肿瘤相关抗原RCAS1重组质粒的构建与GST-RCAS1融合蛋白的表达和纯化及鉴定  

Construction of recombinant GST-RCAS1 fusion gene and its expression in E.Coli

作　　者：洪学军[1] 沈芬平[1] 王青青[1] 

机构地区：[1]浙江大学医学院免疫研究所,浙江杭州310031

出　　处：《浙江大学学报（医学版）》2006年第4期377-383,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金项目(30100163)

摘　　要：目的:构建RCA S1的重组质粒、表达GST-RCA S1融合蛋白并进行纯化和生物学活性鉴定。方法:从M CF-7细胞提取总RNA,通过RT-PCR得到RCA S1的扩增产物,纯化后用E coRⅠ和BamHⅠ双酶切。选择pGEX-2T作为载体,用E coRⅠ和BamHⅠ双酶切后与上述酶切后DNA连接,转化感受态JM 109大肠杆菌,挑单个菌落提取质粒进行双酶切和测序鉴定。选择测序正确的质粒重新转化BL 21大肠杆菌,用终浓度0.1 mm o l/L的IPTG诱导表达GST-RCA S1蛋白,用GST亲和层析纯化并通过SDS-PAGE电泳和W estern印迹试验证实。利用特异性抗RCA S1多抗(N-18和C-20)鉴定所表达的融合蛋白,通过流式细胞仪检测GST-RCA S1融合蛋白诱导活化T细胞凋亡的作用。结果:通过RT-PCR得到大小为642bp的产物,重组质粒通过双酶切和测序鉴定正确。通过IPTG诱导在BL 21大肠杆菌中表达并纯化得到GST-RCA S1蛋白,通过SDS-PAGE电泳鉴定分子量为52 kM r,与预测一致,并由W estern印迹试验证明为GST融合蛋白。该融合蛋白能被特异性抗RCA S1(N-18和C-20)多抗识别。GST-RCA S1对诱导活化的T细胞凋亡有一定的作用。结论:成功构建RCA S1的重组质粒,并表达GST-RCA S1融合蛋白,对其生物学活性作了初步研究。Objective： To construct the recombinant plasmid of RCASI,to express and purify its fusion protein GST-RCAS1, and to investigate its biological function. Methods： RCAS1 encoding gene was amplified by RT-PCR from total RNA extract of MCF-7 cells and was ligated with expression plasmid vector pGEX-2T by T4 DNA ligase after digested by the restricted endonucleases BamH I and EcoR I. Then the ligated products were inserted into competence JM109 E. Coli and the positive recombinants were identified by restriction endonuclease digestion assay and DNA sequencing. The GST-RCAS1 fusion protein expression was induced by IPTG in BL21 E. Coli and was purified with GST column and identified by SDS-PAGE and Western blotting with anti-GST monoclonal antibody, anti-RCAS1 （N-18） and anti-RCAS1 （C-20） polyclonal antibody. The apoptosis of activated T cells induced by GST-RCAS1 fusion protein was detected by flow cytometry with Annexin V and propidium iodide （PI） staining. Results： A 642 bp product was cloned by RT-PCR and the recombinant plasmid was constructed successfully. The GST-RCAS1 fusion protein was recognized by GST monoclonal antibody and RCAS1 （N-18 and C-20） polyclonal antibody. FACS analysis showed that GST-RCAS1 fusion protein induced apoptosis in activated T cells. Conclusion： The recombinant plasmid of RCAS1 has been successfully constructed and the GST-RCAS1 fusion protein expressed and purified. The apoptosis inducing effect of GST-RCAS1 fusion protein on activated T cells is demonstrated.

关 键 词：抗原 肿瘤 RCAS1 重组质粒 GST—RCAS1融合蛋白 细胞凋亡 

分 类 号：R730.231[医药卫生—肿瘤]