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摘要： 目的：建立稳定表达人CD14分子的Hela-CD14细胞系，为研究CD14分子及CD14相关性疾病提供研究材料。 方法：以人CD14mRNA为模板，通过RT-PCR法扩增CD14基因，T-A克隆后测序核实CD14基因序列。通过EcoRⅠ/XbaⅠ双酶切和体外连接法将人CD14基因连接到真核表达载体pcDNA 3.1(+)中 ，通过转化、克隆得到阳性重组质粒pcDNA 3.1（+）/CD14。将pcDNA 3.1（＋）/CD14 转染人宫颈癌Hela细胞，经G418筛选、定量PCR（qPCR）和免疫荧光检测CD14基因和蛋白的表达，筛选出稳定表达人CD14阳性的Hela-CD14细胞系。 结果：测序显示，扩增到的人CD14基因序列正确，双酶切质粒结果表明，CD14基因成功克隆到pcDNA 3.1（＋）载体中；免疫荧光结果显示，人CD14主要以膜蛋白形式在Hela细胞上成功表达。 结论：本研究建立了稳定的以膜蛋白形式表达人CD14抗原的细胞系Hela-CD14。
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| 关键词：
Hela细胞/细胞学； 抗原;
CD14/遗传学； 转染； 重组;
遗传； 质粒； 克隆;
分子； 基因表达； 逆转录聚合酶链反应
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Abstract: Objective： To establish a transgenic cell line with stable expression of CD14. Methods： Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase，the human CD14 gene was cloned and sequenced through the RT-PCR，T-A clone techniques and ABI PRISM377 machine.Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases EcoRⅠ/XbaⅠ and ligating with T4 ligase.The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent.Positive clones were selected by G418 at a concentration of 0.5 μg/μl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay. Results： There was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01).The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells，and no visible fluorescence was observed in blank vector transfected cells. Conclusions： The transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases. |
| Key words:
Hela cells/cytology； Antigens
CD14/genetics； Transfection； Recombination
genetic； Plasmids； Cloning
molecular； Gene expression； Reverse transcriptase polymerase chain reaction
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出版日期: 2012-09-25
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