mcpr1基因的真核表达载体的构建与鉴定

Construction and identification of mcpr1 gene eukaryotic expressing vector

目的 :构建腭裂相关基因mcpr1与pcDNA3 .1/V5 HisB融合的高效真核表达载体 ,为研究mcpr1基因的功能奠定基础。方法 :根据mcpr1基因的核苷酸序列 ,设计并合成引物 ,通过PCR方法 ,从包含有mcpr1基因全长的克隆载体T easy/mcpr1中 ,扩增出该基因外显子片段 ,将扩增产物连接到真核表达载体pcDNA3 .1/V5 HisB中。对该重组体进行PCR和酶切鉴定 ,以及测序验证。结果 :以重组体为模板扩增出40 0bp左右的特异性基因片段 ,与mcpr1基因片段大小一致 ,酶切鉴定也显示有 40 0bp左右的基因片断。测序结果显示与已知基因序列一致。结论 :成功构建mcpr1基因的真核表达载体 ,为下一步研究mcpr1基因的功能奠定了基础。

Objective: To construct a high effective eukaryotic expre ss ing vector containing mcpr1 gene. Methods: mcpr1 gene w as amplified by PCR from the plasmid T-easy/ mcpr1, then PCR product was in serted into eukaryotic expressing vector pcDNA3.1/V5-His B. The positive recomb inant was identified by PCR analysis, HindIII and BamHI restriction analysis and Sequence analysis. Results: A 400 bp DNA fragment was amplified from the recombinant. Sequence analysis and restriction digest demonstrated tha t the mcpr1 gene was successfully inserted into pcDNA3.1/V5-His B plasmid. Conclusion: The eukaryotic expressed vector pcDNA3.1/V5-His B/ mcpr1 has been successfully reconstructed.