(His)6 -eIF3s4融合蛋白在人乳腺癌细胞Bcap37中的表达

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。

Expression of (His)6-eIF3s4 fusion protein in human breast cancer cell Bcap37

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.