甲型流感病毒M_2蛋白胞外功能区真核表达质粒的构建和表达分析

Construction and expression of eukaryotic expressing vector containing M_2e gene of influenza A virus

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(+)-M2e;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蛋白不仅存在于细胞膜上,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。

Objective To construct eukaryotic expressing plasmid containing M2 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 M2 e was inserted into the eukaryotic expressing vector peDNA3. 1 （＋） for constructing the recombinant plasmid peDNA3.1（＋）-M2 e. The recombinant plasmid was identified by restrictive enzyme analysis and sequencing analysis. The correct plasmid DNA was transfected into HEK293 cells with LipofeetamineTM 2000 Transfection Reagent. The expression of M2 e gene was analyzed by Immuno-fluorescence assay and lymphoeytes multiplication test with MTT. Results Restrictive enzyme identification and sequencing analysis demonstrated that the M2 e gene was successfully synthesized and inserted into pcDNA3.1 （＋）. Immuno-fluorescence assay confirmed that the M2 gene could be expressed in HEK293 cells, and expressed M2e could be secreted into culture su pernatants. Conclusion The eukaryotie expressing plasmid containing M2 e gene was constructed successfully. As M2 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.