针对乙酰肝素酶基因的短发夹RNA干扰载体的构建

Construction of short hairpin RNA vector targeted to heparitinase gene

目的构建对人乙酰肝素酶(hpa)基因有特异性抑制作用的短发夹RNA(shRNA)表达载体。方法根据GenBank数据库提供的hpa基因核苷酸序列,按照shRNA设计原则设计5对特异性的寡核苷酸序列及阴性对照,分别克隆到pGPU6/GFP/Neo载体中,酶切鉴定和测序验证阳性克隆。结果经酶切、测序鉴定均证实重组质粒构建成功。结论成功构建了5个靶向人hpa的shRNA表达载体,为进一步研究该载体转染MDA-MB-231细胞后对hpa基因的沉默效果奠定基础。

Objective To construct expression vectors of short hairpin RNA（shRNA） having specific inhibition for heparitinase（hpa） gene.Methods According to nucleotide sequences of hpa gene supplied from GenBank database,five pairs of specific interference sequences and negative control were designed with shRNA principle of design,and were cloned into the vectors of pGPU6/GFP/Neo respectively.Then the masculine clones were confirmed by restriction enzyme digestion and sequencing analysis.Results Both restriction enzyme digestion and sequencing analysis proved that the shRNA expression vectors were constructed successfully.Conclusion Five shRNA expression vectors are successfully constructed,which establishes foundation for further researching the effects of these vectors to hpa gene after transfected into MDA-MB-231 cell.