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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，western blot 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。

英文摘要:

Objective: To construct human lentiviral vector of mir-122, and establish a new hepatoma sub cell line of HepG2 stabilized infected with mir-122 virus. Methods: Primers were designed and synthesized, according to the human has-mir-122 precursor sequence. MiR-122 was amplified using PCR, and connected to the lentiviral expression plasmid pGCSIL-GFP. Lentiviral vector of mir-122 (pGCSIL-GFP-miR-122) was constructed. After infection of HepG2 cells with the miR-122 lentiviral vector, miR-112 expression was confirmed by qPCR and fluorescent protein GFP expression using FACS. Expression of one of mir-122 targeted molecules CAT-1 was validated by Western blot in pGCSIL-GFP-miR-122 HepG2 cells. Results: PGCSIL-GFP-of miR-122 was confirmed by sequencing analysis, indicating that Mir-122 lentiviral expression vector was successfully constructed. miR-122 expression in the lentiviral mir-122 infected cell line was significantly increased comparing to the parent cell line HepG2. Conclusion: Flow cytometry based on GFP fluorescence filter purification, also shows the infection rate of more than 90%. Inhibited the expression of its target molecules was confirmed by Western blot.

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