Ⅱ型登革病毒NS1蛋白的表达及其免疫血清的制备

Expression and preparation of antibody for Dengue virus 2 NS1 protein

目的原核表达、纯化II型登革病毒NS1A蛋白(NS1蛋白1～180氨基酸即DENV2 NS1A),将其免疫动物制备免疫血清并鉴定。方法构建重组融合质粒p ET22b-p64K-L-DENV2 NS1A,转化至大肠杆菌BL21(DE3)中,IPTG低温诱导表达。表达的融合蛋白经Hitrap Talon crude柱亲和层析纯化后,免疫雌性日本长耳兔获得抗DENV2 NS1A兔抗血清,用Western blot和免疫荧光检测免疫血清对重组麻疹病毒MV_(S191)-DENV2 NS1 6Xhis的识别和结合特性。结果重组融合质粒p ET22b-p64K-L-DENV2 NS1A经双酶切和测序证明构建正确。表达的重组融合蛋白相对分子质量为100 000,纯化后蛋白纯度在85%以上,免疫获得的抗DENV2 NS1A兔抗血清可识别重组病毒MV_(S191)-DENV2 NS1 6XHis表达的DENV2 NS1蛋白。结论原核表达并纯化了DENV2 NS1A蛋白,建立了基于NS1蛋白的重组病毒MV_(S191)-DENV2 NS1 6XHis的Western blot和免疫荧光检测方法,为登革热疫苗研制中重组病毒MV_(S191)-DENV2 NS1 6XHis的质检奠定了基础。

Objective To express and purify protein NS1 A（ 1-180 amino acid of a non-structural protein NS1） of dengue virus 2 serotype（ DENV2）,and to prepare polyclonal antibody against NS1 A. Methods The recombinant plasmid of p ET22 b-p64 K-L-DENV2 NS1 A was generated by means of the gene encoding DENV2 NS1 A protein,amplified and sequenced,and then cloned into prokaryotic vector p ET22 b-p64 K-L. The constructed recombinant plasmid was transformed to E.coli BL21（ DE3） and induced with IPTG. The expressed recombinant p64 K-L-DENV2 NS1 A was purified by Talon crude Co2＋chromatography and then immunized rabbits in preparation of the polyclonal antibody,and identification was performed by Western blot and immuno-fluorescence in react with prepared DENV2 NS1 A. Results Restriction analysis and sequencing proved that recombinant plasmid p ET22 b-p64 K-L-DENV2 NS1 A was correctly constructed. The expression of p64 K-LDENV2 NS1 A protein was inducted with 0.5 mmol/L IPTG at 20 ℃ for 20 h. The expressed recombinant protein,with a relative molecular weight of 100 000,reached a purity of more than 85% after purification,The protein showed a good reaction with the recombinated DENV2 NS1 and Measle virus（ MV（S191）-DENV2 NS1 6 XHis）. The rabbit sera against recombinant NS1 proteins were successfully prepared. Conclusion DENV2 NS1 A protein and its polyclonal antibodies were successfully produced,and provided a foundation in preparation of detective reagent for detection MV（S191）-DENV2 NS1 6 XHis.