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目的构建人肠道病毒71型3A基因原核表达质粒,制备重组3A蛋白及其抗体。方法 PCR方法扩增人肠道病毒71型3A基因,构建原核表达质粒PET28a-3A并转化大肠埃希菌,诱导表达3A重组蛋白,免疫小鼠制备3A蛋白抗体。通过免疫荧光和Western blot方法鉴定抗体特异性。结果成功构建了PET28a-3A原核表达质粒并表达了3A重组蛋白,3A蛋白以包涵体的形式存在。细胞免疫荧光和Western blot检测显示,制备的鼠源3A蛋白抗体可识别真核表达的EGFP-3A融合蛋白以及EV71感染细胞表达的3A蛋白。结论成功构建了人肠道71型3A基因原核表达质粒,利用该质粒表达的重组蛋白成功制备了特异性的3A蛋白的鼠源抗体。
Objective To construct prokaryotic expression plasmid of human enterovirus 71 3A gene and prepare recombinant 3A protein and its antibody. Methods The 3A gene of human enterovirus 71A was amplified by PCR. The prokaryotic expression plasmid PET28a-3A was constructed and transformed into Escherichia coli. The 3A recombinant protein was induced and immunized to prepare the 3A protein antibody. Antibody specificity was identified by immunofluorescence and Western blot. Results The prokaryotic expression plasmid PET28a-3A was successfully constructed and 3A recombinant protein was expressed. The 3A protein existed in the form of inclusion body. Cell immunofluorescence and Western blot showed that the prepared murine 3A protein antibody could recognize the eukaryotic expressed EGFP-3A fusion protein and the 3A protein expressed by EV71 infected cells. Conclusion The prokaryotic expression plasmid of human intestinal type 71 3A gene was successfully constructed. The specific antibody of 3A protein was successfully prepared using the recombinant protein expressed in this plasmid.