减毒沙门氏菌介导的小鼠肝炎病毒S1基因DNA疫苗的免疫特性分析

Immunogenicity of S1 gene DNA vaccine of mouse hepatitis virus delivered by attenuated Salmonella typhimurium

摘要:

根据GenBank公开序列自行设计一对引物,通过RT-PCR扩增出小鼠肝炎病毒的全长S1基因,并将其插入真核表达质粒pVAX1中,构建出重组真核表达质粒pVAX1-S1。将重组质粒转染COS-7细胞,采用间接免疫荧光检测出S1蛋白的体外表达。将重组质粒转入减毒鼠伤寒沙门氏菌SL7207中,构建出运送DNA疫苗的重组沙门氏菌SL7207(pVAX1-S1)。分别以5×10⁸CFU、1×10⁹CFU、2×10⁹CFU剂量的重组菌口服接种6周龄BALB/c小鼠,试验结果表明,重组菌对小鼠具有良好的安全性。以1×10⁹CFU剂量的重组菌口服免疫小鼠,抗体检测结果显示,在二免后两周和三免后两周,重组菌免疫组的血清抗体水平与SL7207(pVAX1)空载体免疫组间分别存在显著性差异(P<0.05)和极显著性差异(P<0.01)。在三免后两周重组菌免疫组出现了较高水平的肠黏膜抗体。

Abstract:

The complete S1 gene from mouse hepatitis virus (MHV) was amplified by RT-PCR and cloned into the pMD18-T vector. After confirmed by the restriction endonuclease analysis and PCR amplification, the positive clone of S1 gene was sequenced and then was transferred into eukaryotic expressing vector pVAX1. The recombinant plasmid pVAX1-S1 was transfected into COS-7 cells. The expressed S1 protein was successfully detected with indirect immunofluorescent assay. Finally, The recombinant plasmid pVAX1-S1 was transformed by electroporation into attenuated Salmonella typhimurium strain SL7207 and confirmed by PCR and Salmonella agglutination test. The recombinant was named as SL7207(pVAX1-S1). 6-week-old BALB/c mice were inoculated orally with SL7207 (pVAX1-S1) at dosage of 5×10⁸ CFU, 1×10⁹ CFU and 2×10⁹ CFU respectively. The immunized mice showed no clinic symptom. The results suggested that SL7207 (pVAX1-S1) was safe for mice after oral immunization at dosage of 2×10⁹ CFU or below. BALB/c mice were immunized orally with SL7207 harboring recombinant plasmid at the dosage of 109 and boosted two weeks later with the same dose, for a total of three times. The recombinant Salmonella SL7207(pVAX1-S1) could induce significant humoral immune response in mice compared with the control (P<0.05 or 0.01) at 2 w post-boosting and 2 w post-three immunization. The antibodies against MHV were also detected in small intestinal mucosal samples from immunized mice at 2 w post-three immunization. These results indicated that recombinant SL7207(pVAX1-S1) induced both systemic and local mucosal immunity.