载体介导的shRNA抑制猪瘟病毒在PK-15细胞中增殖特性的研究

利用生物学软件分析猪瘟病毒NS3基因,设计针对NS3基因不同位置的干扰序列,化学合成这些序列,然后退火连接为双链干扰片段,再定向克隆到干扰载体中,将酶切和序列测定鉴定正确的重组载体命名为pGene-NS3-1、pGene-NS3-2、pGene-NS3-3、pGene-NS3-Negative。重组干扰载体经纯化后转染PK-15细胞,并进行G418抗性筛选。克隆细胞扩大培养后,接种猪瘟病毒Shimen株,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,与对照细胞比较,pGene-NS3-1、pGene-NS3-2、pGene-NS3-3转录产生的shRNA分子均在一定程度上沉默了病毒的基因,抑制率约为63%、46%、49%。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。

Inhibition of CSFV''''s Propagation in PK-15 Cells by shRNA Expression Vector

Interference sequences toward different positions of CSFV NS3 gene were designed,these sequences were annealed and cloned into interferece plasmid.Recombinant interferencing plasmids were named pGene-NS3-1,pGene-NS3-2,pGene-NS3-3 and pGene-NS3-Negative(negative control) by restriction analyzing and sequencing.These recombinant plasmids were transfected into PK-15 cells and were screened by G418.CSFV were inoculated in cultured cells,transfected by interference plasmid.Collected cells were analyzed by Real-time PCR and ELISA.The results of Real-time PCR indicated that gene were silenced by pGene-NS3-1,pGene-NS32 and pGene-NS3-3,and inhibition ratio were 63%,46%,49% respectively.The results of ELISA indicated that CSFV's propagation were inhibited in PK-15 cells by pGene-NS3-1,pGene-NS3-2 and pGene-NS3-3 in different extent.