牛病毒性腹泻病毒Erns蛋白的中华仓鼠卵巢细胞表达及免疫原性分析

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis

摘要:

本研究利用中华仓鼠卵巢(Chinese hamster ovary, CHO)细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV) E^rns蛋白，并分析其免疫原性。以BVDV-1 NADL标准毒株基因序列为基础，构建BVDV E^rns蛋白重组真核表达质粒pcDNA3.1-BVDV-E^rns，转染悬浮培养的CHO细胞，进行上清分泌表达。SDS-PAGE分析E^rns蛋白的表达和纯化，并用抗His单克隆抗体和BVDV阳性血清进行Western blotting鉴定纯化蛋白；进一步使用纯化的E^rns蛋白免疫新西兰大白兔，通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)和细胞间接免疫荧光(indirect immunofluorescence, IFA)实验检测血清抗体水平及其免疫反应活性，用病毒中和实验测定免疫兔血清的中和抗体滴度。BCA蛋白定量试剂盒检测纯化的E^rns蛋白浓度为0.886 mg/mL，Western blotting结果显示，抗His单克隆抗体和BVDV阳性血清均能够与纯化的E^rns蛋白发生特异性免疫反应。间接ELISA和IFA实验结果显示，一免后第7天血清抗体呈阳性，并持续至免疫后第28天，血清抗体效价水平可达1:128 000，且该血清抗体可以与MDBK细胞中感染的BVDV病毒发生特异性免疫反应。重组E^rns蛋白免疫兔可诱导动物机体产生病毒中和抗体，中和效价为log₁₀=2.71。本研究利用CHO细胞表达系统成功制备了纯化的BVDV E^rns蛋白，并通过体内和体外实验证明，该重组蛋白具有良好的免疫原性，为牛病毒性腹泻(bovine viral diarrhea, BVD)诊断方法及新型亚单位疫苗的研制奠定了基础。

Abstract:

The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the E^rns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the E^rns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log₁₀ was induced in rabbits. In this study, purified BVDV E^rns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.