| 摘要: |
| ［摘要］ 目的 构建人CYB5R2基因真核表达载体，并观察其在人鼻咽癌细胞HONE1中的表达。方法 根据表达载体pCMV-Tag3A上的多克隆位点和CYB5R2基因CDS序列设计引物，应用RT-PCR从人睾丸cDNA中克隆出CYB5R2的CDS，并进行TA克隆。对经PCR、双酶切和双向测序验证正确的质粒，进行CDS目的片段的回收，将其连接于pCMV-Tag3A载体的多克隆位点内构建真核载体，然后进行PCR、双酶切和双向测序验证。脂质体法转染鼻咽癌细胞HONE1细胞，荧光显微镜观察和RT-PCR验证该基因的表达。结果 PCR、双酶切和双向测序结果显示pCMV-Tag3-CYB5R2-CDS真核表达载体构建成功，荧光显微镜和RT-PCR的结果显示该重组质粒在HONE1细胞中正确表达。结论 成功构建了pCMV-Tag3-CYB5R2-CDS真核表达载体，并在鼻咽癌细胞HONE1中表达，为进一步验证其抑癌作用及探索其抑癌机制做准备。 |
| 关键词: 鼻咽癌 CYB5R2基因 真核表达载体 |
| DOI：10.3969/j.issn.1674-3806.2011.08.01 |
| 分类号:R 739.6 |
| 基金项目:国家自然科学基金资助项目（编号：30960416）；国家重点基础研究发展计划（973计划）资助项目（编号：2011CB504301） |
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| Construction of human eukaryotic expression vector with CYB5R2 gene |
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YU Na-na， DU Chun-ping，XIAO Xue, et al.
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Department of Otolaryngology-Head & Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
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| Abstract: |
| ［Abstract］ Objective To construct a eukaryotic expression vector with human CYB5R2 gene, which is high frequency methylation in nasopharyngeal carcinoma(NPC). Methods According to the sequence of the multiple clone sites of the vector pCMV-Tag3A and the sequence of CYB5R2 gene, a pair of primers were designed. RT-PCR amplification of CYB5R2 gene CDS from human testis tissue cDNA was achieved. Then, the fragment was cloned into T-A coloning vector. The recombined vector confirmed by PCR, sequencing and double digested by Sal I/BamH I to obtain the gene cDNA fragment, and subcloned into the multiple sites of pCMV-Tag3A vector. The recombined vector confirmed by PCR, sequencing and double digested by Sal I/BamH I and BamH I/EcoR V. The recombinant plasmid was transfected into HONE1 cells by FuGENE HD Transfection Reagent. The result and expression were examined using fluorescent microscopy and RT-PCR. Results PCR, sequencing and restriction enzyme digestion analysis indicated that the eukaryotic vector pCMV-Tag3-CYB5R2-CDS was constructed successfully. The expression of vector was confirmed by fluorescent microscopy and RT-PCR. Conclusion The eukaryotic expression vector pCMV-Tag3A with CDS of gene CYB5R2 is successfully constructed, and with efficient expression in HONE1 cells, which serve a good basis for futher study on the relationship of methylation CYB5R2 gene with NPC. |
| Key words: Nasopharyngeal carcinoma CYB5R2 gene Eukaryotic expression vector |
