小鼠IL-39的克隆及真核表达载体的构建

Cloning of mouse interleukin-39 and construction of its eukaryotic expression vector

[目的]构建小鼠白介素(interleukin,IL)-39单链融合基因及其真核表达载体。[方法]通过RT-PCR得到小鼠EB病毒诱导基因3(Epstein-Barr virus-induced gene 3,EBI3)及IL-23p19基因的全长编码区。通过重叠延伸PCR和编码疏水性多肽接头(linker)(Gly4Ser)3的DNA序列将小鼠EBI3全长编码区及IL-23p19成熟肽编码区连接起来,构建小鼠IL-39单链融合基因,并将其克隆至真核表达载体pc DNA3.1/V5-His中,通过限制性内切酶双酶切及基因测序鉴定阳性重组载体。[结果]通过重叠延伸PCR得到1 254 bp大小的目的条带,测序分析显示,小鼠IL-39单链融合基因中EBI3、linker和IL-23p19的基因序列及连接顺序和方向均完全正确。[结论]成功构建了小鼠IL-39单链融合基因及其真核表达载体。

[Objective]To construct mouse single chain interleukin-39（ IL-39） fusion gene and its eukaryotic expression vector. [Methods]The mouse Epstein-Barr virus-induced gene 3（ EBI3） c DNA and IL-23p19 c DNA were amplified by RT-PCR. EBI3 whole coding sequence and IL-23p19 mature peptide coding sequence were fused via a hydrophobic polypeptide linker（ Gly4Ser） 3 by overlap extension PCR to obtain mouse single chain IL-39 fusion gene. The mouse single chain IL-39 fusion gene was cloned into eukaryotic expression vector pc DNA3. 1/V5-His and the positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing. [Results] Overlap extension PCR results showed that1 254 bp target band was obtained. The sequence analysis showed that the sequence,splicing order and orientation of mouse single chain IL-39 fusion gene were completely correct. [Conclusion] The research has successfully constructed the mouse single chain IL-39 fusion gene and its eukaryotic expression vector.