甲型流感病毒M2蛋白胞外功能区真核表达质粒的构建和表达分析

目的 构建流感病毒M2蛋白胞外功能区（M2e）真核表达质粒，并研究其在真核细胞中的表达。方法 根据Genbank（NCBI ISDN13425）中查得的M2e编码序列，设计并合成两条DNA单链;在两条链两端添加碱基构成酶切位点的粘性末端，退火后使之互补结合成为M2e编码序列；然后将其插入到经双酶切的真核表达载体pcDNA3．1（＋）中，构建重组真核表达质粒pcDNA3．1（＋）-M2ef经酶切和DNA测序鉴定后，转染HEK293细胞。用免疫荧光技术检测pcD—NA3．1（＋）-M2e在HEK293细胞的表达。并通过MTT检测刺激淋巴细胞增殖及M2e蛋白的分泌情况。结果 合成的寡核苷酸链经退火形成双链，插入酶切的真核表达载体后构建成pcDNA3．1（＋）-M2e。免疫荧光技术证实该质粒表达的M2e蛋白定位于细胞膜上，转染细胞的培养上清经MTT实验证实能刺激淋巴细胞增殖，表明表达的M2e蛋白也可分泌至细胞外。结论成功构建了流感病毒M2e的真核表达质粒pcDNA3．1（＋）-M2e，表达的M2e蛋白不仅存在于绳胞膜上，也可分泌到细胞外。流感病毒M2e基因真核表达质粒的构建及成功表达为流感病毒基因工程疫苗、通用疫苗和核酸疫苗的研究奠定了基础。

Construction and expression of eukaryotic expressing vector containing M2e gene of influenza A virus

Objective To construct eukaryotic expressing plasmid containing M_(2)e gene of influenza A virus(H1N1),transfer it into HEK293 eukaryotic cell line and detect the expression of target protein.Methods Two oligonucleotides were synthesized and annealed.The annealed DNA fragment encoding for M_(2)e was inserted into the eukaryotic expressing vector pcDNA3.1(+) for constructing the recombinant plasmid pcDNA3.1(+)-M_(2)e.The recombinant plasmid was identified by restrictive enzyme analysis and sequencing analysis.The correct plasmid DNA was transfected into HEK293 cells with Lipofectamine~(TM) 2000 Transfection Reagent.The expression of M_(2)e gene was analyzed by Immuno-fluorescence assay and lymphocytes multiplication test with MTT. Results Restrictive enzyme identification and sequencing analysis demonstrated that the M_(2)e gene was successfully synthesized and inserted into pcDNA3.1(+).Immuno-fluorescence assay confirmed that the M_(2) gene could be expressed in HEK293 cells,and expressed M_(2)e could be secreted into culture supernatants.Conclusion The eukaryotic expressing plasmid containing M_(2)e gene was constructed successfully.As M_(2) protein of influenza A virus was a kind of highly conserved protein in amino acid sequence,this study provided a foundation for exploring genetic engineering vaccine,DNA vaccine and broad-spectrum vaccine against for influenza A virus.