捻转血矛线虫AK,ES-15,TpMy,STP-1,EF-1α,MTF-12蛋白的克隆表达及其对山羊PBMCs功能的影响
发布时间：2022-01-16 22:02
1.捻转血矛线虫精氨酸激酶体外抑制山羊外周血单核细胞（PBMC）增殖并促进其凋亡精氨酸激酶（AK）是磷酸原激酶家族的重要成员,在脊椎动物和无脊椎动物中得到广泛研究。AKs是身体的重要组成部分,参与各项细胞生物学过程,并被认为是有效的免疫调节剂和促炎性细胞因子。然而,捻转血矛线虫（HHaemonchus contortus,Hc）的AK触发的宿主细胞免疫调节反应仍是未知的。本研究对Hc-AK进行克隆、表达,并评价其在体外条件下对山羊外周血单个核细胞（PBMCs）的细胞因子产生、细胞增殖、细胞迁移、NO产生和细胞凋亡等的调节作用。本章应用逆转录聚合酶链式反应（RT-PCR）扩增了 Hc-AK基因全长,将其亚克隆至原核表达载体pET-32a。通过亲和层析纯化重组Hc-AK蛋白,并对其进行酶学测定。通过免疫荧光试验（IFA）验证体外rHc-AK与PBMC的结合。通过免疫组化检测Hc-AK在成虫中的定位。通过rHc-AK与山羊PBMC共孵育研究rHc-AK对细胞因子分泌,细胞增殖,细胞迁移,一氧化氮产生和凋亡的免疫调节作用。本章成功克隆了完整的Hc-AK基因（1,080bp）,并且重组蛋白以Hi...
【文章来源】：南京农业大学江苏省 211工程院校 教育部直属院校
【文章页数】：291 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Introduction
PART Ⅰ: REVIEW OF LITERATURE
CHAPTER 1
1. Background
2. Developmental cycle of H contortus
3. Pathogenicity
4. Importance
5. Anti-parasitic control strategies
6. Helminths-associated immunity
7. Immunoregulatory properties of H contortus ES proteins on goat PBMC
References
CHAPTER 2: Host-Parasite interacting proteins and their immunological importance
1. Parasite evasion and immune regulation
2. Arginine kinase is an immune modulatory protein of parasites
3. Tropomyosins and their related immune functions
4. Small size proteins and their related immune regulatory functions
5. Serine/threonine-protein phosphatases and their related immune regulatory functions
6. Elongation factor 1 alpha, and its immune regulatory functions
7. Methyltransferase like proteins and their associated functions
References
PART Ⅱ: RESEARCH WORK
CHAPTER 3: Arginine kinase from Haemonchus contortus decreased the proliferation andincreased the apoptosis of goat PBMCs in vitro
1. Introduction
2. Material and Methods
2.1 Animals
2.2 Parasite collection
2.3 PBMCs isolation and culture
2.4 RNA isolation and construction of cDNA from H contortus
2.5 PCR amplification, cloning and expression of Hc-AK gene
2.6 Bioinformatics analysis and phylogenetic tree
2.7 Expression of H contortus AK protein
2.8 Production of antibodies against recombinant protein
2.9 Western-blot analysis of rHc-AK
2.10 Enzyme assay for AK protein
2.11 Detection of rHc-AK binding to goat PBMC
2.12 Localization of Hc-AK in adult H contortus (male/female) worms
2.13 Detection of cytokines level by Enzyme Linked Immunosorbent Assay (ELISA)
2.14 Cell proliferation assay
2.15 Cell migration assay
2.16 Nitric oxide production Assay
2.17 Cell apoptosis assay
2.18 Statistical analysis
3. Results
3.1 Molecular cloning and sequence analysis of Hc-AK gene
3.2 Expression, purification and immunoblot analysis of rHc-AK
3.3 Enzyme activity assay
3.4 Confirmation of rHc-AK binding to PBMCs
3.5 Immunohistochemical study of Hc-AK in adult H contortus (m/f) worms
3.6 Effect of the rHc-AK on individual cytokine secretion in PBMCs
3.7 PBMCs proliferation
3.8 Cell migration assay
3.9 Nitric oxide production
3.10 rHc-AK protein enhance apoptosis of goat PBMCs
4. Discussion
Reference
CHAPTER 4: Characterization of the Haemonchus contortus excretory/secretory antigenrHcES-15 and its effects on goat PBMCs in vitro
1. Introduction
2. Material and Methods
2.1 Ethics statement
2.2 Animals and parasites
2.3 Isolation of PBMCs and monocytes
2.4 Synthesis of H contortus cDNA
2.5 Molecular cloning of HcES-15 gene and expression of rHcES-15 protein
2.6 Alignments and phylogenetic analysis
2.7 Production of antibodies
2.8 Immunoblot analysis for the rHcES-15
2.9 Binding of rHcES-15 to goat PBMCs
2.10 Localization of HcES-15 in adult H contortus worms
2.11 Analysis of cytokine levels of PBMCs treated with rHcES-15
2.12 Cell migration assay
2.13 Cell proliferation assay
2.14 Nitric oxide production assay
2.15 Cell apoptosis assay
2.16 Cell phagocytosis activity
3. Results
3.1 Cloning of HcES-15 gene
3.2 Construction and identification of the recombinant pET-32a(+)-HcES-15
3.3 Sequence and phylogenetic analysis of HcES-15
3.4 Expression and purification of rHcES-15 protein
3.5 Western blot analysis of rHcES-15 protein
3.6 Binding of rHcES-15 to goat PBMCs
3.7 Expression of HcES-15 in adult worms of H contortus
3.8 Detection of the cytokine levels by ELISA
3.9 rHcES-15 effected the proliferation of goat PBMCs
3.10 Cell migration assay
3.11 Nitric oxide production assay
3.12 rHcES-15 protein enhance apoptosis of goat PBMCs
3.13 rHcES-15 stimulated phagocytosis of goat monocytes
4. Discussion
Reference
CHAPTER 5: Recombinant tropomyosin from Haemonchus contortus mediatedsuppressive effects on goat PBMCs in vitro
1. Introduction
2. Materials and Methods
3. Results
3.1 Amplification, cloning and sequence analysis of Hc-TpMy
3.2 Expression, purification and immuno-blot of rHc-TpMy protein
3.3 rHc-TpMy binding confirmation on surface of goat PBMCs
3.4 Immunohistochemical location of rHc-TpMy in adult H contortus
3.5 Cytokines level in PBMCs detected by ELISA
3.6 PBMCs proliferation effected by rHc-TpMy
3.7 Cell migration assay
3.8 H contortus TpMy decreased NO production in goat PBMCs
3.9 H contortus TpMy induced apoptosis of PBMCs
4. Discussion
References
CHAPTER 6: Characterization of serine/threonine-protein phosphatase 1 fromHaemonchus contortus and its effects on functions of goat PBMCs in vitro
1. Introduction
2. Materials and methods
2.1 Ethics statement
2.2 Animals, parasites and PBMC isolation
2.3 Cloning and sequence analysis of H contortus STP-1 gene
2.4 Expression and purification of H contortus STP-1 gene
2.5 Immuno-blot analysis
2.6 Localization assay
2.7 Binding of rHcSTP-1 on the surface of goat PBMCs
2.8 Detection of cytokine transcripts by real-time PCR
2.9 Analysis of MHC Ⅰ and Ⅱ molecule expression
2.10 Cell proliferation assay
2.11 Cell migration assay
2.12 Intracellular nitrite production by PBMCs
2.13 Apoptotic efficiency of rHcSTP-1 on PBMCs
2.14 Statistical analysis
3. Results
3.1 Cloning and protein expression of H contortus STP-1
3.2 Localization of HcSTP-1 within H contortus parasite
3.3 Binding confirmation of rHcSTP-1 protein to goat PBMCs
3.4 Quantification of cytokine mRNA expressions in cultured PBMCs
3.5 rHcSTP-1 Inhibited MHC class-Ⅰ and Ⅱ Expression on Goat Monocytes
3.6 Recombinant STP-1 protein affected PBMCs proliferation
3.7 PBMC migration assay
3.8 rHcSTP-1 involved in intra-PBMCs NO production
3.9 rHcSTP-1 dramatically modulated early and late apoptosis of PBMC
4. Discussion
References
CHAPTER 7: Characterization of Elongation factor 1 alpha of Haemonchus contortus andits effects on goat PBMCs in vitro
1. Introduction
2. Materials and methods
3. Results
3.1 Amplification and recombinant plasmid confirmation for HcEF-1α gene
3.2 Sequence and phylogenetic analysis
3.3 Prediction of B and T cell epitopes
3.4 Expression and purification of rHcEF-1α protein
3.5 Immunoblot analyses for recombinant and native protein of HcEF-1α
3.6 rHcEF-la was localised on the surface of goat PBMCs
3.7 Immunohistochemical localisation of rHcEF-1α protein in adult H contortus
3.8 Cytokine transcription levels by PBMCs in response to rHcEF-1α
3.9 Change in MHC-Ⅰ and Ⅱ molecules mediated by rHcEF-1α
3.10 PBMC proliferation assay
3.11 PBMC migration assay
3.12 Measurement of intracellular NO production
3.13 rHcEF-1α potentially induced the apoptosis of PBMCs
4. Discussion
References
CHAPTER 8: Identification of a novel protein Methyltransferase-Type 12 fromHaemonchus contortus and its effects on goat PBMCs in vitro
1. Introduction
2. Materials and methods
3. Results
3.1 Amplification and cloning of HcMTF-12 gene
3.2 Bioinformatics analysis and phylogenetic tree
3.3 rHcMTF-12 protein expression and purification
3.4 Antibodies detected through western blot analysis
3.5 Detection of rHcMTF-12 on surface of goat PBMCs
3.6 H contortus worm's structure enriched with MTF-12 protein
3.7 rHcMTF-12 mediated cytokines expression in goat PBMCs
3.8 rHcMTF-12 down-regulated multiplication of goat PBMCs
3.9 rHcMTF-12 promoted migration efficiency of PBMCs
3.10 rHcMTF-12 mediated NO Production in goat PBMCs
4. Discussion
References
Summary
Publications
Acknowledgements
DEDICATION
【参考文献】：
期刊论文
[1]Helminth infections and intestinal inflammation[J]. Li Jian Wang, Yue Cao, Hai Ning Shi, Mucosal Immunology Laboratory, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, United States. World Journal of Gastroenterology. 2008(33)
本文编号：3593502
【文章来源】：南京农业大学江苏省 211工程院校 教育部直属院校
【文章页数】：291 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
Introduction
PART Ⅰ: REVIEW OF LITERATURE
CHAPTER 1
1. Background
2. Developmental cycle of H contortus
3. Pathogenicity
4. Importance
5. Anti-parasitic control strategies
6. Helminths-associated immunity
7. Immunoregulatory properties of H contortus ES proteins on goat PBMC
References
CHAPTER 2: Host-Parasite interacting proteins and their immunological importance
1. Parasite evasion and immune regulation
2. Arginine kinase is an immune modulatory protein of parasites
3. Tropomyosins and their related immune functions
4. Small size proteins and their related immune regulatory functions
5. Serine/threonine-protein phosphatases and their related immune regulatory functions
6. Elongation factor 1 alpha, and its immune regulatory functions
7. Methyltransferase like proteins and their associated functions
References
PART Ⅱ: RESEARCH WORK
CHAPTER 3: Arginine kinase from Haemonchus contortus decreased the proliferation andincreased the apoptosis of goat PBMCs in vitro
1. Introduction
2. Material and Methods
2.1 Animals
2.2 Parasite collection
2.3 PBMCs isolation and culture
2.4 RNA isolation and construction of cDNA from H contortus
2.5 PCR amplification, cloning and expression of Hc-AK gene
2.6 Bioinformatics analysis and phylogenetic tree
2.7 Expression of H contortus AK protein
2.8 Production of antibodies against recombinant protein
2.9 Western-blot analysis of rHc-AK
2.10 Enzyme assay for AK protein
2.11 Detection of rHc-AK binding to goat PBMC
2.12 Localization of Hc-AK in adult H contortus (male/female) worms
2.13 Detection of cytokines level by Enzyme Linked Immunosorbent Assay (ELISA)
2.14 Cell proliferation assay
2.15 Cell migration assay
2.16 Nitric oxide production Assay
2.17 Cell apoptosis assay
2.18 Statistical analysis
3. Results
3.1 Molecular cloning and sequence analysis of Hc-AK gene
3.2 Expression, purification and immunoblot analysis of rHc-AK
3.3 Enzyme activity assay
3.4 Confirmation of rHc-AK binding to PBMCs
3.5 Immunohistochemical study of Hc-AK in adult H contortus (m/f) worms
3.6 Effect of the rHc-AK on individual cytokine secretion in PBMCs
3.7 PBMCs proliferation
3.8 Cell migration assay
3.9 Nitric oxide production
3.10 rHc-AK protein enhance apoptosis of goat PBMCs
4. Discussion
Reference
CHAPTER 4: Characterization of the Haemonchus contortus excretory/secretory antigenrHcES-15 and its effects on goat PBMCs in vitro
1. Introduction
2. Material and Methods
2.1 Ethics statement
2.2 Animals and parasites
2.3 Isolation of PBMCs and monocytes
2.4 Synthesis of H contortus cDNA
2.5 Molecular cloning of HcES-15 gene and expression of rHcES-15 protein
2.6 Alignments and phylogenetic analysis
2.7 Production of antibodies
2.8 Immunoblot analysis for the rHcES-15
2.9 Binding of rHcES-15 to goat PBMCs
2.10 Localization of HcES-15 in adult H contortus worms
2.11 Analysis of cytokine levels of PBMCs treated with rHcES-15
2.12 Cell migration assay
2.13 Cell proliferation assay
2.14 Nitric oxide production assay
2.15 Cell apoptosis assay
2.16 Cell phagocytosis activity
3. Results
3.1 Cloning of HcES-15 gene
3.2 Construction and identification of the recombinant pET-32a(+)-HcES-15
3.3 Sequence and phylogenetic analysis of HcES-15
3.4 Expression and purification of rHcES-15 protein
3.5 Western blot analysis of rHcES-15 protein
3.6 Binding of rHcES-15 to goat PBMCs
3.7 Expression of HcES-15 in adult worms of H contortus
3.8 Detection of the cytokine levels by ELISA
3.9 rHcES-15 effected the proliferation of goat PBMCs
3.10 Cell migration assay
3.11 Nitric oxide production assay
3.12 rHcES-15 protein enhance apoptosis of goat PBMCs
3.13 rHcES-15 stimulated phagocytosis of goat monocytes
4. Discussion
Reference
CHAPTER 5: Recombinant tropomyosin from Haemonchus contortus mediatedsuppressive effects on goat PBMCs in vitro
1. Introduction
2. Materials and Methods
3. Results
3.1 Amplification, cloning and sequence analysis of Hc-TpMy
3.2 Expression, purification and immuno-blot of rHc-TpMy protein
3.3 rHc-TpMy binding confirmation on surface of goat PBMCs
3.4 Immunohistochemical location of rHc-TpMy in adult H contortus
3.5 Cytokines level in PBMCs detected by ELISA
3.6 PBMCs proliferation effected by rHc-TpMy
3.7 Cell migration assay
3.8 H contortus TpMy decreased NO production in goat PBMCs
3.9 H contortus TpMy induced apoptosis of PBMCs
4. Discussion
References
CHAPTER 6: Characterization of serine/threonine-protein phosphatase 1 fromHaemonchus contortus and its effects on functions of goat PBMCs in vitro
1. Introduction
2. Materials and methods
2.1 Ethics statement
2.2 Animals, parasites and PBMC isolation
2.3 Cloning and sequence analysis of H contortus STP-1 gene
2.4 Expression and purification of H contortus STP-1 gene
2.5 Immuno-blot analysis
2.6 Localization assay
2.7 Binding of rHcSTP-1 on the surface of goat PBMCs
2.8 Detection of cytokine transcripts by real-time PCR
2.9 Analysis of MHC Ⅰ and Ⅱ molecule expression
2.10 Cell proliferation assay
2.11 Cell migration assay
2.12 Intracellular nitrite production by PBMCs
2.13 Apoptotic efficiency of rHcSTP-1 on PBMCs
2.14 Statistical analysis
3. Results
3.1 Cloning and protein expression of H contortus STP-1
3.2 Localization of HcSTP-1 within H contortus parasite
3.3 Binding confirmation of rHcSTP-1 protein to goat PBMCs
3.4 Quantification of cytokine mRNA expressions in cultured PBMCs
3.5 rHcSTP-1 Inhibited MHC class-Ⅰ and Ⅱ Expression on Goat Monocytes
3.6 Recombinant STP-1 protein affected PBMCs proliferation
3.7 PBMC migration assay
3.8 rHcSTP-1 involved in intra-PBMCs NO production
3.9 rHcSTP-1 dramatically modulated early and late apoptosis of PBMC
4. Discussion
References
CHAPTER 7: Characterization of Elongation factor 1 alpha of Haemonchus contortus andits effects on goat PBMCs in vitro
1. Introduction
2. Materials and methods
3. Results
3.1 Amplification and recombinant plasmid confirmation for HcEF-1α gene
3.2 Sequence and phylogenetic analysis
3.3 Prediction of B and T cell epitopes
3.4 Expression and purification of rHcEF-1α protein
3.5 Immunoblot analyses for recombinant and native protein of HcEF-1α
3.6 rHcEF-la was localised on the surface of goat PBMCs
3.7 Immunohistochemical localisation of rHcEF-1α protein in adult H contortus
3.8 Cytokine transcription levels by PBMCs in response to rHcEF-1α
3.9 Change in MHC-Ⅰ and Ⅱ molecules mediated by rHcEF-1α
3.10 PBMC proliferation assay
3.11 PBMC migration assay
3.12 Measurement of intracellular NO production
3.13 rHcEF-1α potentially induced the apoptosis of PBMCs
4. Discussion
References
CHAPTER 8: Identification of a novel protein Methyltransferase-Type 12 fromHaemonchus contortus and its effects on goat PBMCs in vitro
1. Introduction
2. Materials and methods
3. Results
3.1 Amplification and cloning of HcMTF-12 gene
3.2 Bioinformatics analysis and phylogenetic tree
3.3 rHcMTF-12 protein expression and purification
3.4 Antibodies detected through western blot analysis
3.5 Detection of rHcMTF-12 on surface of goat PBMCs
3.6 H contortus worm's structure enriched with MTF-12 protein
3.7 rHcMTF-12 mediated cytokines expression in goat PBMCs
3.8 rHcMTF-12 down-regulated multiplication of goat PBMCs
3.9 rHcMTF-12 promoted migration efficiency of PBMCs
3.10 rHcMTF-12 mediated NO Production in goat PBMCs
4. Discussion
References
Summary
Publications
Acknowledgements
DEDICATION
【参考文献】：
期刊论文
[1]Helminth infections and intestinal inflammation[J]. Li Jian Wang, Yue Cao, Hai Ning Shi, Mucosal Immunology Laboratory, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, United States. World Journal of Gastroenterology. 2008(33)
本文编号：3593502
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