| Asthma is a serious public health problem, affecting people of all ages. The morbidity and mortality of asthma is increasing throughout the world in recent years. Although glucocorticoid can effectively reduce its symptoms, the strong side effects limited its use in medical practice. Furthermore, it can not adjust immunological disorders and impede the progressing of asthma. So, it is urgent in clinical practice to find effective ways to prevent the occurrence and development of this allergic disease. Studies showed that there was excessive Th2 immune response in patients with allergic disease, while mycobacterium such as mycobacterium tuberculosis might induce predominant Th1 immune response. In order to change the predominant Th2 immune response induced by some anaphylactogen, we constructed recombinant BCG by transferring Der p2 into BCG in our lab. The recombinant Der p2-BCG could stimulate Th1 predominant immune response in mice, when injected intraperitoneally or subcutaneously. The results indicated that antigen recombinant BCG could be used to treat asthma which was hypersensitive to some specific antigen.Given that the repeated injection of rBCG vaccine could induce serious local delayed-type hypersensitivity, which might influence the therapeutic efficacy, oral vaccine was administered to mice. Results showed that it could also induce antigen specific Th1 predominant immune response. Related studies indicated that oral vaccination of mycobacterium was not only effective in stimulating ideal immune response, but also economic and convenient. Mycobacterium usually doesn't inhabit in the intestinal tract because of its low affinity to intestinal epithelia. Furthermore, a large dosis of mycobacteria could cause dysbacteria and oropharynx adenitis. So its immune efficacy is limited. Protein PEB1 was found to be one of the major adhesion molecules which played an important role in adhesion between Campylobacter jejuni (C.jejuni) and intestinal mucosa. These findings provide us with a convincing theory of constructing antigen specific oral vaccine of recombinant mycobacterium.AIM1. To construct the oral recombinant mycobacterium smegmatis (M.S) vaccine expressing the fusion protein of Der p2 and PEB1 on its cell wall.2. To Compare Der p2-PEB1-rM.S vaccine with Der p2-rM.S vaccine by evaluating their differential biological behaviors and immunological properties.METHODS AND RESULTS1. Expression and purification of protein PEB1PEB1 gene was amplified by polymerase chain reaction (PCR) from genomic DNA of M.S, and cloned into vector pUC19. The DNA sequence of PEB1 was identical with that published in GenBank. After confirmed by sequencing, the target gene was subcloned into prokaryotic expression vector pProEXHTb and identified by restrictive enzyme digestion. The recombinant plasmid was then transformed into E.coli DH5αstrain and induced by IPTG. The analysis of SDS-PAGE showed that there was a specific protein expression at 28kD molecular marker just as we anticipated, and the protein was further identified by Western-blot using anti-His mAb. The fusion protein was purified by Ni-NTA purification system under native condition and its purity was about ninety percent.2. Preparation of PEB1 polyclonal antibody in miceBALB/c mice were immunized subcutaneously three times at 2-week intervals, by embedded in fold groin with recombinant protein loaded to nitrocellulose filter. The control group were given 0.9% saline. Antibody titers of PEB1 protein group increased the maximum at 1: 204800 after 6 weeks of the first vaccination detected by ELISA.3. Construction and expression of the recombinant M.S expressing Der p2-PEB1 fusion proteinThe genes of Der p2 and PEB1 were first amplified by PCR respectively, then ligated into pUC19 vector. At last, the Der p2-PEB1 fused gene was subcloned into the E.coli-mycobacterium shuttle plasmid pCW. The recombinant plasmid was identified by restrictive enzyme digestion and the positive plasmid was named pCW-Der p2-PEB1. The plasmid was transformed into M.S competent cells and selected by hygromycine. The positive rM.S was identified by PCR and the result showed that there was specific amplification of the target gene. Indirect immunofluorescence was used to detect the fusion protein expressed in bacterium body of rM.S, and specifc green immunofluorescence was observed, indicating that rM.S could express the goal protein on its surface.4. Influence of intestinal tract environment on biological behavior of rM.S vaccineBALB/c mice were immunized continuously with 100μl pCW-Der p2-PEB1-rM.S, pCW-Der p2-rM.S and M.S(1×1013 CFU·L-1) via oral administration for 5 days respectively. Feces of every group were collected at 1, 6, 8, 10, 14, 28, 56 days after the first vaccination. The handled feces were plated and incubated on 7H10 agar plates, and the bacteria numbers were counted. Mice were killed at 2, 14, 28, 56 days after vaccination. Organs such as GALT, lungs and spleens were isolated aseptically, then homogenized and plated on 7H10 agar plates, at last the bacteria numbers were counted. The results showed that M.S were found in feces of every group mice in the second day after oral immunization. The numbers of M.S achieved the maximum at the sixth day, and then gradually decreased in the following days, at last disappeared in the second week after immunization. There was no significant difference among the three groups. M.S could be identified from homogenates of GALT at 2, 14 and 28 days after immunization, and the numbers of pCW-Der p2-PEB1-rM.S group were higher than that of pCW-Der p2-rM.S group at all time points ( P<0.05 ) . Few bacteria were identified from homogenates of lungs in all groups at all time points except 56 days after immunization. No live mycobacterium was recovered from the spleens in all groups. These results indicated that pCW-Der p2-PEB1-rM.S could be easily intaken by intestinal epithelia and could reach into GALT.5. The immune responses induced by oral rM.S with different affinity in miceBALB/c mice were immunized continuously with 100μl pCW-Der p2-PEB1-rM.S, pCW-Der p2-rM.S, M.S(1×1013CFU·L-1) and glycerol via oral administration for 5 days respectively. Mice blood sera were collected and culture supernatant of spleen lymphocytes were prepared at 14, 28, and 56 days after the last immunization, and the levels of IFN-γ, IL-2 and IL-4 were detected respectively by ELISA. Results showed that the levels of IFN-γand IL-2 from sera and the supernatant of cultured spleen lymphocytes induced by these two rM.S vaccines were significantly high, while the levels of IL-4 were significantly low. Similarly, the Der p2 stimulation could induce higher levels of IFN-γ, IL-2 and lower levels of IL-4 than those of M.S group in culture supernatant of spleen lymphocytes. Comparing the two rM.S groups, we also found that the concentrations of IFN-γand IL-2 in pCW-Der p2-PEB1-rM.S group were significantly higher than those in pCW-Der p2-rM.S group, but the concentrations of IL-4 had no difference between the two groups. We can conclude that -11- the vaccination of rM.S could induce the Der p2 specific Th1 predominant immune response, and the transformation of PEB1 to rM.S could increase the efficacy of oral vaccination of rM.S.CONCLUSION1. Both of the two rM.S vaccines with different affinity could be intaken by intestinal epithelia and reach into GALT in mice after oral vaccinition. Intestinal epithelial cells could ingested more rM.S, when it was fused with PEB1 protein.2. Both of the rM.S vaccines could induce Der p2 specific Th1 predominant immune response in mice after oral vaccinition, and the transformation of PEB1 to rM.S could elevate the efficacy of oral rM.S vaccines.
|
PDF Full Text Request