Ⅱ型登革病毒NS1基因的克隆及其在昆虫细胞中的表达

Cloning and expression of the nonstructure 1 gene of dengue virus in insect cell

目的获得II型登革热病毒NS1基因并在昆虫细胞中表达,为进一步研发登革热血清学检测试剂盒提供抗原。方法RT-PCR扩增II型登革热病毒NS1基因并测序列,并定向克隆入杆状病毒转移载体pFastBac-HTA,获得重组转移载体pFastBac-NS1。并将pFastBac-NS1转化到DH10Bac感受态细胞中,筛选到重组转座子rBacmid-NS1。在脂质体介导下将rBacmid-NS1转染Sf9昆虫细胞获得重组杆状病毒rBV-NS1。rBV-NS1感染Sf9细胞后,通过SDS-PAG和Western blot检测NS1重组蛋白。结果成功克隆II型登革病毒NS1基因,SDS-PAGE、间接免疫荧光和Western blot检测表明NS1基因在昆虫细胞得到表达,能与登革患者血清发生特异性免疫反应,具有免疫原性。结论II型登革病毒非结构蛋白NS1在昆虫中表达,为进一步研究NS1的生物学特性和血清学检测奠定基础。

By obtaining the NS1 genes of dengue virus and expressing it in Sf9 cell, to study the antigenicity and establish the basis for developing the diagnosis kit. The NS1 eDNA was amplified with RT-PCR and cloned into the transfer plasmid pFastBac-HTA,then the recombianat transfer plasmid pFastBac-NS1 was transformed into DH10Bac competent cells. The recombinant transposition rBacmid-NS1 was obstained by screening of white plaque and was identified by PCR. After the rBacmid- NS1 transfected into Sf9 cells,the recombinant baculovirus rBV-NS1 was harvested. The SDS-PAGE and Western blot showed the NS1 gene was well expressed ,and the expressed product had antigenicity. The stable expression of this protein and the analysis of its antigenic specificity provide the foundation to develop the diagnostidc kit for dengue virus.