猪伪狂犬病病毒（浙江株）感染性细菌人工染色体克隆的构建
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摘要
伪狂犬病(Pseudorabies virus,PRV)又称Aujeszky氏病,是由疱疹病毒科(Herpesviridae)α疱疹病毒亚科中的猪I型疱疹病毒(suiderpesvirus l)所引起的猪、牛、羊等多种家畜及野生动物的一种以发热、奇痒(除猪外)、脑脊髓炎为主要症状的急性传染病。伪狂犬病是猪的主要传染病之一,给世界养猪业造成了巨大的经济损失。伪狂犬病毒基因组是线性双链DNA,全长约143 kb,可编码70-100种蛋白,与α疱疹病毒亚科的其它成员一样,PRV具有潜伏感染和神经嗜性等α疱疹病毒的典型特征。
构建重组病毒突变体将基因置于病毒基因组的背景中进行研究,是解析疱疹病毒基因功能和研究病毒感染和致病机理的主要方法。传统的同源重组方法获得病毒突变株难度大、无法对必需基因进行缺失或者突变操作,因此将国内强毒分离株PRV-ZJ克隆为细菌人工染色体(bacterial artificial chromosome,BAC),可以在大肠杆菌质粒中开展病毒基因组的修饰,提高构建重组病毒的效率,为进一步研究其基因功能和新型疫苗载体奠定基础。
本研究将强毒分离株PRV-ZJ的UL22和UL24基因片段作为同源臂,先后克隆到pMD-18T载体,构建了pUL22-24;再将携带BAC载体和绿色荧光(GFP)标记的pHA2质粒插入到pUL22-24,构建了转移载体pUL22-gfp-24-HA2;将PRV基因组与转移质粒共转染Vero细胞,获得携带BAC载体的PRV重组病毒PRV-ZJ-HA2;进而将在真核细胞内环化的重组病毒基因组转化到大肠杆菌内,通过限制性酶切图谱筛选BAC克隆;BAC克隆转染Vero细胞可以获得拯救的病毒recPRV-ZJ,通过噬斑大小测定、病毒增殖曲线测定和动物实验等对野生病毒PRV-ZJ、重组病毒PRV-ZJ-HA2及拯救病毒recPRV-ZJ生长特性进行研究,结果发现PRV和PRV-ZJ-HA2在病毒粒子增殖数量上无明显差异,但在病毒的增殖速度上PRV-ZJ-HA2变慢,掩蔽期延长,这说明TK基因的缺失及大片段的插入没有影响病毒的增殖能力,但TK基因的缺失影响了病毒潜伏感染状态的激活;PRV-ZJ-HA2和recPRV-ZJ无论在病毒增殖速度还是病毒滴度上均无明显差别,这表明PRV-HA2经过一个从真核细胞—大肠杆菌—真核细胞的过程后,无论在真核或原核细胞都能稳定增殖,因此包含PRV-ZJ基因组的感染性BAC成功克隆到大肠杆菌DH10B细胞中。PRV-ZJ感染性BAC克隆的成功构建,将提供一种可以对病毒基因组进行快速、准确操作的技术平台。
Pseudorabies virus(PRV),or“Aujeszky”s virus, is a member of alpha herpesvirinae, Herpesviridae,which can cause pseudorabies disease in swine,bovine,sheep and wild animals. Pseudorabies is an acute infection diease and characterized by high fever, extreme itching (except swine) and encephalomyelitis.Especially, porcine pseudorabies has become one of the most economically important diseases in worldwide swine industry. PRV genome is a double-stranded DNA of approximately 143 kb and encodes 70-100 proteins.Like other alpha herpesvirus, PRV has some typical chatacteristics of alpha herpesvirus such as latent infection and neurotropism.
The main method studying on the fuctional genes of herpesvirus and pathogenesis was to construct genetic-deleting mutants in the viral genome. It’s difficult to obtain the genetic-deleting mutants in the traditional method of homologous recombination mutants, which can not delet the essential gene. So that infectious bacterial artificial chromosome (BAC) clone of pseudorabies virus (PRV) strain ZJ, which can carry out the modification of the virus genome in E.coli and improve efficiency of recombinantion was constructed to serve as foundation for further study on PRV genes functions and viral vector.
In this study, a transfer plasmid pUL22-gfp-24-HA2, consisted of pHA2 (including the GFP tag and BAC-vector) flanked by two homologous arms of PRV strain ZJ UL22 and UL24 gene segments, was constructed. The recombinant virus (PRV-ZJ-HA2) was observed in fluorescence microscope and then isolated by plaque-forming assay after co-transfection of transfer plasmid and PRV genome into Vero cell. The circular genome of the recombinant virus was electroporated into E.coli DH10B and a PRV BAC clone was screened by Restriction Fragment Length Polymorphism (RLFP). The recombinant virus (recPRV-ZJ) were reconstituted after PRV BAC transfected into Vero cells.The reproduction feature of PRV, PRV-ZJ-HA2 and recPRV was investigated by a comparison of relative plaque size, growth curve and animal experiment, the results showed that the reproduction rate but not viral titers of PRV-HA2 was indistinguishable to that of parental virus PRV. It indicated that the 8.8kb-HA2 sequnce inserted in the TK gene didn’t influence the viral reproduction rate, but deletion of TK gene affects the activation state of virus infection. The reproduction rate and viral titers of recPRV was indistinguishable to that of PRV-ZJ-HA2, the results showed that PRV-ZJ-HA2 can be stable replicated either in E.coli (eukaryotic) or Vero cell (prokaryotic). The PRV BAC system will be allow for quick and reliable manipulation of the viral genome in the functional research of the genes and in the development of PRV vector in vaccine.
构建重组病毒突变体将基因置于病毒基因组的背景中进行研究,是解析疱疹病毒基因功能和研究病毒感染和致病机理的主要方法。传统的同源重组方法获得病毒突变株难度大、无法对必需基因进行缺失或者突变操作,因此将国内强毒分离株PRV-ZJ克隆为细菌人工染色体(bacterial artificial chromosome,BAC),可以在大肠杆菌质粒中开展病毒基因组的修饰,提高构建重组病毒的效率,为进一步研究其基因功能和新型疫苗载体奠定基础。
本研究将强毒分离株PRV-ZJ的UL22和UL24基因片段作为同源臂,先后克隆到pMD-18T载体,构建了pUL22-24;再将携带BAC载体和绿色荧光(GFP)标记的pHA2质粒插入到pUL22-24,构建了转移载体pUL22-gfp-24-HA2;将PRV基因组与转移质粒共转染Vero细胞,获得携带BAC载体的PRV重组病毒PRV-ZJ-HA2;进而将在真核细胞内环化的重组病毒基因组转化到大肠杆菌内,通过限制性酶切图谱筛选BAC克隆;BAC克隆转染Vero细胞可以获得拯救的病毒recPRV-ZJ,通过噬斑大小测定、病毒增殖曲线测定和动物实验等对野生病毒PRV-ZJ、重组病毒PRV-ZJ-HA2及拯救病毒recPRV-ZJ生长特性进行研究,结果发现PRV和PRV-ZJ-HA2在病毒粒子增殖数量上无明显差异,但在病毒的增殖速度上PRV-ZJ-HA2变慢,掩蔽期延长,这说明TK基因的缺失及大片段的插入没有影响病毒的增殖能力,但TK基因的缺失影响了病毒潜伏感染状态的激活;PRV-ZJ-HA2和recPRV-ZJ无论在病毒增殖速度还是病毒滴度上均无明显差别,这表明PRV-HA2经过一个从真核细胞—大肠杆菌—真核细胞的过程后,无论在真核或原核细胞都能稳定增殖,因此包含PRV-ZJ基因组的感染性BAC成功克隆到大肠杆菌DH10B细胞中。PRV-ZJ感染性BAC克隆的成功构建,将提供一种可以对病毒基因组进行快速、准确操作的技术平台。
Pseudorabies virus(PRV),or“Aujeszky”s virus, is a member of alpha herpesvirinae, Herpesviridae,which can cause pseudorabies disease in swine,bovine,sheep and wild animals. Pseudorabies is an acute infection diease and characterized by high fever, extreme itching (except swine) and encephalomyelitis.Especially, porcine pseudorabies has become one of the most economically important diseases in worldwide swine industry. PRV genome is a double-stranded DNA of approximately 143 kb and encodes 70-100 proteins.Like other alpha herpesvirus, PRV has some typical chatacteristics of alpha herpesvirus such as latent infection and neurotropism.
The main method studying on the fuctional genes of herpesvirus and pathogenesis was to construct genetic-deleting mutants in the viral genome. It’s difficult to obtain the genetic-deleting mutants in the traditional method of homologous recombination mutants, which can not delet the essential gene. So that infectious bacterial artificial chromosome (BAC) clone of pseudorabies virus (PRV) strain ZJ, which can carry out the modification of the virus genome in E.coli and improve efficiency of recombinantion was constructed to serve as foundation for further study on PRV genes functions and viral vector.
In this study, a transfer plasmid pUL22-gfp-24-HA2, consisted of pHA2 (including the GFP tag and BAC-vector) flanked by two homologous arms of PRV strain ZJ UL22 and UL24 gene segments, was constructed. The recombinant virus (PRV-ZJ-HA2) was observed in fluorescence microscope and then isolated by plaque-forming assay after co-transfection of transfer plasmid and PRV genome into Vero cell. The circular genome of the recombinant virus was electroporated into E.coli DH10B and a PRV BAC clone was screened by Restriction Fragment Length Polymorphism (RLFP). The recombinant virus (recPRV-ZJ) were reconstituted after PRV BAC transfected into Vero cells.The reproduction feature of PRV, PRV-ZJ-HA2 and recPRV was investigated by a comparison of relative plaque size, growth curve and animal experiment, the results showed that the reproduction rate but not viral titers of PRV-HA2 was indistinguishable to that of parental virus PRV. It indicated that the 8.8kb-HA2 sequnce inserted in the TK gene didn’t influence the viral reproduction rate, but deletion of TK gene affects the activation state of virus infection. The reproduction rate and viral titers of recPRV was indistinguishable to that of PRV-ZJ-HA2, the results showed that PRV-ZJ-HA2 can be stable replicated either in E.coli (eukaryotic) or Vero cell (prokaryotic). The PRV BAC system will be allow for quick and reliable manipulation of the viral genome in the functional research of the genes and in the development of PRV vector in vaccine.
引文
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