基于Fosmid文库表达猪瘟病毒E2蛋白和猪圆环病毒2型衣壳蛋白的重组伪狂犬病病毒的构建及免疫原性评价

论文摘要

伪狂犬病（Pseudorabies,PR）、猪瘟（Classical swine fever,CSF）和猪圆环病毒病（Porcine circovirus associated disease,PCVAD）在全世界范围广泛分布,三者常常混合感染,对养猪业造成巨大的经济损失。CSF是由猪瘟病毒（Classical swine fever virus,CSFV）引起的,而E2蛋白作为猪瘟病毒的囊膜糖蛋白,常用于猪瘟亚单位疫苗的研制。猪圆环病毒病是由猪圆环病毒2型（Porcine circovirus type 2,PCV-2）感染所致,病毒衣壳蛋白（Capsid,Cap）是猪圆环病毒病亚单位疫苗开发的主要抗原蛋白。2011年,Bartha-K61疫苗免疫的猪场爆发伪狂犬病;前期研究表明,用新分离的PRV变异株PRV TJ攻毒时,Bartha-K61疫苗无法对猪只提供完全的保护;而gE/gI/TK基因缺失PRV TJ疫苗安全性能高,能够提供完全保护。多价疫苗,尤其是表达外源蛋白的病毒载体疫苗,是抵抗多种猪病混合感染的有效策略。因此,基因缺失PRV开发多价活病毒载体疫苗具有良好的应用前景。在本研究中,利用Fosmid文库平台构建了共表达CSFV E2蛋白和PCV 2 Cap蛋白的gE/gI/TK三基因缺失重组PRV,利用间接免疫荧光技术（IFA）和免疫印迹法（WB）验证E2蛋白和Cap蛋白可在PK-15细胞上高水平表达。重组病毒rPRVTJ-delgE/gI/TK-E2-Cap在pK-15细胞上传代20代后,其生长曲线与亲本病毒rPRV-delgE/gI/TK无明显差别,说明基因缺失和外源基因的插入不影响PRV病毒粒子的形成和增殖能力。以P5、P10、P15和P20代病毒的基因组为模板,PCR扩增均能获得比亲本毒对应基因分子量减小的gE/gI/TK基因以及外源基因E2和Cap,说明该重组病毒具有良好的遗传稳定性。在家兔和猪只上的免疫评价结果表明,rPRVTJ-delgE/gI/TK-E2-Cap对家兔和猪是安全的,没有引起任何PRV特异性的临床症状,包括发热。从免疫后家兔和猪只的血清中均能检测出抗PRV的抗体,但没有抗CSFV和PCV 2的抗体,可能插入外源基因的条件不合适或E2和Cap蛋白的表达量太低不足以诱导产生抗体。本研究为病毒载体疫苗的开发提供了一个新的平台,并为优化设计后的rPRVTJ-delgE/gI/TK-E2-Cap有希望作为一种的三价候选疫苗,以对抗PRV、PCV 2和CSFV混合感染提供了新的见解。

论文目录

• 附件
• 摘要
• Abstract
• Abbreviations
• CHAPTER 1 Introduction
•   1.1 Pseudorabies（PR）
•     1.1.1 Overview of PR
•     1.1.2 Pseudorabies virus（PRV）
•     1.1.3 Epidemiology of PRV in China
•     1.1.4 Vaccines against PR
•     1.1.5 Use of PRV as a vaccine vector
•   1.2 Classical swine fever（CSF）
•     1.2.1 Overview of CSF
•     1.2.2 Classical swine fever virus（CSFV）
•     1.2.3 The distribution of CSF
•     1.2.4 Vaccines against CSF
•   1.3 Porcine circovirus-associated disease（PCVAD）
•     1.3.1 Porcine circovirus type2 （PCV2）
•     1.3.2 Vaccines and control of PCV2 in China
•   1.4 Co-infections of PRV,CSFV,and PCV2
•   1.5 Importance of vaccines and the need for developing novel vaccines
•   1.6 Live virus-vectored vaccines
•   1.7 Fosmid Library and Red/ET
•     1.7.1 Mechanism/steps for Copy Control?Cloning System
•   1.8 Modification of fosmids by Red/ET mediated recombination
•     1.8.1 Red/ET Recombination
•     1.8.2 The rps L-neo counter-selection system
•   1.9 Research Purpose and significance
• CHAPTER 2 Establishment of fosmid library for the PRV TJ Strain
•   Background
•   2.1 Materials and methods
•     2.1.1 Key reagents and equipments
•     2.1.2 Virus strain and cells
•     2.1.3 Extraction of high-quality genomic DNA from PRV-TJ strain
•     2.1.4 Assessment of quality and integrity of genome by Virus rescue
•     2.1.5 Shearing of purified DNA into25-40 kb sized DNA fragments
•     2.1.6 End repairing of DNA fragments5’phosphorylated blunt ends
•     2.1.7 Separation and isolation of25-40 kb end-repaired DNA fragments by LMP gold agarose pulse field gel electrophoresis（PFGE）
•     2.1.8 Ligation of purified end-repaired DNA fragments to PCC1FOS vector
•     2.1.9 Packaging of PCC1FOS ligated DNA into lambda phage and plating on EPI300?cells and incubation of clones for overnight
•     2.1.10 Picking single clones and induce the fosmids to high-copy number
•     2.1.11 Fosmid Extraction Protocol
•     2.1.12 Sequencing
•     2.1.13 Preparation of bacterial stock of fosmids
•     2.1.14 Sequencing Results and analysis
•     2.1.15 Alignment of different fragments to get complete genome of PRV TJ-strain
•     2.1.16 Arrangement of fosmids into library of various sized fosmids
•     2.1.17 Arrangement of fosmids into various groups
•     2.1.18 Culturing of fosmid containing on agar plates and LB medium
•     2.1.19 Extraction of fosmids
•     2.1.20 Checking of fosmid concentration
•     2.1.21 Culturing of Vero cells into10 cm petri dishes
•     2.1.22 Rescue of the recombinant PRV by fosmids
•     2.1.23 Immunofluorescence assay（IFA）
•     2.1.24 Polymerase chain reaction（PCR）
•     2.1.25 Pulse field gel electrophoresis（PFGE）
•     2.1.26 Electron microscopy
•     2.1.27 Plaque assay
•     2.1.28 Replication kinetics of the rescued PRV
•   2.2 Results
•     2.2.1 Extraction of high-quality PRV genomic DNA
•     2.2.2 Generation of the fosmid library for PRV
•     2.2.3 Rescue and Characterization of r PRV TJ by fosmid library
•   2.3 Discussion
•   2.4 Summary
• CHAPTER 3 Generation of a recombinant PRV co-expressing CSFV E2 protein and PCV2 capsid protein using fosmid library platform
•   Background
•   3.1 Materials and methods
•     3.1.1 Key reagents and equipments
•     3.1.2 Virus and cells
•     3.1.3 Experimental protocol for Red/ET mediated recombination to generate recombinant fosmid
•       3.1.3.1 Setting up overnight culture of DH10B
•       3.1.3.2 Co-electroporation of fosmid and p Red/ET
•       3.1.3.3 Amplification of rps L-neo cassette flanked by homologous arms
•       3.1.3.4 Precipitation of DNA for electroporation
•       3.1.3.5 Insertion of rps L-neo into desired fosmid
•       3.1.3.6 Picking single colonies for antibiotic selection
•       3.1.3.7 Extraction of fosmids for PCR amplification
•       3.1.3.8 PCR amplification
•       3.1.3.9 Sequencing analysis
•       3.1.3.10 Replacing the rps L-neo cassette in the target fosmid by desired gene
•       3.1.3.11 Picking single colonies
•       3.1.3.12 Extraction of fosmids for PCR verification
•       3.1.3.13 PCR
•       3.1.3.14 Sequencing
•     3.1.4 Construction of recombinant E2 expression cassette
•     3.1.5 Codon optimization of PCV2 Cap gene
•     3.1.6 Construction of recombinant fosmids with TK and g E/g I gene-deletion
•     3.1.7 Construction of recombinant fosmid with E2 and Cap gene insertion
•     3.1.8 Rescue and identification of r PRVTJ-delg E/g I/TK-E2-Cap
•     3.1.9 Immunofluorescence assay（IFA）
•     3.1.10 Western blotting
•     3.1.11 One-step growth curve
•     3.1.12 Genetic stability of the recombinant virus
•   3.2 Results
•     3.2.1 Rescue and characterization of r PRVTJ-delg E/g I/TK-E2-Cap
•     3.2.2 Confirmation of E2 and Cap expression
•     3.2.3 Growth kinetics and stability of recombinant viruses
•   3.3 Discussion
•   3.4 Summary
• CHAPTER 4 Safety and immunogenicity evaluation in rabbits and pigs
•   4.1 Materials and Methods
•     4.1.1 Ethics approval for animal experiments
•     4.1.2 Experimental design for immunization of rabbits
•     4.1.3 Experimental design for immunization of pigs
•     4.1.4 Blocking ELISA
•     4.1.5 Statistical analysis
•   4.2 Results
•     4.2.1 Safety of r PRVTJ-delg E/g I/TK-E2-Cap for rabbits
•     4.2.2 Immunogenicity of r PRVTJ-delg E/g I/TK-E2-Cap in rabbits
•     4.2.3 Immunogenicity of r PRVTJ-delg E/g I/TK-E2-Cap in pigs
•   4.3 Discussion
•   4.4 Summary
• CHAPTER 5 CONCLUSION
• REFERENCES
• APPENDIX
• ACKNOWLEDGEMENTS
• AUTHOR BIOGRAPHY

文章来源

类型: 博士论文

作者: Muhammad Abid

导师: 仇华吉

关键词: 伪狂犬病病毒,猪瘟病毒,猪圆环病毒型,疫苗

来源: 中国农业科学院

年度: 2019

分类: 基础科学,农业科技

专业: 生物学,畜牧与动物医学

单位: 中国农业科学院

基金: National Natural Sciences Foundation of China (Nos.31870152,31802163 and 31702279)

分类号: S852.651

DOI: 10.27630/d.cnki.gznky.2019.000074

总页数: 116

文件大小: 5177k

下载量: 3