| Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) is a gram-negative enteropathogen, parasitic in human and animal intestines, which could bring an important public problem worldwide. Vaccination is one of effective measures to control salmonellosis at present. T3SS (type Ⅲ secretion/translocation system, T3SS) encoded by SPI-1 and SPI-2 is an important virulence factor for Salmonella pathogenicity. The gene engineering techniques allow us to selectively knock out concrete virulence genes to make attenuated live Salmonella vaccine candidates, which have been received considerable attention. It has not been reported that SPI-1 T3SS effectors sopE2 and sptP were used to be the target genes of attenuated Salmonella.In this study we have constructed two deleted mutant strains which were named as C50336AsopE2 and C50336△sptP by homologous recombination system, and identified their biological characteristics. Moreover, the immunological properties of the C50336AsptP were tested for the potential as live attenuated vaccine.1. Construction and identification the biological characteristics of the deleted mutant strains C50336AsopE2 and C50336△sptP of S. EnteritidisIn this study, we constructed the prokaryotic expression plasmid pET30a-sopE2 and pET30-sptP, and then transformed into E.coli BL21(DE3), respectively. The proteins were expressed efficiently in the recombinant bacteria induced by IPTG, which confirmed by SDS-PAGE and Western blotting ananlysis. The results showed that the proteins were expressed in the form of soluble. BALB/c mice were immunized with purified recombinant proteins for further preparation of polyclonal antibodies.The deletion strains of SE were constructed by X,-Red recombination system, named as C50336△sopE2 and C50336△sptP. The gene sopE2 of C50336△sopE2 could not be transcribed and C50336△sopE2 could not react with anti-SopE2 polyclonal antibodies, as well as the gene sptP to C50336AsptP. Then their biological characteristics and virulence were determined. Among the three strains C50336, C50336AsopE2 and C50336AsptP, there are no obviously differences in growth capacity and biochemical properties. The BALB/c mouse lethal test indicated that the LD50 of C50336AsopE2 was consistent with that of the parent strain C50336. However, the LD50 of the C50336AsptP was 100 times higher than that of C50336. This study would pave a way for developing C50336ΔsptP as a live attenuated vaccine strain.2. Analysis on immunobiological characteristics of S. Enteritidis C50336ΔsptP attenuated strainThe macrophage cell line RAW264.7 and epithelial cell line Caco-2 were used as the models to investigate the characteristics of C50336ΔsptP. The results explained that the invasion rate of C50336AsptP within RAW264.7 and Caco-2 were significantly lower compared with C50336(p<0.05). The replication efficiency of deletion mutant was obviously declined in RAW264.7(p<0.05).To determine the safety of C50336AsptP, it was inoculated with mice at the dose of 1 × 107 CFU and 1 X 108 CFU for observing the number of bacteria colonization and the weight change among 21 days. At 21 day post immunization (dpi), the attenuated strain has gradually been cleared in every mice organs of 1 ×107 CFU immunized group, but liver, spleen and mLNs have been still isolated a small amount of bacteria from just one mouse of 1 × 108 CFU immunized group. However, no bacteria colonized in vaccinated mice feces and cecal tissues from 3 dpi until to 21 dpi. Besides, the results indicated that there was no significant effect on mice’s body weight after immunization.In order to evaluate the protective efficacy of Salmonella Enteritidis C50336AsptP attenuated strain as a live vaccine,6-8-week-old mice were orally immunized with C50336AsptP for the first time and were subsequently boosted after 14 days with the same doses and route (vaccinated group), while control mice were immunized with PBS (control group). Specific humoral and cellular immune responses were significantly induced.To evaluate the protective capacity of C50336AsptP, mice immunized orally with C50336AsptP were challenged with C50336. A complete protection was yielded in 1 X 108 CFU immunized group. Even in 1 X 107 CFU immunized group, a protection percent of 62.5% was yielded. In contrast, just a protection percent of 25% was yielded in the control. The bacteria were aslo isolated from the survival mice after challenged C50336. No bacteria have been found in most of the immunized mice after challenged. In contrast, a large number of bacteria were isolated from the control. Thus, this shows that C50336△sptP can be helpful for the removal of virulent strain after immunization. |
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