Effect Of Zmp1 On The Immunogenicity Of PPE68/PPE68 Peptide Of Mycobacterium

Objective:1. To observe the difference of immunogenecity between PPE68 and PPE68 peptides in BALB/c mice by oral adminstation of attneuated salmonlla, which harbour plasmid pBudCE4.1-PPE68 or pBudCE4.1-PPE68-peptide.2. To observe effect of Zmp1 on immunogenicty of PPE68 or PPE68 peptide in BALB/c mice by oral adminstation of attneuated salmonlla, which harbour plasmid pBud CE4.1-PPE68-zmp1 or pBudCE4.1-PPE68-peptide-zmp1.Methods:1. According to the PPE68 gene sequence（NC₀00962） in GenBank and its antigen protein coding sequence, its T cell epitopes were screened through online antigen peptide analysis software（http://www.cbs.dtu.dk/ Services/BepiPred/） and the pBudCE4.1-PPE68-peptide plasmids was constructed.2.The pBud CE4.1-PPE68 and pBud CE4.1-PPE68-peptide recombinant plasmids were transformed into the attenuated Salmonella strain SL7207 by electrotransformation and recombinant Salmonella were identified by PCR. The recombinant Salmonella were used to inoculate BALB/c mice by oral administration. Immunogenicity of PPE68 and PPE68 peptide were observed by screening splenocyte proliferation,IL-12 and IFN-γ level in serum,and production of sIgA in mice.3. Plasmid p Bud CE4.1-PPE68-Zmp1 and pBudCE4.1-PPE68 peptide-Zmp1 were constructed by inserting the Zmp1 gene into its another multiple cloning site. Both were transfected into RAW264.7 cells to observe the mRNA expression of Zmp1 gene by real-time quantitative PCR（qRT-PCR）.4.The pBudCE4.1-PPE68-Zmp1 and p BudCE4.1-PPE68 peptide-Zmp1 plasmid were transformed into the attenuated Salmonella SL7207 to construct corresponding recombinant Salmonella. The recombinant Salmonella were used to inoculate BALB/c mice by oral administration. The effect of Zmp1 on immunogenicity of PPE68 and PPE68 peptide were observed by screening splenocyte proliferation,IL-12 and IFN-γ level in serum,and production of sIgA in mice.Results:1. PPE68 antigen peptide were screened out successfully. The constructed recombinant pBudCE4.1-PPE68-peptide was correctly identified by PCR and DNA sequencing.2. The recombinant plasmid pBudCE4.1-PPE68 and pBudCE4.1-PPE68-peptide were transformed into the attenuated Salmonella by electrotransformation. The recombinant salmonella were identified correctly by PCR.3. The level of cytokines（IL-12 and IFN-γ in serum, sIgA in Small intestine and lung） were assessed by ELISA after last oral immunization. PPE68 and PPE68 peptide all can induce immune response, the level of IL-12 and IFN- γ were higher than control group（P<0.05）; The level of IL-12 and IFN- γ in PPE68 peptide group were higher than the PPE68 group（P<0.05）; Compared with control group,the level of specific spleen lymphocyte proliferation were higher in PPE68 group and PPE68 peptide group（P<0.05）,but it didn’t show the significant difference（P>0.05） between PPE68 group and PPE68 peptide group;The level of s IgA in the intestinal mice and pulmonary lavage was higher than control group and showed the significant difference（P<0.05）, but it didn’t show the significant difference（P>0.05） between PPE68 group and PPE68 peptide group.4.The recombinant plasmid p BudCE4.1-PPE68-Zmp1 and pBud CE4.1-PPE68peptide-Zmp1 were digested by Bgl II and Kpn I, and restriction fragment appeared agreement with the theory. DNA sequencing analysis was consistent with Zmp1 gene in GenBank. The mRNA expression of Zmp1 gene was detected in macrophages cell RAW264.7 by qRT-PCR.5.The recombinant plasmid pBudCE4.1-PPE68-Zmp1 and pBudCE4.1-PPE68peptide-Zmp1 were transformed into the attenuated Salmonella. Both recombinant salmonella were identified correctly by PCR.6.The level of IL-12 and IFN-γ in serum and s Ig A in mice were detected by ELISA after last oral immunization. The level of IL-12 in serum was higher in PPE68 group than the PPE68/Zmp1 group（P<0.05）. The level of IL-12 in serum was higher in PPE68 peptide group than the PPE68peptide/Zmp1 group（P<0.05）. The level of IFN-γ in serum was higher in PPE68 group than the PPE68/Zmp1 group（P<0.05）. The level of IFN-γ in serum was no significant difference between PPE68 peptide group and PPE68 peptide/Zmp1 group（P>0.05）. The level of sIg A in Small intestine was higher in PPE68 group than the PPE68/Zmp1 group（P <0.05）. The level of sIgA in Small intestine was no significant difference between PPE68 peptide group and PPE68 peptide/Zmp1 group（P>0.05）. The level of sIgA in lung was no significant difference between PPE68 group and PPE68/Zmp1 group（P>0.05）. The level of sIgA in lung was no significant difference between PPE68 peptide group and PPE68 peptide/Zmp1 group（P>0.05）. There was significant difference between experimental groups and control group in the Spleen lymphocyte proliferation（P <0.05）, but there was no significant difference when compared between experimental groups.Conclusion:1. Using attenuated Salmonella as vector, both PPE68 and PPE68 peptide can induce distinct cellular immune response, immune effect of PPE68 peptide is superior to PPE68 protein.2. Zmp1 protein may inhibit PPE68 digestion in immune process in the body. Zmp1 affect the secretion of inflammatory chemokines and affect the immune response.3. Using attenuated Salmonella as vector, both PPE68-zmp1 and PPE68peptide-zmp1 can induce distinct mice immune response, Zmp1 has certain effect on mucosal immunity.