Construction of the surface display vector for lactobacilli and the effect of porcine epidemic diarrhea viral (PEDV) antigens expressed on the surface of Lactobacillus plantarum SK156 on the immune responses of mice

다국어 초록 (Multilingual Abstract)

Neonatal diarrhea is the main health problem among suckling and weaned piglets around the world. PEDV is one of the major causes of diarrhea in pigs, and is a disease that has been rapidly increasing in recent years in the United States and South Korea.

Lactobacilli is the largest genus in the lactic acid bacteria (LAB) group. The LAB group consists considerably of probiotic microbes that are effective candidates for a vaccine antigen delivery system in the intestinal mucosa.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection. Lactobacilli were isolated in order to construct a novel plasmid-based surface display system. Novel plasmid DNA was isolated from L. plantarum LP27 to construct pULP3, a lactic acid bacterial expression vector with an increased expression rate under bile conditions. β-glucuronidase assay showed a 15% ~ 48% increase in expression level of pLDH1, pEⅡDM, pPTSA and pHPRK promoter harbored vectors under the bile condition. In order to express PEDV antigens on the surface of recombinant SK156, the fusion gene was constructed by linking the signal peptide (SP) and cell wall anchor domain (CWA) of the slpA gene derived from L. acidophilus ATCC4356 strain with the selected PEDV membrane protien antigen determinants M1, M2 and M3. The surface-displayed recombinant plasmid pULP3:SPepitope:CWA was electroporated into L. plantarum, where expression and localization of SP-M1-CWA, SP-M2-CWA and SP-M3-CWA protein was confirmed by immunofluorescence assay, respectively. After intragastric administration to Balb/C mice, it was observed that live L. plantarum-expressing M1 and M3 epitope revealed stronger immunogenicity than the other epitopes (versus wild type SK156, p < 0.05). This means that the PEDV M1 and M3 epitopes can induce a much stronger mucosal and systemic immune response than other epitopes and wild-type SK156.

This study confirmed that epitopes M1 and M3 displayed on L. plantarum SK156 surface could be utilized as new mucosal vaccines, even though Balb/C mice are not strongly susceptible animal models for PEDV. This therefore offers an important opportunity for vaccine development.

목차 (Table of Contents)

• I. Introduction 1
• II. Review of Related Literature 4
• 1. Porcine epidemic diarrhea virus (PEDV) 4
• 2. Expression vectors and surface display for delivering antigens 14
• 3. Lactic acid bacteria as antigen delivery system 20
• III. Materials and Methods 24
• 1. Characterization and construction of E. coli-Lactobacillus shuttle-cloning vector and selection of Lactobacillus host for gene delivery 24
• 1) Source of samples 24
• 2) Bacterial strains, plasmids and culture conditions 24
• 3) Isolation of lactic acid bacteria (LAB) 26
• 4) Isolation of plasmid DNA 26
• 5) Identification of plasmid-harboring LAB 29
• 6) Sequence analysis of the backbone plasmid DNA 30
• 7) Cloning of antibiotic selection marker and reporter genes 31
• 8) Selection of LAB host for transformation 34
• 9) Bacterial transformation and LAB transformation efficiency verification 34
• 10) Plasmid stability 35
• 2. Construction of bile-inducible expression vector in Lactobacillus plantarum SK 156
• 1) Bacterial strains, plasmids and culture conditions 36
• 2) Isolation of chromosomal DNA from L. johnsonii PF01 for putative bile inducible promoter gene amplification 38
• 3) PCR and DNA manipulations 38
• 4) Cloning of putative bile inducible promoter and reporter genes 38
• 5) Bacterial transformation 40
• 6) β-glucuronidase (Gus) activity detection and assay 41
• 3. Bacterial surface display of porcine epidemic diarrhea (PED) virus antigen gene in L. plantarum SK156
• 1) Bacterial strains, plasmids and culture conditions 42
• 2) Selection of porcine epidemic diarrhea (PED) virus antigen gene 44
• 3) PCR and DNA manipulations 44
• 4) Construction of cell surface display system 45
• 5) Bacterial transformation 48
• 6) Detection of cell surface displayed GFP and PEDV antigens by immunofluorescence assay 48
• 4. Comparison of the immune responses of cell surface displayed PEDV antigen on L. plantarum SK 156
• 1) Intestinal epithelial cell line and recombinant L. plantarum SK156 surface displayed PEDV antigen 49
• 2) Mouse immunization and sample collection 50
• 3) Evaluation of humoral immune responses by enzyme-linked immunosorbent assay 50
• 4) Cytokine ELISA 51
• 5) Statistical analysis 51
• IV. Results and Discussion
• 1. Characterization and construction of E. coli-Lactobacillus shuttle-cloning vector and selection of Lactobacillus host for gene delivery 52
• 1) Isolation and characterization of Lactobacillus plantarum LP27 52
• 2) The identification of the isolated Lactobacillus sp. 54
• 3) Isolation and sequence analysis of pLP27 57
• 4) Construction of E. coli-Lactobacillus shuttle-cloning vector using pUC19 and pLP27 63
• 5) Selection of LAB host for transformation and transformation efficiency verification 66
• 2. Construction of bile-inducible expression vector in Lactobacillus plantarum SK 156
• 1) Cloning of putative bile inducible promoter and reporter genes 71
• 2) The β-glucuronidase (Gus) activity detection and assay 77
• 3. Bacterial surface display of porcine epidemic diarrhea (PED) virus antigen gene in L. plantarum SK 156
• 1) Construction of Lactobacillus surface display system 80
• 2) Selection of porcine epidemic diarrhea (PED) virus antigen gene and construction of cell surface display system 87
• 3) Surface display of PEDV membrane antigen in L. plantarum SK156 90
• 4. Comparison of the immune responses of cell surface displayed PEDV antigen on L. plantarum SK 156
• 1) Porcine small intestinal epithelial cell line (IPEC-J2) of immune responses to PEDV membrane antigens 92
• 2) Evaluation of humoral immune responses by enzyme-linked immunosorbent assay in mice 95
• V. Conclusion 100
• VI. References 102