真核表达质粒pcDNA3.1(+)-h/mCD44v6的构建和鉴定

目的构建含人/鼠嵌合粘附分子CD44拼接变异体6(chimeric human and mouse CD44 splice variant 6,h/mCD44v6)基因真核表达质粒pcDNA3.1(+)-h/mCD44v6,并进行表达。方法通过PCR扩增h/mCD44v6的全基因cDNA,将扩增的cDNA克隆至PGM-Teasy,测序后插入pcD-NA3.1(+)真核表达质粒,构建重组真核表达质粒pcD-NA3.1(+)-h/mCD44v6,经限制内切酶酶切分析及测序鉴定后,用脂质体转染B16细胞,通过RT-PCR扩增出B16/pcDNA3.1(+)-h/mCD44v6细胞株中h/mCD44v6全段基因cDNA。结果经4轮PCR,成功扩增出h/mCD44v6的cD-NA全长基因,成功构建了真核表达质粒pcDNA3.1(+)-h/mCD44v6,通过RT-PCR方法证实该质粒能在B16真核细胞中正确表达;建立了稳定的细胞株B16/pcDNA3.1(+)-h/mCD44v6。结论成功克隆和构建了h/mCD44v6的真核表达质粒pcDNA3.1(+)-h/mCD44v6,为进一步研究h/mCD44v6的新功能和免疫治疗奠定了基础。

Construction and determination of a recombinant eukaryotic plasmid pcDNA3.1(+)-h/mCD44v6

Aim To construct a eukaryotic plasmid containing the chimeric human and mouse CD44 splice variant 6 gene(h/mCD44v6)and test the expression of the plasmid in the eukaryotic cell.Methods The full-length cDNA of h/mCD44v6 encoding gene was obtained by PCR.Then the cDNA was inserted into the eukaryotic expression vector pcDNA3.1(+),the resultant recombinant plasmid was confirmed by restriction endonuclease and sequencing,then designated as pcDNA3.1(+)-h/mCD44v6.The recombinant eukaryotic plasmid pcDNA3.1(+)-h/mCD44v6 was transfected into eukaryotic cell B16 by LIPOFECTIN.RT-PCR was used to certify whether the purpose gene was correctly expressed in B16/pcDNA3.1(+)-h/mCD44v6 cell.Results About 358 bp cDNA of h/mCD44v6 was obtained by four steps of overlapping PCR.The recombinant eukaryotic expression plasmid pcDNA3.1(+)-h/mCD44v6 was successfully constructed and expressed in the B16 cells.Conclusion This recombinant pcDNA3.1(+)-h/mCD44v6 plasmid will provide a basis for further study on the unknown function of h/mCD44v6.