CAJ | 학술논문

目的:构建人源microRNA-455(miR-455)慢病毒载体,并鉴定成熟has-miR-455在细胞内的表达水平.方法:提取SiHa细胞中的人基因组DNA,设计并合成人miR-455的上下游引物,PCR扩增目的基因,将其中表达miR-455的结构经酶切后插入慢病毒转移质粒pWPT-GFP,构建成pWPT-GFP-pri-miR-455,在293T细胞中与pMD2G、pSPAX2包装产生慢病毒,并用含慢病毒的上清感染SiHa细胞.结果:测序结果证明插入质粒载体中的miR-455前体序列完全正确,慢病毒载体构建成功并获得相应的慢病毒;重组慢病毒质粒pWPT-GFP-pri-miR-455感染SiHa细胞后上调miR-455的表达近40倍.结论:构建了miR-455的慢病毒载体,并能在293T细胞中表达,产生的慢病毒能成功感染SiHa细胞.为进一步研究miR-455的功能,以及利用慢病毒进行基因治疗奠定了基础.
목적:구건인원microRNA-455(miR-455)만병독재체,병감정성숙has-miR-455재세포내적표체수평.방법:제취SiHa세포중적인기인조DNA,설계병합성인miR-455적상하유인물,PCR확증목적기인,장기중표체miR-455적결구경매절후삽입만병독전이질립pWPT-GFP,구건성pWPT-GFP-pri-miR-455,재293T세포중여pMD2G、pSPAX2포장산생만병독,병용함만병독적상청감염SiHa세포.결과:측서결과증명삽입질립재체중적miR-455전체서렬완전정학,만병독재체구건성공병획득상응적만병독;중조만병독질립pWPT-GFP-pri-miR-455감염SiHa세포후상조miR-455적표체근40배.결론:구건료miR-455적만병독재체,병능재293T세포중표체,산생적만병독능성공감염SiHa세포.위진일보연구miR-455적공능,이급이용만병독진행기인치료전정료기출.
Objective: To construct a lentivirus vector expressing microRNA-455(miR-455) and transfect the vector into SiHa cell line. Methods: Human miR-455 gene with its promoter was amplified by PCR from human SiHa cells genomic DNA. We constructed a lentivirus vector pWPT-GFP containing pri-miR-455 transcript. After having been conformed, the pWPT-GFP-pri-miR-455 plasmid was co-transfected with framework plasmid pMD2G, pSPAX2 into 293T cells to obtain the miR-455 lentivirus vector. After the lentivirus expression vector pWPT-GFP-pri-miR-455 was transduced into SiHa cells, the transduction efficiency was determined by real-time PCR. Results: The inserted pre-miR-455 sequence of plasmid vectors was absolutely correct confirmed by sequencing re?sults. miR-455 was notable up-regulation after transfecting SiHa cells with recombinant lentivirus vector pWPT-GFP-pri-miR-455. Conclusion: The recombinant lentivirus vector for miR-455 was successfully constructed which expressed in 293T cells, and then infected with SiHa cells. The study may lay a foundation on further researching the function of miR-455 and gene therapy by lentivirus .