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| dc.contributor.advisor | Bülent, Bozdoğan | |
| dc.contributor.author | Salih Doğan, Hanife | |
| dc.date.accessioned | 2024-07-30T12:28:33Z | |
| dc.date.available | 2024-07-30T12:28:33Z | |
| dc.date.issued | 2024-07-30 | |
| dc.date.submitted | 2024-11-23 | |
| dc.identifier.uri | http://hdl.handle.net/11607/5198 | |
| dc.description.abstract | Purpose: It was aimed to develop a recombinant antibiotic originating from the VH sequence of antibodies obtained from PBP5-immunized mice Materials and Methods: The gene encoding soluble PBP5 was cloned into the pET-30a(+) vector, produced recombinantly, and purified by IMAC. CD-1 mice were immunized with sol-PBP5, and the immune response was confirmed by ELISA. RNA from immunized mice spleens was used for cDNA synthesis and VH amplification. A VH-displaying phage library was created by cloning VH amplicons into pComb3-hy phagemid. Phages binding to sol-PBP5 were selected via biopanning, and their VH sequences were identified. The 3D structure of VH43 and its interaction with sol-PBP5 were modeled, and VH43 was produced recombinantly. Binding tests were performed with BOCILLIN-FL. Synergistic effects of VH43 with cephalosporin were tested using bacterial growth analysis and modified Kirby-Bauer/spot tests. Results: Cloning of sol-PBP5 into the pET-30a(+) vector was confirmed by sequence analysis. Expression and purification were validated via SDS-PAGE, with BOCILLIN-FL treatment confirming the protein's native conformation. Immunization was confirmed by ELISA (p-value = 9.02e-05). cDNA from total RNA of immunized mice spleens showed 500-600 bp bands on agarose gel. VH amplicons were cloned into the phagemid, and a phage library was produced via M13K07 infection. Affinity phages for sol-PBP5 were selected by biopanning, and VH sequences were determined after the third biopanning cycle. VH43's amino acid sequence and CDRs were identified, with a molecular weight of 10.3 kDa, 12 alpha helices, and 61 beta chains. Seven hydrogen bonds were found between VH43 and sol-PBP5. Recombinant VH43, confirmed via SDS-PAGE to be 20.3 kDa including His-tags, showed reduced brightness in BOCILLIN FL binding experiments. Combining VH43 with cephalosporin significantly inhibited bacterial growth, with increased zone diameter in modified Kirby-Bauer tests compared to cephalosporin alone. Conclusion: The increase in antibiotic-resistant E. faecium strains requires the development of new molecules. VH43 discovered in this thesis study is the first antibody/antibody fragment developed against E. faecium and whose sequence was determined. Combining recombinant VH43 with cephalosporin showed synergetic effect and significantly inhibited bacterial growth. This result underscores the potential of VH43 to enhance the effectiveness of cephalosporins that are normally ineffective, offering a promising approach for combating bacterial infections. | tr_TR |
| dc.description.sponsorship | This thesis was supported by Aydın Adnan Menderes University Scientific Research Projects Unit with project number TPF-22024. | tr_TR |
| dc.description.tableofcontents | ACKNOWLEDGEMENT iii TABLE OF CONTENTS iv ABBREVIATIONS viii LIST OF FIGURES ix LIST OF TABLES xi ABSTRACT xii ÖZET xiv 1. INTRODUCTION 1 2. GENERAL INFORMATIONS 2 2.1. Antibiotic resistance concerns 2 2.2. Enterococcus faecium 2 2.2.1. General information 2 2.2.2. Antimicrobial resistance of E. faecium 3 2.2.3. The cell wall structure 4 2.2.4. Penicillin Binding Proteins 4 2.2.5. Genetic determinants of pbp5 5 2.3. Current strategies to fight bacterial infections 6 2.3.1. Bacteriophages 6 2.3.2. Antimicrobial peptides 6 2.3.3. Antibodies 7 2.3.4. CRISPR-Cas Technology 8 2.3.5. Nanoparticles 8 2.3.6. Other approaches 9 2.4. Antibody Phage Display Technology 9 2.4.1. Antibody based therapies 9 2.4.2. Antibody structure 9 2.4.3. Antibody libraries 11 2.4.4. Innovational antibody fragments 12 2.4.5. Phage Display library for VH 14 3. MATERIALS AND METHOD 17 3.1. MATERIALS 17 3.1.1. Devices 17 3.1.2. Chemicals 17 3.1.3. Bacteria and phage strains 17 3.1.4. Media and Solutions 18 3.1.5. Plasmids 19 3.1.6. Enzymes 19 3.1.7. Protein experiments 20 3.1.8. Animals 20 3.1.9. Antibiotics 20 3.1.10. Markers 20 3.1.11. Primers 21 3.2. Method 21 3.2.1. The cloning of sol-pbp5 21 3.2.2. The expression and purification of sol-pbp5 25 3.2.3. Confirmation of Recombinant sol-PBP5 Protein in Native Conformation 29 3.2.4. Immunization of mice with recombinant sol-PBP5 29 3.2.5. Confirmation of the Immune Response Against Recombinant sol-PBP5 using ELISA 30 3.2.6. The construction of VH library and the cloning of variable heavy (VH) chain library into pComb3-hy phagemid 30 3.3. The phage display of VH library obtained from sol-PBP5 immunized mice 33 3.3.1. The infection of E. coli XL1-Blue cells with M13K07 helper phage 33 3.3.2. The infection of VH library in E. coli XL1-Blue cells and Production of VH Phage Library 34 3.3.3. Biopanning for Selection of M13K07 Phages Displaying sol-PBP5-Specific VH Fragment 34 3.3.4. The examination of VH displaying phage affinity to sol-PBP5 35 3.4. The cloning of VH showing affinity to sol-PBP5 36 3.5. Comparative binding studies of VH displaying phages and recombinant antibiotic 36 3.6. Determination of antibacterial activity of the recombinant VH43 with high binding affinity to sol-PBP5 37 3.6.1. Determination of minimum inhibitory concentration of recombinant VH43 for HM1070 37 3.6.2. Determination of minimum inhibitory concentration of cephalosporin for E. faecium HM1070 and clinical E. faecium isolates 37 3.6.3. Synergistic Effects of Combined Use of Recombinant VH43 with antibiotic on Enterococcus faecium HM1070 38 3.6.4. Determination of antibacterial activity of the VH for clinical Enterococcus faecium isolates 39 3.7. Cell cytotoxicity assay 40 4. RESULTS 41 4.1. The cloning of sol-PBP5gene 41 4.1.1. The amplification of sol-PBP5gene 41 4.1.2. The restriction and ligation of sol-PBP5 PCR products and pET-30 a (+) vector 41 4.1.3. The transformation of pET-30 a (+) Ω sol-PBP5 construction into E. coli BL21 (DE3) competent cells 42 4.1.4. The confirmation of cloning of sol-pbp5 gene 43 4.2. The expression and purification of sol-pbp5 46 4.2.1. The expression of recombinant sol-PBP5 protein in E. coli BL21 (DE3) 46 4.2.2. The purification of sol-PBP5protein 47 4.2.3. Confirmation of Recombinant sol-PBP5ef Protein in Native Conformation 48 4.3. Confirmation of immunization of mice with recombinant sol-PBP5 48 4.4. The construction of VH library and the cloning of variable heavy (VH) chain library into pComb3-H phagemid 49 4.4.1. Total RNA extraction and cDNA synthesis 49 4.4.2. The amplification of VH 50 4.4.3. The construction of pComb3-hy phagemid 51 4.4.4. The generation of TA vector from pComb3-hy phagemid and the ligation of VH amplicons and TA vector 53 4.4.5. Characterization of the VH Library Constructed by Cloning VH Amplicons into the pComb3-hy Vector 53 4.5. The phage display of VH library obtained from sol-PBP5 immunized mice 54 4.5.1. Production of M13K07 helper phage and VH Phage Library 54 4.5.2. Biopanning for Selection of M13K07 Phages Displaying sol-PBP5 Specific VH Fragments 55 4.5.3. Production of phages showing affinity to sol-PBP5 57 4.6. The examination of VH displaying phage affinity to sol-PBP5 58 4.6.1. The sequencing of the examined four VHs 58 4.7. The prediction of 3D structure of VH43 and sol-PBP5 and their protein-protein interactions 59 4.8. The expression and purification of recombinant VH43 60 4.8.1. The expression of recombinant VH43 protein in E. coli BL21 (DE3) 60 4.8.2. The purification of recombinant VH43 protein 61 4.9. Comparative binding studies of VH displaying phages and recombinant VH43 62 4.10. Determination of antibacterial activity of the recombinant VH43 with high binding affinity to sol-PBP5 64 4.10.1. Minimum inhibitory concentration of cephalosporin and VH43 for E. faecium HM1070 and clinical E. faecium isolates 64 4.10.2. Synergistic Effects of Combined Use of Recombinant VH43 with antibiotic on Enterococcus faecium HM1070 65 4.11. Determination of antibacterial activity of the VH for clinical Enterococcus faecium isolates 69 4.12. Cell cytotoxicity assay 69 5. DISCUSSION 71 6. CONCLUSION and SUGGESTIONS 78 REFERENCES 79 APPENDIX 87 APPENDIX 1 87 CURRICULUM VITAE 89 | tr_TR |
| dc.language.iso | eng | tr_TR |
| dc.rights | info:eu-repo/semantics/openAccess | tr_TR |
| dc.subject | D,D-transpeptidase | tr_TR |
| dc.subject | Enterococcus faecium | tr_TR |
| dc.subject | PBP5 | tr_TR |
| dc.subject | Recombinant antibiotic | tr_TR |
| dc.title | Development of Recombinant Antibiotics Against Enterococcus Faecium | tr_TR |
| dc.title.alternative | ENTEROCOCCUS FAECİUM’A KARŞI REKOMBİNANT ANTİBİYOTİK GELİŞTİRİLMESİ | tr_TR |
| dc.type | doctoralThesis | tr_TR |
| dc.contributor.department | Aydın Adnan Menderes Üniversitesi, Sağlık bilimleri enstitüsü, moleküler biyoteknoloji (ingilizce) abd | tr_TR |
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