真核表达质粒pcDNA3.1(+)-rmCGβ的构建和鉴定

Construction and determination of a recombinant eukaryotic plasmid pcDNA3.1(+)-rmCGβ

目的构建恒河猴绒毛膜促性腺激素β亚基(macaca mulatta chorionic gonadotrophinβsubunit,rmCGβ)基因真核表达质粒pcDNA3.1(+)-rmCGβ,并了解该质粒能否在真核细胞中表达。方法通过PCR扩增rmCGβ的全段基因cDNA,应用基因工程技术将扩增的cDNA克隆至PGM-Teasy,测序后插入pcDNA3.1(+)真核表达质粒,构建重组真核表达质粒pcDNA3.1(+)-rmCGβ,经限制内切酶酶切分析及测序鉴定正确后,用脂质体转染技术转染B16细胞,通过RT-PCR扩增出B16/pcDNA3.1(+)-rmCGβ细胞株中rmCGβ全段基因cDNA。结果经4轮PCR,成功扩增出rmCGβ的cDNA全长基因,酶切和测序证明正确构建了真核表达质粒pcDNA3.1(+)-rmCGβ,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(+)-rmCGβ。结论成功克隆和构建了rmCGβ的真核表达质粒载体pcDNA3.1(+)-rmCGβ,为进一步研究rmCGβ的新功能和免疫治疗奠定了基础。

Objective To construct an eukaryotic expression plasmid expressing macaca mulatta chorionic gonadotropin hormone subunit（rmCGβ） gene and test whether the gene could be expressed in the cell B16 in vitro. Methods The full-length cDNA of rmCGβ gene was obtained by PCR. Then the cDNA was inserted into the eukaryotie expression vector pcDNA3.1 （＋）. The recom- binant eukaryotic plasmid pcDNA3.1 （＋）-rmCGβ was transfected into eukaryotic cell B16 by Lipofectin. RT-PCR was used to cer- tify whether the gene was correctly expressed in B16/ cDNA3.1（＋）-rmCGβ cell. Results cDNA of rmCGβ was correctly ampli- fied. The recombinant eukaryotic expression plasmid pcDNA3.1 （＋）-rmCGβ was successfully constructed, and it could be correctly expressed in the B16 cells. Conclusion This recombinant plasmid pcDNA3.1（＋）-rmCGβ will provide a basis for further study on unknown functions of rmCGβ.