| 摘要: |
| 【目的】构建gga-miRNA-155前体基因（pre-gga-miRNA-155）真核表达载体，验证gga-miRNA-155在MDCC-MSB1细胞中的表达效果，探究其对MDCC-MSB1细胞增殖的影响。【方法】采用PCR方法从鸡肝脏基因组DNA中扩增gga-miRNA-155前体基因片段pre-gga-miRNA-155，并将其克隆入pMD-18T载体，构建克隆载体pMD-18T-pre-gga-miRNA-155。用EcoRⅠ和XhoⅠ双酶切pcDNA6.2-GW/EmGFP-miRNA真核表达载体和pMD-18T-pre-gga-miRNA-155，将pre-gga-miRNA-155酶切回收片段与pcDNA6.2-GW/EmGFP-miRNA的酶切回收片段进行连接，构建重组真核表达载体pcDNA6.2-pre-gga-miRNA-155，对其进行PCR、EcoRⅠ和XhoⅠ双酶切及DNA测序鉴定。将重组载体转染到MDCC-MSB1细胞中，应用实时荧光定量PCR ( Real-time PCR )检测gga-mi-RNA-155的表达水平，利用MTT法检测细胞增殖能力的变化。【结果】PCR扩增获得了长度约154 bp的鸡pre-gga-mi-RNA-155基因。成功构建了pre-gga-miRNA-155的真核表达载体pcDNA6.2-pre-gga-miRNA-155，瞬时转染MDCC-MSB1细胞后gga-miRNA-155表达水平显著增加，且MDCC-MSB1细胞的体外增殖能力增强。【结论】成功构建了pre-gga-miRNA-155的真核表达载体，其可在MDCC-MSB1细胞中过表达gga-miRNA-155，且可促进MDCC-MSB1细胞的体外增殖。 |
| 关键词: gga-miRNA-155 表达载体 MDCC-MSB1细胞 细胞增殖 |
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| 基金项目:国家自然科学基金项目（U1504308，31702207）；河南科技大学博士科研启动基金项目(13480068)；河南科技大学省部级科技创新平台培育项目（2015SPT004） |
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| Effects of gga-miRNA-155 on proliferation of MDCC-MSB1 cell |
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YU Zuhua,DING Ke,YU Chuan,et al
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| Abstract: |
| 【Objective】This study constructed the eukaryotic expression vector of gga-miRNA-155 precursor gene fragment (pre-gga-miRNA-155) and tested its expression efficiency in MDCC-MSB1 to explore the effects on proliferation of MDCC-MSB1.【Method】The precursor gene fragment of gga-miRNA-155 was amplified by PCR from the genomic DNA of chicken liver tissue,before being cloned into the pMD-18T vector to construct the cloning vector pMD-18T-pre-gga-miRNA-155.The eukaryotic expression vector pcDNA6.2-GW/EmGFP-miRNA and cloning vector pMD-18T-pre-gga-miRNA 155 were digested by the restriction enzymes EcoRⅠ and XhoⅠ,the pre-gga-miRNA-155 and pcDNA6.2-GW/EmGFP-miRNA gene fragments were recycled,and the pre-gga-miRNA-155 gene fragment was connected to the pcDNA6.2-GW/EmGFP-miRNA to construct the recombinant vector pcDNA6.2-pre-gga-miRNA-155.The accuracy of the recombinant eukaryotic expression vector was verified by PCR,EcoRⅠ and XhoⅠ double enzyme digestion,and DNA sequencing.The MDCC-MSB1 cells were transfected with the recombinant vector pcDNA6.2-gga-miRNA-155,and the expression of gga-miRNA-155 was evaluated by real-time quantitative PCR.The proliferation of MDCC-MSB1 cell was also determined by MTT.【Result】The pre-gga-miRNA-155 gene fragment with length of 154 bp was obtained by PCR.The eukaryotic expression vector of gga-miRNA-155（pcDNA6.2-pre-gga-miRNA-155) was successfully constructed.The expression level of gga-miRNA-155 was significantly increased in pcDNA6.2-pre-gga-miRNA-155 transfected MDCC-MSB1 cells.The proliferation of MDCC-MSB1 cells in vitro was also promoted.【Conclusion】The eukaryotic expression vector of pre-gga-miRNA-155 was successfully constructed.It could over-express gga-miRNA-155 in MDCC-MSB1 cell and promote the proliferation of MDCC-MSB1 cell. |
| Key words: gga-miRNA-155 expression vector MDCC-MSB1cell cell proliferation |
