摘要
目的:构建真核细胞翻译起始因子3第4亚单位(eIF3s4)真核表达载体用于进一步功能研究。方法:设计引物,以K562细胞的总RNA为模板,采用RT-PCR方法扩增eIF3s4的全长编码区cDNA,克隆于pGEM-TEasy载体,经DNA测序并与GenBank数据库序列进行比对后,将该cDNA序列亚克隆于真核表达载体pcDNA4/HisMaxB载体,构建成(His)6-eIF3s4融合蛋白的表达载体,用LipofectamineTM2000瞬时转染人乳腺癌细胞株Bcap37,利用Westernblot方法检测(His)6-eIF3s4融合蛋白的表达。结果:DNA序列测定表明,利用RT-PCR方法克隆的eIF3s4全长编码区cDNA序列与GenBank数据库序列一致,Westernblot结果证实(His)6-eIF3s4融合蛋白在Bcap37细胞中获得预期表达。结论:成功地构建了eIF3s4的真核表达载体并在人乳腺癌细胞Bcap37中表达,为建立稳定的eIF3s4高表达细胞系,进而研究eIF3s4与肿瘤细胞多药耐药相关性打下了基础。
AIM： To construct eukaryotic expression vector of eukaryotic translation initiation factor 3 subunit 4 （eIF3s4） for further functional study, METHODS： The cDNA of eIF3s4 containing full length coding region was amplified by RT-PCR and cloned into pGEM-T Easy. The sequence of the cDNA was verified by DNA sequencing and blast against sequence data in GenBank database. The cDNA was then subcloned into the pcDNA4/HisMaxB to make a （His）6- eIF3s4 fusion protein expression vector. The vector was transfected into human breast cancer cell Bcap37 by LipofectamineTM2000, and the expression of （His）6-eIF3s4 fusion protein was detected by Western blot. RESULTS： DNA sequencing and sequence blast showed that the cDNA amplified by RT-PCR was consistent with the eIF3s4 sequence in GenBank database, and Western blot results showed the expression of the （His）6-eIF3s4 fusion protein in human breast cancer cell Bcap37 as expected. CONCLUSION： The （His）6-eIF3s4 fusion protein expression vector is constructed successfully with expression of the fusion protein in Bcap37 breast cancer cells. This work provides the basis for establishing a stable eIF3s4 expressing cell line for further study on the role of eIF3s4 in cancer multidrug resistance.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第7期627-629,632,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30471955)
关键词
真核翻译起始因子
克隆
融合蛋白
eukaryotic translation initiation factor
cloning
fusion protein
