| Clostridium perfringens is anaerobic bacillus bacterium, it can cause foodborne gastroenteritis. It’s main pathogenic factor is exotoxin, exotoxin is absorbed through the intestinal mucosa and infected people and animals. Therefore, the mucosal immune immune method is an effective measure to prevent the disease.Lactic acid bacteria is a kind of probiotic which in the people and animal’s intestinal tract, respiratory tract and urinary tract. It is a group of spore gram-positive bacteria, mainly contain lactococcus lactis, lactobacillus and bifidobacterium. Long-term application of lactic acid bacteria in food and fermentation industry has proven its food security features. Lactic acid bacteria has many health benefits, it can improve human and animal nonspecific immunity, especially in improving human and animal gastrointestinal mucosal immune function, so it can be used as an endogenous probiotics, play an important role in micro ecological probiotics, lactic acid bacteria can be settled in the intestinal mucosa adhesion, and can carry specific antigen persistence to stimulate mucosal immune response. As a result, which has the function of safe non-toxic and a variety of live lactic acid bacteria, become the most appropriate immune adjuvant and antigen candidate carrier strains.This institute with cheese lactobacillus strains, it is used in food for a long time of probiotics.To develop a stable and marker-free Lactobacillus strain useful for Expression of Vaccines, we developed a temperature-sensitive suicide plasmid with PPaT expression cassettes containing an HCE promoter, a PgsA anchor, a-toxin and an rrnB T1T2terminator(PPaT) pGBHCupp-2A2B-PPaT that use a5-FU counter-selectable marker for Lactobacillus casei. Three strains containing the correct integron were produced the selective pressure of5-FU screening. We confirmed that the upp gene was deleted and the PPaT expression cassettes were inserted into the upp site in L.casei ATCC393by genomic PCR amplification and sequencing. The integrons encoding5-FU resistance could be stably inherited for as long as fifty generations following insertion. Bacteria containing the integrated DNA did not change morphologically, as determined by gram staining, but did grow more slowly than wild-type L.casei. The protein indirect enzyme-linked immunosorbent assay result show a-toxin gene was expressed, and expression protein existed on the surface of L.casei cells by laser confocal microscopy. Integron may be safe and effective vaccine candidate strain. To confirm expression of PgsA-toxin protein, western blotting, indirect enzyme-linked immunosorbent (ELISA) and laser confocal technology were used. The results of western blotting showed PgsA-toxin fusion protein of three strains recombinant bacteria were expressed. ELISA results showed that OD490absorbance value of three strains recombinant bacteria were0.6962±0.1145,0.4935±0.0239,0.5455±0.0318and the negative control group was0.2468±0.0282. Between groups were significant different (p<0.01). Results showed that the alpha toxin proteins were expressed, and have biological activities. Laser confocal showed the protein of three strains recombinant bacteria located on the surface of the lactobacillus bacteria. Above results showed that we successfully obtained vaccine candidate strains, it has nonselective markers, conform to the standard of biological safety.BALB/c mice for six to eight weeks were randomly divided into four groups, each group twenty, two hundreds microliter PBS, two hundreds microliter L.casei, two hundreds microliter△upp L.casei or two hundreds microliter PPαT△upp L.casei containing10live bacteria were gavaged into mice respectively. One time every two weeks, each time was three days, in all four times. In the first immune0,7,12,17,21,25,31,35,40,45,50and56days respectively, serum, excrement, tears and genital tract mucous were collected, IgG of serum and IgA of excrement (tears or genital tract mucous) were detected by ELISA. The results shown antibody level of PPαT△upp L.casei group were significantly higher than the others three groups in the seventh day after immuned and antibody level of others three groups were not obvious difference. Generally, the production of antibodies reach peak the seventh after immuned and soon afterwards antibodies declined, antibody levels rise rapidly after strengthening immune. The duration of antybody was relatively long.The immuned mice spleens were acquired sterilely a in the fifty-sixth days after immunization, spleen cells suspension were prepared, adjust the cell concentration was adjusted into1×106every milliliter, spleen lymphocyte cells were stimulated using different concentration alpha toxin antigen for sixty-six hours, spleen lymphocyte proliferation were detected through ELISA test. The results shown spleen lymphocyte proliferation effect were obvious for the different concentration of antigen stimulation, but effect were better than others three groups. Effect of low dose antigen stimulation was better than that of high dose in the PPαT△upp L.casei group.Spleen cells separated were inoculated into the cells culture plate, and were added fifty nanograms every milliliter PMA and five hundred nanograms every milliliter ionomycin and zero point seven microliter termination agent (Golgistop), and incubated at thirty-seven degrees Celsius for four hours.Lymphoid cells were collected, CD4cells were markered using CD4antibody or CD4cells were reverse markered by CD3and CD8antibody, cells were washed using PBS, stationary liquid and Fix&Perm were added into cells. The fluorescent tag antibody of corresponding cytokines was added and cytokine levels of spleen cells were detected using flow cytometry. The results showed that interleukin2(IL-2) and interferon gamma (IFN-gamma) were increased in PPαT△upp L.casei. But the rise of interleukin2was more apparent. At the same time, interleukin-4and interleukin-10were also increased, but interleukin-4rise was more apparent. The results showed that PPαT△upp L.casei group not only stimulate the body to produce the cellular immune but also stimulate the body to produce the humoral immunity.The mice were immuned four times, different minimum lethal dose alpha toxin protein were injected into immuned mice by intraperitoneal injection. The results showed that PPαT△upp L.casei group could completely resist one point five times MLD alpha toxin attack, and protection rate was one third when the mice were injected using double MLD alpha toxin attack. The others three group mice all died within twenty-four hours. The results implied that recombinant bacteria can let mice produce immune protection.Serum of immuned mice were mixed with triple MLD alpha toxin at thirty-seven degrees celsius for one hour and injected into healthy and non-immuned mice by intraperitoneal injection. The results showed that all mice injected serum of the PPαT△upp L.casei group were healthy, and all mice injected the others three groups serum were died within24hours.The foreign genes were integrated into L.casei and foreign proerin were expressed through genome in this experiment for the first time. Expressed protein product could stimulate the body to produce humoral immunity and could also stimulate the body to produce cellular immunity. These provide experimental data for development of clostridium perfringens a toxin vaccine, provide also a new idea for other microbial food grade vaccine. |
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