论文部分内容阅读
目的构建人PIF1基因5端、3端及全长基因的真核表达载体。方法从pCR2.1-TOPO-PIF1原始质粒中,采用PCR方法扩增目的基因PIF1,PIF1N及PIF1C,将目的基因和目的载体pcDNA3.1(-)分别酶切;纯化酶切产物后定向连接,并将其转化大肠杆菌DH5α,对生长出的克隆行特异限制性内切酶酶切鉴定,鉴定为阳性的克隆送样测序。结果凝胶成像结果显示重组基因pcDNA3.1(-)-PIF1,pcDNA3.1(-)-PIF1N及pcDNA3.1(-)-PIF1C酶切后条带大小分别为1 926,540,1 428bp。结论成功构建hPIF1基因5端、3端及全长基因表达载体,分别为pcDNA3.1(-)-PIF1N,pcDNA3.1(-)-PIF1C及pcDNA3.1(-)-PIF1。
Objective To construct eukaryotic expression vector of 5 ’end, 3’ end and full length gene of human PIF1 gene. Methods The target genes PIF1, PIF1N and PIF1C were amplified by PCR from the pCR2.1-TOPO-PIF1 original plasmid. The target gene and the target vector pcDNA3.1 (-) were respectively digested with restriction endonucleases. The recombinant plasmid was transformed into E.coli DH5α. The clones were identified by restriction endonuclease digestion, and the positive clones were sequenced. Results The results of gel imaging showed that the size of recombinant plasmid pcDNA3.1 (-) - PIF1, pcDNA3.1 (-) - PIF1N and pcDNA3.1 (-) - PIF1C were 1 926,540 and 1 428 bp, respectively. Conclusion The 5 ’end, 3’ end and full length gene of hPIF1 gene were successfully constructed and expressed as pcDNA3.1 (-) - PIF1N, pcDNA3.1 (-) - PIF1C and pcDNA3.1 (-) - PIF1, respectively.