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目的:探讨提高丹毒丝菌表面抗原A(spaA)N端核酸疫苗免疫应答的策略。方法:通过基因重组构建含人α胰岛素抑制剂(AAT)的信号肽、大鼠寡聚软骨基质蛋白(COMP)片段、丹毒丝菌spaA基因N末端及3分子的C3d序列的真核细胞表达质粒pcDNA3-AAT-COMP-spaAN-C3d3(pcD-ACSC)和pcDNA3-spa(pcD-S)。肌注免疫LCR小鼠后,RT-PCR检测小鼠体内丹毒丝菌spaAN基因的瞬时表达,ELISA检测小鼠血清spaA抗体水平的变化,以丹毒丝菌XJ1249评价免疫小鼠的保护效果。结果:免疫第4周时pcD-ACSC核酸疫苗激发了较高水平的SpaAN抗体而pcD-S核酸疫苗激发的抗体水平与对照相比差异不显著;同时pcD-ACSC核酸疫苗具有70%保护效果,pcD-S核酸疫苗则不具保护效果。结论:通过融合高表达分泌蛋白信号肽、增加抗原溶解度的序列和分子佐剂C3d3能显著提高spaAN的抗体水平,为丹毒丝菌spaA核酸疫苗的应用奠定基础。
Objective: To explore strategies to improve the immune response of N-terminal nucleic acid vaccine of Erysipelasmus toxin A (spaA). Methods: Eukaryotic expression plasmids containing signal peptide of human α-insulin inhibitor (AAT), rat oligomeric cartilage matrix protein (COMP) fragment, N-terminal of the genus erythorrhizal bacteria spa A gene and 3 molecules of C3d sequence were constructed by gene recombination pcDNA3-AAT-COMP-spaAN-C3d3 (pcD-ACSC) and pcDNA3-spa (pcD-S). After intramuscular injection of LCR mice, the transient expression of spaAN gene was detected by RT-PCR in mice. The level of spaA antibody in serum was detected by ELISA, and the protective effect was evaluated by XJ1249. Results: At 4 weeks after immunization, pcD-ACSC DNA vaccine stimulated a higher level of SpaAN antibody and pcD-S DNA vaccine did not show any significant difference compared with the control. At the same time, pcD-ACSC DNA vaccine had a protective effect of 70% pcD-S nucleic acid vaccine is not protective effect. CONCLUSION: The fusion antibody of C3d3 with high expression of secreted protein signal peptide and increased antigen solubility can significantly increase the antibody level of spaAN, which lays the foundation for the application of the nucleic acid vaccine of sparia.