hsa-miR-150-5p抑制序列慢病毒载体的构建、转染和表达  

Construction,transfection and expression of hsa-miR-150-5p-down lentivirus vector

作　　者：叶文广[1] 王景杰[1] 张明鑫[1] 周苏娜[2] 

机构地区：[1]第四军医大学唐都医院消化内科,西安710038 [2]第四军医大学唐都医院放疗科

出　　处：《山西医科大学学报》2018年第1期6-10,共5页Journal of Shanxi Medical University

基　　金：唐都医院创新基金资助项目(2015JCYJ006)

摘　　要：目的构建携带抑制表达hsa-miR-150-5p的慢病毒载体,建立稳定表达该载体的HT-29细胞。方法设计并合成hsa-miR-150-5p抑制序列,并克隆到工具载体GV-280(h U6-MCS-Ubiquitin-EGFP-IRES-puromycin)构建重组载体,将重组载体与p Helper1.0载体和p Helper2.0载体共转染293T细胞,得到所需的病毒液LV-hsa-miR-150-5p-down,最后感染HT-29细胞,并通过嘌呤霉素筛选出稳定感染的细胞株。荧光显微镜观察HT-29细胞中绿色荧光蛋白(GFP)的表达,RT-PCR检测hsa-miR-150-5p在HT-29细胞中的表达水平。结果测序结果证明成功构建重组质粒,并成功包装成慢病毒,实验组(转染过表达LV-hsa-miR-150-5p-down病毒)病毒滴度为5×10~8TU/ml,阴性对照组(转染阴性LV-NC病毒)病毒滴度为8×10~8TU/ml。所得病毒液感染HT-29细胞并筛选出稳定细胞株HT29-150-5p-down。荧光显微镜观察带有绿色荧光蛋白的目的重组质粒在人结肠癌HT-29细胞中高表达,PCR进一步验证了这一结果。结论 LV-hsa-miR-150-5p-down慢病毒载体构建成功,HT-29细胞稳定表达该载体,为后续miR-150的功能研究奠定基础。Objective To construct HT-29 cell lines with stable down-expression of hsa-miR-150-5p. Methods The suppression sequences of hsa-miR-150-5p was obtained by PCR amplification, and then inserted into the lentiviral vector GV-280 （hU6-MCS-Ubiquitin-EGFP-IRES-puromycin）. The recombinant lentivirus plasmid was confirmed by DNA sequencing and extracted. The recombinant lentivirus plasmid and packaging plasmid pHelper1. 0 and pHelper2. 0 were co-transfected into 293T cells. The virus yield-ed by 293T cell was transfected into HT29 ceils. Tthe status of transfection was observed by fluorescence microscope and the expression of miR-150 was detected by real-time quantitative PCR before and after transfection. Results The results of DNA sequencing showed that the recombinant plasmid was successfully constructed and successfully packaged into lentivirus. The viral titer was 5 × 10^8 TU/ml in experimental group（ transfected with overexpression of LV-hsa-miR-150-Sp-down-recombined virus） , and 8 × 10^8 TU/ml in negative control group （transfected with LV-NC-recombined virus）. The recombinant plasmid with green fluorescent protein was highly expressed in human colon cancer HT-29 cells by fluorescence microscopy, and this result was verified by PCR. Conclusion The LV-has-miR-150- down lentiviral vector was successfully constructed, which lays a foundation for its function research of the role of miR-150.

关 键 词：miR-150 慢病毒载体 炎症性结肠病 

分 类 号：R735.35[医药卫生—肿瘤]