【作者】 孙学强；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2010， 博士

【摘要】 马疱疹病毒1型(Equine herpesvirus type1, EHV-1)是马科动物的重要病原之一,临床上多引起呼吸道、流产及神经系统疾病。疱疹病毒拥有庞大的双股DNA基因组,由于其基因组中存在大量非必需基因,有大约30kbp的序列可以被外源基因取代,并且不会严重影响病毒在体外细胞培养中增殖,EHV-1可作为表达外源蛋白活疫苗载体,不但可以用于马病的预防,而且最近的研究显示EHV-1有发展为通用载体或者基因治疗载体的潜力。本实验以ATCC标准株438/77株基础,分别克隆其ORF19和20,与含有绿色荧光蛋白和氯霉素抗性报告基因的pHA2相连构建转移载体后,与母本病毒共转染以传统的同源重组方法在ORF19和20之间插入mini-F序列,构建了含有GFP和氯霉素基因的重组病毒,提取重组病毒基因组环状中间体电转至大肠杆菌DH10B感受态细胞,经过PGR和RFLP筛选到多株阳性细菌人工染色体(BAC)克隆,以磷酸钙法转染RK13细胞,结果均拯救出病毒。拯救病毒与转移载体p1920XM通过同源重组进一步删除含有报告基因mini-F序列构建了无痕迹有标记的恢复病毒。野生株、拯救株和恢复株经过噬斑测定和一步生长曲线测定证明各毒株体外培养特性差异不显著,说明本实验所构建的BAC具有感染性,而且mini-F的插入和删除均没有改变病毒的体外培养特性。猪链球菌2型作为一种人畜共患病病原日益受到关注,而溶血素又是其分泌到细胞外的外毒素,是公认的重要毒力因子之一,具有良好的免疫原性。通过对其基因序列的分析发现5’约81bp编码的27个氨基酸残基为信号肽序列。本试验通过PCR及融合PCR方法构建载体在大肠杆菌中成功的表达了带有信号肽和无信号肽的SLY,以及将463和464位色氨酸突变为丙氨酸的带有信号肽和无信号肽的溶血素突变体-SLYm。经过蛋白杂交试验证明,表达的重组SLY和SLYm与提取的SS2分泌的SLY分子量完全一致,而且带有的信号肽的SLY和SLYm均能在细菌上清中检测到杂交条带,说明SS2sly基因的信号肽序列同样能够引导在大肠杆菌中成熟的SLY或者SLYm进行跨膜转运,而且转运至细胞外的SLY和SLYm的分子量与SS2的分子量一致,说明其信号肽序列已被切除,而无信号肽的SLY和SLYm的上清中均未检测到。同时通过血平板和96孔板溶血试验证明溶血素突变体失去溶血活性,而野生型SLY不仅能够溶解红细胞而且对PK15.RK13.SUVEC细胞具有毒性,突变体则不能引起任何细胞发生病变。小鼠毒力实验证明所表达的SLY通过腹腔注射和静脉注射均能致死小鼠,而SLYm在同样条件下对小鼠安全。用SLY和SLYm免疫小鼠制备的抗血清均能抑制SS2SLY的溶血活性,提示了463和464为氨基酸的突变虽然灭活其溶血活性,但是仍具备免疫原性其抗血清能够封闭SLY的膜结合结构域阻止其细胞膜的结合。瞬时表达及免疫转印试验证明未经优化的SS2溶血素突变体全基因在PK15细胞中不能被Pcmv启动子启动表达,而优化合成的slyom则能够表达,所表达的溶血素蛋白在PK15细胞中不需要信号肽序列也能实现跨膜转运。MRP被认为是猪链球菌的重要的毒力因子,从病猪体内分离的菌株常常含这种分子。对链球菌致病菌株的MRP进行了克隆、测序,显示N端前47个氨基酸是典型的信号肽,游离于细菌表面,N端有7个带电荷的残基,后面是一个21个氨基酸的疏水孔和一个公认的信号肽酶切位点。剪切掉信号肽得到一个分子量为131094的成熟蛋白,它与136kD的MRP接近。C端有一类似于A群链球菌M蛋白基因的锚式序列,本试验通过构建含有mrp片段的真核表达质粒转染细胞后瞬时表达MRP并经免疫转印确定能够在Pcmv启动子启动下表达。应用疫苗预防目前仍旧是防控猪链球菌病有效措施,传统的灭活疫苗仍旧具有需多次注射的局限,新型高效的猪链球菌2型疫苗仍有待于进一步的研究。本试验在所构建的p438/77的基础上,以两步法无痕迹的将mrp基因片段和溶血素突变体分别插入EHV-1基因组内构建了重组病毒,经过免疫转印鉴定能够表达MRP和SLY。重组病毒通过传统的同源重组方法删除了含有氯霉素抗性基因以及gfp的mini-F序列获得4M和4S株。免疫小鼠后以间接ELISA方法检测到4M能够诱导小鼠产生抗MRP特异性抗体,4M株免疫小鼠后能够抵抗致死量的SLY静脉注射的攻击。更多还原

【Abstract】 Equine herpesvirus type1（EHV-1） is a major cause of respiratory and reproductive disease in horses worldwide.The genome of EHV-1strain438/77isolated from aborted equine fetus was cloned as bacterial artificial chromosomes （BAC） in E. coli without any gene deletion. Mini-F sequence was inserted in the middle of ORF19and20via homologous recombination upon co-transfection of viral DNA and plasmid pE19₂0/HA into RK13cells. Circular viral DNA was extracted from RK13cells infected with purified recombinant virus expressing green fluorescent protein （GFP） and electrophorated into E. coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLP. Reconstitution of the recombinant virus was achieved successfully by transfection of the BAC DNA into RK13cells. The mini-F sequence in the reconstituted virus was further removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild type viral genome demonstrated that no unexpected change occurred during mutagenesis. In vitro replication assay showed that the BAC-reconstituted virus mutant was able to grow with plaque size and kinetics indistinguishable with those of wild type.Streptococcus suis serotype2（SS2） is a zoonotic pathogen causing diseases in swine and streptococcal toxic shock syndrome in humans.An effective swine or human SS2vaccinewithout side effects does not exist. Suilysin （SLY） is an extracellularly secreted exotoxin produced by SS2that has been shown to protect vaccinated pigs challenged with SS2. The aim of this study was to construct an avirulentSLY mutant （SLYm）, keeping the immunogenicity of wild type SLY, as a subunit vaccine to protect againstSS2infection. Recombinant SLY and SLYm, with Trp-463and Trp-464substituted with Ala residues, were made and reacted with anti-SLY monoclonal antibody by westernblot analysis. The hemolytic and cytopathogenic activities and toxicity of SLYm and SLY were examined, confirming that SLYm lostits perforationability towards eukaryotic cellswhile wild type SLY did not. Both anti-serum of mice vaccinated with SLYm and SLY inhibited the hemolytic activity of wild type SLY.The hemolytic activities were indistinguishable from each other （p>0.05）. Both mice vaccinated with SLY and SLYm were protected from the challenge of a lethal dose ofwild type SLY（p>0.05）. Most importantly, mice vaccinated with SLYm gained body weight similar to control mice, while those vaccinated with SLY gained less weight （p<0.001）. Additionally, SLYmlacked perforation activity towards eukaryotic cellsand was verified to be avirulent to the host cell and animal but kept the immunogenicity of SLY that would make the subunit vaccine effective against the pathogen andsafer to the host.Application of vaccine is one of the effective methods to control the SS2, killed vaccines are limited torepeated immunizationand whether these vaccines confer protection against challenges with heterologous strains has not been evaluated. An effective SS2vaccine without side effects that is convenient to use in swine or humans should be developed. Based on the BAC p438/77, mrpfragment and sly were introduced to the genome of EHV-1by the two-step Red E/T, respectively. The recombinant virus were comfired by western blot and could express SLY and MRP. The virus4M and4S strains were generated by homologous recombination with the removal of mini-F sequence harbouring CAM and gfp. The MRP antibody titers of miace inoculated with4M strain were assayed and the4S strain could protect the mice against the lethal dosage of recombinant SLY.更多还原