Red蛋白抗血清的制备及其亚细胞定位研究

Preparation of anti-Red antisera and its subcellular localization

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。

AIM: To prepare rabbit anti-Red antisera. METHODS: The bet, exo and gam genes of lambda phage were amplified by PCR from genomic DNA and cloned into the expression vector pDH2, respectively. Red proteins were induced to express at 42℃. The expressed proteins were analyzed by PAGE and thin-layer scanning. The antisera were prepared by immunizing rabbits with the three Red proteins, respectively. The titers and specificities of the antisera were detected by Western blot. RESULTS: Beta, Exo and Gam proteins accounted for about 40.3%, 49.2% and 73.4% of total bacterial protein, respectively. The titers of the antisera were about 1∶2 000. Western blot analysis indicated that the three antisera all had good specificities to the corresponding proteins. CONCLUSION: Specific anti-Red antisera are prepared successfully.