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目的:原核表达EV71结构蛋白VP1并制备其单克隆抗体。方法:以肠道病毒71型(EV71)P1基因为模板,设计引物扩增出目的片段VP1,将其连接至大肠杆菌表达载体pET-32a(+)上,转化大肠杆菌TG1,筛选出阳性克隆后进行测序。将重组表达载体pET-32a(+)-VP1转化大肠杆菌表达菌株BLgold(DE3),对该工程菌进行诱导表达,SDS-PAGE电泳分析,表达产物以包涵体的形式存在。包涵体用6 mol/L盐酸胍溶解,经过Ni-NTA亲和层析法纯化,获得了纯度较高的目的蛋白。用纯化的VP1蛋白免疫小鼠,制备单克隆抗体(mAb)。结果:获得了24株mAb,其中1株EV71 Westernblot法鉴定呈阳性,5株EV71间接免疫荧光法(IFA)鉴定阳性。结论:成功地制备了VP1的mAb。
AIM: To prokaryotic express EV71 structural protein VP1 and prepare its monoclonal antibody. Methods: The VP1 gene of EV71 P1 was amplified by PCR and ligated into E. coli expression vector pET-32a (+). The recombinant plasmid was transformed into E. coli TG1 and positive clones were screened out After sequencing. The recombinant expression vector pET-32a (+) - VP1 was transformed into E. coli expression strain BLgold (DE3). The recombinant strain was induced and expressed by SDS-PAGE electrophoresis. The expressed product was in the form of inclusion bodies. The inclusion body was dissolved in 6 mol / L guanidine hydrochloride and purified by Ni-NTA affinity chromatography to obtain the target protein with high purity. Mice were immunized with the purified VP1 protein to prepare monoclonal antibodies (mAbs). RESULTS: Twenty-four mAbs were obtained, of which one was positive for EV71 Western blot and the other was positive for five EV71 indirect immunofluorescence assays (IFA). Conclusion: VP1 mAb was successfully prepared.