禽白血病病毒p27蛋白在大肠杆菌中的表达和多克隆抗体的制备

摘要

p27 protein gene from avian leukosis virus was cloned and constructed with plasmid pET-28a(+) . The recombinant protein p27 was expressed in an E. coli strain BL21(DE3) with IPTG induction and detected by Western blot with horseradish peroxidase labeled rabbit-anti-p27 antibodies. After that,the protein was purified to above 95% of purity by affinity chromatography with Ni-NTA agarose column. The anti-sera against p27 protein was obtained by immunizing rabbit with the purified recombinant p27 protein. The specificity of polyclonal antibody was about 1 ∶ 256000 after detected by ELISA. The results show that the p27 protein expressed by E. coli has good antigenicity and can replace the purified virus protein for detection of avian leukosis virus.%将禽白血病病毒p27基因克隆并构建重组表达质粒pET28a-p27，在大肠杆菌BL21中经IP TG诱导后产生可溶性表达蛋白。表达的蛋白用Western blot进行活性检测，其能与辣根过氧化物酶标记的兔抗p27抗体发生特异性反应；采用金属螯和层析对表达蛋白进行纯化，其纯度约为95％；用纯化的表达蛋白p27免疫家兔制备抗血清，ELISA抗体效价可达1∶25600。研究结果表明，大肠杆菌表达的p27蛋白具有良好的抗原性，可以替代纯化病毒蛋白用于禽白血病病毒检测。