携带TGEV DNA疫苗减毒沙门氏菌的构建及安全性与免疫性分析

Construction and safety,immunogenicity analysis of attenuated Salmonella typhimurium harbouring TGEV DNA vaccine

摘要:

【目的】探讨以减毒沙门氏菌为载体，进行TGEV DNA疫苗口服免疫可行性。【方法】通过RT-PCR扩增TGEV四川株（SC-H）S基因5’端约2.1 kb的主要抗原位点片段，将其插入真核表达载体pVAX1，构建重组质粒pVAX-S，体外转染COS7细胞，间接免疫荧光检测S基因表达。通过电转化将pVAX-S转入减毒鼠伤寒沙门氏菌SL7207，构建SL7207（pVAX-S）重组菌，并在体外感染小鼠腹腔巨噬细胞，以RT-PCR、间接免疫荧光检测细胞内S基因的转录与表达情况。将SL7207（pVAX-S）重组菌以5×108、1×109、2×109CFU剂量口服接种BALB/c小鼠，分析其安全性，并以1×109CFU剂量的重组菌3次免疫BALB/c小鼠，通过间接ELISA检测免疫小鼠的血清IgG与肠道粘膜IgA抗体。【结果】成功构建重组质粒pVAX-S，且重组质粒能在COS7细胞中表达。重组菌SL7207（pVAX-S）感染巨噬细胞后检测到目的基因的转录、表达。小鼠口服接种不同剂量重组菌，具有良好的安全性。免疫小鼠于二免后两周可检测到针对TGEV S蛋白的特异性血清IgG与肠道粘膜IgA抗体，且三免后两周与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）。【结论】携带TGEV DNA疫苗的减毒沙门氏菌小鼠试验显示了良好的免疫原性与安全性。

Abstract:

[Objective] To investigate the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of TGEV DNA vaccine. [Methods] 2.1 Kb fragments of the TGVE SC-H strain S gene that encompasses all the four major antigenic domains was amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transfected into COS7 cells and the expression of recombinant plasmids was identified by indirect immunofluorscence assay. Then pVAX-S was transformed by electroporation into attenuated Salmonella typhimurium SL7207, The recombinant was screened and designated as SL7207(pVAX-S). Mouse peritoneal macrophages were infected with SL7207(pVAX-S), the transcription and expression of S gene were detected by RT-PCR and indirect immunofluorscence . BALB/c mouse were inoculated orally with SL7207(pVAX-S) at dosage of 5×108、1×109、2×109CFU for safety analysis. In a vaccination test, BALB/c mouse were immunized orally with recombinant bacterial at dosage of 1×109 CFU, for three times and specific serum IgG and intestinal mucosa1 IgA antibody were detected by indirect ELISA. [Results] Recombinant plasmid pVAX-S was constructed correctly and expressed in COS7 cells. The transcription and expression of S gene was detected after mouse peritoneal macrophages were infected with SL7207(pVAX-S). The recombinant bacterium was safe to mouse at dosage of 2×109CFU. Specific serum IgG and intestinal mucosa1 IgA antibody against TGEV S protein were detected in SL7207 (pVAX-S) immunized group at 2 weeks post-boosting, and there were significant difference (P<0.05) in serum IgG and most significant difference (P<0.01) in intestinal mucosa1 IgA at 2 weeks post-three immunization compared with SL7207(pVAX) control group. [Conclusion] The recombinant Salmonella carried TGEV S gene DNA vaccines had good immunogenicity and safety in mouse.