中文摘要：

目的重组表达登革1型病毒NS1蛋白并制备多克隆抗体。方法通过RT-PCR扩增编码登革1型病毒NS1蛋白的基因序列,将其克隆到原核表达载体pET-32a（＋）后经酶切测序鉴定,获得重组质粒pET-NS1。进一步使用IPTG诱导表达,经免疫印迹鉴定后,采用蛋白浸提方法从SDS-PAGE胶中纯化回收融合蛋白,并通过透析对目的蛋白进行复性;纯化复性后的重组蛋白免疫BALB/c小鼠制备多克隆抗体,通过间接免疫荧光法测定抗体效价。结果成功构建了高效表达登革1型病毒NS1蛋白的原核表达载体,表达的重组NS1蛋白占菌体蛋白总量的50%以上,以包涵体形式存在;免疫印迹表明表达的重组NS1蛋白能够为登革病毒兔抗血清识别,纯化后纯度可达95%以上;免疫小鼠后获得了效价〉1∶1000的抗登革病毒NS1蛋白的鼠多克隆抗体。结论原核表达的重组NS1蛋白具有良好的免疫原性,诱导产生的多克隆抗体能够有效识别天然NS1蛋白,为进一步研究登革病毒NS1蛋白及其抗体的生物学功能奠定了基础。

英文摘要：

Objective To express the nonstructural protein 1 （NS1） of dengue virus type 1 in E. coli,and prepare the corresponding polyclonal antibodies in mice. Methods The full-length coding sequence of NS1 protein was amplified by RT-PCR,and cloned into the prokaryotic expression vector pET-32a（＋） to construct the desired plasmid pET-NS1. Recombinants were identified by restrict enzyme digestion and DNA sequencing,then transformed into E. coli BL21 （DE3）,followed by induction of the engineered BL21/pET-NS1 with IPTG. The expressed NS1 protein was subsequently identified by SDS-PAGE and Western blotting. The recombinant NS1 protein was further extracted and purified directly from the polyacrylamide gels,followed by renaturation of the purified protein by dialysis. BALB/c mice of 6 weeks old were hypodermically immunized and boosted with the recombinant NS1 protein at 2 weeks interval. Anti-sera against NS1 were finally collected and characterized by indirect immunofluorescence assays. Results The recombinant plasmid that expressed the NS1 protein was successfully constructed. The NS1 protein was expressed as inclusion bodies in E. coli,and the proportion of which in total protein of E. coli was about 50%. The expressed recombinant NS1 protein could be recognized by rabbit anti-sera to dengue virus in Western blot assay. The purity of NS1 protein which was used for immunization was over 95% after extraction from polyacrylamide gels. Polyclonal antibodies against NS1 of dengue virus type 1 with the titer of 1：1000 were finally obtained in mice. Conclusions Highly expressed recombinant NS1 protein from E. coli is good in immunogenicity,and can be used to generate polyclonal antibodies that react with natural NS1 protein of dengue virus. These findings are helpful for better understanding the functions of NS1 protein and anti-NS1 antibodies of dengue virus.