D-氨基酸营养缺陷型减毒鼠伤寒沙门氏菌口服疫苗的构建与评价

Construction and evaluation of a D-amino acid auxotrophic attenuated oral vaccine against Salmonella typhimurium

目的研究D-谷氨酸和D-丙氨酸营养缺陷型鼠伤寒沙门氏菌的安全性和免疫原性,探讨D-氨基酸营养缺陷株作为口服减毒活疫苗或载体的可行性.方法通过聚合酶链式反应(PCR)扩增并测序进行基因型分析,同时利用生长曲线实验、扫描电镜和透射电镜观察菌株生长状态和形态,确认敲除相应基因后D-氨基酸营养缺陷型鼠伤寒沙门氏菌表型稳定;药敏试验探讨外源补充D-氨基酸后,营养缺陷株对作用于细胞壁的青霉素和氨苄青霉素的敏感性;安全性评价有设置不同免疫剂量攻毒,脏器病理切片,小鼠活体成像,菌株计数和粪便16s rRNA全长测序,以检测D-氨基酸营养缺陷型菌株的定植和侵袭能力;免疫小鼠测抗体滴度检测疫苗引起的免疫应答,免疫后攻毒测定D-氨基酸营养缺陷株的保护效力.结果D-氨基酸营养缺陷型鼠伤寒沙门氏菌生长稳定,口服有效减毒,在小鼠体内停留时间缩短,激发的炎症反应较低.尤其是D-谷氨酸营养缺陷株,即使在外源补充D-谷氨酸的条件下耐药性也明显降低,既减少了对小鼠机体的损伤又能激发有效的保护性免疫应答.结论D-谷氨酸营养缺陷型鼠伤寒沙门氏菌有希望作为减毒活疫苗或载体递送抗原,预防病原体感染.

OBJECTIVE To study the safety and immunogenicity of D-glutamate(D-glu)and D-alanine(D-ala)aux-otrophic Salmonella typhimurium and explore the feasibility of D-amino acid auxotrophic strain as an oral live at-tenuated vaccine or carrier.METHODS Polymerase chain reaction(PCR)amplification and sequencing were per-formed for evaluation of genotypes.The growth status and morphological features of the strain were observed by growth curve experiment,scanning electron microscopy and transmission electron microscopy.The phenotype of D-amino acid auxotrophic S.typhimurium was confirmed stable after knocking out the corresponding genes.The sensitivities of the auxotrophic strains to penicillin and ampicillin acting on the cell walls were explored by drug susceptibility testing after exogenous supplement of D-amino acid.Safety evaluation was carried out by counterac-ting toxic test with various immunizing doses,pathological sections of organs,living imaging of rats,counting of strains and 16s rRNA full-length sequencing were performed to test the colonization and invasion capacity of the D-amino acid auxotrophic strains.The antibody titers of the immune rats was tested to detect the immune response induced by the vaccine,and the protective effect on the D-amino acid auxotrophic strains was determined by coun-teracting toxic test after immunization.RESULTS The growth of D-amino acid auxotrophic S.typhimurium was stable,which was effectively attenuated by oral administration and stayed for short period of time in the rats,and the trigged inflammatory response was lower.The D-glutamate auxotrophic strains,in particularly,showed the remarkable decline of drug resistance even under the exogenous supplement of D-glutamate,and they reduced the damage to the bodies of the rats and triggered the effective protection of immune response.CONCLUSION The D-glutamate auxotrophic S.typhimurium is expected to be the attenuated live vaccine or carrier for delivery of anti-gen so as to prevent pathogens infections.