【作者】 程胖；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2010， 硕士

【摘要】 疯牛病是牛海绵状脑病(Bovine spongiform Eneephalopathy,BSE)的俗称,是由朊病毒引起的一种传染性的,慢性的,致死性的中枢神经系统退行性疾病。疯牛病是由Prnp基因编码的朊病毒蛋白PrPc发生构象变化为PrPsc而引起的传染性疾病, PrPsc在中枢神经的聚集引发了动物的传染性海绵状脑病。因此,降低(下调)PrPc的表达量使其减少向PrPsc转化,是抑制疯牛病的有效途径。RNAi是一种高效的特异性强的基因阻断技术,它可以特异性的降解细胞内与其序列同源的mRNA,封闭内源性基因表达,实现类似基因敲除的效果。因此本实验采取了RNA干涉的方法对疯牛病进行了基础研究。siRNA表达载体法能够在产生细胞内持续产生siRNA,介导较长时间的基因沉默,且带有抗生素标记的基因,能够进行药物筛选,可以获得稳定转染的细胞,适合长期的实验研究。本文利用RNAi技术,针对牛源cDNA序列设计了3段PrPc干扰序列及一个阴性对照,经反转处理并加酶切位点和loop环等相关处理后连到空载体pGenesil-1上,构建了针对prnp的干扰载体,分别命名为Prnp1,Prnp2,Prnp3和Prnp-NC。然后将4个干扰载体分别转染牛胎儿成纤维细胞,转染后的第2天,第3天分别收集瞬时转染的细胞,利用无菌通道的流式细胞仪分选出带荧光的细胞。用实时荧光定量PCR方法和进行Western blot法对其进行干扰效率的检测。结果表明,3个干扰片段的抑制效率分别是51.2%、85.7%、74.6%,阴性对照的干扰效率是2.05%,因而得到了一个较为有效的干扰片段prnp2。然后用700μg/mL G418对转染prnp2的牛胎儿成纤维细胞进行药物筛选,15 d之后获得了稳定转染的细胞单克隆。本实验结合无菌通道的流式细胞仪技术,在分选出瞬时转染的阳性细胞后进行干扰效率的检测,避免了细胞转染效率低对干扰效率检测的影响。在筛选出有效的干扰片段后进行了稳定转染,筛选出的细胞可以用于后续长期的实验研究。更多还原

【Abstract】 The mad cow disease is the Bovine spongiform Eneephalopathy’s(BSE) popular name. It is an infectious, chronic, lethal central nervous system recession disease which is caused by the prion protein– PrPsc. PrPsc is the isoform of normal prion protein PrPc, they are coded by prnp gene. Along with the aggregation of PrPsc in the central nervous system, it can result to the transmissible bovine spongiform encephalopathy. By cutting down PrPc’s expression to reach the purpose of decreasing its conversion into PrPsc is an effective way to inhibit BSE. RNAi is a highly efficient and specific gene knockdown technology, it can specially degrad it’s homology sequence’s mRNA in the cells, inhibit endogenous gene expression, then achieve the effect similar to gene knockout. So we employed RNA interference technology to knockdown prnp expression, it will contribute to deep research of this gene.SiRNA expression vector can continuously generate siRNA in the cells, make a long time gene silencing. And because it carries the antibiotic resistance gene, we can conduct drug screening to get the stably transfected cells, this is suitable for long time research. In this article, we used the technology of RNAi to cut down PrPc’s expression, three candidate shRNAs targeting the CDS of bovine prnp gene and a negative control were designed, after reverse transcription, restriction enzyme cutting site and loop structure introduction, they were ligated into the vector of pGenesil-1 respectively, then we successfully constructed 4 RNAi-vector targeted to prnp, they were termed Prnp1, Prnp2, Prnp3 and Prnp–NC respectively. We transfected the four vectors into bovine fibroblast cells separately, 2 days and 3 days after transfection, we collected the transient transfected cells with fluorescence by flow cytometry(FCM)in sterile channel, then examined their suppression efficiency by the means of Real-time PCR and Western blot, the results showed that the interference effects of the three shRNA vectors were 51.2%、85.7% and 74.6%, the negative control’s efficiency is 2.05%, so we obtained a more efficacious shRNA―Prnp2. After that, we used 700μg/mL G418 selected the fibroblast cells transfected with Prnp2, after 15 days, the positive cell clones were obatained.In this study, we combined the technology of FCM selected the transient transfected positive cells, then detected their interference efficiency, this can avoid the side effect of lower transfection efficiency. Except that, we obtained positive cell clones by the means of G418 selection, it provides a way for later study.更多还原