【作者】 翁育伟； 林梅清； 王金章； 黄萌； 严延生；

【机构】 福建省疾病预防控制中心； 福建省人兽共患病研究重点实验室； 福建医科大学公共卫生学院；

【摘要】 目的克隆、表达4种血清型登革病毒(Dengue virus,DENV)非结构蛋白1(Non-structural protein 1,NS1)基因,为今后临床诊断应用提供材料。方法从含有4种型别NS1基因的克隆载体中PCR扩增完整的NS1编码区,测序后插入pET30a(+)载体并转化BL21(DE3)宿主菌。SDS-PAGE电泳分析诱导后的细菌裂解物,WB法分析重组蛋白的免疫反应性。结果构建的4种血清型病毒NS1重组质粒经诱导后均可表达相应的蛋白,重组蛋白的分子量符合预期。Western blot结果表明,4种重组NS1蛋白均能与DENV2型病毒感染者血清反应,证实重组蛋白具有免疫反应活性。结论成功表达4种血清型登革病毒的重组NS1蛋白,重组蛋白具有开发检测试剂等临床诊断应用价值,并可用于进一步研究该蛋白的结构与功能。更多还原

【Abstract】 Objective To clone and to express non-structual protein 1(NSl) of dengue virus of four distinct serotypes for potential diagnostic utilization. Method The encoding region of NSl was amplified by using plasmids containing NS1 gene fragments as templates. PCR products were inserted into the pET30a(+) and subsequently confirmed by endonuclease digestion and sequencing.The recombinant plasmids transformed into E.coli and induced to express the target proteins. SDS-PAGE and WB were used to analyze the expression profile and immunoreactivity of the recombinant proteins. Results SDS-PAGE results showed the four distinct rNSls could be synthesized in host bacterial and WB results showed the rNSls could be recognized by antibody from patients infected with DENV2. Conclusion The rNS1s possessed potential utilization for clinical diagnostic and could be used for further study the structure and function of viral NSl protein.更多还原