摘要
目的:克隆小鼠白细胞介素17A(mIL-17A)基因,构建mIL-17A原核表达质粒在E.coil Rosetta(DE3)PlysS表达,得到有活性的mIL-17A蛋白。方法:提取人IL-1β免疫的小鼠胸腺总RNA,反转录成cDNA,以此为模板,根据GenBank报道的mIL-17A序列设计引物,进行巢式PCR,得到成熟mIL-17A的编码序列并构建到pMD18-T载体中,再亚克隆至原核表达载体pHisSUMO Express中,经DNA测序鉴定后转化Rosetta感受态细胞,IPTG诱导表达,Western blot鉴定。纯化后的蛋白处理3T3-L1前体脂肪细胞,real time PCR鉴定白细胞介素6(IL-6)的表达变化。结果:DNA测序证明所克隆基因序列与GenBank报道的完全一致。成功构建了pHisSUMO Express-mIL-17A原核表达质粒以包涵体形式表达了重组mIL-17A蛋白,且Western blot证实为目的蛋白。所得蛋白上调3T3-L1细胞表达IL-6。结论:获得具有生物活性的mIL-17A蛋白,为进一步研究其蛋白特性及其生物活性奠定基础。
Objective：To clone mouse interleukin-17A （mIL-17A）,construct mIL-17A prokaryotic expression plasmid and express bioactive mIL-17A protein in E.coil Rosetta （DE3）PlysS.Methods：The total RNA of the mouse thymus stimulated by human IL-1β was abstracted,and then the RNA was reverse-transcripted into cDNA that was used as the template for PCR reaction.The primers were designed according to the published mIL-17A sequence.The cDNA coding for mature mIL-17A was cloned by nested PCR.The mIL-17A cDNA was subcloned to the prokaryotic expression vector pHisSUMO Express and confirmed by DNA sequencing.The pHisSUMO Express-mIL-17A was transformed into the ROSETTA competence cells.The recombinant bacteria were induced by IPTG for protein expression,and the Western blot was used to confirm the protein.The purified protein was used to stimulate the pre-adipocyte 3T3-L1 cells.Expression of IL-6 was detected by real time PCR.Results：The mIL-17A was cloned from the mouse thymus stimulated by human IL-1β.The recombinant mIL-17A was expressed from prokaryotic system and purified as an inclusion body.The mIL-17A was confirmed by Western blot using conmmerially available antibody against mIL-17A.The purified mIL-17A upregulated the expression of IL-6 in pre-adipocytes 3T3-L1 cells.Conclusion：The mouse IL-17A is cloned from thymus and bioactive mIL-17A is purified for further study of the cytokine.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2010年第9期783-786,共4页
Chinese Journal of Immunology
基金
哈尔滨市科技创新人才研究专项资金(2006RFXXS002)
东北农业大学创新团队项目(CXZ001)
