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目的构建人偏肺病毒融合蛋白亚单位F1、F2原核表达系统,初步获得抗F1、F2重组蛋白的多克隆抗体,为相关疫苗的研究奠定基础。方法聚合酶链反应(PCR)法扩增F1、F2基因片段,经T-A克隆确保其准确性,双酶切后插入原核表达载体p ET32a(+)中,转化大肠埃希菌BL21(DE3),按优化后条件大量诱导表达,纯化后经Western Blot检测特异性。取纯化蛋白免疫BALB/c小鼠制备多克隆抗体,ELISA法检测效价,间接免疫荧光法(IFA)检测多克隆抗体是否能与人偏肺病毒发生特异性反应。结果 F1、F2基因均正确插入pET32a(+)中,并具有正确的读码框架。0.5 mmol/L IPTG 37℃诱导培养5 h可获得大量带His标签的重组蛋白,纯化后浓度分别达到200μg/ml和300μg/ml。Western Blot结果显示,重组蛋白可被抗His标签抗体特异识别;免疫小鼠获得多克隆抗体效价分别为抗pET32a-F1最高1∶640,抗pET32a-F2最高1∶40 960。IFA显示抗p ET32a-F1、抗pET32a-F2多克隆抗体均可与人偏肺病毒作用产生特异性荧光。结论成功构建了F1、F2的原核表达质粒,并在大肠埃希菌中获得高效表达,该蛋白具有良好的抗原性;免疫小鼠获得特异性多克隆抗体,有利于人偏肺病毒的深入研究。
Objective To construct the prokaryotic expression system of human metapneumovirus fusion protein subunits F1 and F2 and obtain the polyclonal antibodies against the F1 and F2 recombinant proteins preliminarily, which laid the foundation for the research of related vaccines. Methods The F1 and F2 gene fragments were amplified by polymerase chain reaction (PCR) and confirmed by TA cloning. The two fragments were inserted into prokaryotic expression vector p ET32a (+) and transformed into E. coli BL21 (DE3) After optimization, the expression was induced in large quantities, and the specificity was detected by Western Blot after purification. The purified protein was used to immunize BALB / c mice to prepare polyclonal antibody. ELISA was used to detect the titer. Indirect immunofluorescence assay (IFA) was used to detect whether the polyclonal antibody reacted specifically with human metapneumovirus. Results The F1 and F2 genes were correctly inserted into pET32a (+) and had the correct reading frame. A large number of His-tagged recombinant proteins were obtained after incubation with 0.5 mmol / L IPTG at 37 ℃ for 5 h. The purified recombinant proteins reached 200 μg / ml and 300 μg / ml, respectively. Western Blot results showed that the recombinant protein was specifically recognized by anti-His-tag antibody. The titer of the polyclonal antibody obtained from the immunized mice was 1: 640 for anti-pET32a-F1 and 1:40 for anti-pET32a-F2. IFA showed that anti-p ET32a-F1 and anti-pET32a-F2 polyclonal antibodies all produced specific fluorescence with human metapneumovirus. Conclusion The prokaryotic expression plasmids of F1 and F2 were successfully constructed and expressed highly in Escherichia coli. The protein has good antigenicity. The specific polyclonal antibody was obtained from immunized mice, which is in favor of the further study of human metapneumovirus .