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目的原核细胞中表达、纯化新疆出血热病毒BA88166毒株核蛋白(NP)并制备及鉴定抗NP蛋白的多克隆抗体。方法采用逆转录-聚合酶链式反应(RT-PCR)扩增出BA88166毒株S基因的cDNA片段,将其构建到原核表达载体pET-32a上,形成重组原核表达质粒pET-88166S。构建好的质粒在大肠杆菌BL21(DE3)中进行诱导表达,经镍柱亲和层析法纯化NPHis融合蛋白,SDS-PAGE分析蛋白相对分子质量(M r)。用该纯化蛋白免疫新西兰白兔制备抗血清,ELISA和Western blot法检测血清效价和特异性。结果双酶切鉴定和DNA测序证实构建的pET-88166S重组表达载体正确,目的基因序列与GenBank中公布的序列一致,在E.coli BL21中表达的NP-His融合蛋白经SDS-PAGE分析,其M r约为66 000。ELISA检测抗体效价高于1∶25 600,蛋白免疫印迹实验结果表明抗体能特异性识别新疆出血热病毒YL04057毒株的NP蛋白及其截短蛋白。结论成功获得新疆出血热病毒NP-His融合蛋白,得到了特异性兔抗NP蛋白多克隆抗体。
Objective To express and purify the nucleocapsid protein (NP) of Xinjiang hemorrhagic fever virus BA88166 strain and to prepare and identify the polyclonal antibody against NP protein. Methods The cDNA fragment of S gene of BA88166 strain was amplified by reverse transcription polymerase chain reaction (RT-PCR) and inserted into prokaryotic expression vector pET-32a to construct recombinant prokaryotic expression vector pET-88166S. The constructed plasmid was induced in E. coli BL21 (DE3), purified by nickel column affinity chromatography NPHis fusion protein, SDS-PAGE analysis of protein relative molecular mass (M r). The purified protein was used to immunize New Zealand white rabbits to prepare antiserum. The serum titer and specificity were determined by ELISA and Western blot. Results Double enzyme digestion and DNA sequencing confirmed that the constructed recombinant plasmid pET-88166S was correct. The sequence of the target gene was identical with the sequence published in GenBank. The NP-His fusion protein expressed in E. coli BL21 was analyzed by SDS-PAGE. M r is about 66 000. The titer of antibody was higher than 1:25 600 by ELISA. Western blotting results showed that the antibody could specifically recognize NP protein and truncated protein of Xinjiang hemorrhagic fever virus YL04057 strain. Conclusion NP-His fusion protein of Xinjiang hemorrhagic fever virus was successfully obtained, and specific polyclonal antibody to rabbit anti-NP protein was obtained.