中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2009年
11期
1014-1018
,共5页
孙梅%李金东%姜睿%高楠%金成彦%罗树立%王荣有%张兴义
孫梅%李金東%薑睿%高楠%金成彥%囉樹立%王榮有%張興義
손매%리금동%강예%고남%금성언%라수립%왕영유%장흥의
CD80基因%RNAi%慢病毒
CD80基因%RNAi%慢病毒
CD80기인%RNAi%만병독
CD80%RNA interference%Lentivirus
目的:构建大鼠CD80基因的RNAi慢病毒载体并在大鼠NRK和IEC6细胞上鉴定其沉默效率.方法:将筛选获得的大鼠CD80基因特异性siRNA靶点,合成短发卡结构shRNA并退火成双链DNA,与pGCSIL-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,随后将其感染大鼠NRK细胞、IEC6细胞和大鼠DC细胞(树突状细胞,Dentritic cell),分别采用Real-time PCR和Western blot方法检测靶基因在mRNA和蛋白质水平的沉默效率.结果:构建的慢病毒载体shRNA的PCR鉴定和测序正确,包装病毒后滴度达到4×10~8 TU/ml.shRNA慢病毒颗粒感染NRK和IEC6细胞后CD80基因的 mRNA表达量较阴性对照载体慢病毒感染组分别下降了66.9%和63.5%.结论:成功构建了大鼠CD80基因的shRNA慢病毒表达载体,能够在细胞水平有效沉默靶基因.
目的:構建大鼠CD80基因的RNAi慢病毒載體併在大鼠NRK和IEC6細胞上鑒定其沉默效率.方法:將篩選穫得的大鼠CD80基因特異性siRNA靶點,閤成短髮卡結構shRNA併退火成雙鏈DNA,與pGCSIL-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,隨後將其感染大鼠NRK細胞、IEC6細胞和大鼠DC細胞(樹突狀細胞,Dentritic cell),分彆採用Real-time PCR和Western blot方法檢測靶基因在mRNA和蛋白質水平的沉默效率.結果:構建的慢病毒載體shRNA的PCR鑒定和測序正確,包裝病毒後滴度達到4×10~8 TU/ml.shRNA慢病毒顆粒感染NRK和IEC6細胞後CD80基因的 mRNA錶達量較陰性對照載體慢病毒感染組分彆下降瞭66.9%和63.5%.結論:成功構建瞭大鼠CD80基因的shRNA慢病毒錶達載體,能夠在細胞水平有效沉默靶基因.
목적:구건대서CD80기인적RNAi만병독재체병재대서NRK화IEC6세포상감정기침묵효솔.방법:장사선획득적대서CD80기인특이성siRNA파점,합성단발잡결구shRNA병퇴화성쌍련DNA,여pGCSIL-GFP만병독재체중조형성shRNA표체재체,이용PCR화측서감정획득련접정학적극륭.경유293T세포포장shRNA만병독과립,수후장기감염대서NRK세포、IEC6세포화대서DC세포(수돌상세포,Dentritic cell),분별채용Real-time PCR화Western blot방법검측파기인재mRNA화단백질수평적침묵효솔.결과:구건적만병독재체shRNA적PCR감정화측서정학,포장병독후적도체도4×10~8 TU/ml.shRNA만병독과립감염NRK화IEC6세포후CD80기인적 mRNA표체량교음성대조재체만병독감염조분별하강료66.9%화63.5%.결론:성공구건료대서CD80기인적shRNA만병독표체재체,능구재세포수평유효침묵파기인.
Objective:To construct a RNAi lentiviral vector targeting rat CD80 gene and detect its effect of gene silencing in NRK and IEC6 cells.Methods:The effective sequence of siRNA targeting rat CD80 gene was confirmed in our previous work.Oligo-DNA fragment containing short hairpin frame was synthesized and reannealed,and then cloned into pGCSIL-GFP lentiviral expression vector.PCR and sequencing analysis were made for verifying the positive clones.The virus packaging plasmids were transfected into 293T cells to harvest shRNA lentivirus.After infection in NRK and IEC6 cells,Real-time PCR was performed to determine the expressing level of CD80.Results:PCR and sequencing revealed that shRNA plasmids was correctly constructed.Virus with a titer of 4×10~8 TU/ml was successfully packaged.CD80 expression in NRK and IEC6 cells could be knockdown by virus infection as characterized by 66.9% and 63.5% decrease of CD80 mRNA in NRK and IEC6 cells respectively,compared with negative control lentivirus.Conclusion:The recombinant lentiviral shRNA expressing vector targeting rat CD80 gene has been successfully constructed and packaged.CD80 mRNA could be down-regulated availably in NRK and IEC6 cells.
