人树突状细胞特异性细胞间黏附分子-3-结合非整合素基因RNAi慢病毒载体的构建及外源性筛选与鉴定

Construction, exogenous selection and identification of lentiviral vector targeting human dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintergrin gene

背景:慢病毒载体是一个能够在原代培养的人树突状细胞中进行RNA干扰的有效工具,但目前国内对RNA干扰技术应用于人树突状细胞特异性细胞间黏附分子-3-结合非整合素分子的研究却少有报道。目的:拟构建人树突状细胞特异性细胞间黏附分子-3-结合非整合素基因RNA干扰慢病毒载体,为调控人树突状细胞特异性细胞间黏附分子-3-结合非整合素表达水平提供有利的工具。方法:根据人树突状细胞特异性细胞间黏附分子-3-结合非整合素基因的mRNA序列选择4条靶序列,设计合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeⅠ和EcoRⅠ双酶切后的pGCSIL-GFP载体连接产生DC-SIGN慢病毒载体,采用PCR及测序鉴定,应用Westernblot技术外源性筛选干扰效率高的有效靶序列。用慢病毒包装系统pGCSIL-GFP、pHelper1.0和pHelper2.0共转染包装293T细胞,包装产生慢病毒,以系列稀释法测定病毒滴度。结果与结论:PCR扩增和测序结果显示,人树突状细胞特异性细胞间黏附分子-3-结合非整合素核苷酸链序列插入正确,外源性筛选确定两条干扰效率高的有效靶序列。利用筛选确定的有效靶序列,包装产生慢病毒,其浓缩病毒悬液的滴度为1×109TU/mL。证实实验成功构建了DC-SIGN基因慢病毒载体。

BACKGROUND： The lentiviral vector is an effective tool to execute RNAi in the human dendritic cells. However, there are few reports addressing applying RNAi technology to the human dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintergrin （DC-SIGN）. OBJECTIVE： To construct a lentiviral vector expressing small-hairpin RNA （shRNA） targeting human DC-SIGN gene, and to provide a useful tool to regulate the expression level of DC-SIGN. METHODS： Four target sequences were selected according to human DC-SIGN mRNA sequence.The cDNA target sequence containing both sense and antisense Oligo DNA were designed. The obtained lentiviral vector containing DC-SIGN shRNA was confirmed by PCR, sequencing and exogenous selection. 293T cells were cotransfected with lentiviral vector pGCSIL-GFP, pHelper1.0 and pHelper 2.0. The titer of virus was tested by serial dilution. RESULTS AND CONCLUSION： PCR and DNA sequencing demonstrated that the inserted sequences were correct. Two effective targeting sequences were determined by exogenous selection. The titer of concentrated virus was 1×109T U/mL. The lentivirus vector targeting DC-SIGN has been successfully constructed.