小鼠IL-27真核表达载体的构建及其在RAW264.7细胞中的表达

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

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。

Objective： To construct a eukaryotic expression plasmid encoding mouse single chain interleukin-27 （msclL-27） fusion gene and verify its expression in RAW264.7 cells. Methods： The mouse EBI3 eDNA and p28 eDNA were amplified by RT-PCR from the total RNA extracted from spleen ceils of Kunming mice. EBI3 and p28 mature peptide gene were fused via a hydrophobie polypeptide linker （Gly4Ser）3 by SOE PCR （splicing by overlap extension PCR） to obtain msclL-27 fusion gene. The msclL-27 fusion gene was cloned into eukaryotic expression vector pcDNA3.1 （＋） and the positive recombinant clone was analyzed by digestion of restriction en- donuclease and DNA sequencing. The recombinant plasmid was transfeeted 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 mselL-27 fusion gene were completely correct. It was found that the expression of murine 1/,-27 mRNA could be detected in RAW264.7 cells. Conclusions： The mselL-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.