利用转录组测序技术研究鸡lambda干扰素在鸡细胞及器官中介导的免疫信号通路
发布时间：2024-02-14 03:42
杆状病毒表达载体系统是一种高效、快速、成本低廉的能够大规模生产的表达系统,广泛用于表达外源蛋白,尤其是真核蛋白。应用最广泛、研究最深入的杆状病毒是家蚕核型多角体杆状病毒(Bm NPV)和苜蓿银纹夜蛾核型多角体杆状病毒(AcNPV)。此外,基于BmNPV和AcNPV的杆状病毒-细胞/幼虫表达系统在研究和生产应用中有着广泛的应用。干扰素(IFNs)是一种多效细胞因子,是脊椎动物抵御病毒感染的第一道防线。目前已经鉴定出几种类型的干扰素;但是在家禽中,特别是动物实验中的研究有限。IFN-lambda(IFN-λ)最近被证明在哺乳动物中能够针对病原体产生重要的抗病毒作用。为了研究IFN-λ在鸡体内和体外是否能够引起先天性免疫和有潜在的抗病毒作用,我们利用家蚕杆状病毒表达了chIFN-λ,并用其对鸡成纤维细胞(CEF)及白羽肉鸡进行了处理,并对相关结果进行了研究分析。首先,我们使用了一种失活拯救型杆状病毒穿梭质粒BmBac(reBmBac)方法来构建重组杆状病毒并在家蚕杆状病毒表达系统中表达鸡干扰素lambda基因。为了保证reBmBac的成功构建和表达,采用荧光素酶报告基因作为标记基因。其次,在...
【文章页数】：120 页
【学位级别】：博士
【文章目录】：
摘要 ABSTRACT CHAPTER I INTRODUCTION
1.1 RECEPTOR-LIGAND INTERACTION
1.2 TRANSCRIPTIONAL ACTIVATION OF IFNS
1.3 INTERFERONS
1.3.1 Type-I Interferons
1.3.2 Type-II interferons
1.3.3 Type-III Interferons
1.4 MEDIATION OF IFN SIGNALING
1.4.1 First Level Signaling Pathway
1.4.2 Second Level Signaling Pathways
1.4.3 Third Level of Signaling Pathways
1.5 SIGNAL TRANSDUCTION
1.6 MOLECULAR ACTIONS OF IFNS
1.6.1 Myxovirus resistance proteins
1.6.2 Protein Kinase R
1.6.3 2′-5′-oligoadenylate synthetase
1.6.4 IFN-inducible transmembrane proteins (IFITM)
1.6.5 Viperin
1.6.6 CCCH-Type Zinc Finger Antiviral Protein (ZAP)
1.7 BACULOVIRUS EXPRESSION VECTOR SYSTEM (BEVS)
1.7.1 Baculovirus
1.7.2 Baculovirus promoter
1.7.3 Baculovirus expression vector system (BEVS)
1.7.4 Construction of recombinant baculovirus
1.7.5 Application of BEVS
1.7.5.1 Expression of recombinant proteins by employing BEVS
1.7.5.2 Baculovirus surface display technology
1.7.5.3 BEVS in gene therapy
1.8 RESEARCH PURPOSES AND SIGNIFICANCE
1.8.1 Research objective
1.8.2 Research significance CHAPTER II EXPRESSION OF RECOMBINANT CHICKEN INTERFERON LAMBDA(RCHIFN-Λ) IN BACULOVIRUS EXPRESSION VECTOR SYSTEM
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.2.1 Experimental materials and reagents
2.2.2 Instruments
2.2.3 Common reagent configuration methods
2.2.3.1 Media preparation for E. coli
2.2.3.2 Reagents for plasmid extraction
2.2.3.3 DNA fragment recovery reagent
2.2.3.4 Nucleic Acid and Protein Electrophoresis Buffer
2.2.3.5 Cell culture medium
2.3 EXPERIMENTAL METHODS
2.3.1 Data mining and Bioinformatics Analysis of Chicken IFN-lambda (ch IFN-λ)
2.3.2 Construction of baculovirus expression vector
2.3.2.1 Plasmid extraction of p UC-ch IFN-λ
2.3.2.2 Separation of ch IFN-λ fragments
2.3.2.3 Preparation of competent cells
2.3.2.4 Construction of recombinant plasmid p VL-ch IFN-λ
2.3.2.6 ch IFN-λ gene is linked downstream of the pph promoter
2.3.2.7 Preparation of recombinant baculovirus and expression of ch IFN-λ in silkworm
2.3.2.7.1 Revival and passage of Bm5 cells
2.3.2.7.2 Co-transfection
2.3.2.7.3 Infection of the recombinant virus to silkworm
2.3.3 Preparation of chicken embryo fibroblasts (CEF)
2.3.4 Screening, purification, and amplification of recombinant viruses
2.3.5 Preliminary purification of ch IFN-λ expressed by the silkworm
2.4 RESULTS AND ANALYSIS
2.4.1 Analysis and optimization of ch IFN-λ
2.4.2 Restriction of the target gene fragment Ch IFN- λ
2.4.3 Detection of antiviral activity of ch IFN-λ in silkworm
2.4.4 Screening and purification of recombinant virus
2.5 DISCUSSION CHAPTER III CHARACTERIZATION OF CHICKEN INTERFERON LAMBDA IN PRIMARY FIBROBLASTS AND BROILER CHICKEN
3.1 INTRODUCTION
3.2 EXPERIMENTAL MATERIALS AND INSTRUMENTS
3.2.1 Materials and reagents
3.2.2 Instruments
3.2.3 Common reagent configuration method
3.3 EXPERIMENTAL METHODOLOGY
3.3.1 Preparation of chicken embryo fibroblasts (CEF)
3.3.2 ch IFN-λ treatment to the CEF
3.3.3 Egg Inoculation
3.3.4 Washing of RBCs
3.3.5 Hemagglutination test (HA)
3.3.6 Introduction of NDV to cells
3.3.7 Antiviral potential of Chicken Interferon Lambda against NDV
3.3.8 Birds and Management
3.3.9 Sample collection
3.3.10 Total RNA Extraction and Sequencing
3.3.11 RNA Extraction Protocol from Tissue Samples
3.3.12 RNA-Seq Quality
3.3.13 Gene Ontology (GO) and KEGG Enrichment Analysis
3.4 RESULT AND ANALYSIS
3.4.1 Dynamics of weight gain post ch IFN-λ treatment
3.4.2 Characterization of ch IFN-λ-induced Gene Expression in Chicken
3.4.3 Confirmation of DEGs by q PCR
3.4.4 Upregulation of crucial genes in response to ch IFN-λ treatment
3.4.5 Functional Analysis of DEGs
3.4.6 KEGG Pathway Enrichment
3.5 DISCUSSION CHAPTER IV CONCLUSION REFERENCES APPENDIX
CODON OPTIMIZED SEQUENCE OF CHIFN-LAMBDA3
SEQUENCES FOR PROTEIN MODELING OF CHICKEN INTERFERONS ACKNOWLEDGEMENTS CURRICULUM VITAE PUBLICATION FROM DOCTORAL THESIS
本文编号：3897687
【文章页数】：120 页
【学位级别】：博士
【文章目录】：
摘要 ABSTRACT CHAPTER I INTRODUCTION
1.1 RECEPTOR-LIGAND INTERACTION
1.2 TRANSCRIPTIONAL ACTIVATION OF IFNS
1.3 INTERFERONS
1.3.1 Type-I Interferons
1.3.2 Type-II interferons
1.3.3 Type-III Interferons
1.4 MEDIATION OF IFN SIGNALING
1.4.1 First Level Signaling Pathway
1.4.2 Second Level Signaling Pathways
1.4.3 Third Level of Signaling Pathways
1.5 SIGNAL TRANSDUCTION
1.6 MOLECULAR ACTIONS OF IFNS
1.6.1 Myxovirus resistance proteins
1.6.2 Protein Kinase R
1.6.3 2′-5′-oligoadenylate synthetase
1.6.4 IFN-inducible transmembrane proteins (IFITM)
1.6.5 Viperin
1.6.6 CCCH-Type Zinc Finger Antiviral Protein (ZAP)
1.7 BACULOVIRUS EXPRESSION VECTOR SYSTEM (BEVS)
1.7.1 Baculovirus
1.7.2 Baculovirus promoter
1.7.3 Baculovirus expression vector system (BEVS)
1.7.4 Construction of recombinant baculovirus
1.7.5 Application of BEVS
1.7.5.1 Expression of recombinant proteins by employing BEVS
1.7.5.2 Baculovirus surface display technology
1.7.5.3 BEVS in gene therapy
1.8 RESEARCH PURPOSES AND SIGNIFICANCE
1.8.1 Research objective
1.8.2 Research significance CHAPTER II EXPRESSION OF RECOMBINANT CHICKEN INTERFERON LAMBDA(RCHIFN-Λ) IN BACULOVIRUS EXPRESSION VECTOR SYSTEM
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.2.1 Experimental materials and reagents
2.2.2 Instruments
2.2.3 Common reagent configuration methods
2.2.3.1 Media preparation for E. coli
2.2.3.2 Reagents for plasmid extraction
2.2.3.3 DNA fragment recovery reagent
2.2.3.4 Nucleic Acid and Protein Electrophoresis Buffer
2.2.3.5 Cell culture medium
2.3 EXPERIMENTAL METHODS
2.3.1 Data mining and Bioinformatics Analysis of Chicken IFN-lambda (ch IFN-λ)
2.3.2 Construction of baculovirus expression vector
2.3.2.1 Plasmid extraction of p UC-ch IFN-λ
2.3.2.2 Separation of ch IFN-λ fragments
2.3.2.3 Preparation of competent cells
2.3.2.4 Construction of recombinant plasmid p VL-ch IFN-λ
2.3.2.6 ch IFN-λ gene is linked downstream of the pph promoter
2.3.2.7 Preparation of recombinant baculovirus and expression of ch IFN-λ in silkworm
2.3.2.7.1 Revival and passage of Bm5 cells
2.3.2.7.2 Co-transfection
2.3.2.7.3 Infection of the recombinant virus to silkworm
2.3.3 Preparation of chicken embryo fibroblasts (CEF)
2.3.4 Screening, purification, and amplification of recombinant viruses
2.3.5 Preliminary purification of ch IFN-λ expressed by the silkworm
2.4 RESULTS AND ANALYSIS
2.4.1 Analysis and optimization of ch IFN-λ
2.4.2 Restriction of the target gene fragment Ch IFN- λ
2.4.3 Detection of antiviral activity of ch IFN-λ in silkworm
2.4.4 Screening and purification of recombinant virus
2.5 DISCUSSION CHAPTER III CHARACTERIZATION OF CHICKEN INTERFERON LAMBDA IN PRIMARY FIBROBLASTS AND BROILER CHICKEN
3.1 INTRODUCTION
3.2 EXPERIMENTAL MATERIALS AND INSTRUMENTS
3.2.1 Materials and reagents
3.2.2 Instruments
3.2.3 Common reagent configuration method
3.3 EXPERIMENTAL METHODOLOGY
3.3.1 Preparation of chicken embryo fibroblasts (CEF)
3.3.2 ch IFN-λ treatment to the CEF
3.3.3 Egg Inoculation
3.3.4 Washing of RBCs
3.3.5 Hemagglutination test (HA)
3.3.6 Introduction of NDV to cells
3.3.7 Antiviral potential of Chicken Interferon Lambda against NDV
3.3.8 Birds and Management
3.3.9 Sample collection
3.3.10 Total RNA Extraction and Sequencing
3.3.11 RNA Extraction Protocol from Tissue Samples
3.3.12 RNA-Seq Quality
3.3.13 Gene Ontology (GO) and KEGG Enrichment Analysis
3.4 RESULT AND ANALYSIS
3.4.1 Dynamics of weight gain post ch IFN-λ treatment
3.4.2 Characterization of ch IFN-λ-induced Gene Expression in Chicken
3.4.3 Confirmation of DEGs by q PCR
3.4.4 Upregulation of crucial genes in response to ch IFN-λ treatment
3.4.5 Functional Analysis of DEGs
3.4.6 KEGG Pathway Enrichment
3.5 DISCUSSION CHAPTER IV CONCLUSION REFERENCES APPENDIX
CODON OPTIMIZED SEQUENCE OF CHIFN-LAMBDA3
SEQUENCES FOR PROTEIN MODELING OF CHICKEN INTERFERONS ACKNOWLEDGEMENTS CURRICULUM VITAE PUBLICATION FROM DOCTORAL THESIS
本文编号：3897687
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