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目的 :构建人的ureb1(hureb1)原核高效表达重组质粒 ,以此表达的外源蛋白为抗原制备抗ureb1的抗体。方法 :用XhoI/NotI从pGU 2质粒酶切得到hureb1的ORF与pGEX 4T 2的XhoI/NotI大片段连接 ,构建表达GST hureb1融合蛋白的重组载体。转化大肠杆菌BL2 1(DE3) ,IPTG诱导表达。以粗制的GST hureb1融合蛋白分别免疫大白兔和小鼠 ,制备抗hureb1的多克隆抗体和单克隆抗体 ,采用WesternBlot进行特异性鉴定。结果 :构建了高效表达GST hureb1融合蛋白的原核表达载体 ,融合蛋白的表达量占菌体蛋白质总量的 33 45 %。以大肠杆菌表达的GST hureb1融合蛋白免疫动物制备了高滴度、高特异性的抗hureb1的抗体。结论 :利用重组GST hureb1融合蛋白免疫动物可获得高滴度的抗hureb1抗体。重组GST hureb1融合蛋白和抗hureb1的抗体可用于hureb1的生物学功能研究
OBJECTIVE: To construct a prokaryotic recombinant plasmid expressing human ureb1 (hureb1) and use the expressed foreign protein as antigen to prepare anti-ureb1 antibody. Methods: The ORF of hureb1 digested with XhoI / NotI from plasmid pGU 2 was ligated into the large XhoI / NotI fragment of pGEX 4T 2 to construct a recombinant vector expressing GST hureb1 fusion protein. The transformed E. coli BL21 (DE3) was induced by IPTG. Rabbit and mouse were respectively immunized with crude GST hureb1 fusion protein to prepare polyclonal and monoclonal antibodies against hureb1, and then identified by Western blot. Results: Prokaryotic expression vector expressing GST hureb1 fusion protein was constructed. The fusion protein accounted for 33 45% of the total bacterial protein. High titers, high specific anti-hureb1 antibodies were prepared from animals immunized with E. coli expressed GST hureb1 fusion protein. Conclusion: High titers of anti-hureb1 antibodies can be obtained by immunizing animals with recombinant GST hureb1 fusion protein. Recombinant GST hureb1 fusion protein and anti-hureb1 antibody can be used in the biological function of hureb1