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目的利用Bac-to-Bac表达系统和分批补料式培养技术在草地夜蛾昆虫细胞(Sf9)中高效表达重组单纯疱疹病毒Ⅱ型糖蛋白D(HSV-2 gD2)。方法 以HSV-2基因组DNA为模板克隆全长gD2基因,利用Bac-to-Bac表达系统构建含目的基因的重组杆状病毒。同时采用分批补料式培养技术高密度培养Sf9细胞实现重组gD2蛋白的高效表达。构建豚鼠生殖器疱疹模型,研究重组HSV-2 gD2蛋白的免疫活性。结果在一个5 L生物反应器内,在15 mmol/L葡萄糖、0.4 g/L谷氨酰胺以及45%溶解氧的培养条件下,重组gD2蛋白的产量高达192 mg/L。此外,纯化后的重组gD2免疫能显著降低豚鼠生殖器疱疹模型的外阴皮损症状。结论 重组HSV-2 gD2蛋白利用杆状病毒和分批补料发酵联用得到高效表达,并表现出良好的免疫原性,为发展HSV-2疫苗奠定了基础。
Objective To express recombinant herpes simplex virus type 2 glycoprotein D (HSV-2 gD2) efficiently in Bacillus thuringiensis insect cell (Sf9) using Bac-to-Bac expression system and fed-batch culture technique. Methods The full-length gD2 gene was cloned by using HSV-2 genomic DNA as a template. The Bac-to-Bac expression system was used to construct a recombinant baculovirus containing the gene of interest. At the same time, fed-batch culture technique was used to culture Sf9 cells in high density to achieve high expression of recombinant gD2 protein. Guinea pig genital herpes model was constructed to study the immunological activity of recombinant HSV-2 gD2 protein. Results The yield of recombinant gD2 protein was as high as 192 mg / L in a 5 L bioreactor with 15 mmol / L glucose, 0.4 g / L glutamine and 45% dissolved oxygen. In addition, purified recombinant gD2 immunization can significantly reduce genital lesions in guinea pig genital herpes lesions. Conclusion The recombinant HSV-2 gD2 protein was highly expressed by baculovirus combined with fed-batch fermentation and showed good immunogenicity, which laid the foundation for the development of HSV-2 vaccine.