小鼠IL-17基因的克隆及其真核表达载体的构建

Molecular cloning of mouse IL-17 gene and construction of its eukaryotic expression vector.

目的克隆小鼠IL-17(mIL-17)基因编码区并构建其真核载体。方法将10 ug LPS和等体积的完全福氏乳化后免疫C57BL/6小鼠,7 d后,取小鼠脾脏,提取脾细胞总RNA,通过RT-PCR扩增mIL-17基因全长编码区并将其克隆至pcDNA3.1载体。菌落PCR筛选阳性克隆,限制性内切酶消化和序列分析进行鉴定。结果构建的重组载体中含有mIL-17基因编码区的全长序列,与NCBI公布的序列一致。结论获得mIL-17基因并构建了其真核表达载体,为研究IL-17在自身免疫性疾病中的作用奠定了基础。

Objective To clone the mouse IL - 17 gene and construct its eukaryotic expression vector. Methods The cDNA encoding mIL - 17 was amplified by RT - PCR using the total RNA extracted from spleen cells Of C57BL/6 mice immunized with LPS in CFA. The PCR product was cloned into pcDNA3.1 vector and then transformed into E. eoli DHSet. The positive recombinant clone was analyzed by PCR, digestion of restriction endonuclease and DNA sequencing. Results The recombinant pcDNA3.1 - mIL - 17 had a complete open reading frame of mIL - 17 and shared 100% homology with the sequence of mRNA for raiL - 17 in gene bank. Conclusion The cDNA of raiL - 17 is cloned successfully, which will be helpful for the further research on its biological function in autoimmune.