小鼠IL-27真核表达载体的构建及其在RAW264.7 细胞中的表达

目的:构建小鼠白介素27(Interleukin 27,IL-27)单链融合基因的真核表达载体并检验其在RAW264.7细胞中的表达情况。方法:提取小鼠脾细胞总RNA,通过RT-PCR扩增出小鼠EBI3和p28 c DNA。采用重叠延伸PCR(splicing by overlap extension PCR,SOE PCR)通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3和p28基因片段,构建小鼠IL-27单链融合基因(mouse single chain IL-27,msc IL-27),并将其克隆至pc DNA3.1(+)载体。通过酶切和测序鉴定阳性重组载体,将重组质粒pc DNA3.1-IL-27通过脂质体转染法转染小鼠巨噬细胞株RAW264.7,通过RT-PCR方法检测目的基因的表达。结果:测序分析表明,小鼠IL-27单链融合基因中EBI3、linker和p28的连接顺序、方向及碱基序列与预期相符。在转染后的RAW264.7细胞中检测到了小鼠IL-27 m RNA的表达。结论:成功构建了小鼠IL-27单链融合基因及其真核表达载体,并在RAW264.7细胞中实现表达,为进一步探讨IL-27的生物学功能奠定了基础。

Construction of Mouse Single Chain Interleukin-27 Fusion Gene and Its Expression in RAW264.7 Cells

Objective:To construct a eukaryotic expression plasmid encoding mouse single chain interleukin-27 (mscIL-27) fusion gene and verify its expression in RAW264.7 cells.Methods:The mouse EBI3 cDNA and p28 cDNA were amplified by RT-PCR fromthe total RNA extracted from spleen cells of Kunming mice. EBI3 and p28 mature peptide gene were fused via a hydrophobic polypeptide linker (Gly4Ser)3 by SOE PCR (splicing by overlap extension PCR) to obtain mscIL-27 fusion gene. The mscIL-27 fusion gene was cloned into eukaryotic expression vector pcDNA3.1 (+) and the positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing. The recombinant plasmid was transfected into RAW264.7 cells and the expression of target gene was detected by RT-PCR.Results:Sequence analysis showed that the splicing order, orientation and sequence of mscIL-27 fusion gene were completely correct. It was found that the expression of murine IL-27 mRNA could be detected in RAW264.7 cells.Conclusion:The mscIL-27 fusion gene was constructed successfully, and was detected in RAW264.7 cells, which will be helpful for the further research on its biological function.