【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。更多还原

【Abstract】 Objective: To construct the recombinant plasmid of RCAS1,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 BamHⅠ and EcoRⅠ.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.更多还原