信号肽序列对禽流感病毒H5N1 HA 蛋白GFP融合分子在真核细胞中表达的影响

目的]构建绿色荧光蛋白(GFP)与禽流感病毒(AIV)HA基因的融合表达载体,观察信号肽序列的有无及位置对HA-GFP在293T细胞中的表达影响.方法]应用PCR方法从H5N1亚型AIV质粒DNA中扩增出完整的或除去信号肽序列的HA基因片段,PCR产物经Xho Ⅰ和SmaⅠ双酶切后定向插入绿色荧光蛋白真核表达载体pEGFP-C1的不同位点,将得到的重组体M1,M2和M3分别转化宿主菌DH5a,经双酶切及.DNA测序鉴定分析后,采用脂质体转染法将pEGFF-C1,M1,M2和M3转染人胚肾细胞系293T细胞,荧光显微镜下观察HA-GFP的表达,流式细胞仪检测表达HA蛋白的细胞百分数.结果]经酶切及测序鉴定成功构建了HA-GFP重组表达载体M1,M2和M3,荧光显微镜及流式细胞仪检测到重组质粒转染的293T细胞表达强的荧光蛋白,信号肽的有无对HA-GFP融合蛋白在293T细胞中的表达有显著影响,信号肽序列使HA-GFP融合蛋白在293T细胞中的表达减少,而信号肽的位置对融合蛋白表达量的影响不显著.结论]信号肽的有无对HA-GFP融合蛋白在细胞中的表达有显著影响.

Effects of signal peptide sequence of hemagglutinin on the expression of green fluorescent protein and hemagglutinin fusion gene

Abstract: Objective] Purified avian influenza virus (AIV) hemagglutinin (HA) gene fragment was inserted into green fluorescent protein (GFP) expression vector, pEGFP-C1. The role of signal peptide in the HA-GFP expression in 293T cells was investigated by cutting the signal peptide or placing it in different locations of HA-GFP fusion gene. Methods] The expression of GFP in 293T cells was examined directly with fluorescence microscope and flow cytometry analysis. Results] After transfected with pEGFP-C1, M1, M2 and M3 plasmid, under fluorescent microscope the HA-GFP fusion protein was successfully expressed. Fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the empty vector pEGFP-C1, whereas signal peptide could obviously reduced the expression of GFP-HA compared with recombinant plasmid without signal peptide. In addition, we found that there was no significant effect of the location of signal peptide on the expression of HA-GFP fusion protein.