介导大鼠结缔组织生长因子基因沉默的慢病毒载体转移质粒的构建及鉴定

目的：构建介导大鼠结缔组织生长因子（CTGF）基因沉默的慢病毒载体转移质粒pGCL-CTGF，为进一步包装慢病毒载体奠定基础。方法：以大鼠CTGF基因为靶基因，根据RNA干扰（RNAi）序列设计原则，设计4对有小发夹结构的RNAi靶点序列，退火形成双链DNA，双酶切后定向克隆到慢病毒载体转移质粒pGCL-GFP中，构建4个含靶基因片段的重组慢病毒载体转移质粒pGCL-CTGF，并对质粒进行PCR及测序鉴定。结果：CTGF的短发夹RNA（shRNA）片段被成功克隆到慢病毒载体转移质粒pGCL-GFP中，4个插入序列与设计的靶基因片段完全一致。结论：构建了能够表达4个含CTGF靶基因片段的慢病毒载体转移质粒，为进一步包装介导CTGF基因沉默的慢病毒载体奠定了基础。

Construction and Identification of the Lentiviral Vector Transferred Plasmid Mediating Rat Connective Tissue Growth Factor Gene Silencing

Objective： To construct the lentiviral vector transferred plasmid pGCL-CTGF mediating rat connective tissue growth factor（CTGF） gene silencing and set up the basis for further packaging the lentiviral vector. Methods： Taking rats CTGF as a target gene and according to the principle of designing RNA interference sequence, four pairs of two DNA sequences containing small hairpin structure were designed, and then were formed into double-stranded DNA by annealing. The obtained products were cloned into the lentiviral vector transferred plasmid pGCL-GFP after double digestion. The re- combinant lentiviral vector transferred plasmids pGCL-CTGF containing four target gene fragments were constructed. Finally the plasmids that were identified by PCR were used for sequence analysis. Results： The CTGF shRNA fragments were successfully cloned into the lentiviral vector transferred plasmid pGCL-GFP. And the shRNA coding sequences of the four obtained recombinant plasmids were exactly consistent with the designed fragments. Conclusion： The four recombinant lentiviral vector transferred plasmids of CTGF shRNA were successfully constructed. It laid the foundation for further packaging the lentiviral vector mediated CTGF gene silencing.