人源化BPVL1-GFP融合基因的构建以及在哺乳动物细胞中的瞬时表达

Construction and transient expression in mammalian cells of humanized BPVL 1-GFP fusion gene

目的:构建牛乳头瘤病毒晚期基因L1-GFP融合基因真核表达质粒,自细胞水平探讨优化密码对晚期基因L1蛋白瞬时表达的影响。方法:用PCR方法扩增获得优化密码人源化BPVL1(hL1)基因。TA克隆后,酶切释放hL1基因,定向插入质粒pET30a/NM-GFP的BamHI、SacII位点,获得正确框架的hL1-GFP融合基因。再用BamHI、NotI酶切释放hL1-GFP融合基因,与pcDNA3载体定向重组构建真核表达质粒pcDNA3/hL1-GFP。同法克隆获得含野生型BPVL1-GFP融合基因的表达质粒pcDNA3/wL1-GFP。用质粒DNA分别在体外转染哺乳动物细胞COS-1细胞和Wish细胞,荧光显微镜下观察L1-GFP融合蛋白的瞬时表达情况。结果:酶切鉴定表明成功构建了含wL1-GFP、hLI-GFP的真核表达载体,与pcDNA3/wL1-GFP相比,pcDNA3/kL1-GFP在COS-1细胞、WISH细胞中获得有效表达。结论:优化密码能提高乳头瘤病毒L1基因在哺乳动物细胞内的蛋白表达。

Objective:To construct bovine papi llomavirus (BPV)L1-GFP eukaryotic expression vector and investigate the effe ct of optimal codon usage on expression of L1gene in cells.Methods:HBPVL1(h L1)gene,whose codon usage has been optimized,was amplified by PCR.After TA cloning,hL1gene was cut with BamHI and SacII and inserted into pET30a/NM-GFP.The hL1-GFP DNA frag-ment was digested with BamHI and NotI,and inse rted into pcDNA3to construct eukaryotic expression vector pcDNA3/hL1-GFP.p cDNA3/wL1-GFP vector was obtained by the same methods.The plasmids were tr ansfected into COS-1cells and WISH cells in vitro.The transient expression o f L1-GFP was detected under fluorescence microscope.Results:According to the results of enzyme digestion,eukary-otic expression vector pcDNA3/hL1-GFP and pcDNA3/wL1-GFP were constructed successfully.Com-pared with wL1-GFP gene,hL1-GFP expressed much effectively in COS-1cells and WISH cells.Con-clusion:The codon usage optimization contributes to the enhancement of BPV L 1gene expression.