登革病毒NS1蛋白的原核表达及其在登革热快速诊断中的应用  

Prokaryotic Expression of NS1 Protein of Dengue Virus and Application in Rapid Diagnosis of Dengue Fever

作　　者：傅强[1] 田疆[1] 方丹云[1] 周俊梅[1] 庞贤武[1] 李兴华[1] 江丽芳[1] 

机构地区：[1]中山大学中山医学院微生物学教研室//教育部热带病防治研究重点实验室,广东广州510080

出　　处：《中山大学学报（医学科学版）》2012年第3期316-321,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(NSFC)-广东联合基金(U1132002)

摘　　要：【目的】重组表达1型登革病毒(DENV-1)和3型登革病毒(DENV-3)NS1蛋白,制备多克隆抗体,建立特异、敏感的登革病毒NS1抗原ELISA检测法。【方法】RT-PCR扩增DENV-1和DENV-3 NS1全长基因,经T-A克隆后,将目的基因分别与质粒PET30a(+)连接构建重组质粒,转化大肠杆菌BL21感受态细胞。阳性克隆经IPTG诱导表达,并对目的蛋白进行纯化。用纯化的NS1蛋白免疫BALB/c小鼠,制备鼠抗NS1多克隆抗体。应用NS1多克隆抗体建立登革病毒NS1抗原双抗体夹心ELISA检测法。【结果】成功构建了高效表达DENV-1和DENV-3 NS1蛋白的原核表达系统,获得纯化的NS1蛋白;用重组NS1蛋白免疫BALB/c小鼠后获得高效价(>1:30 000)的抗DENV-1和DENV-3 NS1的特异性抗体;用NS1多克隆抗体建立了登革病毒NS1抗原双抗体夹心ELISA检测法,该法可特异性检出登革病毒NS1抗原,敏感性达到1 ng/mL,与黄病毒的其它成员无交叉反应,表明具有良好的特异性。【结论】本研究成功构建了DENV-1和DENV-3 NS1蛋白的原核表达系统,高效表达了具有良好免疫原性及免疫反应性的DENV-1和DENV-3重组NS1蛋白;建立了检测登革病毒NS1抗原的双抗体夹心法,并证明该检测法具有较高的灵敏度和特异性,具有良好的应用前景。[Objective] To express the nonstructural protein 1 （NS1） of Dengue virus type 1 （DENV-1） and Dengue virus type 3 （DENV-3） in E.coli, and prepare polyclonal antibodies against the NS1 proteins in vivo and establish a high sensitivity and specificity ELISA of DENV NS1 antigen. [ Methods ] The full-length DENV-1 and DENV-3 NS1 genes were amplified by RT-PCR, and then cloned into pGEM-T vector. After the NSlegenes were cloned into expression plasmid PET30a （＋） respectively, the recombinant plasmids were transformed into E. coli BL21. The recombinant NS1 proteins were expressed by induction with IPTG, and purified. For production of anti-NS1 polyclonal antibodies, we immunized mice with the purified recombinant proteins. The polyclonal antibodies were used to establish a double-antibody sandwich ELISA for detecting Dengue NS1 antigen. [ Results ] Successfully constructed highly efficient prokaryotic expression system which expressed DENV-1 or DENV-3 NS1 protein and obtained purified NS1 proteins. The recombinant proteins were used to immunize BALB/c mice. Polyclonal antibodies against NS1 of DENV-1 or DENV-3 with high titer （〉1：30000） were finally obtained. With these polyclonal antibodies, we detected i ng/ml homo-typic DENV NSI protein by using double-antibody sandwich ELISA. There was no cross-reactivity with other members of flavivirus, indicating a good specificity. [Conclusion] In this study, we succeeded in constructing highly efficient prokaryotic expression system of DENV-1 or DENV-3 NS1 protein, and demonstrated that the expressed NS1 proteins had good immunogenicity and immune reactivity. Established a doubleantibody sandwich ELISA for detecting Dengue NS1 antigen, and proved the assay had high sensitivity and specificity, with a good application prospect.

关 键 词：登革病毒 NS1蛋白 原核表达 多克隆抗体 

分 类 号：R373.3[医药卫生—病原生物学]