| 路晓红 付玉荣 伊正君 路静 张德峰.小鼠IL-27真核表达载体的构建及其在RAW264.7 细胞中的表达[J].,2016,16(13):2425-2429 |
| 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 ItsExpression in RAW264.7 Cells |
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| DOI： |
| 中文关键词: 白介素27 融合基因 重叠延伸PCR 真核表达载体 RAW264.7 |
| 英文关键词: Mouse interleukin-27 Fusion gene Splicing by overlap extension PCR Eukaryotic expression vector RAW264.7 |
| 基金项目:国家自然科学基金项目（81170080，81470001）；潍坊医学院2011 年科技创新前沿探索重点研究项目（K11TS1004） |
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| 中文摘要: |
| 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 cDNA。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, mscIL-27），并将其克隆至pcDNA3.1(+)载体。通过酶切和测序鉴定阳性重组载体，将重组质粒pcDNA3.
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 mRNA 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。 |
| 英文摘要: |
| 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. |
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