摘要
目的克隆人IL-31基因,构建原核表达载体,并诱导其在大肠杆菌中表达,研究hIL-31蛋白与皮肤炎症的关系。方法采用RT-PCR克隆人IL-31基因,将其克隆到原核表达载体pET28a(+),并通过IPTG诱导目的基因在大肠杆菌中表达,用该目的蛋白刺激人表皮角质细胞HaCaT,RT-PCR检测趋化因子MIP-3β、TARC及TCA-3(I-309)mRNA的表达;小鼠皮内注射该目的蛋白,观察局部炎症表现,皮肤标本HE染色观察皮肤炎性特征,采血进行白细胞计数及分类,ELISA法检测血清IL-1β、IL-6和TNF-α水平,流式细胞仪分析胸腺及脾脏T细胞亚群。结果成功获得495bp的人IL-31基因,原核表达质粒构建正确,IPTG诱导目的基因在大肠杆菌中大量表达;该目的蛋白可刺激HaCaT表达趋化因子MIP-3β、TARCI、-309;小鼠皮肤注射部位有脱毛,HE染色可见炎性细胞浸润,外周血白细胞总数增加,中性粒细胞比例增高,血清IL-1β、IL-6和TNF-α水平增高,脾脏CD4+T细胞百分比增高,CD8+T细胞百分比减少。结论人IL-31基因克隆及原核表达已获成功,hIL-31蛋白可刺激HaCaT细胞表达MIP-3β、TARC、I-309,诱导小鼠皮肤炎症反应。
Objective To construct prokaryotic expression vector for human IL-31 gene, and study its relationship with skin inflammation. Methods Full-length human IL-31 cDNA was amplified by RT-PCR. The functional fragment of human IL-31 gene without signal peptide was amplified by PCR and cloned into the pET28（ ＋ ） vector. The recombinant protein was achieved in E. coli through IPTG induction. The effects of the recombinant protein on the production of MIP-3β, TARC, and I-309 were detected by RT-PCR. The levels of serum IL-1β, IL-6 and TNF-α were detected by ELISA. Cell suspensions prepared from thymus and spleens were analyzed by flow cytometry. Balb/c mice was injected intradermally with hIL-31. Histopathology of paraffin-embedded skin section from the Balb/c mice was stained with hematoxylin and eosin, and then observed with microscope. The blood cells were counted.Results The obtained full encoding sequence of hIL-31 was identical with that included in Genbank, and the prokaryotic expression vector pE-T28a（ ＋ ）-hIL31 was construct correctly. IL-31 could be expressed in E. coli after IPTG induction. Soluble IL-31 could stimulate the expressions of MIP-3β, TARC, and I-309 in HaCaT. Mice treated with IL-31 had hair-loss and inflammatory cell infiltration. The number of peripheral white blood cells, the percentage of the neutrophil, and the serum IL- 1β, IL-6, and TNF-α were increased. The data from flow cytometric analysis indicated that proportion of spleens CD4^＋ T lymphocytes was increased. Conclusion Human IL-31 is cloned and expressed successfully in E. coll. IL-31 can stimulate the expressions of MIP-3β, TARC, and I-309 in HaCaT cells and induce skin inflammation in mice.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第3期306-310,共5页
Immunological Journal
关键词
人IL-31
克隆
表达
皮肤炎症
Human IL-31
Cloning
Expression
Skin inflammation
