【作者】 邓文；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2008， 硕士

【摘要】 microRNA是一种调节性的非编码小分子RNA,其在生命活动中起着重要的作用。microRNA一般作用于靶标分子mRNA的非编码区,通过不完全互补配对的方式抑制靶标mRNA的翻译,从而对靶标基因在转录后水平进行调控。研究表明,microRNA在多种病毒感染过程中起着重要的调节作用,很可能在对猪瘟病毒(CSFV)的感染起着直接的调节作用。这种调节可能是由microRNA与CSFV的3’非编码区相互作用来实现的。通过生物信息预测,发现多种microRNA可能在CSFV上有作用位点。为研究microRNA在猪瘟病毒感染中可能起到的作用,本研究构建了绿色荧光蛋白报告系统,以利用绿色荧光蛋白来筛查microRNA在CSFV 3’非编码区的作用位点;同时还建立了表达miR-150 shRNA的PK-15细胞系,为研究microRNA对CSFV感染中的潜在作用打下基础。本研究获得以下结果:1.在猪脐静脉血管内皮细胞(SUVEC)上增殖CSFV石门株,对其增殖状况进行检测;设计引物,通过RT-PCR方法成功地扩增出了与预期大小相符的CSFV 3’UTR片段,与克隆载体pMD18-T载体连接后,经鉴定得到了猪瘟病毒石门株的3’UTR区域;重组克隆载体经酶切回收3’UTR片段,并将其连入真核表达载体pEGFP-C1,经酶切以及测序鉴定正确,表明成功地构建了融合猪瘟病毒石门株3’UTR片段的绿色荧光蛋白报告基因质粒载体;质粒用QIAGENE Plasmid Mini Kit纯化,获得了较高纯度的质粒DNA。2.采用RNA22和miRanda等软件,分别用已知的猪源microRNA和人源microRNA对猪瘟病毒基因组进行扫描,总结了其中可能作用于CSFV基因组上的microRNA,并重点对可能作用于CSFV非编码区的microRNA进行了关注,发现了猪的11个可能与CSFV基因组直接作用的microRNA。3.设计并合成了表达miR-150的两条shRNA序列的DNA单链,将其退火后与干扰载体pGenesil-1连接并测序鉴定了该重组载体的正确性,提取并纯化质粒后,由脂质体转染方法高效地转入PK-15细胞;通过G418抗性筛选获得了转染pGenesil和pGene-miR-150的阳性细胞,再由有限稀释法分别获得了转染pGenesil和pGene-miR-150质粒的PK-15细胞株,细胞株能分别持久转录表达miR-150的shRNA。更多还原

【Abstract】 microRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional gene regulation. miRNAs regulate their targets by imperfect complimentary match sequences in target site and result in translational repression. It is reported that miRNAs play important roles in virus infection and it maybe have direct regulatory functions in CSFV infection by interaction with 3’-noncoding region of CSFV RNA genome. Bioinformatics analysis shows that several miRNAs potentially targeted to CSFV genome. To investigate the function of cellular miRNAs in CSFV infection, we constructed a GFP report vector with a CSFV 3’UTR sequences at the 3 prime end of GFP mRNA to find the potential miRNAs binding sites on CSFV 3’UTR. And we also constructed a PK-15 cell line that express miR-150 shRNA to research the miRNA function in CSFV infection.1. Identification the replication of CSFV Shimen strain on SUVEC, and according to the 3’UTR sequences of the CSFV Shimen strain published, a pair of primers were designed to get the 3’UTR gene by RT-PCR. After enzymatic assay and recovering the products of PCR, the 3’UTR was cloned to the pEGFP-C1,named pEGFP-3’UTR and the recombination plasmid pEGFP-3’UTR was also identified by sequencing and enzymatic digestion. The the pEGFP -3’UTR was extracted and purified by QIAGENE Plasmid Mini Kit.2. Using the software RNA22 and miRanda, we predicted the target sites of swine and human miRNAs on CSFV genome, and get several miRNAs that may target on the genome RNA. Specially, we focused on the 11 miRNAs that may target to the un-translation region of the genome.3. Two pairs of single DNA chain encoding short hairpin RNA(shRNA)of miR-150 were designed and synthesized, and were ligated to the interference vector pGenesil-1. After sequencing identification and purification, recombination plasmid pGene-miR-150 were transfected into the PK-15 cells. The positive cells selected by optimal concentration of G418 were expanded and the monoclonal culture was carried out though the twice limiting dilution assay. Monoclonal strains were subcultured which can transcribe the short hairpin RNA persistently.更多还原