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目的用基因重组技术构建LV3-人类EGFL7基因的小发卡RNA(LV3-h EGFL7-shRNA)慢病毒表达载体。方法设计靶向h EGFL7-shRNA序列,建立LV3-h EGFL7-shRNA,在慢病毒载体p GLV3/H1/GFP+Puro中放置h EGFL7-shRNA基因片段,DNA测序以及酶切的方式进行检测h EGFL7片段,转染293T细胞后对上清液进行浓缩,最后在对病毒滴度进行检测。结果 EGFL7-shRNA核苷酸链有正确的插入顺序,包装慢病毒产生病毒悬液的滴度为2×108TU·m L~(-1)。结论 h EGFL7-shRNA慢病毒表达载体构建成功,鉴定可满足试验需求。
Objective To construct LV3-h EGFL7-shRNA lentiviral expression vector of LV3-human EGFL7 gene by gene recombination technology. Methods h EGFL7-shRNA sequence was designed and targeted. LV3-h EGFL7-shRNA was constructed and h EGFL7-shRNA gene fragment was placed in lentiviral vector pGLV3 / H1 / GFP + Puro. DNA sequencing and restriction enzyme digestion were used to detect h EGFL7 Fragment, the supernatant was concentrated after 293T cells were transfected, and finally the virus titer was tested. Results The correct insertion sequence of EGFL7-shRNA nucleotides was obtained. The titer of virus-enveloped lentivirus was 2 × 108TU · m L -1. Conclusion h EGFL7-shRNA lentiviral vector was constructed successfully, which can meet the experimental needs.