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目的:原核表达EV71结构蛋白VP0(VP2+VP4)并制备其多克隆抗体。方法:以肠道病毒71型(EV71)全基因组为模板,设计引物扩增出目的片段VP0,将其克隆至表达载体pET-30a(+),并转化大肠杆菌TG1,筛选出阳性克隆后进行测序。将重组表达载体pET-30a(+)-VP0转入大肠杆菌表达菌株Rosetta中。该重组菌经过IPTG诱导表达并通过SDS-PAGE电泳和Western Blot验证后,有与预期分子量大小一致的蛋白条带,并且主要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。将纯化的蛋白免疫新西兰大白兔制备了VP0多克隆抗体,并对该抗体进行了细胞免疫荧光分析。结果:经过大肠杆菌重组表达并纯化得到了纯度较高的VP0蛋白,制备的多克隆抗体经过细胞免疫荧光的验证表明反应性良好。结论:成功地表达VP0蛋白并制备了其多克隆抗体,有利于EV71病毒的检测及下一步对其疫苗的研究。
OBJECTIVE: To prokaryotic express EV71 structural protein VP0 (VP2 + VP4) and prepare its polyclonal antibody. Methods: The full-length genomic DNA of EV71 was used as a template to amplify VP0. The recombinant plasmid was cloned into expression vector pET-30a (+) and transformed into E. coli TG1. The positive clones were screened out Sequencing. The recombinant expression vector pET-30a (+) - VP0 was transformed into Escherichia coli expression strain Rosetta. After induced by IPTG and verified by SDS-PAGE electrophoresis and Western Blot, the recombinant strain has a protein band consistent with the expected molecular weight and mainly exists as an inclusion body. 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. The purified protein was immunized New Zealand white rabbits VPO polyclonal antibody, and the antibody was analyzed by immunofluorescence. Results: The VP0 protein with high purity was obtained by recombinant expression in Escherichia coli and purified. The prepared polyclonal antibody was verified by immunofluorescence and showed good reactivity. Conclusion: The VP0 protein was successfully expressed and its polyclonal antibody was prepared, which is beneficial to the detection of EV71 virus and the further study of its vaccine.