论文部分内容阅读
目的构建HIF-1α基因RNA干扰慢病毒载体。方法选定HIF-1α基因RNAi靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经XhoⅠ和HpaⅠ双酶切后的慢病毒载体pLentilox3.7(pLL3.7)连接,产生pLL3.7HIF-1α慢病毒载体,转化大肠杆菌DH5α菌株,抽取质粒后,用限制性内切酶XbaⅠ和NotⅠ进行酶切鉴定,将阳性克隆送金斯特公司进行测序鉴定;用PHR、pVSVG和pLL3.7慢病毒载体共转染包装细胞293T细胞(磷酸钙转染法),包装产生慢病毒,将收集的病毒上清经高速离心浓缩后,按一定比例稀释,然后感染293T细胞,用流式细胞仪检测293T细胞GFP蛋白的表达水平,从而测定病毒滴度。结果酶切和测序证实,构建出HIF-1αshRNA的慢病毒载体pLL3.7HIF-1α;包装慢病毒,测得离心的浓缩病毒悬液的滴度为2×108TU/ml。结论成功构建HIF-1α基因RNAi慢病毒载体。
Objective To construct RNA interference lentiviral vector of HIF-1α gene. Methods RNAi target sequence of HIF-1α gene was selected and OligoDNA of target sequence was synthesized. After double annealing, double-stranded DNA was ligated with lentiviral vector pLentilox3.7 (pLL3.7) digested with XhoⅠ and HpaⅠ to generate pLL3.7HIF -1α lentiviral vector was constructed and transformed into E. coli DH5α. Plasmids were extracted and digested with restriction endonucleases XbaⅠand NotⅠ. The positive clones were submitted to Kingston for sequencing and identification. Using PHR, pVSVG and pLL3.7 The viral vector was co-transfected into 293T cells (calcium phosphate transfection) and packaged to produce lentivirus. The collected virus supernatant was concentrated by high speed centrifugation and then diluted to a certain ratio. 293T cells were then infected and detected by flow cytometry 293T cells GFP protein expression levels, thereby measuring the virus titer. Results Enzyme digestion and sequencing confirmed that the lentivirus vector pLL3.7HIF-1α with HIF-1α shRNA was constructed. The titer of the concentrated virus suspension was 2 × 108TU / ml. Conclusion The RNAi lentiviral vector of HIF-1α gene was successfully constructed.