引用本文:[点击复制]
[点击复制]
【打印本页】 【在线阅读全文】【下载PDF全文】 查看/发表评论下载PDF阅读器关闭

←前一篇|后一篇→

过刊浏览    高级检索

本文已被:浏览 46次   下载 17 本文二维码信息
码上扫一扫!
人真核翻译起始因子3C基因的克隆及表达
赵谦,孙潇,罗真真,董怡萍,韩苏夏
0
(西安交通大学第一附属医院肿瘤放疗科)
摘要:
【摘要】 目的 构建人真核翻译起始因子3C(Eukaryotic Translation Initiation Factor 3 Subunit C, EIF3C)编码区全长的真核表达载体,转染FaDu人下咽鳞癌细胞系,为探讨EIF3C的生物学功能奠定基础。方法 从人下咽鳞癌细胞系中提取总RNA,反转录成cDNA后用特异性引物PCR扩增EIF3C基因编码区全长(NM_0010378082),双酶切后将EIF3C基因编码区全长克隆到载体pCMVBlank上,构建真核表达载体pCMVEIF3C。转染FaDu细胞系,realtimePCR和Western blot检测EIF3C表达水平。将未加质粒转染FaDu细胞作为未加质粒转染组,加质粒pCMVBlank作为pCMVBlank转染组,以及加质粒pCMVEIF3C作为pCMVEIF3C转染组。分别将pCMVEIF3C转染组EIF3C的mRNA和蛋白表达水平与未加质粒转染组、pCMVBlank转染组进行比较,以及比较未加质粒转染组和pCMV-Blank转染组。通过生物信息学网站和软件分析人EIF3C氨基酸序列的理化性质、磷酸化位点、二级结构和三维结构。结果 测序结果表明构建的真核表达载体pCMVEIF3C中人EIF3C序列正确。转染实验表明载体pCMVEIF3C转染组EIF3C的mRNA水平相比载体pCMVBlank转染组升高708倍,未加质粒转染组升高802倍(P<0.001),且载体pCMVEIF3C转染组蛋白表达水平相比于载体pCMVBlank转染组和未加质粒转染组也显著升高(P<0.05)。EIF3C蛋白质由913 个氨基酸组成,预测含有27个磷酸化位点,其二级结构以α螺旋和无规则卷曲为主。结论 成功构建真核表达载体pCMVEIF3C,并使EIF3C在下咽鳞癌细胞系中过表达。
关键词:  真核翻译起始因子3C  人下咽鳞癌细胞  基因克隆  真核表达
DOI:
基金项目:陕西省创新人才推进计划 重点科技创新团队
Cloning and expression analysis of eukaryotic translation initiation factor 3 subunit C
ZHAO Qian,SUN Xiao,LUO Zhenzhen,DONG Yiping,HAN Suxia
(Department of Radiation Oncology, The First Affiliated Hospital of Xi'an Jiaotong University)
Abstract:
【Abstract】 Objective To construct an eukaryotic expression vector including the fulllength encoding sequences of human eukaryotic translation initiation factor 3 Subunit C (EIF3C) and to transfect it into FaDu cell line, which provides a foundation for studying the biological function of EIF3C.Methods Total RNA was extracted from FaDu cell line and the fulllength encoding sequences of EIF3C (NM_0010378082) was amplified from cDNA fragment by specific PCR primers. After double enzyme digestion, the digested PCR products were cloned into vector pCMVBlank, and construct the eukaryotic expression vector pCMVEIF3C. The expression of EIF3C in FaDu cell line was detected by realtime PCR and Western blot after the recombinant plasmid transfected into FaDu cell line The FaDu cell line was transfected with vectorfree as the vectorfree group, vector pCMVBlank as the pCMVBlank transfected group, and vector pCMVEIF3C as the pCMVEIF3C transfected group. The mRNA and protein expression levels of EIF3C in pCMVEIF3C transfected group were compared with vectorfree group and pCMVBlank transfected group, as well as vectorfree group and pCMVBlank transfected group. The physical and chemical properties of amino acid sequences, phosphorylation sites, secondary structure and threedimensional structure of human EIF3C were analyzed by bioinformatics website and software. Results The sequencing results indicated that the human EIF3C sequence was correct in the constructed eukaryotic expression vector pCMVEIF3C. Transfection experiments showed that the mRNA level of EIF3C in vector pCMVEIF3C transfected group was 708 and 802 times higher than vector pCMVBlank transfected group and vectorfree group (P<0.001), and protein expression levels of EIF3C were significantly increased than these groups. EIF3C protein is composed of 913 amino acids. It is predicted to contain 27 phosphorylation sites. The secondary structure of EIF3C protein is mainly alphahelix and random coil. Conclusion The recombinant eukaryotic expression vector of pCMVEIF3C was successfully constructed, and overexpressed in cell line of human squamous cell carcinoma.
Key words:  Eukaryotic translation initiation factor 3 subunit C  Human hypophryngeal cell  Gene cloning  Eukaryotic expression

用微信扫一扫

用微信扫一扫