CAJ | 학술논문

目的：在293T细胞中获得可溶性表达的1型登革病毒（DENV1）包膜（E）蛋白。方法PCR扩增DENV1-E蛋白功能区（1～394 aa）基因并克隆到Psectag2B-Fc真核表达载体中构建表达质粒，脂质体法转染293T细胞，经2 mg／ml Zeocin筛选，免疫荧光法鉴定所获细胞克隆；Protein-G亲和层析纯化DENV1-E-Fc重组融合蛋白，间接免疫荧光和蛋白免疫印迹法鉴定蛋白的完整性和抗原性。结果获得稳定可溶性表达重组融合蛋白的细胞克隆，蛋白产量约为25μg／1×107个细胞，纯化得到的DENV1-E-Fc蛋白相对分子质量约90×103，该蛋白可与识别天然构象E蛋白的单克隆抗体结合，并与免疫动物血清有良好的反应性。结论在真核细胞中成功获得可溶性表达的DENV1-E-Fc重组融合蛋白，E蛋白功能区保持完整且具备天然的构象表位和良好的抗原性，为包膜蛋白的保护性抗原表位和功能研究奠定了基础。
목적：재293T세포중획득가용성표체적1형등혁병독（DENV1）포막（E）단백。방법PCR확증DENV1-E단백공능구（1～394 aa）기인병극륭도Psectag2B-Fc진핵표체재체중구건표체질립，지질체법전염293T세포，경2 mg／ml Zeocin사선，면역형광법감정소획세포극륭；Protein-G친화층석순화DENV1-E-Fc중조융합단백，간접면역형광화단백면역인적법감정단백적완정성화항원성。결과획득은정가용성표체중조융합단백적세포극륭，단백산량약위25μg／1×107개세포，순화득도적DENV1-E-Fc단백상대분자질량약90×103，해단백가여식별천연구상E단백적단극륭항체결합，병여면역동물혈청유량호적반응성。결론재진핵세포중성공획득가용성표체적DENV1-E-Fc중조융합단백，E단백공능구보지완정차구비천연적구상표위화량호적항원성，위포막단백적보호성항원표위화공능연구전정료기출。
Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.