Ⅰ型登革病毒ns1基因克隆及其表达产物的免疫原性研究

目的克隆并表达Ⅰ型登革病毒非结构蛋白1(NS1)基因,初步鉴定其免疫原性。方法登革热Ⅰ型病毒标准株感染C6/36细胞后,抽提病毒RNA,经RT-PCR方法扩增出NS1全长基因片段,经T-A克隆后,在QIA表达系统中表达,表达产物用Ni柱亲和层析纯化后,用兔抗Ⅰ型登革病毒免疫血清及登革热患者血清对重组蛋白进行Western Blot及ELISA鉴定。结果构建的重组质粒pQE-30/DV1NS1经IPTG诱导,重组蛋白NS1高效表达并纯化成功,经Western Blot及ELISA证实重组蛋白NS1可以被免疫血清和病人血清特异识别。结论Ⅰ型登革病毒非结构蛋白NS1表达载体在大肠杆菌M15中高效表达。纯化产物具有较强的免疫原性,为进一步研究NS1的生物学特性和血清学检测奠定了基础。

Cloning and expression of the ns1 gene encoding the non-structural protein of dengue virus serotype 1 and identification of its immunogenicity

To clone and expression of the ns1 gene encoding the non-structural protein of dengue virus serotype 1(DEN1)and to investigate its immunogenicity of the recombinant protein,the NS1 cDNA was amplified with RT-PCR from the C6/36 cells infected with DEN1 and was cloned into pMD18-T vector.Then the ns1 gene was inserted to the multi-cloning sites of plasmid pQE30 and expressed with induction of IPTG.After purification under denatured condition,the immunogenicity of the recombinant protein was identified with rabbit antiserum against DEN1 and sera of patients with DEN1 infection by Western blot assay and ELISA method.Experimental results demonstrated that the recombinant NS1 protein was highly expressed in E.coli M15 and the purified NS1 protein could be recognized by and combined with the sera against DEN1 from rabbits and patients infected with DEN1. In conclusion,it is evident that the expressed NS1 protein shows good immunogenicity,which may provide for a potential source to study the function of the NS1 protein and to be used as an antigen to detect the presence of the dengue virus infection.