Preliminary Investigation Of Membrane Proteins NmpC, PagC And OmpW Of Salmonella Paratyphi A As Vaccine Candidates

Since chloramphenicol resistant strains of Salmonella paratyphi A came into being, the treatment of paratyphoid fever became much more difficult, resulting in a severe health threat for human being in developing countries, especially in China. Currently, situation for prevention and control of paratyphoid fever faced a great challenge due to the absence of vaccines for that fever. Therefore, development of safe, efficient, convenient and economical paratyphoid vaccine is warranted and urgent.In this study, probability of using NmpC, PagC and OmpW, the membrane proteins, as vaccine candidates, was preliminarily studied. More details were summarized as follows.1. Preliminary study on new vaccines candidates of membrane proteins of NmpC and PagC of S. paratyphi A. According to previous study, NmpC and PagC showed strong immunogenicity and immune protection. Four vaccine candidates, nmpC*-asd/FWL01,PagC*-asd/FWL01, nmpC-lpp-ompA/FWLO1 and pagC-lpp-ompA/FWL01, were constructed by applying the host-plasm id balanced-lethal system. Biochemical properties were confirmed to be the same as the host FWL01 according to the results conducted by automatic bacterial identification instrument. Results of plasmid stability experiment indicated that four recombinant plasmids stabilized the genetic without affecting the host’s growth. Immune response was observed by immunization of rabbits with recombinant bacteria. The results of ELISA showed that the specific IgG antibody titer of nmpC*-asd/FWL01, pagC*-asd/FWL01 and nmpC-lpp-ompA/FWL01,pagC-lpp-ompA/FWL01 was 6400, 6400 and 12800,6400, respectively.2. Immunogenicity study of sPagC, a tandem-expressed protein of hydrophilic epitopes of PagC. Recombinant antigen sPagC was derived from PagC by picking out the hydrophilic epitopes and connecting them by linking peptide sequence. The whole sequence of spagC was obtained by whole gene synthesis which carried a His-tag; and pagC was amplified from the genome of S. paratyphi A CMCC 50973 which carried a His-tag located on the 5’terminal as well. Recombinant bacteria pagC-pET-30a/BL21 and spagC-pET-30a/BL21 were obtained by connecting the pagC and spagC fragments with the expression plasmid pET-30a, and transforming them into the host BL21 respectively. The fusion proteins with molecular weight of 19.4 and 8.9 kDa were highly expressed by inducing with IPTG, and PagC was expressed in the form of inclusion bodies, while sPagC was water-soluble. The PagC and sPagC were purified by immobilized metal ion affinity chromatography, then PagC was successfully renatured in Tirs buffer by the method of gradient dialysis, and imidazole was removed from PagC and sPagC solution with the same method, and final purity of proteins reached up to 90%. Mice were immunized with two respective purified proteins using aluminum hydroxide as adjuvant. The immunogenicity of PagC and sPagC was confirmed by the total IgG antibody titers. The results showed that there was good immunogenicity for PagC, while non-significant immunogenicity for sPagC.3. The study on the correlation between the ompWgene and the virulence in Salmonella paratyphi A. S. paratyphi A CMCC 50973 gene deletion mutant strain 50973△ompW::kan was constructed using X-Red recombinant system. The reverse mutant strain ompW+ was recombined by using low-copy plasmid pACU184. The growth curve of ompW and ompW+ as well as wild strain was depicted, and that curve of mutant strains were similar to that of wild strain (P>0.05). Biochemical properties of mutant strains ompW and ompW+, and the wild strain, were identified by the automatic bacterial identification instrument, and their behaviour were the same. The LD5o value of the wild strain and the mutant strains ompW and ompW+ was 3.16×106 CFU/mL,2.37×106 CFU/mL, and 4.22×106 CFU/mL, respectively, by intraperitoneal injection(1×109 CFU/mL, 1×108 CFU/mL, 1×107 CFU/mL, 1×106 CFU/mL, 1×105 CFU/mL)with recombinant bacteria.In summary, four recombinant bacteria could induce gentle immunoreaction according to the study of vaccine candidates NmpC and PagC, which met the low immunogenicity character of oral live vaccine. sPagC, tandem-expressed protein of hydrophilic epitopes of PagC, showed poor immunogenicity, which may result from hydrophobic substance in that protein. ompW showed no significant corelation with the virulence in S. paratyphi A, its protein might be developed into a potential vaccine. The data obtained in this study can lay foundation for the subsequent study and contribute to the development of vaccines of paratyphoid fever.