禽网状内皮组织增生病病毒gp90全长蛋白的原核表达、纯化及其免疫原性分析

Prokaryotic expression, purification and identification of the recombinant gp90 protein of Reticuloendotheliosis virus

摘要:

摘要：【目的】原核表达免疫原性良好的禽网状内皮组织增生病病毒（( Reticuloendotheliosis virus, REV )gp90蛋白，并制备抗gp90蛋白高效价多克隆血清。【方法】利用PCR技术，以pMD18T-env为模板，扩增得到REV的gp90蛋白编码基因，将其克隆入表达载体pET-28a(+)中，将构建的原核表达质粒pET28-gp90，转化大肠杆菌（Escherichia coli ) BL21 (DE3) 感受态细胞，经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后进行gp90蛋

Abstract:

Abstract: [Objective] To obtain the recombinant gp90 protein of Reticuloendotheliosis virus (REV) and the anti-gp90 serum with high titer. [Methods] Using the plasmid pMD18T-env as template, we amplified the gp90 gene and then cloned it into pET-28a (+). The recombinant plasmid pET28a-gp90 was transformed into Escherichia coli BL21 (DE3) ,which was induced with isopropylthio-β-D-galactoside(IPTG). After identification by SDS-PAGE and Western blotting, the purified gp90 protein was injected into Balb/c mice to prepare anti-gp90 serum. The specificity and titer of the antiserum were evaluated by IFA and the enzyme-linked immunosorbant assay (ELISA). [Results] SDS-PAGE and Western blotting showed that the gp90 protein was expressed successfully in the form of inclusion body in the recombinant E coli. ELISA showed the mouse anti-gp90 serum had a titer of 1:12800. Successful expression of recombinant gp90 protein and preparation of its antiserum laid the foundation for the development of diagnostic reagent of REV.