【摘要】 目的建立稳定高效表达正反义A lu-Sx的细胞系。方法根据A lu亚家族Sx序列合成两对两端带有限制性酶切位点的引物,提取HEK293细胞的总DNA后PCR扩增,产物连接到真核表达载体pcDNA3.1/myc-H is A中构建成重组体。经酶切及测序鉴定后,阳离子脂质体转染法转染HEK293细胞后G418筛选稳定表达的细胞克隆,Northern杂交检测并挑选出表达最强的亚克隆。结果成功构建A lu亚家族Sx的正反义真核表达载体并获得高效稳定表达的HEK293细胞亚克隆。结论高效稳定表达正反义A lu Sx的HEK293细胞亚克隆可用于下一步研究。更多还原

【Abstract】 Objective To establish a HEK293 cell line stably and highly expressing sense and antisense Alu-Sx.Methods According to Alu subfamily Sx sequence,a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized.PCR of the total DNA extracted from HEK293 cell line was performed,the products of which were cloned into a highly efficient eukaryotic expression vector pcDNA3.1/myc-His A.The recombinants were sequenced and identified by restrictive endonuclease digestion and then transfected into the HEK293 cell line by lipofectamine2000.The stable transfectants were screened by G418.Cell subclones were isolated by gradient dilution.The highly expressing clones were identified by Northern blotting.Results Eukaryotic expressing vectors stably and efficiently expressing sense and antisense Alu-Sx were constructed and cell subclones stably and efficiently expressing sense and antisense Alu-Sx were established.Conclusion Cell subclones stably and efficiently expressing sense and antisense Alu-Sx can used for our further study.更多还原