CAJ | 학술논문

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72 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最为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.
목적 구건인IgG Fcγ1편단Fcγ여분진만Ⅱ류항원Der f2감합기인진핵표체재체pDisplay-Fcγ-Der f2,병전염입HEK293T세포계순시표체,획득Fcγ-Der f2융합단백.방법 이pMD19-T-Der f2재체위모판,설계인물병가입linker서렬,경PCR확증득도linker-Der f2 DNA편단.경한제성내절매매절후,선후장인Fcγ급linker-Der f2기인편단접입pDisplay진핵표체재체.용Attractene전염시제장기전염지HEK293T세포사지표체융합단백.면역형광검측전염후72 h적HEK293T세포병렬해세포진행Western Blot검측.결과 pDisplay-Fcγ-Der f2질립경쌍매절감정급DNA측서감정증실서렬완전정학,진핵표체재체구건성공.면역형광감정전염세포가견명현홍색형광.Western Blot검측증명융합단백상대분자질최위40×10~3,여이론예기치상부합,병증명료Fcγ여Der f2쌍공능특성.결론 구건적융합단백Fcγ-Der f2부합목적요구.
Objective To construct the human Fcγ-Der f 2 chimeric gene,and to study the expression and effect of Fcγ-Der 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. Fcγ and linker-Der f2 was inserted into pDisplay expression vector. The new pDisplay-Fcγ-Der f2 vector was transfected into HEK293T cells by Attractene reagent. And the expression of fusion Fcγ-Der f 2 protein was determined by immunofluorescence and western blot. Results The results of the sequence and restriction enzyme analysis showed that pDis-play-Fcγ-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 fusion protein and the molecular weight of this protein was 40 × 10~3. Conclusion The Fcγ-Der f 2 fusion gene was constructed successfully and the fusion protein is of biological activity.