小鼠β-防御素6与甲型流感病毒M2e基因融合表达及其免疫特性研究

目的构建小鼠β防御素6（mBD6）与甲型流感病毒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与免疫特性。方法通过重叠延伸PCR法（overlap-PCR）将M2e与mBD6通过一段多肽接头Gly4Ser融合成为mBD6-M2e。将其插入载体pcDNA3．1（＋），构建成pcDNA3．1（＋）／mBD6-M2e。鉴定正确后，转染MDCK细胞，免疫荧光、MTT检测mBD6-M2e表达和分泌。免疫小鼠，半定量PCR检测脾细胞因子IL-2、IFN-γ、TNF-α、CCR7表达。结果融舍基因mBD6-M2e真核表达载体pcDNA3．1（＋）／mBD6-M2e构建成功，在MDCK细胞膜上成功表达融和蛋白，转染细胞的培养上清刺激淋巴细胞增殖。免疫小鼠细胞因子表达改变。结论融合基因mBD6-M2e真核表达载体成功构建，为研究防御素在流感病毒核酸疫苗中的佐剂作用奠定了基础。

Construction and expression of M2e and immunity of fused protein mBD6 and H1N1

Objective To construct the eukaryotic expression vector for mouse 66 defensin （mBD6） and M2e gene of influenza A virus （H1N1） and detect the expression of target protein, and immunity of recombinant protein. Methods A polypeptide linker G1y4Ser was used to splice mBD6 and M2e gene by overlap-PCR for constructing the mBD6-M2e fusion gene. It was inserted into eukaryotie expressing plasmid peDNA3.1 （＋）. The plasmid DNA of pcDNA3.1 （＋）/ mBD6-M2e was transfected into MDCK. The expression of mBD6-M2e gene was analyzed by immunofluorescence assay and lymphocytes multiplication test with MTT. Cytokine isolated from the spleen of BALB/e mice which were injected with plasmids in advance, and analyzed by semi-quantitative PCR. Results Expression vector pcDNA3.1（＋） including mBD6-M2e fusion gene was constructed successfully. The fusion protein could be detected in MDCK transfected with pcDNA3. 1（＋）/mBD6-M2e, and expressed mBD6-M2e could be secreted into culture supernatants. The expression pat- tern of cytokines changed obviously. Conclusion The success in the construction of eukaryotie expressing plasmid for mBD6-M2e fusion gene set up a solid foundation for further exploring the function of defensin against influenza virus through DNA vaccine.