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目的:构建缺失免疫抑制位点的人早孕因子基因的原核表达载体pGEX6P-EPF11,诱导GST-EPF11融合蛋白在大肠杆菌中表达,并免疫小鼠,测定效价。方法:以pMD18T-HSPE1模板,PCR扩增缺失免疫抑制位点的人早孕因子基因片段,经限制性内切酶BamHI和XhoI双酶切后,连接到原核表达载体pGEX-6P-1中,构建重组表达质粒pGEX6P-EPF11。将表达质粒转化大肠杆菌BL21,以1 mmol/L IPTG进行诱导表达GST-EPF11融合蛋白,Western blot鉴定。以表达的GST-EPF11融合蛋白作为免疫原免疫小鼠,抗体效价用ELISA检测。结果:在大肠杆菌中成功表达出相对分子质量约35 000的融合蛋白GST-EPF11。ELISA法检测免疫小鼠的抗体血清可达1∶12 800。结论:在大肠杆菌中成功表达出GST-EPF11融合蛋白,并免疫了小鼠,测定了抗体效价,为下步制备缺失免疫抑制位点的人早孕因子的单克隆抗体(mAb)奠定了基础。
OBJECTIVE: To construct a prokaryotic expression vector pGEX6P-EPF11 for human pregnancy induced by immunosuppression site and express GST-EPF11 fusion protein in E. coli and immunize mice to determine the titer. Methods: The human early pregnancy factor gene fragment lacking immunosuppressive sites was amplified by PCR using pMD18T-HSPE1 template. After digested with restriction endonucleases BamHI and XhoI, the fragment was ligated into prokaryotic expression vector pGEX-6P-1 to construct Recombinant expression plasmid pGEX6P-EPF11. The expression plasmid was transformed into E. coli BL21 and induced by 1 mmol / L IPTG to express GST-EPF11 fusion protein, and identified by Western blot. Mice were immunized with the expressed GST-EPF11 fusion protein and the antibody titers were detected by ELISA. Results: The fusion protein GST-EPF11 with a relative molecular mass of about 35 000 was successfully expressed in E. coli. Antibody sera of mice immunized with ELISA assay reached 1:12 800. CONCLUSION: The GST-EPF11 fusion protein was successfully expressed in E. coli and the mice were immunized to determine the antibody titers, which laid the foundation for the next step in the preparation of monoclonal antibodies (mAbs) for human pregnancy induced by immunosuppressive sites .