人CYB5R2基因真核表达载体的构建  

Construction of human eukaryotic expression vector with CYB5R2 gene

作　　者：余娜娜[1] 杜春平[1] 肖雪[1] 侯波[1] 黄光武[1] 张哲[1] 

机构地区：[1]广西医科大学第一附属医院耳鼻咽喉头颈外科,南宁530021

出　　处：《中国临床新医学》2011年第8期695-699,共5页CHINESE JOURNAL OF NEW CLINICAL MEDICINE

基　　金：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)

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。Objective To construct a eukaryotlc expression vector with human CYBSR2 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-CYBSR2-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 CYBSR2 gene with NPC.

关 键 词：鼻咽癌 CYB5R2基因 真核表达载体 

分 类 号：R739.6[医药卫生—肿瘤]