1型登革病毒包膜蛋白功能区在293T细胞中的表达与鉴定  

Expression and identification of the functional domains of dengue virus type 1 envelope protein in 293T cells

作　　者：郭勇晖[1] 易海粟[1] 陈静[1] 丁细霞[1] 狄飚[2] 车小燕[1] 温坤[1] 

机构地区：[1]南方医科大学珠江医院检验医学部临床医学实验中心,广州510282 [2]广州市疾病预防控制中心病毒科

出　　处：《中华微生物学和免疫学杂志》2015年第6期459-463,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81201296）;NSFC-广东联合基金（U1132002）

摘　　要：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（1～394aa）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2mg／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。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 ceils. 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 expression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

关 键 词：1型登革病毒 包膜蛋白 真核表达 

分 类 号：R373[医药卫生—病原生物学]