连续3次基因抓捕构建人EEF1A1-人凝血因子Ⅶ杂合基因座  

Construction of human eukaryotic translation elongation factor 1A1-human coagulation factor Ⅶ hybrid gene locus by three-step gap-repair

作　　者：刘蓥灿[1,2] 林艳丽[2] 吴晓洁[2] 熊福银[2] 周艳荣[2] 孙晓艳[1,2] 李力[1] 陈红星[2] 

机构地区：[1]陕西师范大学生命科学学院,西安710062 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《中国生物制品学杂志》2012年第12期1627-1630,共4页Chinese Journal of Biologicals

摘　　要：目的利用人类真核翻译延伸因子1A1(Human eukaryotic translation elongation factor 1A1,hEEF1A1)基因座完整的上下游调控序列和人凝血因子Ⅶ(Human coagulation factorⅦ,hFⅦ)基因组序列构建hEEF1A1-hFⅦ杂合基因座。方法首先构建连入6个同源臂的pBR322作为连续3次基因抓捕的载体;然后通过Red同源重组系统,在大肠杆菌内进行缺口修复。第一步将hEEF1A1基因3′端侧翼序列亚克隆至抓捕载体上,第二步将hFⅦ完整基因组序列亚克隆至抓捕载体上,第三步将hEEF1A1基因5′端完整侧翼序列亚克隆至抓捕载体上。结果经3次基因抓捕,3个基因片段在抓捕载体上无痕连接,形成一条长约50 000 bp的hEEF1A1-hFⅦ杂合基因座。经PCR、酶切及测序验证,构建的hEEF1A1-hFⅦ杂合基因座中,原hEEF1A1基因组编码序列从起始密码子到终止密码子被hFⅦ基因组序列精确置换。结论成功构建了hEEF1A1-hFⅦ杂合基因座,为哺乳动物细胞表达大载体的制备提供了实验依据。Objective To construct a human eukaryotic translation elongation factor 1A1-human coagulation factor Ⅶ（hEEF1A1-hFⅦ）hybrid gene locus by using the intactupstream and downstream regulatory sequences of hEEF1A1 gene locus and the genome sequence of hFⅦ.Methods A gap-repair vector based on pBR322 vector backbone was constructed by inserting six joint homologous arms,then gap repair was performed in E.coli by Red homologous recombination.The flanking region at 3′-terminus of hEEF1A1 gene was subcloned to the gap-repair vector at first,followed by the whole hFⅦ genome sequence,and the flanking region at 5′-terminus of hEEF1A1 gene.Results After three-step gap-repair,the three DNA fragments were automatically combined together without any gap on the gap-repair vector to construct a hEEF1A1-hFⅦ hybird gene locus at a length of about 50 000 bp.PCR,restriction analysis and sequencing proved that the encoding sequence of hEEF1A1 genome in the constructed hEEF1A1-hFⅦ hybird gene locus,from the starting codon to the termination codon,was substituted exactly with hFⅦ genome sequence.Conclusion A hEEF1A1-hFⅦ hybird gene locus was successfully constructed,which provided an experimental basis for preparation of large vectors for expression in mammal cells.

关 键 词：基因抓捕 人真核翻译延伸因子1A1 人凝血因子Ⅶ 杂合基因座 RED同源重组 

分 类 号：Q781[生物学—分子生物学]