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目的分析埃博拉病毒核蛋白(NP)抗原表位,克隆表达埃博拉病毒NP抗原,为免疫学诊断试剂的研究奠定基础。方法采用BioSun生物学软件预测分析埃博拉病毒NP抗原的氨基酸表位区间,采用大肠杆菌优势密码子反向翻译成基因序列,采用PCR退火合成法合成NP优势密码子抗原基因,并利用载体pBVIL1进行克隆表达。采用间接ELISA技术评价获得NP抗原的特异性。结果确定埃博拉病毒NP抗原的氨基酸表位位于360~739 aa,共设计36条引物,扩增合成1140 bp的NP抗原基因,克隆表达后,融合蛋白相对分子质量为58×103,测序结果显示插入的NP基因正确。初步结果显示,NP抗原的特异性为99.24%(130/131)。结论获得埃博拉病毒NP抗原,为进一步研制特异的埃博拉病毒快速诊断试剂提供了抗原储备。
Objective To analyze the epitopes of Ebola virus nucleoprotein (NP) and clone and express Ebola virus NP antigen, and lay the foundation for the study of immunological diagnostic reagents. Methods BioSun biological software was used to predict the amino acid epitope of Ebola virus NP antigen. The dominant codons of E. coli were reverse translated into gene sequences. The NP dominant codon antigen gene was synthesized by PCR annealing synthesis. The pBVIL1 Clone expression. Indirect ELISA was used to evaluate the specificity of obtaining NP antigen. Results The amino acid epitopes of Ebola virus NP antigen were located at 360-739 aa. A total of 36 primers were designed to amplify 1140 bp NP antigen gene. After cloning and expression, the relative molecular mass of the fusion protein was 58 × 103. Sequencing results The inserted NP gene is shown to be correct. Preliminary results showed that the specificity of NP antigen was 99.24% (130/131). Conclusion Ebola virus NP antigen was obtained, which provided antigenic reserve for the further development of specific Ebola virus rapid diagnostic reagent.