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目的:在大肠杆菌中分别表达3种Red蛋白,并制备兔抗Red蛋白的抗体。方法:从λ噬菌体基因组中,通过PCR分别扩增gam、bet和exoDNA的全长序列。将PCR产物克隆入非融合表达载体pDH2中,构建Red蛋白的高表达工程菌。通过温敏诱导表达3种Red蛋白(Bet、Exo和Gam),用PACTE薄层扫描检测目的蛋白含量。用3种Red蛋白分别免疫家兔制备抗血清,并以Westernblot鉴定抗体的效价和特异性。结果:大肠杆菌中表达的Bet、Exo和Gam蛋白,分别占菌体总蛋白量的40.3%、49.2%和73.4%。3种抗血清的效价约为1∶2000。Westernblot分析显示抗血清具有较好的特异性。结论:成功地获得Bet、Exo和Gam3种蛋白,并分别制备了相应的抗血清。使用这3种抗血清,检测了3种蛋白在真核细胞中的表达和定位,为研究Red蛋白的同源重组奠定了基础。
OBJECTIVE: To express three kinds of Red proteins respectively in E. coli and prepare rabbit anti-Red protein antibody. Methods: The full-length sequences of gam, bet and exoDNA were amplified by PCR from lambda phage genome. The PCR product was cloned into the non-fusion expression vector pDH2 to construct a highly expressed engineered Red protein. Three kinds of Red proteins (Bet, Exo and Gam) were expressed by temperature-sensitive induction, and the content of the target protein was detected by TLC scanning with PACTE. Rabbits were immunized with three kinds of Red proteins respectively to prepare antiserum. The antibody titer and specificity were identified by Western blot. Results: The Bet, Exo and Gam proteins expressed in E. coli accounted for 40.3%, 49.2% and 73.4% of the total bacterial proteins, respectively. The titer of the three antisera was about 1: 2000. Western blot analysis showed that the antiserum has good specificity. Conclusion: Three kinds of Bet, Exo and Gam proteins were successfully obtained and corresponding antisera were prepared respectively. Using these three kinds of antiserum, we detected the expression and localization of three proteins in eukaryotic cells, which laid the foundation for the study of homologous recombination of Red protein.