昆虫杆状病毒表达系统表达TCRγ9/δ2-Fc融合蛋白及其生物学功能的初步鉴定

Expression and biological function identification of TCRγ9/δ2-Fc protein in baculovirus vector expression system

目的建立昆虫杆状病毒表达系统表达TCRγ9/δ2-Fc融合蛋白技术平台并鉴定其表达产物的生物学功能。方法用搭桥PCR获得γ9Fc和δ2(OT3)Fc基因片段,构建成pBACp10ph-γ9/δ2(OT3)-Fc重组质粒,与AcNPV昆虫病毒DNA共转染昆虫sf9细胞,表达TCRγ9/δ2-Fc融合蛋白;Western blot鉴定TCRγ9/δ2-Fc蛋白表达位置;胞内流式细胞仪检测γ9Fc和δ2(OT3)Fc两基因表达效率;蛋白G亲和层析后,SDS-PAGE鉴定蛋白纯度;激光共聚焦扫描显微镜技术和生物传感器技术检测了TCRγ9/δ2-Fc蛋白与SKOV3细胞和MNS蛋白的结合特性。结果成功构建pBACp10ph-γ9/δ2(OT3)-Fc表达载体,经鉴定发现TCRγ9/δ2-Fc蛋白在sf9细胞内表达,但γ9Fc和δ2(OT3)Fc两基因的表达效率有较大差异。纯化得到一定纯度的TCRγ9/δ2-Fc蛋白,并且具有与SKOV3细胞和MNS蛋白结合的特性。结论成功建立昆虫杆状病毒表达系统表达TCRγ9/δ2-Fc融合蛋白的技术平台,表达产物TCRγ9/δ2-Fc可以在体外模拟TCRγδ识别抗原的特性。

Objective To establish an expression system of TCRγ9/δ2-Fc protein by baculovirus vector expression system and identify biological function of expressed TCRγ9/δ2-Fc protein.Methods γ9Fc and δ2（OT3）Fc gene fragments were amplified by overlap PCR and inserted into expression vector pBACp10ph.The recombinant plasmid pBACp10ph-γ9/δ2（OT3）-Fc and the baculovirus DNA were co-transfected into sf9 cells.The expression position of TCRγ9/δ2（OT3）-Fc was identified by Western blot and the expression efficiency of γ9Fc and δ2（OT3）Fc was tested by flow cytometry（FCM）.Furthermore,the binding activity of TCRγ9/δ2（OT3）-Fc protein with SKOV3 cells and MNS protein was evaluated with laser scanning confocal microslopy and surface plasmon resonance（SPR）.Results The recombinant vector pBACp10ph-γ9/δ2（OT3）-Fc was constructed and TCRγ9/δ2（OT3）-Fc protein was expressed in sf9 cells.However,the expression efficiency of γ9Fc and δ2（OT3）Fc was quite different.It was proved that purified TCRγ9/δ2（OT3）-Fc protein can bind with SKOV3 cell and MNS protein.Conclusion TCRγ9/δ2-Fc protein is successfully expressed in baculovirus vector expression system and TCRγ9/δ2-Fc protein can simulate the binding activity of TCR in vitro.