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To develop a host-vector balanced lethal system of Salmonella typhimurium adenylate cyclase mutant, and detect its biological characteristics.We constructed SL1344ΔcyaΔasd mutant strain by recombinant suicide plasmid (pREAasd), and screened by two-step method, transformed pYA3493 plasmid containing the asd gene without resistance electric into the lack of SL1344 AcyaΔasd, then the recombinant strains SL1344 ΔcyaΔasd (pYA3493) was constructed successfully.The biochemical characteristics and growth rate of the mutant were different from that of the wild strain SL1344, but almost the same as that of the parent strain SL1344Δcya. The mutant strain could neither ferment maltose, lactose, and sorbitol, nor decompose H2S, galactose and rat lee sugar, but still retained the ability to use glucose. The one-day chicken lethal test showed that SL1344ΔcyaΔasd (pYA3493) mutant was at least 104 times lower than SL1344 strain. The protection rate induced by the SL1344ΔcyaΔasd (pYA3493) mutant was 62. 5%.The SL1344ΔcyaΔasd (pYA3493) mutant was successfully constructed, and had good immune protection, it laid a foundation for developing potential oral vaccines.

In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica (Bb) challenge.The complete coding sequence (2040 bp) of the prn gene (PRN) and its fragments,5'-terminal 1173 bp fragment (PN),3'-terminal 867 bp fragment (PC), two copies of region I (654 bp; PR I) in PN, and 2 copies of region II (678 bp; PR II) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by isopropyl-beta-D-thiogalactopyranoside. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PR I and GST-2PR II. All five recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PR I group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN,GST-PC or GST-2PR II serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PR I, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5).In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge. Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN. In addition, GST-2PR II and GST-2PR I provided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis on the basis of these five pertactin recombinants.