The Study Of Bacillus Subtilis Recomment Sporessurfacedisplaying Porcine Epidemic Diarrhea Viral (PEDV) Protective Antigen Segment By CotB As Carrier Molecules

The study was to construct a gene fusion between protective antigen segment COE1 of Porcine epidemic diarrhea virus (PEDV) S protein and Bacillus subtilis spore coat gene Cot B, through double crossing-over event constructed a recombinant Bacillus subtilis with the ability to display the protective antigen segment of protein S on Bacillus subtilis spore surface. Mice as animal model, the recombination spores were fed to mice by gavage and mixing materials, observing mice immune protection against PEDV and the partial of the micro ecological probiotic function by administration with the recombinant spores, we hope to obtain an effective gene engineering oral vaccine, depend on the probiotic function of Bacillus subtilis combined with the specific mucosal immune response by the stimulation of PEDV protective antigen epitope, to reduce the PEDV influence on animal husbandry. The test content and results:(1) In this study, we choose the protective antigen segment COE1 of protein S by reference the existing research of the PEDV protein S antigen epitope, further segment CotB、COE1 were integrated in plasmid pDG364 locus to construct the recombinant pDG364-CotB-COE1. The recombinant pDG364-CotB- COE1 was verified by PCR、 double-digest and DNA sequencing, the result indicated that CotB and COE1 was integrated in pDG364 amylase gene locus, successfully construct the recombinant plasmid pDG364-CotB-COE1.(2) CotB-COE1 fusion gene is inserted into B. subtilis 168 chromosomal to construct the recombinant B. subtilis (PBE) by the double crossing-over event between the recombinant plasmid pDG364-CotB-COE1 and the B.subtilis168 genome. The result of amylase activity and PCR amplification indicated that CotB-COE1 was successfully integrated in B. subtilis chromosomal;the result of Western-blot analysis showed a specific band about 67.78 kDa which indicated the segment CotB-COE1 was displayed on B.subtilis surface; significant rad fluorescence was observed on recombinant spores surface under fluorescent microcopy, which suggested the partial protective gene COE;of S protein of PEDV was displayed on B.subtilis spore surface successfully.(3) There are 120 male mouse(ICR), averagely was divided into 5groups:recombinant spores intragastric gavage group(gb), recombinant spores mix the material feeding groups (bb),wild type spores intragastric gavage groups (g1), wild type spores mix the material feeding groups(b1) in randon and PBS control groups (K).g1 and gb groups was gavaged each the suspension of spore 100μL and consisted of 1.0×1010 spores dose;b1 and bb groups was performed by mix the material feeding in the whole process and the per gram consisted of 1.0×106 spores dose;K group was PBS control groups by fed basal diet. Blood samples and intestinal content were taken, serum was analyzed against PEDV specific IgG antibody levels and intestinal content was analyzed against PEDV specific sIgA antibody levels. The result indicated that the mice of gb and bb groups induced clear PEDV specific IgG responses and specific IgA antibody responses, the clear specific antibody levels of PEDV was detected on days 21, antibody level has reached the highest on days 35, higher antibody level still be detected on days49. The resulted that the antibody levels of recombination spores groups were proved to be observably higher than those of the control groups and wild type spores groups(p<0.01),and the antibody and sIgA antibody levels of recombinant spores mix the material feeding groups were proved to be higher than gavage groups.(4) The proliferation test of splenic lymphocytes were tested by MTT,INF-y and IL-4 levels in spleen cell were tested by ELISA.The MTT result suggested that the spleen cell proliferation stimulation index of each mice group were not significant different(p>0.05). The parameters of splenic cell IL-4 and INF-y levels suggested that the recombinant spores gavage group was remarkable higher than wild type spore gavage group and control group(p<0.01); the IL-4 and INF-y levels of recombinant spores mix the material feeding group were remarkable higher than wild type spore mix the material feeding control group(p<0.01); the IL-4 levels of recombinant spores mix the material feeding group were higher than recombinant spores gavage group(p<0.01),and the INF-y levels of recombinant spores gavage group were less higher than recombinant spores mix the material feeding group(p>0.05).(5) The cecum contents were analyzed by viable count and PCR-DGGE on days 49. The result suggested that the weight increment of spore group mice were higher than control group and remarkable differences (p<0.05), but than the spores groups were not remarkable differences (p>0.05), the number of E.coli (g1、gb、 b1 and bb groups) were lower than control groups; the number of lactic acid bacteria(g1、 gb、b1 and bb groups) were higher than control group, the result of PCR-DGGE suggested that the number and density of the bacteria flora of the spore groups mouse has increased in cecum contents. the result of immune organ index showed that the thymus index were slightly difference between groups, but no significant different(p>0.05); the spleen index of recombinant spore groups mice were higher than wild type spore groups and control group, extremely differences(p<0.01).In summary, the present experiment, porcine epidemic diarrhea virus protective antigen S protein fragment COE1 integrated in Bacillus subtilis genome successfully with Bacillus subtilis outer protein gene CotB. By the fluorescence immunoassay and Western-blot identification of recombinant Bacillus subtilis has good immunogenicity. Animal test results showed that the recombinant Bacillus subtilis can induce mice to produce a certain level of anti-porcine epidemic diarrhea virus humoral immuneresponse and cellular immune response. Another Bacilus subtilis recombinant can promote mice growth, increase the number of cecal flora advantages, increase the diversity, abundance and stability of the flora of intestinal microflora and improve the spleen index.