[Construction and immunogenicity analysis of the attenuated recombinant Salmonella typhimurium strains expressing Echinococcus granulosus Eg95 antigen].

Objective: To study the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of Eg95 antigen of Echinococcus granulosus.

Methods: The recombinant plasmid pYA3341-Eg95 was constructed by inserting the Eg95 gene into expression vector pYA3341, and identified by the methods of PCR and enzyme digestion. The recombinant plasmid pYA3341-Eg95 was electro-transformed into attenuated S. typhimurium strains X3730 and X4550 one by one to construct the recombinant strain St-Eg95. The expression of recombinant Eg95 protein in the recombinant strains St-Eg95 was analyzed by Western blotting. The strains of St-Eg95 were passaged 10 times in vitro and the recombinant plasmids were extracted at one generation interval. The genetic stability of recombinant plasmids was identified by PCR. BALB/c mice were randomly divided into six groups (five mice per group) and inoculated orally with St-Eg95, 100 µl/mouse, at dosage of 1 x 10(9), 1 x 10(10), 1 x 10(11), and 1 x 10(12) cfu/ml, wild-type S. typhimurium strain(l x l0(7)cfu/ml), and PBS, respectively. The survival rate was monitored daily for 30 days. Another 15 mice were divided into three groups and inoculated orally with St-Eg95(5 x 10(5) cfu/ml), X4550(pYA3341)(5 x 10(5) cfu/ml), and PBS, respectively, for 2 times, 0.5 ml/mouse/time, at biweekly intervals. On weeks 0, 2, 4, and 6 after the second immunization, sera were collected and tested for the presence of Eg95 antibody titers using commercially Eg antibody detection ELISA kit. The splenic lymphocyte proliferation was detected by MTT assay at 6 weeks after the second immunization.

Results: The constructed recombinant plasmid pYA3341-Eg95 was identified by enzyme digestion and PCR identification. The Eg95 protein (M, 18000) was expressed in the recombinant strains St-Eg95. After the recombinant strains St-Eg95 were passaged 10 times, the Eg95 gene (about 486 bp) was still amplified from St-Eg95. Safety results showed that mice inoculated orally with the St-Eg95 or PBS were all survival on the 30th day after immunization. However, all mice taking wild virulent S. typhimurium strain diedr within 4 days. The Eg95-specific antibodies examined by indirect ELISA were significantly higher in mice immunized with St-Eg95 than that of mice immunized with X4550 (pYA3341) or PBS at 2 weeks after the second immunization (P<0.05). The average Eg95-specific antibody titers reached up to the highest value of 1:1700 in mice immunized with St-Eg95 at 4 weeks after the second immunization. The lymphocyte proliferation test showed that the stimulation index value was significantly higher(P<0.05) in mice immunized with the St-Eg95(reached up to 1.94±0.15) than that in mice immunized with X4550 (pYA3341) or PBS at 6 weeks after the second immunization.

Conclusion: The recombinant oral attenuated S. typhimurium St-Eg95 was successfully constructed, and has a good safety and immunogenicity profile in mouse.