过表达EB病毒潜伏期膜蛋白1(LMP1)的人B淋巴细胞株的建立

Establishment of human B lymphocyte strain overexpressing Epstein-Barr virus latent membrane protein 1(LMP1)

目的构建EB病毒潜伏期膜蛋白1(LMP1)基因的真核表达载体,通过电穿孔法转染人B淋巴瘤细胞株Ramos细胞,建立稳定的LMP1表达细胞株。方法扩增带有EcoRⅠ与BamHⅠ酶切位点的LMP1编码基因,克隆至pcDNA3.1-EF1a-mcs-3FLAG-CMV-EGFP真核表达载体,挑取阳性单克隆感受态细胞行PCR以及测序后将重组质粒通过电穿孔法转染Ramos细胞, G418筛选稳定表达LMP1的细胞株, PCR和Western blot法分别检测LMP1 mRNA和蛋白在Ramos细胞中的表达。结果 PCR与测序证实成功构建了LMP1重组质粒,建立了稳定转染的Ramos细胞,且LMP1蛋白可在细胞中成功表达。结论成功建立了稳定表达LMP1的Ramos细胞。

Objective To establish a human B lymphoma cell line which can stably express Epstein-Barr virus latent membrane protein 1(LMP1). Methods The LMP1 coding gene with EcoRⅠ and BamHⅠ restriction sites was amplified, cloned into pcDNA3.1-EF1a-mcs-3FLAG-CMV-EGFP plasmid, and positive monoclonal competent cells were picked for PCR and sequencing. The recombinant plasmid was transfected into Ramos cells by electroporation, and the cell line stably expressing LMP1 was picked by G418. The expression of LMP1 in Ramos cells was detected by PCR, Western blot analysis and green fluorescent protein Tag. Results PCR and sequencing confirmed that the LMP1 recombinant plasmid was successfully constructed and a stably transfected Ramos cell line was established. Western blot analysis confirmed that LMP1 protein was successfully expressed in this cell line. Conclusion The Ramos cells stably expressing LMP1 have been successfully established.