中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2009年
7期
660-663
,共4页
孙梅%李金东%姜睿%高南%王荣有%金成彦%张兴义
孫梅%李金東%薑睿%高南%王榮有%金成彥%張興義
손매%리금동%강예%고남%왕영유%금성언%장흥의
CD86基因%RNAi%慢病毒载体%树突状细胞
CD86基因%RNAi%慢病毒載體%樹突狀細胞
CD86기인%RNAi%만병독재체%수돌상세포
CD86 gene%RNA interference%Lentivirul vector%Dendritic cells
目的 构建大鼠CD86基因的RNAi慢病毒载体并在大鼠原代培养树突状细胞(DC)上鉴定其基因沉默效率.方法 将筛选获得的大鼠CD86基因特异性siRNA靶点,合成短发卡结构shRNA序列并退火成双链DNA,与pgC-GFP慢病毒载体重组形成shRNA表达载体,利用PCR和测序鉴定获得连接正确的克隆.经由293T细胞包装shRNA慢病毒颗粒,随后将其感染原代培养的大鼠Dc细胞,采用real-time PCR和Western blot的方法检测靶基因在mRNA和蛋白水平的沉默效率.结果 构建的慢病毒载体shRNA的PcR鉴定和测序正确,shRNA慢病毒颗粒感染大鼠DC细胞后CD86基因的mRNA表达量较阴性对照载体慢病毒感染组下降了90.6%;蛋白表达显著抑制.结论 成功构建了大鼠CD86基因的shRNA慢病毒表达载体,能够在大鼠DC细胞上有效沉默靶基因.
目的 構建大鼠CD86基因的RNAi慢病毒載體併在大鼠原代培養樹突狀細胞(DC)上鑒定其基因沉默效率.方法 將篩選穫得的大鼠CD86基因特異性siRNA靶點,閤成短髮卡結構shRNA序列併退火成雙鏈DNA,與pgC-GFP慢病毒載體重組形成shRNA錶達載體,利用PCR和測序鑒定穫得連接正確的剋隆.經由293T細胞包裝shRNA慢病毒顆粒,隨後將其感染原代培養的大鼠Dc細胞,採用real-time PCR和Western blot的方法檢測靶基因在mRNA和蛋白水平的沉默效率.結果 構建的慢病毒載體shRNA的PcR鑒定和測序正確,shRNA慢病毒顆粒感染大鼠DC細胞後CD86基因的mRNA錶達量較陰性對照載體慢病毒感染組下降瞭90.6%;蛋白錶達顯著抑製.結論 成功構建瞭大鼠CD86基因的shRNA慢病毒錶達載體,能夠在大鼠DC細胞上有效沉默靶基因.
목적 구건대서CD86기인적RNAi만병독재체병재대서원대배양수돌상세포(DC)상감정기기인침묵효솔.방법 장사선획득적대서CD86기인특이성siRNA파점,합성단발잡결구shRNA서렬병퇴화성쌍련DNA,여pgC-GFP만병독재체중조형성shRNA표체재체,이용PCR화측서감정획득련접정학적극륭.경유293T세포포장shRNA만병독과립,수후장기감염원대배양적대서Dc세포,채용real-time PCR화Western blot적방법검측파기인재mRNA화단백수평적침묵효솔.결과 구건적만병독재체shRNA적PcR감정화측서정학,shRNA만병독과립감염대서DC세포후CD86기인적mRNA표체량교음성대조재체만병독감염조하강료90.6%;단백표체현저억제.결론 성공구건료대서CD86기인적shRNA만병독표체재체,능구재대서DC세포상유효침묵파기인.
Objective To construct an RNAi lentiviral vector targeting rat CD86 gene and detect its effect of gene silencing in dendritic cells. Methods The effective sequence of siRNA targeting rat CD86 gene was confirmed in our previous work. DNA oligo containing short hairpin frame was synthesized and re-annealed, and then cloned into pGCL-GFP lentivind expression vector. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids were transfected into 293T cells to har-vest shRNA lentivirus. After infection in dendritic cells, real-time PCR and Western blot were performed to determine the expression level of CD86 at mRNA and protein of NC( negative control virus). Results PCR and sequencing analysis revealed that shRNA plasmid was correctly constructed. Virus with a titer of 2×108 TU/ml was successfully packaged. CD86 expression in dendritic cells can be knockdown at both mRNA and protein level by virus infection as characterized by 90.6% decrease of mRNA and significant inhibition of protein compared with NC. Conclusion The recombinant lentiviral shRNA expressing vector targeting rat CD86 gene has been successfully constructed and packaged. CD86 mRNA and protein can be effectively down-regulated in dendritic cells.
