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

基础医学与临床 ›› 2009, Vol. 29 ›› Issue (12): 1268-1272.

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

郭阳郑静胡愉崔莲仙何维

中国医学科学院基础医学研究所北京协和医学院基础学院中国医学科学院基础医学研究所北京协和医学院基础学院中国医学科学院基础医学研究所北京协和医学院基础学院中国医学科学院基础医学研究所北京协和医学院基础学院中国医学科学院基础医学研究所北京协和医学院基础学院

收稿日期:2009-09-01 修回日期:2009-09-23 出版日期:2009-12-20 发布日期:2009-12-20
通讯作者: 何维

Expression and biological function identification of TCR 9/ 2-Fc protein in baculovirus vector expression system

Yang GUO, Jing ZHENG, Yu HU, Lian-xian CUI, Wei HE

IBMS & School of Basic Medicine, CAMS & PUMC IBMS & School of Basic Medicine, CAMS & PUMC

Received:2009-09-01 Revised:2009-09-23 Online:2009-12-20 Published:2009-12-20
Contact: Wei HE

摘要/Abstract

摘要： 目的建立昆虫杆状病毒表达系统表达TCR 9/ 2-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 Fc和 Fc基因片段，构建成pBACp10ph- 9/ 2(OT3)-Fc重组质粒，与AcNPV昆虫病毒DNA共转染昆虫sf9细胞，表达TCR 9/ 2-Fc融合蛋白；Western blot鉴定TCR 9/ 2-Fc蛋白表达位置；胞内流式细胞仪检测 Fc和 Fc两基因表达效率；proteinG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confocal和生物传感器（Surface Plasmon Resonance SPR）技术检测了TCR 9/ 2-Fc蛋白与SKOV3细胞和MNS蛋白的结合特性。结果成功构建pBACp10ph- 9/ 2(OT3)-Fc表达载体，经鉴定发现TCR 9/ 2-Fc蛋白在sf9细胞内表达，但 Fc和 Fc两基因的表达效率有较大差异。纯化得到一定纯度的TCR 9/ 2-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TCR 9/ 2-Fc融合蛋白的技术平台，表达产物TCR 9/ 2-Fc可以在体外模拟TCR 识别抗原的特性。

关键词: TCRγ9/δ2-Fc, 昆虫杆状病毒表达系统

Abstract: Objective To establish an expression system of TCR 9/ 2-Fc protein by baculovirus vector expression system and identify biological function of expressed TCR 9/ 2-Fc protein. Methods Fc and Fc gene fragments were amplified by overlap PCR and inserted into expression vector pBACp10ph. The recombinant plasmid pBACp10ph- 9/ 2(OT3)-Fc and the baculovirus DNA were co-transfected into sf9 cells. The expression position of TCR 9/ 2(OT3)-Fc was identified by Western blot and the expression efficiency of 9Fc and 2(OT3)Fc was tested by FCM. Furthermore, the binding activity of TCR 9/ 2(OT3)-Fc protein with SKOV3 cells and MNS protein was evaluated with confocal and SPR. Results The recombinant vector pBACp10ph- 9/ 2(OT3)-Fc was constructed and TCR 9/ 2(OT3)-Fc protein was expressed in sf9 cells. However, the expression efficiency of 9Fc and 2(OT3)Fc was quite different. It was proved that purified TCR 9/ 2(OT3)-Fc protein can bind with SKOV3 cell and MNS protein. Conclusions TCR 9/ 2-Fc protein is successfully expressed in baculovirus vector expression system and TCR 9/ 2-Fc protein can mimic the binding activity of TCR in vitro

Key words: TCRγ9/δ2-Fc, baculovirus vector expression system

郭阳郑静胡愉崔莲仙何维. 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[J]. 基础医学与临床, 2009, 29(12): 1268-1272.

Yang GUO; Jing ZHENG; Yu HU; Lian-xian CUI; Wei HE. Expression and biological function identification of TCR 9/ 2-Fc protein in baculovirus vector expression system[J]. Basic & Clinical Medicine, 2009, 29(12): 1268-1272.