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Position: Home > Articles > Prokaryotic expression and immune function analysis of giant panda interleukin-18 Chinese Veterinary Science 2014 (10) 1084-1088

大熊猫白细胞介素-18的原核表达及免疫功能分析

作  者:
王强;严悦;邓家波;牛李丽;余建秋
单  位:
成都动物园;成都动物园四川大学生命科学学院生物资源与生态环境教育部重点实验室四川省分子生物学及生物技术重点实验室;成都大熊猫繁育研究基地;四川省分子生物学及生物技术重点实验室四川大学生命科学学院成都市九眼桥望江路29号
关键词:
大熊猫;白细胞介素-18;原核表达;免疫功能;小鼠
摘  要:
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。
译  名:
Prokaryotic expression and immune function analysis of giant panda interleukin-18
作  者:
WANG Qiang;YAN Yue;DENG Jia-bo;NIU Li-li;YU Jian-qiu;YIN Meng-meng;ZHANG Yi-zheng;TAN Xue-mei;Chengdu Zoo;Key Laboratory for Bio-Resources and Eco-Environment,Ministry of Education/Sichuan Key Laboratory of Molecular Biology and Biotechnology/College of Life Sciences,Sichuan University;
关键词:
giant panda;;interleukin-18;;prokaryotic expression;;immune function;;mouse
摘  要:
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.

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