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目的在大肠埃希菌中表达登革病毒(Dengue virus,DENV)1型(DENV-1)E蛋白第三结构域(EⅢ),并分析重组蛋白的免疫原性。方法利用PCR技术从质粒p MD-D1pr M/E中扩增DENV-1的EⅢ序列,插入原核表达载体p ET-32a(+),构建重组表达质粒p ET-D1EⅢ,转化至E.coli Rosetta2(DE3)中,IPTG诱导表达。表达的重组蛋白经亲和层析和阴离子交换层析纯化后,免疫家兔,制备多克隆抗体,采用Western blot法检测抗体的特异性,间接ELISA法检测抗体滴度,蚀斑减少中和试验(plaque reduction neutralization test,PRNT)检测中和抗体效价。用纯化的重组蛋白免疫经腹部皮下多点注射BALB/c小鼠3次,末次免疫2周后,用DENV-1(Hawaii株)经脑内攻击,分析重组蛋白对小鼠的免疫保护力。结果重组原核表达质粒p ET-D1EⅢ经双酶切和测序证实构建正确;表达的重组蛋白相对分子质量约为33 000,主要以可溶性形式表达,占重组菌裂解上清蛋白量的52.37%;纯化的纯度可达90%以上,浓度达2 mg/ml;制备的多克隆抗体特异性强,效价高;重组蛋白能保护57.1%的小鼠免受致死剂量DENV的攻击。结论成功在E.coli Rossetta2(DE3)中高效表达了DENV-1 EⅢ蛋白,纯化的重组蛋白免疫家兔获得的多克隆抗体具有较强的特异性和较高的效价,且在小鼠免疫保护试验中显示出较好的保护力。本实验为进一步研究E蛋白的功能和登革新型疫苗的开发奠定了基础。
Objective To express the third domain of Dengue virus (DENV) type 1 (DENV-1) E protein in Escherichia coli and analyze the immunogenicity of the recombinant protein. Methods The EⅢ sequence of DENV-1 was amplified from plasmid pMD-D1pr M / E by PCR and inserted into the prokaryotic expression vector p ET-32a (+). The recombinant plasmid p ET-D1EⅢ was constructed and transformed into E.coli Rosetta2 (DE3), IPTG induced expression. The expressed recombinant protein was purified by affinity chromatography and anion exchange chromatography, then immunized rabbits to prepare polyclonal antibody. The specificity of the antibody was detected by Western blot. The antibody titer and plaque reduction neutralization test (plaque reduction neutralization test, PRNT). BALB / c mice were injected subcutaneously with BALB / c mice by immunization with purified recombinant protein for three times. After two weeks of last immunization, the mice were immunized with DENV-1 (Hawaii strain) in intracerebral challenge. Results The recombinant plasmid p ET-D1EⅢ was confirmed by double enzyme digestion and sequencing. The recombinant protein was expressed in soluble form, accounting for 52.37% of the total amount of the recombinant protein. The purified recombinant Purity of more than 90%, the concentration of 2 mg / ml; prepared polyclonal antibodies strong specificity, high titer; recombinant protein can protect 57.1% of mice from lethal doses of DENV attack. Conclusion The DENV-1 EⅢ protein was successfully expressed in E.coli Rossetta2 (DE3). The polyclonal antibody obtained from the purified recombinant protein was more specific and potent than the rabbit immunized with the recombinant protein. Protection test showed a better protection. This experiment lays the foundation for the further study of the function of E protein and the development of new dengue vaccine.