杆状病毒和分批补料发酵联用高效表达单纯疱疹病毒Ⅱ型糖蛋白D  

Recombinant functional herpes simplex virus type 2 glycoprotein D expressed in baculovirus system with fed batch culture

作　　者：刘涛[1] 董惠钧 黄志成[1] 郑伟[1] 祝水芬[1] 

机构地区：[1]杭州市疾病预防控制中心,浙江杭州310021 [2]临沂市鲁南制药集团股份有限公司

出　　处：《中国预防医学杂志》2011年第8期653-657,共5页Chinese Preventive Medicine

基　　金：杭州市科技局重点专病专科计划项目(20080333Q29);杭州市医药卫生科技计划项目(2008A029)

摘　　要：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 functional recombinant gD2 in high quantities using Bac-to-Bac expression system with fed batch culture.Methods The full-length gD2 protein coding cDNA of HSV-2 was subcloned into pFastBac HTb vector and the recombinant protein was expressed in Spodoptera frugiperda（Sf9） cells with fed-batch culture.Genital herpes model was constructed to study the immunogenicity of recombinant HSV-2 gD2 protein in guinea pig.Results The yield of recombinant biological gD2 protein was up to 192 mg/L in cell cultures under the optimized conditions of 15 mmol glucose,0.4 g/L glutamine and 45% dissolved oxygen in a 5 liter stirred bioreactor.Immunization with the recombinant gD2 significantly reduced the mean lesion scores of genital in guinea pigs infected with HSV-2（P0.05）.Conclusion The efficient production of recombinant HSV-2 gD2 protein will provide a solid basis for the development of further application in future.

关 键 词：昆虫杆状病毒表达系统 单纯疱疹病毒Ⅱ型 糖蛋白D 分批补料发酵 

分 类 号：R392[医药卫生—免疫学]