Construction of fused plasmid containing protective antigen V of Yersinia pestis and signal peptide of tPA and its immuno genicity

To construct recombination secretory eukaryotic expression vector tPA-pVAX1/V based on the coding-gene of V an tigen and signal of tPA and then investigate its immunogenicity. Yersinia pestis V gene was amplified from EV76 line by PCR, and it was inserted into the eukaryotic expression vector pVAXl, tPA signal encoding sequence was amplified by PCR method and then was inserted to the 5'terminus of V to construct secretory eukaryotic expression plasmid tPA-pVAX1/V. Then the recombinant plasmid was injected into BALB/c mice via an intramuscular route together with plasmid DNA carrying GM-CSF gene as an adjuvant. Mice were challenged via subcutaneous injection of virulent Yersinia pestis 10 days after the fourth immunization and observed twice daily for morbidity and mortality. The recombinant plasmid tPA-pVAX1/V was successfully constructed; Immunocytohist ochemistry method analysis indicated that V protein was expressed in COS-7 cells; An imals immunized with the recombinant plasmid induced antigen specific antibody; Furthmore, the recombinant plasmid enhanced typical T-helper 1-dominated immune response as determined by immunoglobulin G isotype and cytokine analysis. It was able to induce an effective immuno protection against subcutaneous challenge of virulent Yersinia pestis of 400 LD50, and the survival rate was 80%. It is concluded that tPA pVAXl/V can induce both humoral and cellular immune responses, it could be used as potential candidate vaccine for the control of Yersinia pestis infections.