HIV-1糖蛋白gp120在昆虫细胞中的表达、纯化、性质鉴定以及免疫原性分析

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells

目的 利用杆状病毒-昆虫细胞表达系统建立高效分泌表达人类免疫缺陷病毒（HIV）-1 Env gp120糖蛋白的方法,并对其理化性质及免疫原特性进行分析.方法 选择B亚型HIV-1 NL4-3 gp120进行蛋白克隆设计,将目的蛋白基因序列克隆到杆状病毒转移载体pAcgp67B中pH启动子的下游,构建成重组转移载体pAc-gp120,利用同源重组的方式在宿主细胞内获得重组杆状病毒,并在High FiveTM昆虫细胞中表达gp120蛋白.之后使用酶联免疫吸附试验、分析超离和分子排阻色谱进行理化性质分析,并通过小鼠免疫实验分析其免疫原性.结果 建立昆虫细胞表达系统制备和纯化重组HIV-1 gp120蛋白的方法,最终获得纯度约90％的gp120蛋白,多批次纯化后鉴定产量约为13 mg/L;酶联免疫吸附测定、分析超离和分子排阻色谱性质分析试验显示,重组HIV gp120蛋白在溶液有良好的抗原性并且在溶液中较为均一;通过弗氏佐剂与目的蛋白混合免疫BALB/c小鼠,免疫血清对NL4-3病毒具有一定程度中和活性,表明gp120蛋白能有效刺激机体产生免疫应答.结论成功建立了简便、可放大的HIV gp120蛋白表达和纯化方法,获得较为均一的、免疫原性良好的gp120抗原,为进一步开展HIV疫苗研究工作奠定了基础.

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 （ HIV-1 ） envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90% and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.