| 摘要: |
| 【目的】构建含有let-7c前体基因的重组质粒pGene-pre-let-7c，建立稳定表达猪miRNA let-7c细胞株，研究let-7c对猪瘟病毒(CSFV)复制的调控作用。【方法】利用RNA22软件筛选出CSFV基因组3′-UTR 潜在的let-7c结合位点，PCR扩增出let-7c前体片段，连接到表达载体pGenesil-1.1-dBm2上,构建重组质粒pGene-pre-let-7c，采用脂质体法将重组质粒转染猪脐静脉血管内皮细胞（SUVEC），经G418抗性压力筛选获得阳性细胞株。对获得的阳性细胞株进行接毒试验，检测CSFV的复制情况。用MTT比色法检测阳性细胞的增殖情况。【结果】成功扩增出let-7c前体基因，并构建了pGene-pre-let-7c重组质粒。重组质粒转染SUVEC细胞后筛选获得了稳定表达let-7c的细胞株。let-7c可下调CSFV在细胞中的复制。【结论】let-7c可下调CSFV的RNA复制水平。 |
| 关键词: 猪瘟病毒 猪脐静脉血管内皮细胞 miRNA let-7c |
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| 基金项目:国家自然科学基金项目(30771607);家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2007KFKT012) |
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| Establishment of cell line expressing miRNA let-7c and regulation on classical swine fever virus |
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| Abstract: |
| 【Objective】The study was to construct a recombinant vector bearing the let-7c gene and to research on potential regulation of classical swine fever virus（CSFV）by let-7.【Method】let-7c was selected by Bioinformatics of RNA22.Two pairs of single DNA chain encoding pre-let-7c were synthesized,and were ligated to the expression vector pGenesil-1.1-dBm2.The recombinant plasmid pGene-pre-let-7c was constructed and transfected into the swine umbilicus vein endothelial cells（SUVEC）by liposome after extraction and purification.The positive clones were selected by G418 screening.We also analyzed the expression of CSFV through the approach of real time fluorescence quantitative RT PCR and MTT was performed to detect cell proliferatime.【Result】The pGene-pre-let-7c was successfully constructed and pre-let-7c was expressed in porcine vascular endothelial cell line.【Conclusion】let-7c can inhibit the duplication of CSFV. |
| Key words: classical swine fever virus(CSFV) swine umbilicus vein endothelial cells（SUVEC） miRNA let-7c |
