【作者】 冶贵生；

【导师】 张彦明；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2007， 博士

【摘要】 猪瘟病毒具有对猪专一性致病的特性,病毒虽能在多种体外培养的猪源细胞中增殖,但只引起猪血管内皮细胞(SVEC)发生细胞病变效应(CPE),而被感染的其他细胞不能够产生眼观的病理变化。产生这种现象的原因可能是猪血管内皮细胞被猪瘟病毒感染后刺激某种/某些敏感基因(我们将其称之为猪瘟病毒“敏感基因”)表达了某种/某些蛋白酶,将猪瘟病毒的p125蛋白裂解成为p80蛋白所致。本文在前期研究工作的基础上,采用RNA干扰技术,对11个候选猪瘟病毒“敏感基因”中的4个基因采用生物学软件对每一条基因设计、合成不同位置的shRNA干扰序列,以载体法介导实现shRNA分子在内皮细胞中的有效转录,筛选、鉴定差异基因的功能,为猪瘟病毒对猪专一性致病机理的研究提供科学资料。以RACE法延伸差异基因的编码序列,为差异基因在PK-15细胞中的表达及功能研究提供序列基础。将靶向猪瘟病毒NS3基因的shRNA重组干扰表达载体导入PK-15细胞中,为猪瘟病毒的抗感染以及基因功能的研究奠定基础。本研究获得了以下结果:1.应用在线RNA干扰设计软件针对4个差异基因中每一条基因设计不同位置的小发卡RNA干扰序列,合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到RNA干扰载表达体中,转化DH5α大肠杆菌感受态细胞,对转化产物进行卡那霉素筛选,碱裂解法提取重组质粒,将酶切鉴定为阳性的质粒进行核苷酸序列的测序,序列结果表明,重组载体构建成功,插入的干扰序列位置、大小、序列均正确。鉴定为阳性的重组质粒纯化后转染体外分离培养的猪血管内皮细胞,接种猪瘟病毒石门株并与不同时间断观察内皮细胞的病变情况。结果表明,转染了靶向差异基因Y8的shRNA重组干扰载体的内皮细胞,相对于对照细胞及其他的差异基因转染组,细胞的形态较好、存活率较高,差异基因Y8可能是所寻找的与猪瘟病毒致内皮细胞病变有关的“敏感基因”。2.为了验证差异基因Y8能否使猪瘟病毒致对PK-15产生细胞病变效应,以5’端标记磷酸基团的反转录引物反转录差异基因Y8的cDNA序列,然后用两对反式PCR引物以环化的cDNA序列模板进行5’端上游未知序列的扩增。以特异性上游引物与3’接头引物对差异基因Y8的cDNA序列3’端下游未知序列进行延伸。扩增的目的片断在T4 DNA酶的作用,分别连接到pMD18-T与pMD19-T simple vector载体中,转化DH5α大肠杆菌感受态细胞,挑取的重组质粒经酶切鉴定后进行核苷酸序列的测序。结果表明,通过5’RACE与3’RACE分别对差异基因Y8的cDNA序列5’端上游未知序列及3’端下游未知序列进行了延伸。3.应用软件分析猪瘟病毒NS3基因,设计针对NS3基因不同位置的小发卡RNA干扰序列,化学合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到pGenesil-1 RNA干扰表达载体中,构建重组载体pGene-NS3-1、pGene-NS3-2、pGene-NS3-3及阴性对照pGene-NS3-Neg,转化DH5α大肠杆菌感受态细胞,对转化产物进行卡那霉素筛选,碱裂解法提取重组质粒,将酶切、测序鉴定为阳性的重组干扰载体经Plasmid mini kit纯化后在脂质体的作用下转染PK-15细胞。G418抗性筛选获得稳定转染的阳性细胞,克隆细胞扩大培养后,接种猪瘟病毒Shimen株,72h时收集细胞分别进行实时定量PCR和ELISA光吸收度分析。Real-time PCR分析表明,pGene-NS3-1、pGene-NS3-2、pGene-NS3-3转录产生的shRNA分子与对照细胞比较,均在一定程度上沉默了病毒基因;ELISA检测结果表明,转染pGene-NS3-1、pGene-NS3-2、pGene-NS3-3重组载体的PK-15细胞均在不同程度上抑制了猪瘟病毒粒子的增殖。更多还原

【Abstract】 Classical swine fever virus(CSFV) effected specificly pathopoiesis for swine,CSFV could propagate in more swine cells in vitro culture without cytopathic effect(CPE),but had CPE in swine vascellum endothelial cells(SVEC).It’s that one or some protease which one or some“sensitive genes”(they were named with CSFV“sensitive genes”) expressed splited p125 protein to p80 protein after CSFV infected in SVEC possibly.On basis of previous study, we designed small hairpin RNA(shRNA) for four genes of eleven CSFV“sensitive genes”by biology software and synthetized them.We Screened and identified“different genes”by vector transcribing shRNAs in SVEC. The“different genes”sequences by Rapid Amplification of cDNA Ends(RACE) extending provided a basis for researching functions of different genes in PK-15 cells.It was a basis for gene functions and anti-virus by transfecting shRNAs recombinant vectors targeting CSFV NS3 into PK-15 cells.This study obtained following results:1. We designed shRNAs for four“different genes”by biology software and synthetized them.we annealed double-strands and ligated them into RNAi vector and transformed competent cell of E.coli DH5α.We screened and indentified recombinant plasmids, the results indicated that recombinant vectors were constructed successfully.These recombinant plasmids were transfected into SVEC and inoculated CSFV and observed cells pathological changes during different times.The results indicated that SVEC’s appearance and survival rate was better than control cells and other cells by transfecting recombinant vectors targeting for Y8“different gene”.The Y8“different gene”is CSFV“sensitive gene”with SVEC CPE possibly.2. Did CSFV infect PK-15 cells with CPE by“different genes”?cDNA sequcnce of“different gene”was transcribed reversely by RT primers. 5’-uptream unknows sequenc of Y8“different gene”was amplificated by two inverse PCR primers.3’-downstream unknows sequence of Y8“different gene”was amplificated by specific upstream primer and 3’-adaptor primer.Objective genes was ligated into pMD18-T and pMD19-T simple vector and transformed competent cell of E.coli DH5α.We identified recombinant plasmids by restriction analysis and sequence analysis.The results indicated that 5’upstream unknows sequence and 3’downstream unknows sequence of Y8“different gene”were extended by 5’RACE and 3’RACE.3. We analyzed and designed shRNAs for CSFV NS3 gene by biology software and synthetized them.We annealed double-strands and ligated them into pGenesil-1 vector, the constructing recmbinant vectors for pGene-NS3-1,pGene-NS3-2,pGene-NS3-3 and pGene-NS3-Neg negative control were transformed competent cell of E.coli DH5α. Recombinant vectors were transfected into PK-15 cells by liposome and screened positive cells.Cells were collected for Real-Time PCR and ELISA after CSFV were inoculated into cells in 72h.Real-Time PCR results indicated that shRNAs that were transcribed by pGene-NS3-1 and pGene-NS3-2 and pGene-NS3-3 silenced CSFV gene partly;ELISA results indicated that shRNAs inhibitted CSFV propagation partly.更多还原