人结缔组织生长因子小干扰RNA真核表达载体的构建

Construction of Small Interfering RNA of Human Connective Tissue Growth Factor

目的结缔组织牛长因子(connective tissue growth factor,CTGF)是近年发现的一种重要促纤维化细胞因子,本实验旨在构建以人CTGF基因为靶点的siRNA(small interfering RNA,siRNA)表达载体,进一步探索肝纤维化的基因治疗提供有力工具。方法首先将pGenesil-1质粒的EGFP部分基因片段切除,成为新的pG质粒,然后根据人结缔组织生长因子的基因序列,及互不配对原则,设计3条针对人CTGF的siRNA DNA片段,分别命名为CTGF1-3,同时设无关序列为阴性对照,命名为CTGF0,然后将CTGF l-3及CTGF0克隆到siRNA载体pG质粒中,转化大肠杆菌DH5 Q菌株,挑取阳性菌落进行测序鉴定。结果经测序,成功构建了3条CTGF-siRNA重组质粒。结论成功构建靶向人CTGF基因的siRNA真核表达载体,为进步进行CTGF的功能研究奠定基础。

Objective Connective tissue growth factor（CTGF）is an important promoting fibrosiscell factor in recent years.This experiment aims to build a CTGF gene as the small interfering RNA（siRNA）that targets in expression vector.and provides a powerful tool to explore the gene therapy of liver fibrosis in the future.Method First,the gene of EGFP was cut off from pGenesil-I plasmid.which then was transferred into new a plasmid named pG.Next,according to the gene sequence of connective tissue growth factor and the matching principle,three candidate shRNAs targeting the CTGF gene（CTGF1-3）and a negative control（CTGF0）were synthesized.Then,clone the CTGF1-3 and CTGF0 into pG plasmid from the siRNA in order to transfer into the Escherichia coli DH5 alpha strains.Positive colonies were picked up to determine the enzyme digestion and sequencing identification.Results After the examining,succeeded in building three CTGF-siRNA of recombinant plasmid.Conclusion This successful construction of siRNA targeting people CTGF gene eukaryotic expression vector lax＇s a foundational study of CTGF foundation.