siRNA表达载体对乙酰肝素酶基因有效序列的筛选

Screening of effective sequence of heparanase gene-targeted by small interfering RNA

目的:根据基因库中的人乙酰肝素酶(HPSE)基因序列,构建针对乙酰肝素酶基因的小干扰RNA(siRNA)及其表达载体,转染人乳腺癌MDA-MB-231细胞后观察其对HPSE基因的干扰作用。方法:设计HPSE靶向的发夹状siRNA,合成两条互补的寡核苷酸链(共4对),退火后连接入pG-PU6/GFP/Neo载体,转化后进行序列鉴定,脂质体转染MDA-MB-231细胞株,免疫蛋白印迹(West-ernblot)检测细胞蛋白质的表达。结果:设计出4条针对HPSE的siRNA,将退火后的双链寡核苷酸片段克隆到pGPU6/GFP/Neo载体,经过阳性菌落酶切鉴定与测序,结果正确,并通过脂质体转染方法转染到MDA-MB-231细胞株,Westernblot检测细胞蛋白质的表达明显降低。结论:成功构建了针对HPSE基因的siRNA载体,转染MDA-MB-231细胞后,HPSE蛋白的表达降低,其中HPSE-A和HPSE-B可明显抑制HPSE的表达。

Objective： According to heparanase’s gene sequence of gene bank, to construct heparanase gene-targeted small interfering RNA （siRNA）and its expression vector and to observe its interfering effect on the expression of heparanase gene in human malignant breast cancer MDA-MB-231 cells. Methods： Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strand was synthesized and inserted into pGPU6/GFP/Neo vector,which was identified by sequence identify. Human malignant breast cancer MDA-MB-231 cells were transfected with the constructed vector with lipofectamine method. Western blot was per-formed to evaluate the expression of heparanase protein. Results： Four kinds of heparanase gene-targeted hairpin siRNA were designed, and were inserted into pGPU6/GFP/Neo vector after annealing. The vector containing siRNA was proved to be right by sequencing. The result of Western blot indicated that the expression of heparanase could be degraded by siRNA. Conclusion： The expression of heparanase can be degraded by siRNA method, and HPSE-A and HPSE-B showed the best results.