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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。
OBJECTIVE: To construct an eukaryotic expression vector expressing B0AT1, a human neutral amino acid carrier, and to establish a Hela cell line transfected with the recombinant plasmid, and to screen out a cell strain stably expressing human B0AT1. Methods: Total RNA was extracted from human normal intestinal epithelial cells. The B0AT1 gene fragment was amplified by RT-PCR and digested with EcoRI and XbaI. The recombinant plasmid was inserted into eukaryotic expression vector pcDNA3.1 to construct recombinant plasmid pcDNA3. 1-B0AT1 was transformed into competent cells of E.coli DH5α strain. The positive clones were transfected into Hela cells by lipofectamine 2000. The stable expression strains were screened by G418. The mRNA expression of B0AT1 gene was detected by RT-PCR. Results: The human B0AT1 gene was successfully cloned from intestinal epithelial cells. Digestion and sequencing showed that the eukaryotic expression vector pcDNA3.1-B0AT1 was successfully constructed. The Hela cells transfected with the recombinant plasmid could stably express human B0AT1 gene and screened the Hela cell line stably expressing B0AT1. Amino acid uptake experiments confirmed that the Hela cells transfected pcDNA3.1-B0AT1 B0AT1 gene has the biological activity. Conclusion: The recombinant human neutral amino acid vector B0AT1 was successfully cloned and stably expressed in Hela cells.