脯氨酰羟化酶RNA干扰慢病毒载体的构建与鉴定

背景：慢病毒载体可稳定介导基因沉默且具有较高的转染效率。目的：构建并鉴定脯氨酰羟化酶RNA干扰慢病毒载体。方法：针对脯氨酰羟化酶2基因序列设计RNA干扰靶序列,合成靶序列的寡聚DNA,退火形成双链DNA,与经AgeⅠ和EcoRⅠ酶切的pGCSIL-GFP连接、转化大肠杆菌感受态细胞,产生重组RNA干扰慢病毒表达载体,PCR筛选阳性克隆,测序鉴定。结果与结论：PCR和DNA测序证实合成的含脯氨酰羟化酶短发卡RNA慢病毒载体寡核苷酸链正确插入pGCSIL-GFP载体。说明实验成功构建脯氨酰羟化酶基因RNA干扰慢病毒载体。

Construction and identification of lentiviral vector of RNA interference of prolyl-4 hydroxylase-2 gene

BACKGROUND：Lentiviral vector can stably mediate gene silencing with high transfection efficiency.OBJECTIVE：To construct and identify a lentiviral vector of RNA interference（RNAi） of prolyl-4 hydroxylase-2（P4HA2） gene.METHODS：The effective sequence of siRNA targeting P4HA2 gene was confirmed in our previous study.The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pGCSIL-GFP vector to construct a lentiviral vector which expressed short hairpin RNA（shRNA）,and it was identified by PCR and DNA sequencing.RESULTS AND CONCLUSION：PCR identification and DNA sequencing demonstrated that insertion of oligonucleotide of the lentivirus RNAi vector containing P4HA2 shRNA was right.Results suggested that the lentivirus RNAi vector of P4HA2 was constructed successfully.