Wei Qun, Zhu Feng-jia, Wang Yi, Chen Ping, Wang Lin-bo, Cao Jiang
Sir Run Run Shaw Hospital Clicinal Research Institute, College of Medicine, Zhejiang University, Hangzhou 310016, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Jul;23(7):627-9.
To construct eukaryotic expression vector of eukaryotic translation initiation factor 3 subunit 4(eIF3s4) for further functional study.
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 Lipofectamine 2000, and the expression of (His)(6)-eIF3s4 fusion protein was detected by Western blot.
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.
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.
构建真核翻译起始因子3亚基4（eIF3s4）的真核表达载体，用于进一步的功能研究。
通过RT-PCR扩增包含全长编码区的eIF3s4 cDNA，并克隆到pGEM-T Easy载体中。通过DNA测序验证cDNA序列，并与GenBank数据库中的序列数据进行比对。然后将该cDNA亚克隆到pcDNA4/HisMaxB中，构建（His）6-eIF3s4融合蛋白表达载体。利用Lipofectamine 2000将该载体转染到人乳腺癌细胞Bcap37中，通过蛋白质免疫印迹法检测（His）6-eIF3s4融合蛋白的表达。
DNA测序和序列比对显示，RT-PCR扩增得到的cDNA与GenBank数据库中的eIF3s4序列一致，蛋白质免疫印迹结果表明，（His）6-eIF3s4融合蛋白在人乳腺癌细胞Bcap37中如预期表达。
成功构建了（His）6-eIF3s4融合蛋白表达载体，并在Bcap37乳腺癌细胞中表达了融合蛋白。本研究为建立稳定表达eIF3s4的细胞系奠定了基础，以便进一步研究eIF3s4在癌症多药耐药中的作用。
