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浙江大学学报(医学版)  2012, Vol. 41 Issue (5): 506-511    DOI: 10.3785/j.issn.1008-9292.2012.05.007
浙江大学附属儿童医院血液肿瘤科,浙江 杭州 310003
Establishment of a transgenic cell line with stable expression of human CD14
NING Bo-tao,SONG Hua,YANG Shi-long,XU Wei-qun TANG Yong-min
Department of Hematology and Oncology,the Children′s Hospital,Zhejiang University School of Medicine,Hangzhou 310003,China
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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。
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    
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
出版日期: 2012-09-25
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宁铂涛;宋华;杨世隆;徐卫群;汤永民. 稳定表达人CD14细胞系Hela-CD14的建立[J]. 浙江大学学报(医学版), 2012, 41(5): 506-511.


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