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  

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

作　　者：冯伟[1] 李婉宜[1] 李明远[1] 张强[1] 巩天祥[1] 刘冯欢[1] 罗俊[1] 邝玉[1] 王保宁[1] 蒋忠华[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,四川成都610041

出　　处：《西部医学》2010年第3期400-402,405,共4页Medical Journal of West China

基　　金：国家自然基金资助项目(NO:30671964)

摘　　要：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。结果融合基因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。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.

关 键 词：β-防御素6 甲型流感病毒 mBD6-M2e融合基因 淋巴细胞增殖实验 

分 类 号：R446.9[医药卫生—诊断学] R373.13[医药卫生—临床医学]