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

Chuan YU; Chongkai ZHAI; Chengshui LIAO; Zuhua YU; Lei HE; Yanyan JIA; Jing LI; Chunjie ZHANG; Xiangchao CHENG

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

Author: Chuan YU ¹ ; Chongkai ZHAI ¹ ; Chengshui LIAO ¹ ; Zuhua YU ¹ ; Lei HE ¹ ; Yanyan JIA ¹ ; Jing LI ¹ ; Chunjie ZHANG ¹ ; Xiangchao CHENG ¹
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1. The Key Laborary of Animal Disease and Public Health, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, Henan, China.
Publication Type:Journal Article
Keywords: attenuated Salmonella typhimurium; balance-lethal system; live vaccine vector; sopE gene; type III secretion system
MeSH: Animals; Bacterial Proteins; genetics; Cercopithecus aethiops; Mice; Plasmids; Promoter Regions, Genetic; Salmonella typhimurium; genetics; Type III Secretion Systems; genetics; Vaccines, Attenuated; genetics; Vero Cells; Virulence
From: Chinese Journal of Biotechnology 2016;32(12):1664-1675
CountryChina
Language:Chinese
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.