减毒鼠伤寒沙门菌三型分泌表达系统的构建及其特性

Construction and characterization of type III secretion system of attenuated Salmonella typhimurium

摘要:

为开发新型重组减毒鼠伤寒沙门菌口服活疫苗载体，本研究以pYA3493质粒为基础，用鼠伤寒沙门菌sopENt100基因及其启动子替代原有的Ptrc启动子，构建沙门菌三型分泌表达载体pYA-sopENt100；再将质粒pYA-sopENt100电转入沙门菌ΔcrpΔasdSL1344，构建减毒鼠伤寒沙门菌ΔcrpΔasdSL1344 (pYA-sopENt100) 三型分泌表达系统，研究其生物学特性，进一步将报告基因egfp克隆入sopE基因下游，构建重组菌株ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)，感染Vero细胞，用Western blotting分析该系统递呈外源抗原的能力。PCR、酶切及测序结果表明，减毒鼠伤寒沙门菌ΔcrpΔasdSL1344 (pYA-sopENt100) 三型分泌表达系统构建成功；生物学特性鉴定结果表明，其血清型与亲本株Δcrp SL1344及野生株SL1344保持一致；其生化特性与亲本株基本相近，但与野毒株相比发生明显变化；生长速度也更为缓慢；重组菌株ΔcrpΔasdSL1344 (pYA-sopENt100) 的LD50较野生株SL1344降低了7.0×104倍；Western blotting结果发现，重组菌培养上清中能检测到SopENt100-egfp融合蛋白 (37 kDa)；重组菌株感染Vero细胞后，可以同时检测到SopENt100-egfp融合蛋白 (37 kDa) 和EGFP蛋白 (27 kDa)。以上结果证实，本研究成功构建了新型减毒鼠伤寒沙门菌ΔcrpΔasd (pYA-sopENt100) 三型分泌表达系统，其能够有效递呈外源抗原，该重组菌株有潜力作为安全、稳定、高效表达外源基因的口服重组活疫苗载体。

Abstract:

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD50) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×104 times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344(pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.