桿狀病毒表達(dá)載體系統(tǒng)是一種高效、快速、成本低廉的能夠大規(guī)模生產(chǎn)的表達(dá)系統(tǒng),廣泛用于表達(dá)外源蛋白,尤其是真核蛋白。應(yīng)用最廣泛、研究最深入的桿狀病毒是家蠶核型多角體桿狀病毒(Bm NPV)和苜蓿銀紋夜蛾核型多角體桿狀病毒(AcNPV)。此外,基于BmNPV和AcNPV的桿狀病毒-細(xì)胞/幼蟲(chóng)表達(dá)系統(tǒng)在研究和生產(chǎn)應(yīng)用中有著廣泛的應(yīng)用。干擾素(IFNs)是一種多效細(xì)胞因子,是脊椎動(dòng)物抵御病毒感染的第一道防線(xiàn)。目前已經(jīng)鑒定出幾種類(lèi)型的干擾素;但是在家禽中,特別是動(dòng)物實(shí)驗(yàn)中的研究有限。IFN-lambda(IFN-λ)最近被證明在哺乳動(dòng)物中能夠針對(duì)病原體產(chǎn)生重要的抗病毒作用。為了研究IFN-λ在雞體內(nèi)和體外是否能夠引起先天性免疫和有潛在的抗病毒作用,我們利用家蠶桿狀病毒表達(dá)了chIFN-λ,并用其對(duì)雞成纖維細(xì)胞(CEF)及白羽肉雞進(jìn)行了處理,并對(duì)相關(guān)結(jié)果進(jìn)行了研究分析。首先,我們使用了一種失活拯救型桿狀病毒穿梭質(zhì)粒BmBac(reBmBac)方法來(lái)構(gòu)建重組桿狀病毒并在家蠶桿狀病毒表達(dá)系統(tǒng)中表達(dá)雞干擾素lambda基因。為了保證reBmBac的成功構(gòu)建和表達(dá),采用熒光素酶報(bào)告基因作為標(biāo)記基因。其次,在...

【文章頁(yè)數(shù)】：120 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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



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