LOX-shRNA慢病毒表达载体的构建与鉴定

目的 构建LOX-shRNA的慢病毒表达载体及其病毒包装鉴定。方法 利用Oligo Designer 3.0,根据人LOX基因序列设计shRNA,并合成包含正、反义靶序列的互补DNA链,退火后插入LOX表达载体中,构建shRNA表达质粒,并转化至E.coli TOP10感受态细胞,抽提质粒后进行测序。将重组质粒转染HEK-293T细胞,应用RT-PCR检测各组LOX基因mRNA的表达水平,筛选出最有效的LOX-shRNA后,通过LipofectAMINETM2000介导转染293T细胞,对慢病毒进行包装并测定慢病毒滴度。结果 利用慢病毒介导将重组质粒LOX-shRNA-3024高效转导入HEK-293T细胞并稳定表达。荧光显微镜显示,转染24h后,HEK-293T细胞即开始发出绿色荧光；72h后,有大量荧光表达,而对照组未转染质粒的HEK-293T细胞不表达,可见慢病毒载体被成功转染。LOX-3024重组慢病毒的滴度为2×1010TU/ml。结论 成功构建LOX-shRNA慢病毒表达载体,并稳定转染HEK-293T细胞,为研究LOX对人阴道壁成纤维细胞生物学行为的影响提供了实验基础。
Objective To construct and identify the LOX-shRNA lentiviral expression vector. Methods According to the sequence of human LOX gene, the shRNA sequence was designed and the sense and antisense oligonucleotides were synthesized. After annealing, these double DNA strands were inserted to the LOX expression vector and then transformed into competent cell E.coli TOP10. The plasmids were extracted and sequenced. The recombinant plasmid was transfected into HEK-293T cells and the expression level of LOX gene mRNA was detected by RT-PCR. After selecting the most effective LOX-shRNA, LipofectAMINETM2000 was used to transfect 293T cells for packing lentivirus and testing the titer of lentivirus. Results Recombinant plasmid LOX-shRNA-3024 was transfected into HEK-293T cells by lentivirus vector, and the recombinant plasmid was stably expressed. The green fluorescence HEK-293T cells was observed by fluorescence microscopy 24h after transfection and reached the peak at 72h after transfection, while there was no fluorescence was observed in untransfected HEK-293T cells. The titer of LOX-3024 recombinant lentivirus was 2×1010TU/ml. Conclusion The LOX-shRNA lentiviral expression vector has been successfully constructed and stably expressed in HEK-293T cells, which can be used in studying the effects of LOX on the biological behavior of human vaginal wall fibroblasts