趋化因子受体CXCR4 miRNA真核表达载体的构建

目的构建趋化因子受体4（CXCR4）的特异RNA干扰载体。方法在GenBank数据库查询CXCR4基因的登陆号为NM-003467。使用Invitrogen公司的在线设计软件Invitrogen’sRNAi Designer，设计miR RNAi，选择713这个位点设计成相应的Oligo DNA，退火DNA oligos生成双链oligos，应用T4DNA连接酶连接双链oligos与表达载体pcDNA^TM6．2-GW／EmGFP-miR。酶切鉴定和测序鉴定。结果合成的基因序列完全正确并成功克隆到真核表达载体上。结论CXCR4miRNA真核表达载体构建成功，可作为进一步研究该基因的功能和探索肿瘤的基因治疗的工具。

Construction of eukaryotic expression vector of miRNA for CXCR4

Objective To construct the eukaryotic expression vector of miRNA for CXCR4.Methods we got the number NM003467 of CXCR4 in the database of GenBank to design single-stranded DNA oligos.We used Invitrogen＇s RNAi Designer, an online tool of Co. Invitrogen to design miR RNAi, and the design rules include a proprietary algorithm. We elected site 713 because of high scores.Annealing was operated in the single-stranded oligos to generate a dsolign. Cloning this ds oligo into peDNA^TM6.2-GW/EmGF- PmiR with T4 DNA Ligase. Enzyme cutting method and sequencing were performed to evaluate the recombinant. Results The synthetic DNA was of the correct sequence and inserted into eukaryotic expression vector suecessfttUy. Conclusion The miRNA eukaryotie expression vector of CXCR4 has been successfully constructed,and it will be a useful tool for investigating the function of CXCR4 and for searching a new gene therapy method of tumour.