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目的在哺乳动物细胞中表达单纯疱疹病毒Ⅱ型(Herpes simplex virus type-2,HSV-2)糖蛋白D(GlycoproteinD,gD)胞外区片段,并分析其免疫活性。方法化学合成HSV-2 G株gD蛋白胞外区片断的基因序列gDt,以pCEP4作为表达载体,构建N-末端带His标签的重组表达质粒pCEP4-gDt,转染至HEK293细胞进行表达。表达的蛋白采用镍离子柱亲和层析纯化,ELISA法检测目的蛋白的抗原性。以纯化的重组蛋白免疫小鼠制备多抗血清,间接ELISA法检测效价。结果重组表达质粒经PCR、双酶切和DNA测序证实构建正确;表达产物经Western blot分析,在相对分子质量约46 000处可见目的蛋白条带;纯化的重组蛋白浓度约为45μg/ml,经ELISA检测证实具有良好的抗原性,免疫小鼠5周后,血清中特异性抗体效价达5×103。结论已在哺乳动物细胞中表达了HSV-2 gD胞外区片段,其具有良好的抗原性和免疫原性,为HSV基因重组亚单位疫苗的研制奠定了基础。
Objective To express the extracellular domain of glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) in mammalian cells and analyze its immunological activity. Methods The gene sequence gDt of the extracellular region of gD protein of HSV-2 G strain was chemically synthesized. The recombinant plasmid pCEP4-gDt with N-terminal His-tag was constructed by using pCEP4 as an expression vector and transfected into HEK293 cells for expression. The expressed protein was purified by nickel ion affinity chromatography and the antigenicity of the target protein was detected by ELISA. Multi-antiserum was prepared by immunizing mice with purified recombinant protein and the titer was tested by indirect ELISA. Results The recombinant plasmid was confirmed by PCR, double enzyme digestion and DNA sequencing. Western blot analysis showed that the recombinant protein was expressed at about 46 000 relative molecular weight. The purified recombinant protein was about 45 μg / ml. ELISA showed good antigenicity. After 5 weeks of immunization, the titer of serum specific antibody reached 5 × 103. Conclusions HSV-2 gD extracellular region has been expressed in mammalian cells, which has good antigenicity and immunogenicity, which lays the foundation for the development of HSV recombinant subunit vaccine.