人白细胞介素-31的真核表达及其对HaCaT细胞IL-6和TNFα表达的影响  

Eukaryotic expression of hIL-31 and effects on the IL-6 and TNFα in HaCaT

作　　者：黄海良[1] 罗欣[1] 顾芳[1] 张胜权[1] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,安徽合肥230032

出　　处：《中国生化药物杂志》2011年第5期345-348,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：安徽省重点实验室优秀中青年科研带头人专项基金;安徽医科大学博士基金

摘　　要：目的构建人白细胞介素-31(hIL-31)基因真核表达载体并将其转染到HaCaT细胞,观察其对HaCaT细胞炎症因子分泌的影响。方法分离人外周血单核细胞,加入终浓度为20 nmol/L的豆蔻佛波醇乙酯(PMA),继续培养24 h收集细胞。RT-PCR克隆hIL-31 cDNA,并将其克隆到真核表达载体pSecTag2/Hygro B上。将构建成功的重组体转染至HaCaT细胞,用Western blot分析hIL-31在细胞中的表达以及RT-PCR方法分析其对IL-6、TNFα表达的影响。结果经双酶切、测序及Blast分析鉴定,我们成功获得hIL-31基因cDNA全长序列和pSecTag2/HygroB-IL-31重组体。转染至HaCaT细胞的外源性hIL-31可以促进IL-6、TNFα的表达。结论 hIL-31可以通过自分泌的方式促进HaCaT细胞炎性相关细胞因子的表达。Purpose To clone the encoding sequence of human IL-31 gene,to construct recombinant eukaryotic expression plasmid vector and to study its effects on the expression of IL-6 and TNFα in HaCaT.Methods Full-length hIL-31 cDNA was amplified by RT-PCR and cloned into the pSecTag2/HygroB plasmid vector.Western-blot analyzes the expression of hIL-31 in HaCaT.RT-PCR analyzes the effects of hIL-31 on the expression level of IL-6 and TNFα.Results The recombinant plasmid was cut with restriction endonuclease HindⅢ and XhoⅠ.By the confirmation by sequencing and nucleotide homology analyzed to GenBank,we obtained the hIL-31 cDNA and the recombinant plasmid pSecTag2/HygroB-Il-31 successfully.And the exogenous hIL-31 can promote the expression of IL-6 and TNFα.Conclusion HIL-31 can promote the expression of inflammation factors by the way of autocrining in HaCaT.

关 键 词：人白介素-31 真核表达 皮肤角质形成细胞 

分 类 号：Q78[生物学—分子生物学]