Fcγ-Der f2载体构建及融合蛋白表达

The eukaryotic expression and identification of Fcγ-Der f2 fusion protein and examination of its biological activity

目的构建人IgGFcγ1片段Fcγ与粉尘螨Ⅱ类抗原Derf2嵌合基因真核表达载体pDisplay-Fcγ-Derf2，并转染人HEK293T细胞系瞬时表达，获得Fcγ-Derf2融合蛋白。方法以pMD19-T-Derf2载体为模板，设计引物并加入linker序列，经PCR扩增得到linke-Der f2 DNA片段。经限制性内切酶酶切后，先后将人Fcγ及linker—Derf2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72h的HEK293T细胞并裂解细胞进行Western Blot检测。结果pDisplay-Fcγ-Derf2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Derf2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 human FcT-Der ff 2 chimeric gene,and to study the expression and effect of FcT-Der f 2 fusion protein. Methods Amplified The cDNA encoding Der f2 from pMD19-T-Der f2 vector was amplified and the flexible linker sequence was set at the 5＇-end. Fcy and linker-Der f2 was inserted into pDisplay expression vector. The new pDisplay-FcT-Der f2 vector was transfected into HEK293T cells by Attractene reagent. And the expression of fusion FcT-Der f 2 protein was determined by imnmnofluorescence and western blot. Results The results off the sequence and restriction enzyme analysis showed that pDisplay-Fc× Der f2vector was successfully constructed. The immunofluorescence demonstrated that the recombinant fusion protein was correctly expressed in HEK293T cells. At the same time, the Western blot showed the antigenicity of Der f2 in the ffusion protein and the molecular weight of this protein was 40 × 10^3. Conclusion The FcT-Der f 2 fusion gene was constructed successfully and the fusion protein is of biological activity.