人PCK1基因克隆及其表达载体的构建

Cloning of Human PCK1 Gene and Construction of Its Expression Vector

目的构建人PCK1基因的真核表达载体。方法以人内脏脂肪细胞cDNA为模板,聚合酶链反应(PCR)扩增PCK1基因编码区的全部序列,克隆入pGEM-T载体中,经限制性内切酶、DNA序列分析鉴定目的基因后,定向亚克隆到真核细胞表达载体pcDNA3.1(+)中,并进行双酶切鉴定。结果PCR扩增的特异性片段长度为1 972 bp,以此构建的pGEM-T-PCK1克隆载体,经限制性内切酶酶切证实载体中带有PCK1基因的目的片段,与GenBank中的人PCK1基因cDNA序列一致。重组质粒pcDNA3.1-PCK1经KpnⅠ/XbaⅠ双酶切后显示5.1 kb和2 000 bp左右的2个条带,证明PCK1基因已成功克隆到了真核细胞表达载体pcDNA3.1(+)中。结论成功构建了野生型pcDNA3.1-PCK1重组真核表达载体。

Objective To construct a eukaryon expression vector of human PCK1 gene. Methods The whole coding sequence of PCK1 gene was amplified by polymerase chain reaction （PCR） applied to human adipose cell cDNA. The fragment was inserted into cloning vector pGEM-T and the recombinant pGEM-T-PCK1 was identified by double digestion with restriction enzymes and sequencing. The PCK1 cDNA fragment was then subcloned into pcDNA3.1（ ＋ ） plasmid. The recombinant pcDNA3.1-PCK1 was identified by double digestion with restriction enzymes Results The length of the specific fragment by PCR was 1 972 bp, and the pGEM-T-PCK1 plasmid was identified by endonuclease digestion and sequencing. The sequence＇was identical to that of PCK1 cDNA in GenBank. The recombinant pcDNA3.1-PCK1 plasmid was separated into two bands, namely 5.1 kb and 2 000 bp, by using respective restriction enzymes Kpn I/Xba I, suggesting that PCK1 gene fragment had been cloned into pcDNA3.1 vector correctly. Conclusion The wild-type recombinant eukaryon expression vector pcDNA3. 1-PCK1 was successfully constructed.