人前脑啡肽原绿色荧光蛋白真核表达载体的构建

Construction of Human PENK and Green Fluorescent Protein Eukaryotic Vector

目的构建人前脑啡肽原(preproenkephalin,PENK)绿色荧光真核表达载体并进行鉴定。方法参照PENK基因全序列,在cDNA两端各设计一条对应引物,并引入各自酶切位点。通过RT-PCR的方法从正常人脑组织中扩增出目的基因,并定向克隆至pEGFP-C3绿色荧光蛋白真核表达载体上。筛选阳性克隆,通过双酶切和测序法鉴定重组质粒。结果双酶切结果与目的基因表达的条带完全吻合,克隆测序结果与NCB I收录的PENK序列完全一致。结论成功构建pEGFP-C3-PENK载体,为进一步研究疼痛的基因治疗奠定基础。

Objective To construct and identify the human PENK and green fluorescent protein eukaryotic vector. Methods To correspond to the PENK gene sequences,introduce respective restriction sites in each end of cDNA;and obtain the target gene from the normal tissue of brain by RT-PCR,then connect the target gene to the pEGFP-C3 green fluorescent protein eukaryotic vector;and choose the positive clones to identify the recombinant plasmids by double disgestion and sequencing. Results Double disgestion results and the expression of target gene band matched completely;the clone sequencing results were the same as the sequence of PENK contained in NCBI results. Conclusion pEGFP-C3-PENK vector is successfully constructed for the further study of the gene therapy of pain.