小鼠Smad6基因RNAi慢病毒载体的构建与鉴定

Construction and identification of lentiviral vector of RNA interference of mouse Smad6 gene

构建小鼠Smad6基因RNA干扰(RNAi)慢病毒载体,有效沉默骨髓树突状细胞(BMDC)的Smad6基因表达,为构建骨髓致耐受DC用于哮喘等自身免疫疾病的研究。设计小鼠Smad6 shRNA序列,合成、退火,得到双链DNA,与经酶切后的Psih1-H1-copGFP shRNA Vector载体连接产生LV-shSmad6慢病毒载体,并测序鉴定。转染293TN细胞,包装产生慢病毒,测定滴度。感染小鼠骨髓树突细胞,检测Smad6基因的表达状况成功构建Smad6 shRNA的慢病毒载体LV-shSmad6。包装慢病毒,并显著抑制Smad6 mRNA水平及蛋白水平的表达。成功构建出小鼠Smad6基因shR-NA慢病毒载体,为后期研究Smad6基因在哮喘发病机制及新治疗方法提供了稳定的转染细胞载体。

To construct a lentiviral vector of RNA interference（RNAi） of mouse Smad6 gene and to effectively silence the Smad6 gene expression of bone marrow derived dendritic cells for construction the bone marrow tolerogenic dendritic cell for providing experimental foundation of athma of autoimmune disease,Smad6 shRNA sequence of mouse was designed by software.The complementary DNA containing both sence and antisence DNAs of the targeting sequence was designed,synthesized and cloned into the Psih1-H1-copGFP shRNA vector containing Smad6 shRNA named LV-shSmad6 which was confirmed sequencing.293TN cells were cotransfected.PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of Smad6 producing Smad6 shRNA was constructed successfully.Smad6 mRNA level and protein level express were kept down notability.