摘要目的:通过基因序列设计，制备不同三聚体基序的呼吸道合胞病毒（respiratory syncytial virus，RSV）融合蛋白（fusion protein，F），评价其免疫原性和保护性差异。方法:通过查询相关专利以及文献，选择T4-foldon、Coronin-1A、T3XV、MTQ、MTI、CAT以及DMPK三聚体基因序列，将其添加到RSV F蛋白序列后，将上述质粒转染至Expi293F细胞，表达目的蛋白。采用SDS-PAGE、Western blot和高效液相色谱（high performance liquid chromatography，HPLC）鉴定表达的7种三聚体重组蛋白。将制备的三聚体重组蛋白经肌肉注射BALB/c小鼠，采集血清，检测2次免疫后结合抗体和中和抗体活性。免疫后第4周采用RSV A2毒株进行攻毒，检测肺部病毒载量。结果:SDS-PAGE、Western blot以及HPLC结果表明，制备并纯化出7种三聚体重组蛋白；微量差示扫描试验结果表明不同的三聚体重组蛋白具有不同的起始构象变化温度（onset temperature， Ton）和熔解温度（thermal melting point， Tm），且 Ton值均高于40℃， Tm值均高于60℃，表明在常规储存条件下，三聚体重组蛋白均较为稳定。小鼠实验以及活病毒中和抗体试验表明，所有的三聚体重组蛋白都能够诱导产生较高滴度的结合抗体以及中和抗体；加强免疫后，T3XV免疫组小鼠血清结合抗体滴度最高。中和试验结果显示，针对RSV A2株，含T4-foldon三聚体基序重组蛋白诱导的中和抗体滴度最高，针对RSV B18537株，含MTI与DMPK三聚体基序重组蛋白诱导的中和抗体滴度最高。肺部病毒载量测定以及肺部炎症因子测定结果表明，所有的三聚体重组蛋白都能够对小鼠产生一定的保护作用。 结论:7种三聚体基序的添加均能够使RSV F蛋白呈现融合前三聚体构象，并能够在小鼠体内产生高滴度结合抗体以及有效的中和抗体，对小鼠产生一定的保护作用。

abstractsObjective:To prepare respiratory syncytial virus（RSV）fusion（F）proteins with different trimeric motifs through gene sequence design and evaluate their differences in immunogenicity and protective efficacy.Methods:By querying relevant patents and literature，the gene sequences of T4-foldon，Coronin-1A，T3XV，MTQ，MTI，CAT and DMPK trimeric motifs were selected and added to the RSV F protein sequence. These plasmids were transfected into Expi293F cells to express the target proteins. The seven trimeric recombinant proteins were identified by SDS-PAGE，Western blot，and high performance liquid chromatography（HPLC）. The prepared trimeric proteins were injected intramuscularly into BALB/c mice，and serum samples were collected to detect the binding antibody and neutralizing antibody activities after two doses of immunization. At the fourth week after immunization，the mice were challenged with RSV A2 strain，and the viral loads in lung tissues were measured.Results:SDS-PAGE，Western blot，and HPLC results showed that seven trimeric recombinant proteins were prepared and purified. Results of the micro differential scanning test indicated that different trimeric recombinant proteins had different onset temperatures（ Ton）and melting temperatures（ Tm）. The Ton values were all higher than 40℃，and the Tm values were all higher than 60℃，indicating that the trimeric recombinant proteins were relatively stable under routine storage conditions. The mouse experiments and live virus neutralizing antibody tests showed that all trimeric recombinant proteins could induce the production of high-titer binding antibodies and neutralizing antibodies. The T3XV immunization group had the highest serum binding antibody titer after booster immunization. The neutralization test results showed that for the RSV A2 strain，the recombinant protein with the T4-foldon trimeric motif induced the highest neutralizing antibody titer，and for the RSV B18537 strain，the recombinant proteins with the MTI and DMPK trimeric motifs induced the higher neutralizing antibody titers. The viral loads and the levels of inflammatory factors in lung tissues indicated that all trimeric recombinant proteins could exert certain protective effects on mice. Conclusions:The addition of seven trimeric motifs enables the RSV F protein to present the pre-fusion trimeric conformation. These trimeric recombinant proteins can elicit high titers of binding antibodies and potent neutralizing antibodies in mice，thereby conferring certain protective effects on mice.