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

作　　者：杨恒[1] 刘佳文[1] 曹三杰[1] 黄小波[1] 文心田[1] 

机构地区：[1]四川农业大学动物医学院,动物传染病与基因芯片实验室,四川省动物疫病与人类健康重点实验室,雅安625014

出　　处：《微生物学报》2009年第1期72-77,共6页Acta Microbiologica Sinica

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT0555-9);四川省教育厅资助科研项目~~

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COS-7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。[Objective] To study the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of TGEV DNA vaccine. [Methods] The 2.1 Kb fragments of the TGVE SC-H strain S gene that encompasses all the four major antigenic domains were amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transfected into COS7 cellsand 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 × 10^8 , 1 × 10^9 and 2 × 10^9 CFU for safety analysis. In a vaccination test, BALB/c mouse were immunized orally with recombinant bacterium at dosage of 1 × 10^9 CFU, for 3 times and specific serum IgG and intestinal mucosal 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 were detected after mouse peritoneal macrophages were infected with SL7207 （pVAX-S）. The recombinant bacterium was safe to mouse at dosage of 2 × 10^9 CFU. Specific serum IgG and intestinal mucosal 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 mucosal IgA at 2 weeks after the third immunization, compared with SL7207 （pVAX） control group. [ Conclusion] The recombinant Salmonella carrying TGEV S gene DNA vaccines had good immunogenicity and safety in mouse.

关 键 词：猪传染性胃肠炎病毒 S基因 减毒沙门氏菌 DNA疫苗 安全性 免疫原性 

分 类 号：S852.5[农业科学—基础兽医学]