【摘要】 目的 构建pET30a-CTLA-4IgV-EgG1Y162-2(4)原核表达质粒，诱导表达后纯化，获得重组蛋白CTLA-4IgV-EgG1Y162-2(4),并对其免疫学特性进行初步鉴定，对其空间结构进行预测。方法 利用DNA重组技术构建重组质粒pET30a-CTLA-4IgV-EgG1Y162-2(4),将测序正确的质粒转化至E.coli BL21(DE)菌株中，分别采用不同浓度的IPTG在不同诱导时间及温度下诱导表达重组蛋白。取菌液超声破碎，SDS-PAGE分析上清及沉淀中蛋白的表达；通过镍柱亲和层析法纯化蛋白并进行Western blot鉴定。通过生物信息学技术，利用SOPMA、I-TASSER在线数据库预测重组蛋白CTLA-4IgV-EgG1Y162-2(4)的空间结构。结果 成功构建pET30a-CTLA-4IgV-EgG1Y162-2(4)重组质粒，转化至E.coli BL21(DE)后诱导表达相对分子质量为53×10~3的重组蛋白CTLA-4IgV-EgG1Y162-2(4)。SDS-PAGE分析CTLA-4IgV-EgG1Y162-2(4)重组蛋白在IPTG浓度为0.7 mmol/L,37℃、4 h条件下上清中表达量较高。镍柱层析纯化用20 mmol/L咪唑浓度洗脱的蛋白浓度及纯度较高。Western blot检测重组蛋白CTLA-4IgV-EgG1Y162-2(4)能被相应抗体识别，反应条带位于53×10~3处，与预期相符。生物信息学在线软件分析CTLA-4IgV-EgG1Y162-2(4)中各蛋白均能正常折叠，未对优势表位产生影响。结论 利用原核表达成功获得CTLA-4IgV-EgG1Y162-2(4)重组蛋白，并预测出该蛋白的三维结构模型，为细粒棘球蚴重组疫苗的研制提供了重要的实验依据。更多还原

【Abstract】 Objective To construct pET30a-CTLA-4IgV-EgG1Y162-2(4) prokaryotic expression plasmid and purify the recombinant protein CTLA-4IgV-EgG1Y162-2(4) after induced expression, and to preliminatively identify the immunological characteristics of recombinant protein CTLA-4IgV-EgG1Y162-2(4) and predict its spatial structure. Methods The recombinant plasmid pET30a-CTLA-4IgV-EgG1Y162-2(4) was constructed by DNA recombination technology, and the correctly sequenced plasmid was transformed into E.coli BL21(DE) strain. The recombinant proteins were induced by IPTG with different concentrations at different induction times and temperature. The bacterial solution was crushed by ultrasound, and the expression of protein in supernatant and precipitation was analyzed by SDS-PAGE. The protein was purified by nickel column affinity chromatography and identified by Western blot. The spatial structure of CTLA-4IgV-EgG1Y162-2(4) was predicted using SOPMA and I-TASSER online database by bioinformatics techniques. Results The recombinant plasmid pET30a-CTLA-4IgV-EgG1Y162-2(4) with the length of 6666bp was successfully constructed, which was transformed into E.coli BL21(DE) and induced to express the recombinant protein CTLA-4IgV-EgG1Y162-2(4) with the relative molecular weight of 53×10~3. SDS-PAGE results showed that the expression of CTLA-4IgV-EgG1Y162-2(4) recombinant protein was higher in the supernatant at 37 ℃ for 4 h at the IPTG concentration of 0.7 mmol/L. The concentration and purity of protein eluted by 20 mmol/L imidazole in nickel column chromatography were higher. Western blot analysis showed that the recombinant protein CTLA-4IgV-EgG1Y162-2(4) could be recognized by the corresponding antibody, and the reaction band was located at 53×10~3,which was consistent with the expectation. Bioinformatics online software results showed that each protein in CTLA-4IgV-EgG1Y162-2(4) could fold normally without affecting the dominant epitope. Conclusion The recombinant protein CTLA-4IgV-EgG1Y162-2(4) was successfully obtained by prokaryotic expression, and the three-dimensional structure model of the protein was predicted, which provided an important experimental basis for the development of recombinant vaccine against Echinococcus granulosus.更多还原