核表达载体,转化大肠杆菌Rosetta(DE3)进行诱导表达,纯化目的蛋白(PGpIL-18)。利用荧光定量PCR检测PGpIL-18蛋白对C57BL/6J小鼠脾细胞中细胞因子的影响。将PGpIL-18蛋白与细菌疫苗同时注射小鼠,检测其免疫增强效应。Western-blot结果表明,IPTG诱导PGpIL-18融合表达;其产物PGpIL-18能刺激小鼠脾细胞大量分泌IFN-γ,增加GM-CSF和TNF-α的表达量。同时注射PGpIL-18蛋白和细菌疫苗的小鼠血清中IgG的质量浓度明显高于对照组。同时,通过金黄色葡萄球菌和大肠杆菌攻毒试验,发现PGpIL-18蛋白可以提高疫苗对小鼠的保护率。PGpIL-18能促进特异性免疫应答的发生,这一结果为重组PGpIL-18蛋白作为佐剂的开发利用奠定了理论基础。

GpIL-18 gene was cloned from pMD-GpIL-18 vector by PCR.The sequence encoding GpIL-18 mature peptide was inserted into pET-28 avector and expressed in Escherichia coli Rosetta(DE3),and purified by His-tag.The biological function of PGpIL-18 was determined by the expression level of cytokines from C57BL/6Jmice splenocytes by quantitative real-time PCR.In order to detect the immune efficacy of PGpIL-18 on antimicrobial,mice were vaccinated with a bacterial vaccine co-administrated with PGpIL-18 protein.It was confirmed that GpIL-18 could be expressed in E.coli by Western-blot.IFN-γ,GMCSF and TNF-αin splenocytes were apparently elevated when stimulated by GpIL-18.In vivo,IgG in mice injected with PGpIL-18 and vaccines dramatically increased.Under protection of PGpIL-18,murine survival rates were larger than that of control groups when attacked by Staphylococcus aureus and E.coli.In conclusion,PGpIL-18 is a potential cytokine to promote the specific immunity,which lay the theoretical foundation for application of recombinant PGpIL-18 as an adjuvant.