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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 eukaryotic expression plasmid of mouse β-defensin 6 (mBD6) and M2 e protein of influenza virus (M2) and study its expression in eukaryotic cells and its immunogenicity. Methods M2e and mBD6 were fused to mBD6-M2e via a polypeptide linker Gly4Ser by overlap-PCR. This was inserted into pcDNA3.1 (+) vector and constructed into pcDNA3.1 (+) / mBD6-M2e. After identification, MDCK cells were transfected, and the expression and secretion of mBD6-M2e were detected by immunofluorescence and MTT. Immunized mice, semi-quantitative PCR detection of spleen cytokines IL-2, IFN-γ, TNF-α, CCR7 expression. Results The mBD6-M2e eukaryotic expression vector pcDNA3.1 (+) / mBD6-M2e was constructed successfully. The fusion protein was successfully expressed on the MDCK cell membrane and the supernatant of the transfected cells stimulated lymphocyte proliferation. Immune mouse cytokine expression changes. Conclusion The fusion gene mBD6-M2e eukaryotic expression vector was successfully constructed, which laid the foundation for the study of adjuvant effect of defensin in influenza virus DNA vaccine.