Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene
Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene
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建立转染人 CD14 基因的 HeLa-CD14 细胞系
DOI：
10.3892/ol.2012.557
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发表时间：
2012-04-01
期刊：
影响因子：
2.9
通讯作者：
Tang, Yong-Min
中科院分区：
文献类型：
--
作者：
Ning, Bo-Tao;Tang, Yong-Min
CD 14是CD 14相关疾病诊断和治疗的关键分子，在菌血症中起重要作用。HeLa细胞系由于其多产特性而被认为是永生的。HeLa细胞系来源于人宫颈癌细胞，已广泛用于癌症研究和基因转染。本研究构建了表达质粒pcDNA3.1（+）-CD 14，并将其转染人宫颈癌细胞系HeLa，建立了稳定表达人CD 14抗原的细胞系（HeLa-CD 14）。通过RT-PCR和T-A克隆技术克隆人CD 14基因并测序后，经双酶切和T4连接酶连接，构建真核表达载体pcDNA3.1（+）CD 14。用Superfect转染试剂将pcDNA3.1（+）-CD 14重组质粒转染HeLa细胞。用G418筛选细胞，RT-PCR和免疫组化证实转染子上人CD 14的表达。空白pcDNA3.1（+）转染组与pcDNA3.1（+）-CD 14转染组CD 14 mRNA表达差异有统计学意义（p
CD14 is the pivotal molecule in the diagnosis and therapy of CD 14-associated diseases, and is important in bacteremia. The HeLa cell line is regarded as immortal due to its prolific character. The HeLa cell line is derived from human cervical cancer cells and has been widely used in cancer research and gene transfection. In the present study, we established the expression plasmid pcDNA3.1(+)-CD14, and transfected it into the human cervical cancer cell line HeLa to establish a stable cell line (HeLa-CD14) expressing human CD14 antigen on the membrane. After the human CD14 gene was cloned and sequenced through RT-PCR and T-A cloning techniques, the eukaryotic expression vector pcDNA3.1(+)CD14 was constructed by cleaving with double restriction endonucleases and ligating with T4 ligase. HeLa cells were transfected with the pcDNA3.1(+)-CD14 recombinant plasmid using Superfect transfection reagent. The cells were selected using G418 and the expression of human CD14 on the transfectant was confirmed by RT-PCR and immunohistochemistry. The expression of CD14 mRNA was significantly different between the blank pcDNA3.1(+)-transfected cell group and the pcDNA3.1(+)-CD14-transfected cell group (p