中文摘要：

目的：构建可表达人中性氨基酸载体B0AT1基因的真核表达载体,建立重组质粒转染的Hela细胞系,筛选出稳定表达人B0AT1的细胞株。方法：提取人正常小肠上皮细胞总RNA,采用RT-PCR方法扩增出B0AT1基因片段,EcoRⅠ和XbaⅠ双酶切后,将其插入至真核表达载体pcDNA3.1中,构建重组表达质粒pcDNA3.1-B0AT1,转化E.coliDH5α菌株感受态细胞,将阳性克隆脂质体法转染Hela细胞,经G418筛选获得稳定表达株,用RT-PCR法检测B0AT1基因的mRNA表达。结果：从小肠上皮细胞中成功克隆到人B0AT1基因。酶切和序列测定表明已成功构建真核表达载体pcDNA3.1-B0AT1,将此重组质粒转染的Hela细胞,可稳定表达人B0AT1基因,并筛选出稳定表达B0AT1的Hela细胞系。氨基酸摄取实验证实,转染pcDNA3.1-B0AT1的Hela细胞具有B0AT1基因的生物学活性。结论：重组人中性氨基酸载体B0AT1克隆成功,并在Hela细胞中获得稳定表达。

英文摘要：

Objective： To construct an eukaryotic expression vector containing human neutral amino acid transporter B0AT1 gene and obtain a stably transfected Hela cell line expressing human B0AT1. Methods ： The human B0AT1 gene was amplified by RT-PCR from human intestinal epithelial cell and di- gested by EcoR I / Xba I . This digested fragment was inserted into eukaryotic expression vector pcD- NA3.1 and then transformed E. coli DH5ct. The positive recombinant plasmid was transfected into Hela cell line by LipolectamineTM 2000. Cells stably expressing human B~AT1 were selected by G418 and confirmed by RT-PCR. The biological activities of B0ATI expressed on Hela cells were confirmed by ami- no acid transport experiment. Results： Human B0AT1 gene was amplified by RT-PCR and cloned into eukaryotic expression plasmid pcDNA3.1 successfully. And the Hela cell line stably expressing human neutral amino acid transporter B0ATI was obtained. Conclusion： The pcDNA3.1-B0AT1 transfected Hela cell line is established successfully, laying a foundation for further study of the function and regula- tion of B~AT1 transporter.