小鼠RelB基因RNAi慢病毒载体的构建与鉴定  

Construction and identification of RNAi lentiviral vector on murine RelB

作　　者：包杰[1] 王前[1] 郑磊[1] 曾方银[1] 刘杰[1] 熊石龙[1] 裘宇容[1] 李娟[2] 

机构地区：[1]南方医院检验医学中心,广州510515 [2]南方医院风湿科,广州510515

出　　处：《解放军医学杂志》2007年第6期585-587,共3页Medical Journal of Chinese People's Liberation Army

基　　金：广东省自然科学基金资助(04020404)

摘　　要：目的构建小鼠RelB基因RNA干扰(RNAi)慢病毒载体,有效沉默骨髓树突细胞(DC)的RelB基因表达,为构建骨髓致耐受DC用于自身免疫病的防治提供研究基础。方法利用Invitrogen公司在线软件设计小鼠RelB基因(NM_009046)shRNA序列,合成、退火形成dsoligo后克隆到pENTRTM/U6载体的黏性末端,测序,得到的阳性重组子再与慢病毒载体进行重组,转化stb13感受态细胞,测序鉴定,在脂质体的介导下将慢病毒的包装混合物和RelB基因重组慢病毒载体导入293FT细胞,包装成病毒后,收集病毒上清,采用系列稀释法测定病毒滴度。结果测序结果显示pENTRTM/U6-RelB-shRNA为阳性克隆,将该阳性重组载体与慢病毒载体重组,转化,氨苄青霉素抗性筛选阳性克隆,U6前引物测序鉴定,测序结果显示该重组慢病毒载体也为阳性克隆,在293FT细胞中进行病毒包装,收集上清并在-80℃贮存。系列稀释法检测病毒悬液的滴度为6×105TU/ml。结论成功构建出小鼠RelB基因shRNA慢病毒RNAi表达载体,为制备致耐受DC和研究DC在自身免疫病中的应用提供了稳定的转染细胞载体。Objective To construct a lentiviral expression vector of murine RelB for RNAi, and then to effectively silence the RelB gene expression of murine bone marrow derived dendritic cells for constructing the bone marrow tolerogenic dendritic cell, in order to provide an experimental foundation for a novel cliriical therapy of autoimmune disease. Methods RelB shRNA sequence of mouse was designed by on-line designer software on Corp. Invitrogen, after synthesis and annealing, double strand oligonucleotides （dsoligoes） were cloned into the pENTR^TM/U6 plasrnid, then after sequencing, a positive clone was further subeloned into pLenti6/BLOCK-iT^TM-DEST vector. It was then transformed into stb13 competent cell, and after sequencing, 293FT cell line was transfected by above positive recombined plasmid and lentiviral packing materials. It was incubated for 48 to 72 h in a 37℃, 5% CO2 incubator. Culture supernatant was harvested and stored at -80℃, then the virus titer was determined by serial dilution assay. Results It was showed from sequencing figures that all the pEN- TR^TM/U6-RelB-shRNA plasrnids were positive clone vector, and this positive recombinant vector was recombined with pLenti6/BLOCK- iT^TM/U6-DEST vector. It was then transformed into stb13 competent cell and screen positive clone by ampicillin, and resequenced by the use of primer forward U6 primer. The results also showed that recombinant lentiviral vector was positive clone. Viral particle was packaged with other packaging material mediated by lipofectamine 2000 in 293FT cell line. Cultural supernatant was collected and stored at -80℃, and lentiviral particle titer was determined by serial dilution assay with 6 × 10^5 / transduced unit. Conclusion Lentiviral shRNA expression vector of murine RelB gene for RNAi was successfully constructed. It might be a rational procedure to make tolerogenic DC, and then to develop a DC vaccine and DC-based immunotherapy for autoimmune diseases.

关 键 词：基因 RelB RNA干扰 慢病毒载体 滴度 

分 类 号：R349.83[医药卫生—基础医学]