目的 通过基因序列设计及优化，构建质粒并表达纯化出4种呼吸道合胞病毒(RSV) PreF蛋白，对其免疫原性进行评价。 方法 分别用Human和CHO密码子优化Coronin-1A和T4三聚体蛋白基因序列，并添加到RSV F蛋白序列后，将上述质粒转染Expi293F细胞进行蛋白质表达，收获上清经镍柱纯化后，制备出4种三聚体蛋白，并进行SDS-PAGE分析及Western blot鉴定。于0周和3周免疫动物，采血后进行结合抗体和中和抗体活性检测。 结果 SDS-PAGE分析及Western blot鉴定结果表明，制备及表达的4种蛋白质具有稳定的三聚体结构；抗原抗体亲和力试验表明，4种三聚体蛋白与RSV特异性单抗8897、D25、Motavizumab、AM14和Palivizumab的亲和力均很强；免疫血清的结合抗体效价和中和抗体效价结果表明，初次免疫后，两种T4三聚体蛋白诱导的抗体水平更高，再次免疫后，Human密码子优化的三聚体蛋白抗体增长幅度较高。 结论 两种不同的异源三聚体基序添加均可产生有效的三聚体PreF蛋白，并能在小鼠体内诱导出有效的结合抗体和中和抗体。 Objective To construct and purify four respiratory syncytial virus (RSV) PreF proteins through gene sequence design and optimization and evaluate their immunogenicity. Methods Coronin-1A and T4 trimer protein gene sequences were optimized with Human and CHO codons, and then added to RSV F protein sequence. The above plasmids were transfected into Expi293F cells for protein expression. After purification by nickel column, four trimer proteins were prepared. SDS-PAGE and Western blot were performed for protein identification. BALB/c mice were immunized at week 0 and week 3, and blood samples were collected to measure the activities of binding and neutralizing antibodies in serum. Results SDS-PAGE and Western blot showed that the four proteins had stable trimer structure. Antigen-antibody affinity test showed that the four trimer proteins had strong affinity with RSV-specific monoclonal antibodies 8897, D25, Motavizumab, AM14 and Palivizumab. The titers of antibodies induced by the two T4 trimers were higher after the initial immunization, while there was a substantial increase in the titers of antibodies induced by Human codon-optimized trimer protein after the second immunization. Conclusions PreF trimer protein can be prepared by adding any of the two different heterotrimer motifs, and induce effective binding and neutralizing antibodies in mice.
Preparation and immunogenicity evaluation of two PreF trimer recombinant protein vaccines against respiratory syncytial virus
Objective To construct and purify four respiratory syncytial virus (RSV) PreF proteins through gene sequence design and optimization and evaluate their immunogenicity. Methods Coronin-1A and T4 trimer protein gene sequences were optimized with Human and CHO codons, and then added to RSV F protein sequence. The above plasmids were transfected into Expi293F cells for protein expression. After purification by nickel column, four trimer proteins were prepared. SDS-PAGE and Western blot were performed for protein identification. BALB/c mice were immunized at week 0 and week 3, and blood samples were collected to measure the activities of binding and neutralizing antibodies in serum. Results SDS-PAGE and Western blot showed that the four proteins had stable trimer structure. Antigen-antibody affinity test showed that the four trimer proteins had strong affinity with RSV-specific monoclonal antibodies 8897, D25, Motavizumab, AM14 and Palivizumab. The titers of antibodies induced by the two T4 trimers were higher after the initial immunization, while there was a substantial increase in the titers of antibodies induced by Human codon-optimized trimer protein after the second immunization. Conclusions PreF trimer protein can be prepared by adding any of the two different heterotrimer motifs, and induce effective binding and neutralizing antibodies in mice.
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