Construction Of Regulated Delayed Attenuated Salmonella Choleraesuise Vector With Enhanced Heterologous Antigen Protein

As a kind of aggressive intracellular bacteria, attenuated Salmonella has excellent ability to invade host with slow growth and stimulating effectively the body to produce strong cellular and humoral immunity, as well as mucosal immunity. Unfortunately, after attenuated, bacteria either make excessively attenuated with low immunogenicity or not fully attenuated with strong immunogenicity and inducing disease symptoms to host. To address these problems, four mutations (△Pcrp::TT araC PBAD crp,△manA,△relA::araC PBAD lacI TT and AasdA) were introduced into the wild type Salmonella choleraesuis C78-3, so that the strains display features of wild-type virulent strains of attenuated Salmonella choleraesuis at the time of inoculation to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms under arabinose and mannose regulation. The strain C78-3with△Pcrp::TT araC PBAD crp,△manA,△relA::araC PBAD lacI TT and△asdA was named χ0011. Then the glucose6-phosphate dehydrogenase gene (6-PGD) of streptococcus suis type2was cloned and expressed in the prokaryotic expression vector pAY3493. Finally, the characteristic of the vaccine candidate χ0011(pYA6-PGD) was evaluated. 1. Construction of regulated delayed attenuated Salmonella choleraesuise χ0011We introduced△manA into the wild Salmonella choleraesuis C78-3to make C78-3attenuatiom based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen when without mannose. The means for achieving regulated delayed attenuation in vivo is based on the substitution of a tightly regulated araC PBAD cassette for the promoters of the crp and relA genes such that expression of these genes is dependent on arabinose provided during strains growth. The AasdA was designed in Salmonella choleraesuis to make strain become the balanced lethal system with protein expression vector pAY3493(AsdA+) which expressed constantly exogenous antigen with vaccine candidate growth.First we constructed four suicide vector containing deletion gene APcrp::TT araC PBAD crp, AmanA,△relA::araC PBAD lacI TT and AasdA respectively by no antibiotic resistance marker of gene deletions method. Briefly, based GenBank sequences, the300bp sequences in the upstream and downstream of the potential deletion gene or promoter sequences of Salmonella choleraesuis were amplified with PCR respectively, which is homologous to parent strain, so there were total500-600bases of homologous sequences to parent strain C78-3in the suicide vector inserted into plasmid pRE112or pRE118. The four suicide vectors were constructed respectively, transformed to engineering bacterium Escherichia coli χ7213, and named χ7213(pYAs001)(APcrp::TT araC PBAD crp), χ7213(pYAs002)(△manA),χ7213(pYAs003)(△relA::araC PBAD lacI TT) and x7213(pYAs004)(AasdA) respectively.2. Evaluation of the characteristic of attenuated Salmonella choleraesuis χ0011carrying plasmid pYA6-PGD.Our goal is to construct a vaccine candidate strain to exhibit a regulated delayed attenuation in vivo. In order to investgate the characteristic of the vaccine candidate χ0011(pYA6-PGD), the LD50and distribution of χ0011(pYA6-PGD) were evaluated in Balb/C mice. The survival ability of Salmonella choleraesuis strains χ0011(pYA6-PGD) in porcine peripheral monocytes and porcine lung macrophages in vitro were measured. The results showed that the LD50of χ0011(pYA6-PGD) was decreased significantly compare to that of parent strain C78-3(lower106times), while similar to that of weak vaccine strains C500. The colonization ability of χ0011(pYA6-PGD) was strong in Pyer’s patches, but was weaker than that of wild type parent strain C78-3in liver and spleen in Balb/C mice. The survival ability of x0011(pYA6-PGD) was significantly higher than that of C500at the24h postinoculation (p<0.01), but was obvously lower than those of wild type parent strain C78-3at the48h and72h after inoculation (p<0.01).3. Construction of attenuated Salmonella choleraesuis vector carrying the6-PGD protein of Streptococcus suisIt is reported that6-phosphate dehydrogenase (6-PGD) protein of Streptococcus suis have strong imuuunogenicity. The primers were desigened based on Streptococcus suis type2(GI:8151617), and the1428bp6-PGD gene was amplified and cloned into the prokaryotic expression vector pET28a. After sequenced, the pET28a with6-PGD gene was transformed into E.coli BL21, and6-PGD protein was purified. The8-week-old New Zealand white rabbits were immunized three times with6-PGD protein, then euthanized to make serum against6-PGD antigen. The6-PGD gene was also inserted into the prokaryotic expression vector pYA3493, named pYA6-PGD, which has Asd+cassette, and could be passed stabily60generations in χ0011. Then pYA6-PGD plasmid was tranformed into regulated delayed attenuated Salmonella choleraesuise χ0011, called x0011(pYA6-PGD). Western-blotting analysis showed that6-PGD protein could be expessed in the cytoplasm and supernatant of x0011(pYA6-PGD). Six-week-old Balb/c mice were orally immunized with x0011(pYA6-PGD), and induced higher IgG antibody against6-PGD. After10weeks immunized, the Balb/c mice were challenged with14×LD50Streptococcus suis in intraperitoneal route, and all mice died although the immunized mice could survive more time.Taking together, the regulated delayed attenuated Salmonella choleraesuise strain x0011(△Pcrp::TT araC PBAD crp, AmanA, ArelA::araC PBAD lacI TT and AasdA) were constructed, and displayed the the regulated delayed attenuated feature and the invasion abilty similar to wild type strain. Further study would be need to improve the protection efficiency of χ0011(pYA6-PGD) as a vaccine candidate.