沉默羊传染性脓疱皮炎病毒DNA聚合酶基因的RNAi载体的构建

Construction of a vector for RNA interference targeting DNA polymerase gene of Orf virus

为了构建用于沉默羊传染性脓疱皮炎病毒(Orf virus,ORFV)DNA聚合酶基因的2种载体,试验选择病毒复制所必需的DNA聚合酶基因作为干扰靶基因,并设计靶基因的扩增引物,经PCR扩增后测序;随后,与Check2载体分别经PmeⅠ和NotⅠ双酶切,酶切后连接产生Check2-靶基因(Check2-D)重组质粒;然后,利用公用网站按照RNAi序列设计的原则,设计RNAi靶点序列并合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoⅠ和HpaⅠ酶切后的pll3.7载体连接产生pll3.7-shRNA质粒;最后,利用磷酸钙法进行pll3.7-shRNA质粒和Check2-D质粒共转染293T细胞,利用双荧光检测系统试剂盒检测其RNA干扰效果。结果表明:成功构建了pll3.7-shRNA质粒和Check2-靶基因重组质粒,干扰效果最好,可达到91.0%。

To construct two vectors for RNA interference targeting DNA polymerase gene of Orf virus （ ORFV）, the ORFV genome was analyzed in order to select the DNA polymerase gene which the virus needed to copy as the targeted gene. The primers of the targeted gene were de- signed, and then the targeted gene which obtained by PCR was sequenced and connected with Check2 vector digested by Pine I and Not I. Check2 - targeted gene vectors were constructed. In accordance with RNAi sequence design principles in the public Web site, RNAi target se- quences were designed, and then the target sequences of Oligo DNA were synthesized and annealed to form double - stranded DNA, which were connected with pll3.7 vector digested by Xho I and Hpa I. pll3.7 - shRNA vectors were constructed. In the end, 293T cells were co - trans- feeted through pll3.7 - shRNA vector and Check2 - targeted gene vector by using the calcium phosphate method. The interference effect was as- sayed by the Dual - GloTM Luciferase Assay System. It is concluded that pll3. 7 - shRNA vectors and Check2 - targeted gene vectors are con- structed successfully, and the interference effect can reach 91.0% for the best case.