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为研究肾综合征出血热病毒疫苗侯选株R22核蛋白的结构与特性,应用逆转录PCR扩增了R22编码区基因,并将扩增产物克隆于pET-3a表达质粒,测得R22株S片段编码区序列为1290个核苷酸。比较分析表明与Seoul型同源性高达96.2%,而与Hantaan型同源性仅为71.0%,与用血清学分型的结果一致。将克隆的pET-R22NP转化到BL21后,IPTG诱导得到较高效的表达,产物纯化后进行Westernblot分析,结果表达的NP仅与NP蛋白特异的单克隆抗体A35和3D9反应,而与G2蛋白特异的3D8不反应。表达的产物为汉坦病毒的诊断提供了特异性抗原。
In order to study the structure and characteristics of R22 nucleoprotein candidate vaccine against hemorrhagic fever with renal syndrome virus, the R22 coding region gene was amplified by RT-PCR and the amplified product was cloned into pET-3a expression plasmid. The fragment coding sequence is 1290 nucleotides. Comparative analysis showed that Seoul homology was as high as 96.2%, while homology with Hantaan type was only 71.0%, which was consistent with serological typing. After the cloned pET-R22NP was transformed into BL21, IPTG induced a more efficient expression. The purified product was analyzed by Western blotting. The expressed NP only reacted with the NP protein-specific monoclonal antibodies A35 and 3D9 and specifically with the G2 protein 3D8 does not respond. The expressed product provides a specific antigen for the diagnosis of Hantavirus.