论文部分内容阅读
目的:构建人mir-122慢病毒表达载体,感染肝癌细胞HepG2,建立稳定表达mir-122的HepG2细胞系。方法:以人has-mir-122成熟序列,设计并合成引物,采用PCR的方法扩增目的基因,并连接到慢病毒表达质粒pGCSIL-GFP(含绿色荧光蛋白GFP基因)中。对重组质粒进行双酶切鉴定后,进行mir-122基因慢病毒(pGCSIL-GFP-miR-122)的包装及病毒滴度测定,用构建好的慢病毒表达载体感染HepG2细胞,qPCR检测感染后细胞中MIR-122的变化。通过流式细胞仪检测荧光蛋白GFP,westernblot检测mir-122靶分子CAT-1,验证pGCSIL-GFP-miR-122在HepG2细胞中的表达效果。结果:pGCSIL-GFP-miR-122经双酶切分析及测序,插入序列正确。qPCR检测显示转入病毒后mir-122在细胞中的表达显著提高。表明mir-122慢病毒表达载体构建成功。流式细胞仪根据GFP荧光筛选纯化感染后细胞,感染率达90%以上。Western blot显示mir-122明显抑制其靶分子表达。进一步验证pGCSIL-GFP-miR-122在细胞中的稳定表达。结论:成功构建mir-122慢病毒表达载体,并建立稳定表达的细胞系,为研究mir-122在人体所起的作用及功能机制打下基础。
OBJECTIVE: To construct human mir-122 lentiviral vector and infect HepG2 hepatocellular carcinoma cells, and establish a HepG2 cell line stably expressing mir-122. Methods: Primers were designed and synthesized based on the human has-mir-122 mature sequence. The target gene was amplified by PCR and ligated into the lentiviral expression plasmid pGCSIL-GFP (including the green fluorescent protein GFP gene). The recombinant plasmids were identified by double enzyme digestion. The packaging and virus titers of mir-122 lentivirus (pGCSIL-GFP-miR-122) were determined. HepG2 cells were infected with the constructed lentiviral vector. After infection by qPCR Changes in MIR-122 cells. Fluorescent protein GFP was detected by flow cytometry and the expression of pGCSIL-GFP-miR-122 in HepG2 cells was detected by western blot. Results: pGCSIL-GFP-miR-122 was double-digested and sequenced, and the inserted sequence was correct. qPCR showed that the expression of mir-122 in cells was significantly increased after transfection into the virus. The results showed that mir-122 lentiviral vector was successfully constructed. Flow cytometry based on GFP fluorescence screening purified infected cells, the infection rate of more than 90%. Western blot showed that mir-122 significantly inhibited its target molecule expression. Further verification of the stable expression of pGCSIL-GFP-miR-122 in cells. Conclusion: The mir-122 lentiviral vector was successfully constructed and a stable cell line was established, which laid the foundation for studying the function and mechanism of mir-122 in human.