拯救GPMV毒株细胞系的构建及靶向沉默P基因抑制GPMV复制研究
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摘要
鹅副黏病毒病是由鹅副黏病毒引起的一种高死亡率的传染病。该病对各种年龄的鹅均具有易感性,其中15日龄以内雏鹅的发病率和死亡率可高达100%。经鉴定,鹅副黏病毒为禽副黏病毒血清I型,因此鹅副黏病毒病又可称为是鹅的新城疫。近几年,该病对我国养鹅业造成了严重的经济损失,威胁着养鹅业的健康发展。鹅副黏病毒病的预防主要依靠弱毒疫苗株,目前尚没有同源疫苗进行鹅副粘病毒病的特异免疫,因此,新型疫苗的创制是该病防控的重要环节之一。通过反向遗传学技术来获得鹅新城疫的疫苗株是国内外研究的热点,其中最重要的基础环节就是需要一个稳定表达T7RNA聚合酶的细胞系。
新城疫病毒的复制主要依靠核糖核蛋白聚合体(RNP),形成RNP的必要条件之一就是需要P蛋白,P蛋白和L蛋白的结合物具有RNA依赖RNA聚合酶活性,所以本研究选择靶向沉默P基因,进而抑制病毒的复制。
对T7RNA聚合酶基因进行克隆,构建真核表达载体PCI-T7。利用脂质体转染法将该载体转染到V7细胞系,构建了表达T7 RNA聚合酶的V7细胞系。并利用Western blot和间接免疫荧光对HA蛋白进行了检测,证实T7 RNA聚合酶基因在VT7细胞系中得到了稳定有效的表达。
根据鹅源副黏病毒NA-1株已发表的P基因序列,siRNA表达载体的要求和RNAi靶位基因序列选择原则,应用BLAST工具筛选出特异性地针对鹅源副黏病毒的siRNA序列,同时设计阴性RNAi靶位。根据所设计的RNAi的靶位分子和表达载体的要求,将RNAi靶位分子经过退火、纯化后连接到酶切后的RNAi专用表达载体上,进而转染鸡胚成纤维细胞(CEF)及鸡胚,6h后接种病毒。接毒后36h时分别用Real Time RT-PCR、病毒滴度测定、间接免疫荧光、细胞病变情况来观察RNAi对GPMV NA-1在CEF中复制和繁殖的影响情况,并用血凝试验检测RNAi在鸡胚中的抑制作用。实验结果显示,针对GPMV NA-1株P基因设计的2个RNAi靶位,在RNAi过程中表现出了很强的抑制效果,其中RNAi-P641的抑制效率更高。说明了P基因的这两个位点是鹅源副黏病毒复制所必需的。
脱氧核酶(Deoxyriozyme DNAzyme DZ)是一种通过对体外合成的随机序列库进行筛选得到的具有RNA切割功能的DNA分子,是目前基因沉默研究的热点之一。本试验应用生物信息学针对鹅源副黏病毒NA-1株P基因设计DZ-P-358、DZ-P-446、DZ-P-7023种10-23脱氧核酶,经体外切割实验筛选得到DZ-P-702、DZ-P-358具有切割活性,DZ-P-702活性较高。在CEF细胞水平通过细胞病变情况、病毒滴度测定、间接免疫荧光方法验证了DZ-P-702、DZ-P-358可以抑制鹅源副黏病毒在CEF细胞的复制；通过Real-time PCR方法检测表明DZ-P-358及DZ-P-702降低CEF细胞中P基因的转录水平,其中DZ-P-702抑制效率可达到78.5%。通过对尿囊液血凝效价的测定表明脱氧核酶可以有效抑制GPMV在鸡胚中的增殖,展现了脱氧核酶在生产实践中的应用前景。
综上所述,本研究成功构建稳定表达T7RNA聚合酶的VT7细胞系,为通过反向遗传学技术来获得新城疫的疫苗株奠定了坚实的基础；筛选出两个有效的抑制鹅副黏病毒P基因表达的干扰靶位。筛选出了针对鹅副黏病毒P基因特异的脱氧核酶DZ-P-702。有效靶位的筛选和确立,为进一步研究该病的抗病毒治疗及抗病育种领域提供了技术基础。
Goose paramyxovirus disease is a high mortality rate of infectious diseases caused by the goose paramyxovirus. The disease was susceptibility to all age of goose.Less 15-day-old goslings' morbidity and mortality can be as high as 100%. It was identified that the goose paramyxovirus is theⅠ-type serum virus, so the goose paramyxovirus virus may also called the Newcastle disease of goose. In recent years, this disease cause serious economic damage to the raising goose industry in China, threatening the healthy development of goose industry. The prevention of goose paramyxovirus mainly relied on the Attenuated vaccine, so the creating of new vaccine is one of the most important links to prevent and control the disease. It's a hot research that to gain the attenuated vaccine of Newcastle disease by reverse genetic manipulation, and one of the most important basic link is that it requires a cell lines to express T7RNA polym erase stably.
The replication of the Newcastle disease virus mainly depend on the RNP, and a necessary condition for the formation of RNP is the P protein needed, the complex of P protein and L protein have a RNA-dependent RNA polymerase activity, so this research select to target to science the P gene, in order to prevent the replication of the virus.
To clone the T7RNA polymerase gene, and construct the eukaryotic expression vector PCI-T7. Transfect the expression vector to the cell lines V7 by the method of Liposome transfection, thus constructing a V7 cell lines which express the T7 RNA polymerase. And detect the HA protein by Western blot and indirect immunofluorescence, which confirmed that the T7 RNA polymerase gene have expressed in VT7 cell lines stably and efficiently.
According to the Published P gene sequence of goose paramyxovirus, requirements of SiRNA expression vector and the selection principle of RNAi targeted sequences, selected the SiRNA sequences specific for goose paramyxovirus by BLAST tools, and design negative RNAi sites in the same time. According to the requirement of the designed target molecule and expression vector of RNAi, the target molecule was connected to the dedicated expression vector of RNAi which was after Enzyme cutting when it has annealed and purified, and be transfected into CEF and chicken embryo,which would be inoculate the virus after 6h. in order to observe the influence of RNAi to the GPMV NA-1 which was replicated and reproduced in CEF 36h post-infection, Real Time RT-PCR, detecting of virus titer, indirect immunofluorescence and cell disease had been used.And testing the inhibition of RNAi in CEF by hemagglutination test. The experimental result shows that, the two RNAi sites aiming at the P protein of GPMV NA-1 showed a strong inhibitory effect, and the inhibition efficiency of RNAi-P641 is stronger. What was proofed that the P gene is necessary to the replication of goose paramyxovirus virus.
DNAzyme is the DNA molecules with the function of RNA cutting, which was selecting from random sequence library composed in vitro, it's one of the hot spot in Gene silence research at present. By the bioinformatics having been used, this test design the DZ-P-358, DZ-P-446, DZ-P-702 three kinds of 10-23 DNAzyme aiming at GPMV NA-1 P protein, the cutting experiments in vitro has shown that the DZ-P-702, DZ-P 358 has cut activity, and DZ-P-702 has higher activity. It is proofed that DZ-P-702、DZ-P-358 can inhibit the replication of the goose paramyxovirus virus by observing the cell disease, detecting of virus titer, indirect immunofluorescence in CEF level; Test indicated that DZ-P-358 and DZ-P-702 may reduce the transcription level of P protein in CEF through the method of Real-time PCR, of which, the inhibition efficiency of DZ-P-702 may reach 78.5%. By determine the Blood clots titer of allantoic fluid, it proved that DZ may inhibit the proliferation of GPMV in CEF, and shown the application prospect of DNAzyme in production practice.
Summary:To have successfully constructed VT7 cell lines that can express T7 RNA polymerase steady, and forming the massy foundation for gaining the AMERVAC PRRS of Newcastle disease by reverse genetic manipulation; and have selected two interference sites which may inhibit the expression of GPMV P gene effectively.Screened out the specific DNAzyme DZ-P-702 that is aiming at the goose paramyxovirus P gene. And the screening and establishment of the effective targets provide the technical foundation for further study of antiviral therapy and resistance of disease breeding areas.
新城疫病毒的复制主要依靠核糖核蛋白聚合体(RNP),形成RNP的必要条件之一就是需要P蛋白,P蛋白和L蛋白的结合物具有RNA依赖RNA聚合酶活性,所以本研究选择靶向沉默P基因,进而抑制病毒的复制。
对T7RNA聚合酶基因进行克隆,构建真核表达载体PCI-T7。利用脂质体转染法将该载体转染到V7细胞系,构建了表达T7 RNA聚合酶的V7细胞系。并利用Western blot和间接免疫荧光对HA蛋白进行了检测,证实T7 RNA聚合酶基因在VT7细胞系中得到了稳定有效的表达。
根据鹅源副黏病毒NA-1株已发表的P基因序列,siRNA表达载体的要求和RNAi靶位基因序列选择原则,应用BLAST工具筛选出特异性地针对鹅源副黏病毒的siRNA序列,同时设计阴性RNAi靶位。根据所设计的RNAi的靶位分子和表达载体的要求,将RNAi靶位分子经过退火、纯化后连接到酶切后的RNAi专用表达载体上,进而转染鸡胚成纤维细胞(CEF)及鸡胚,6h后接种病毒。接毒后36h时分别用Real Time RT-PCR、病毒滴度测定、间接免疫荧光、细胞病变情况来观察RNAi对GPMV NA-1在CEF中复制和繁殖的影响情况,并用血凝试验检测RNAi在鸡胚中的抑制作用。实验结果显示,针对GPMV NA-1株P基因设计的2个RNAi靶位,在RNAi过程中表现出了很强的抑制效果,其中RNAi-P641的抑制效率更高。说明了P基因的这两个位点是鹅源副黏病毒复制所必需的。
脱氧核酶(Deoxyriozyme DNAzyme DZ)是一种通过对体外合成的随机序列库进行筛选得到的具有RNA切割功能的DNA分子,是目前基因沉默研究的热点之一。本试验应用生物信息学针对鹅源副黏病毒NA-1株P基因设计DZ-P-358、DZ-P-446、DZ-P-7023种10-23脱氧核酶,经体外切割实验筛选得到DZ-P-702、DZ-P-358具有切割活性,DZ-P-702活性较高。在CEF细胞水平通过细胞病变情况、病毒滴度测定、间接免疫荧光方法验证了DZ-P-702、DZ-P-358可以抑制鹅源副黏病毒在CEF细胞的复制；通过Real-time PCR方法检测表明DZ-P-358及DZ-P-702降低CEF细胞中P基因的转录水平,其中DZ-P-702抑制效率可达到78.5%。通过对尿囊液血凝效价的测定表明脱氧核酶可以有效抑制GPMV在鸡胚中的增殖,展现了脱氧核酶在生产实践中的应用前景。
综上所述,本研究成功构建稳定表达T7RNA聚合酶的VT7细胞系,为通过反向遗传学技术来获得新城疫的疫苗株奠定了坚实的基础；筛选出两个有效的抑制鹅副黏病毒P基因表达的干扰靶位。筛选出了针对鹅副黏病毒P基因特异的脱氧核酶DZ-P-702。有效靶位的筛选和确立,为进一步研究该病的抗病毒治疗及抗病育种领域提供了技术基础。
Goose paramyxovirus disease is a high mortality rate of infectious diseases caused by the goose paramyxovirus. The disease was susceptibility to all age of goose.Less 15-day-old goslings' morbidity and mortality can be as high as 100%. It was identified that the goose paramyxovirus is theⅠ-type serum virus, so the goose paramyxovirus virus may also called the Newcastle disease of goose. In recent years, this disease cause serious economic damage to the raising goose industry in China, threatening the healthy development of goose industry. The prevention of goose paramyxovirus mainly relied on the Attenuated vaccine, so the creating of new vaccine is one of the most important links to prevent and control the disease. It's a hot research that to gain the attenuated vaccine of Newcastle disease by reverse genetic manipulation, and one of the most important basic link is that it requires a cell lines to express T7RNA polym erase stably.
The replication of the Newcastle disease virus mainly depend on the RNP, and a necessary condition for the formation of RNP is the P protein needed, the complex of P protein and L protein have a RNA-dependent RNA polymerase activity, so this research select to target to science the P gene, in order to prevent the replication of the virus.
To clone the T7RNA polymerase gene, and construct the eukaryotic expression vector PCI-T7. Transfect the expression vector to the cell lines V7 by the method of Liposome transfection, thus constructing a V7 cell lines which express the T7 RNA polymerase. And detect the HA protein by Western blot and indirect immunofluorescence, which confirmed that the T7 RNA polymerase gene have expressed in VT7 cell lines stably and efficiently.
According to the Published P gene sequence of goose paramyxovirus, requirements of SiRNA expression vector and the selection principle of RNAi targeted sequences, selected the SiRNA sequences specific for goose paramyxovirus by BLAST tools, and design negative RNAi sites in the same time. According to the requirement of the designed target molecule and expression vector of RNAi, the target molecule was connected to the dedicated expression vector of RNAi which was after Enzyme cutting when it has annealed and purified, and be transfected into CEF and chicken embryo,which would be inoculate the virus after 6h. in order to observe the influence of RNAi to the GPMV NA-1 which was replicated and reproduced in CEF 36h post-infection, Real Time RT-PCR, detecting of virus titer, indirect immunofluorescence and cell disease had been used.And testing the inhibition of RNAi in CEF by hemagglutination test. The experimental result shows that, the two RNAi sites aiming at the P protein of GPMV NA-1 showed a strong inhibitory effect, and the inhibition efficiency of RNAi-P641 is stronger. What was proofed that the P gene is necessary to the replication of goose paramyxovirus virus.
DNAzyme is the DNA molecules with the function of RNA cutting, which was selecting from random sequence library composed in vitro, it's one of the hot spot in Gene silence research at present. By the bioinformatics having been used, this test design the DZ-P-358, DZ-P-446, DZ-P-702 three kinds of 10-23 DNAzyme aiming at GPMV NA-1 P protein, the cutting experiments in vitro has shown that the DZ-P-702, DZ-P 358 has cut activity, and DZ-P-702 has higher activity. It is proofed that DZ-P-702、DZ-P-358 can inhibit the replication of the goose paramyxovirus virus by observing the cell disease, detecting of virus titer, indirect immunofluorescence in CEF level; Test indicated that DZ-P-358 and DZ-P-702 may reduce the transcription level of P protein in CEF through the method of Real-time PCR, of which, the inhibition efficiency of DZ-P-702 may reach 78.5%. By determine the Blood clots titer of allantoic fluid, it proved that DZ may inhibit the proliferation of GPMV in CEF, and shown the application prospect of DNAzyme in production practice.
Summary:To have successfully constructed VT7 cell lines that can express T7 RNA polymerase steady, and forming the massy foundation for gaining the AMERVAC PRRS of Newcastle disease by reverse genetic manipulation; and have selected two interference sites which may inhibit the expression of GPMV P gene effectively.Screened out the specific DNAzyme DZ-P-702 that is aiming at the goose paramyxovirus P gene. And the screening and establishment of the effective targets provide the technical foundation for further study of antiviral therapy and resistance of disease breeding areas.
引文
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