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目的构建人IgG Fcγ1片段Fcγ与粉尘螨Ⅱ类抗原Der f2嵌合基因真核表达载体pDisplay-Fcγ-Der f2,并转染入HEK293T细胞系瞬时表达,获得Fcγ-Der f2融合蛋白。方法以pMD19-T-Der f2载体为模板,设计引物并加入linker序列,经PCR扩增得到linker-Der f2 DNA片段。经限制性内切酶酶切后,先后将人Fcγ及linker-Der f2基因片段接入pDisplay真核表达载体。用Attractene转染试剂将其转染至HEK293T细胞使之表达融合蛋白。免疫荧光检测转染后γ2 h的HEK293T细胞并裂解细胞进行Western Blot检测。结果 pDisplay-Fcγ-Der f2质粒经双酶切鉴定及DNA测序鉴定证实序列完全正确,真核表达载体构建成功。免疫荧光鉴定转染细胞可见明显红色荧光。Western Blot检测证明融合蛋白相对分子质量为40×10~3,与理论预期值相符合,并证明了Fcγ与Der f2双功能特性。结论构建的融合蛋白Fcγ-Der f2符合目的要求。
OBJECTIVE: To construct the eukaryotic expression vector pDisplay-Fcγ-Der f2, which encodes Fcγ1 fragment of Fcγ1 and derivative Der f2 of Dermatophagoides pteronyssinf2 antigen and transfect it into HEK293T cell line for transient expression to obtain Fcγ-Der f2 fusion protein. Methods The pMD19-T-Der f2 vector was used as a template to design the primer and add the linker sequence. The linker-Der f2 DNA fragment was amplified by PCR. After restriction endonuclease digestion, human Fcγ and linker-Der f2 gene fragments were successively ligated into pDisplay eukaryotic expression vector. It was transfected into HEK293T cells with Attractene transfection reagent to express the fusion protein. HEK293T cells at γ 2 h after transfection were detected by immunofluorescence and cells were lysed by Western Blot. Results The pDisplay-Fcγ-Der f2 plasmid was identified by double enzyme digestion and DNA sequencing. The sequence was confirmed correctly and the eukaryotic expression vector was constructed successfully. Immunofluorescence identified transfected cells showed obvious red fluorescence. The result of Western Blot showed that the relative molecular mass of the fusion protein was 40 × 10 ~ 3, which was consistent with the expected value of the theory. The bifunctional characteristics of Fcγ and Der f2 were also proved. Conclusion The constructed fusion protein Fcγ-Der f2 meets the goal.