| 摘要: |
| ［摘要］ 目的 检测肝癌细胞HepG2中丛生蛋白（CLU）的表达情况并构建肝癌差异性表达基因CLU启动子表达质粒，为后续进行CLU基因差异性表达及机制研究奠定基础。方法 采用实时荧光定量PCR（RT-qPCR）检测CLU mRNA在肝细胞癌（HCC）HepG2细胞和正常肝细胞L02中的表达水平。应用生物信息学分析和序列测序获得CLU基因启动子序列，并将其插入到pGL3-Basic质粒中。通过酶切、琼脂糖凝胶电泳及测序对构建完成的pGL3-CLUP质粒进行验证。结果 RT-qPCR结果表明HepG2细胞CLU mRNA的表达量约是L02细胞的9.38倍，差异有统计学意义（P<0.05）。pGL3-CLUP质粒经单酶切后为一条电泳条带，长度为5 000～6 000 bp；双酶切后的质粒为两条电泳条带，一条为5 000 bp左右，另一条为1 000～1 500 bp，大小与质粒和启动子大小一致。测序结果也表明CLU启动子已插入到pGL3-Basic质粒启动子区。结论 CLU在HCC细胞中高表达。pGL3-CLUP启动子表达质粒构建成功。 |
| 关键词: 肝癌 差异性表达基因 启动子 表达质粒 |
| DOI：10.3969/j.issn.1674-3806.2021.03.15 |
| 分类号:R 73-37 |
| 基金项目:国家自然科学基金资助项目（编号：81660510）；广西自然科学青年基金项目（编号：2014GXNSFBA118206） |
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| Detection of differential expression of clusterin in hepatoma cells and construction of promoter expression plasmid |
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YU Qiu-li, WANG Feng
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Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Guangxi Medical University, Nanning 530021, China
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| Abstract: |
| ［Abstract］ Objective To detect the expression of clusterin(CLU) in liver cancer cell HepG2 and to construct the promoter expression plasmid of liver cancer differential expression gene CLU, so as to lay a foundation for further study on the differential expression and mechanism of CLU gene. Methods The expression levels of CLU mRNA in hepatocellular carcinoma(HCC) cell HepG2 and normal hepatocyte L02 were detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR). The promoter sequence of CLU gene was obtained by bioinformatics analysis and promoter sequencing, and was constructed into pGL3-Basic plasmid. The constructed pGL3-CLUP plasmid was verified by restriction enzyme digestion, agarose gel electrophoresis and sequencing. Results The RT-qPCR results showed that the expression level of CLU mRNA in HepG2 cells was 9.38 times higher than that in L02 cells, and the difference was statistically significant(P<0.05). The pGL3-CLUP plasmid was a single band after single enzyme digestion, and the length was about 5 000-6 000 bp. After double enzyme digestion, two bands appeared, one of which was about 5 000 bp and the other was about 1 000-1 500 bp. The length was consistent with the size of plasmid and promoter. The sequencing results also showed that the CLU promoter had been inserted into the promoter region of pGL3-Basic plasmid. Conclusion CLU is highly expressed in HCC cells. pGL3-CLUP promoter expression plasmid is constructed successfully. |
| Key words: Liver cancer Differential expressed genes Promoter Expression plasmid |
