摘要
目的通过构建登革2型病毒B株E基因区1～476 bp的原核表达载体,进行原核表达,并制备单克隆抗体,为研制登革病毒诊断试剂奠定基础。方法将登革2型病毒B株E基因序列克隆入原核表达载体pET28a(+),构建原核表达重组体pET28a(+)-E,将重组表达载体转化BL21(DE3)菌株进行原核表达。纯化后的蛋白免疫BALB/c小鼠,经融合、筛选制备特异性单克隆抗体。结果成功构建了pET28a(+)-E原核表达重组质粒,SDS-PAGE分析表明,E基因区部分序列获得高效表达,获得了1株持续分泌抗E蛋白抗体的杂交瘤细胞株8B3,该单抗为IgG2a亚类。结论成功制备了抗E蛋白mAb,为对登革病毒引起感染的早期诊断提供了有力的工具。
Objective To prepare the high-specific anti-gene E of dengue virus type 2 strain B antibodies using recombinant expressive E proteins. Methods Gene E partial sequence of dengue virus type 2 strain B was amplified by RT-PCR,inserted into prokaryotic vector pET28a(+),and transformed E.coli BL21 cells.The purified protein was used to immunize Balb/c mice. Results SDS-PAGE assay showed that the gene E partial sequence could be highly expressed in BL21,western-blot indicated that the expression products could specifically react with mono-antibody against dengue virus type 2. Conclusions pET28a(+)-Eb containing gene E partial sequence can be highly expressed in BL21.The antigenicity of the produced proteins provides a potential source of reagent for dengue virus diagnosis.
出处
《公共卫生与预防医学》
2011年第2期12-14,共3页
Journal of Public Health and Preventive Medicine
基金
国家自然科学基金资助项目(30360101)
