The Effects Of Oral Der P2Recombinant Mycobacterium Smegmatis On Immune Response After Drinking Antibiotic Solution

BACKGROUUND:Asthma is a worldwide chronic disease and a serious threat to human health. The increasing morbidity and mortality of asthma cause huge economic burden on patients and society. Asthma is the performance of a pathological state that occurs with some genetic basis of the individual, who repeated exposure to the environment allergen and resulted in the immune disorders, respiratory tract chronic inflammation and clinical symptom. Th2dominant disorder in Th1/Th2balance is the essence of the immunology of allergic asthma. Many studies have shown that Mycobacterium used a variety of immune pathway can control the Th1/Th2balance. In our lab we constructed recombinant BCG by transferring Der p2plasmid into BCG. The recombinant Der p2-BCG could induce Th1predominant immune response in mice by oral this rBCG. Because BCG doesn’t inhabit in the intestinal tract for its low affinity to intestinal epithelia.And if mice were given a large doses of BCG which could cause dysbacteria and oropharynx adenitis, thus the immune efficacy is limited. The researcher found that the normal intestinal flora can competitively inhibit and exclude of foreign bacterial not adhering and colonizing in the intestine and play a role in a biological barrier. We envision using antibiotics to destroy this biological barrier, thereby mucosal vaccines can enhance the immune efficacy of intestinal mucosal immune system.AIM1. To construct recombinant Mycobacterium smegmatis vaccine expressing the house dust mite antigen Der p2on it’s cell wall using electroporation.2. To explore the best time of drinking antibiotics solution to reduce the number of intestinal flora without causing intestinal flora disorder.3. To compare pCW-Der p2-rMSvaccine with MS vaccine by evaluating the effects on immune response after drinking antibiotic solution.METHODS AND RESULTS1. Construction and identification of the recombinant Mycobacterium smegmatis expressing Der p2in form of cell wallTo use the electroporation technology transforming identified Der p2plasmid into MS. Selecting positive clones with hygromycin resistance were identified by PCR and the result showed there was specific target gene in rMS. That was successfully constructed pCW-Der p2-rMS which can express exogenous protein Der p2in form of cell wall.2. The affection on intestinal flora after drinking antibiotics solutionThe mice were reared in SPF level environment and were given10%sucrose solution, the final concentration of2mg/ml tinidazole solution, the final concentration of5mg/ml ampicillin solution to drink for seven days. Mice were killed at3,5,7days to collect mouse intestinal contents, then homogenized and plated on LB agar plate and BHI agar plate (simple anaerobic environment). After culture the numbers of bacteria were counted. The results showed that the number of aerobic in intestinal contents of mice which drunk ampicillin solution were gradually reduced at3,5and7days (CFU3d> CFU5d> CFU7d), and Amp group at each time the number of the aerobic bacteria of the intestinal contents were statistically significant (p<0.05) compared with the Suc group and Tin group.The aerobic numbers were the least at7days.The number of anaerobic bacteria in intestinal contents of mice which drunk tinidazole solution were gradually reduced at3,5and7days (CFU3d> CFU5d> CFU7d),and the anaerobic numbers of tinidazole group at each time were significantly different (p<0.05)with Amp group and Suc group. The anaerobic numbers were the least at7days. The feces of mice which drinking antibiotic solution for7days were changed softer, thinner and more,which were symptoms of intestinal flora disorder, so consider drinking for5days of antibiotic solution is appropriate.3. The effects of oral Der p2recombinant Mycobacterium smegmatis on immune response after drinking antibiotic solutionThe mice were oral inoculated100ul the MS, pCW-Der p2-rMS (1×1010CFU/ml) or glycerol after drinking antibiotic solution for5days. At four weeks and eight weeks after the last immunization murine serum were collected and supernatant of culturing spleen lymphocytes were prepared,in which the levels of IFN-γ, IL-2, IL-4were respectively measured by ELISA. The peripheral blood and spleen lymphocytes were collected at8weeks and the distribution of Thl/Th2were detected by flow cytometry. The results showed that the levels of IFN-γ and IL-2of serum and culture supernatant of spleen lymphocytes were decreased from mice which drunk antibiotic solution, while the levels of IL-4were slightly increased, when compared with mice who drink sucrose solution; The predominant Th2cell were detected in peripheral blood and spleen lymphoid from mice which drunk antibiotic solution by flow cytometry. Compared with oral glycerol, oral MS or pCW-Der p2-rMS at4and8weeks after immunization the levels of IFN-y, IL-2levels in murine serum and spleen lymphocyte culture supernatants were higher, the IL-4levels were lower; the ratios of CD4+T cells which secreting of Th2cytokines IL-4in peripheral blood and spleen lymphoid were decreased, the ratios of CD4+T cells which secreting of Thl cytokine IFN-y were increased, and pCW-Der p2-rMS can induce a stronger Thl immune response compared with MS, and this response was the specific response to Der p2. Mice after drinking the antibiotic (Tin or Amp) solution oral inoculated pCW-Der p2-rMS comparing with the mice oral inoculated MS, the IFN-y, IL-2levels of serum and spleen lymphocyte culture supernatants were higher and the ratios of CD4+T cells which secreting of Thl cytokine IFN-y were increased, but there were not significant differences. PCW-Der p2-rMS can induce a stronger Thl immune response compared with MS and this response was the specific response to Der p2.CONCLUSION1. The best time of drinking low-concentration antibiotic solution to reduce the number of intestinal flora without causing intestinal flora disorder was five days.2. Drinking ampicillin or tinidazole solution inhibits the Thl type immune response and leads to the predominant Th2immune response. Oral MS or pCW-Der p2-rMSinduce predominant Thl immune response, and this response was the specific response to Der p2. Mice after drinking the antibiotic (Tin or Amp) solution oral inoculated pCW-Der p2-rMS comparing with the mice oral inoculated MS, the IFN-y, IL-2levels of serum and spleen lymphocyte culture supernatants were higher and the ratios of CD4+T cells which secreting of Thl cytokine IFN-y were increased, but there were not significant differences,pCW-Der p2-rMS can induce a stronger Thl immune response compared with MS, and this response was the specific response to Der p2.