中文摘要：本研究建立了一种重组PvDBPII；研究基于指数流加策略并通过分批补料发酵的方式在大肠杆菌中构建了一个可以优化PvDBPII表达的合成基因，并采用这个合成基因提高重组PvDBPII的表达量。从内含体中分离得到重组PvDBPII，并通过快速稀释进行再折叠，采用离子交换色谱法进行纯化；采用标准化的释放试验对纯化后的重组PvDBPII进行鉴定、纯化和功能分析。将重组PvDBPII与半乳吡喃糖基脂质A稳定乳剂（GLA-SE）或半乳吡喃糖基脂质A水凝胶等佐剂组合用于临床动物免疫以筛选出一种合适的适应临床应用的佐剂配方。结果表明，相比于GLA-SE与半乳吡喃糖基脂质A水凝胶组合，GLA-SE与PvDBPII联用能够产生更高的ELISA反应和吸附抑制滴度。这些结果为基于PvDBPII- GLA-SE佐剂的间日疟原虫重组疫苗的研制提供了数据支撑。
 
外文摘要：Malaria vaccines, Plasmodium vivax, Duffy binding protein, Host cell invasion, Recombinant vaccines, Adjuvants, Fed-batch fermentation, Protein refolding, Vaccine formulation
Abstract: Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.
作者：Bhardwaj, R; Shakri, AR; Hans, D; Gupta, P; Fernandez-Becerra, C; del Portillo, HA; Pandey, G; Chitnis, CE
作者单位：印度国际遗传工程和生物技术中心
期刊名称：PROTEIN EXPRESSION AND PURIFICATION
期刊影响因子：1.351
出版年份：2017
出版刊次：8
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