| Objective: In this study,we designed an M-cell-targeted lactobacillus surface display mucosal delivery system LL-pl SAM,and LL-pl SAM is used to assist Helicobacter pylori multivalent epitope vaccine CWAE,so that to prepare a surface display with M-cell targeting the sexual recombinant antigen SAM-WAE of the recombinant lactic acid bacteria LL-pl SAM-WAE,finally we identify the immunological properties of LL-pl SAM-WAE and its effect in preventing Helicobacter pylori infection.Methods1.The bioinformatics software MOE,DNAstar and RANKPEP are used to screen hybrid peptides Mtp with M cell targeting properties,polyclonal sites of the C-terminal peptidoglycan binding region of the surface display anchor protein Acm A of lactic acid bacteria,signal peptide SPusp45,and the leader of peptide Ps,etc.And then we analyze and determine the connection order of the above originals in order to design the core component SAM which with a scientific and reasonable structure.2.The gene synthesis method and molecular cloning techniques are used to obtain the amino acid sequence and nucleotide sequence of the core component SAM,which is inserted into the lactic acid bacteria expression vector p NZ8148 system to construct a lactic acid bacteria plasmid pl SAM.Then the constructed lactic acid bacteria plasmid pl SAM is electrotransformed into the L.lactis NZ3900 to obtain the recombinant lactic acid bacteria LL-pl SAM.3.The fusion gene of WAE is extracted from the Hp multivalent epitope vaccine CWAE by PCR technology and then inserted into the lactic acid bacteria plasmid pl SAM that of the recombinant lactic acid bacteria LL-pl SAM to construct a recombinant lactic acid bacteria plasmid pl SAM-WAE.The pl SAM-WAE can Expresses M cell-targeted recombinant antigen SAM-WAE.The constructed pl SAM-WAE is then electrotransformed into the L.lactis NZ9000 expression strain to obtain the recombinant lactic acid bacteria LL-pl SAM-WAE.4.Nisin is used to induce recombinant lactic acid bacteria LL-pl SAM-WAE to express the recombinant antigen protein SAM-WAE,and SAM-WAE is identified by SDS-PAGE,Western blot,and immunofluorescence staining;SAM-WAE is detected by ELISA on how to express in LL-pl SAM-WAE Surface;M-cell targeting experiments to detect whether LL-pl SAM-WAE has M-cell targeting;Recombinant Lactobacillus LL-pl SAM-WAE immunizes mice,and then we detect the number of Hp colonies,the urease activity of Hp in the gastric tissues of mice,the gastric inflammation and infiltration and the presence or absence of Hp-specific antibodies in mice,to identify the effect of recombinant lactic acid bacteria LL-pl SAM-WAE on anti-Helicobacter pylori infection.Results1.The LL-pl SAM,an M-cell-targeted lactobacillus delivery system on the surface,is scientific and reasonable in structure and meets the needs of experimental design.It can be used for subsequent experimental research.2.The recombinant lactic acid bacteria plasmids pl SAM and pl SAM-WAE are identified by PCR technology,double enzyme digestion,gene sequencing and other methods.The results show that the amino acid sequence and nucleotide sequence of the core component SAM in the recombinant lactic acid bacteria plasmid pl SAM is consistent with the designed theoretical values.The amino acid sequence and nucleotide sequence of SAM-WAE in pl SAM-WAE are in accordance with the designed theoretical values.The recombinant lactic acid bacteria LL-pl SAM-WAE is induced by Nisin and analyzed by SDS-PAGE,Western blot and immunofluorescence staining.The results show that LL-pl SAM-WAE can express the recombinant antigen protein SAM-WAE,and the protein size is consistent with the designed theoretical value.3.The obtained recombinant lactic acid bacteria LL-pl SAM-WAE can display the recombinant antigen protein SAM-WAE on the cell wall of the lactic acid bacteria,and make the SAM-WAE have M cell targeting,and increase the SAM-WAE uptake and transport efficiency of M cells.Enhance the gastrointestinal mucosal immune effect of SAM-WAE.4.After immunizing mice with recombinant lactic acid bacteria LL-pl SAM-WAE,the results of Hp colonization and urease activity in gastric tissues of mice showed that the number of Hp colonies and urease activity in gastric tissues of LL-pl SAM-WAE and SAM immunized mice Significantly lowered;HE staining and immunohistochemical staining showed that: the recombinant lactobacillus LL-pl SAM-WAE and SAM-WAE immunized mice had less gastric inflammation and lower inflammation scores;Hp-specific antibody test results in mice showed that:both pl SAM-WAE and SAM-WAE immunized groups can produce Hp-specific Ig G and s Ig A in mice.Conclusion: Recombinant lactic acid bacteria multivalent Hp epitope vaccine LL-pl SAM-WAE can successfully express antigen protein SAM-WAE on the surface of lactic acid bacteria after induced expression,and LL-pl SAM-WAE immunizes mice,which can significantly reduce Hp colonization and Hp urease activity in mouse stomach and can stimulate mice to produce specific antibodies against Hp,reduce gastric inflammation in mice.It can provide a new method and a new idea for clinical treatment of Hp infection and design of therapeutic multivalent epitope vaccines against Hp infection. |
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