PRS载体介导的表达CTGF-SiRNA逆转录病毒载体的构建

Construction of Retrovirus Expressing CTGF siRNA Mediated by PRS Retrovirus Vector

【目的】梅建表达人结缔组织生长因子（CTGF）小分子干扰RNA（small interfering RNA，SiR—NA）的PRS-CTGF-SiRNA逆转录病毒载体。【方法】根据SiRNA靶序列设计要求及PRS逆转录病毒载体特点分别设计含64bpDNA序列的4对寡核苷酸，并在体外合成。PRS载体用BgⅡ、HindⅢ双酶切完全后，分别与退火的4对寡核苷酸进行连接，连接后去载体自连，构建成表达人CTGF小分子干扰RNA的PRS-CTGF-SiRNA逆转录病毒载体。【结果】经酶切、连接后构建成的质粒称之为PRS-CTGF—SiRNA1～4，经酶切与测序证实构建成功，无任何碱基突变。【结论】成功构建了能表达人CTGF-SiRNA的PRS-CTGF—SiRNA逆转录病毒载体，为腹膜透析腹膜纤维化防治的体内、外研究提供一种可能有效的方法。

[Objective]To construct a retrovirus expressing connective tissue growth factor small interfering RNA （CTGFsiRNA） mediated by PRS retrovirus vector. [MethodslAccording to siRNA target design and PRS retrovirus desire , four pairs of oligonucleotides including 64bp DNA were designed. These four pairs of oligonucleotides were synthesized in vitro. After annealing, double stranded DNAs formed and were linked to retrovirus PRS vector separately. Vector self-linkages were removed. Retrovirs producing CTGFsiRNA were constructed from the inverted oligonucleotides. [Results]The constructed retrovirus vetor was called PRS-CTGF-SiRNA1～4 , their successful construction was identified by endonuclease digestion and sequence analysis , without any base pair mutation. [Conclusion]Retrovirus vectors PRS-CTGF-SiRNA1～4 expressing CTGFsiRNA are successfully constructed, which provide a highly effective method for prevention and treatment of peritoneal fibrosis in patients subjected to long-term peritoneal dialysis.