gga-miRNA-155对MDCC-MSB1细胞增殖的影响

Effects of gga-miRNA-155 on proliferation of MDCC-MSB1 cell

【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154bp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MD-CC-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。

【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 EcoRⅠand 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,EcoRⅠand XhoⅠdouble 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.