【摘要】 目的构建并鉴定Tie2基因的RNA干扰慢病毒表达载体。方法先构建pNL-EGFP-U6-Tie2-siRNA慢病毒转移质粒,通过XbaⅠ酶切电泳及测序鉴定,再以pNL-EGFP-U6-Tie2-siRNA慢病毒转移质粒,pVSVG包膜质粒和pHelper包装质粒三质粒共转染293T细胞,产生慢病毒,收集病毒上清液并测定病毒滴度。结果成功构建pNL-EGFP-U6-Tie2-siRNA慢病毒转移质粒2条,通过XbaⅠ酶切及测序鉴定,Tie2-siRNA核苷酸序列插入正确,利用三质粒慢病毒包装系统生产EGFP-Tie2-siRNA病毒,收集病毒上清,并测得病毒滴度为9×103/μL。结论成功构建了Tie2基因的RNA干扰慢病毒表达载体。更多还原

【Abstract】 【Objective】 To construct and identify the RNAi lentiviral expression vector for Tie2 gene.【Methods】 The Lentiviral transfer plasmids pNL-EGFP-U6-Tie2-siRNA were constructed,then identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI and sequencing analysis;then the PNL-EGFP-U6-Tie2-siRNA lentiviral transfer plasmid,pVSVG envelope plasmid and pHelper packaging plasmid were cotransfected into 293T cells,resulting in lentivirus.Collect the virus supernatant and determination the viral titer.【Results】 The lentiviral transfer plasmids of pNL-EGFP-U6-Tie2-siRNA were constructed successfully and identified by using enzyme digestion electrophoresis of XbaI and sequencing analysis.The results fully in line with expectations;EGFP-Tie2-siRNA virus were produced by using the three plasmid lentiviral packaging system.The virus supernatant was collected and viral titers measured for the 9×103/μL.【Conclusion】 The RNAi lentiviral expression vectors for Tie2 gene were constructed successfully.更多还原