鸡痘病毒282E4株基因组和蛋白质组解析、载体构建与HIV重组疫苗研究
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摘要
鸡痘病毒(Fowlpox virus, FPV)是脊椎动物痘病毒科、禽痘病毒属的代表成员,该病毒仅感染禽类,可引起禽类动物的疾病。因禽痘病毒通常不感染人与其他哺乳动物,且能有效表达大量的外源DNA,并可在感染细胞的胞浆中复制等特点而成为活病毒载体研究的热点。然而,目前对FPV的基因组、结构、毒力、致病机制、宿主范围及其免疫逃逸等方面尚无系统深入的研究。为设计出更加安全、有效的FPV表达载体系统,本研究以FPV282E4株为研究对象,开展其基因组背景分析、蛋白质组成、新型表达载体构建及重组鸡痘病毒载体疫苗筛选和实验免疫研究。
本研究首先采用高通量Illumina测序技术对中国弱毒疫苗株FPV282E4株进行全基因组测序,通过对测序数据组装后利用基因预测软件进行基因de novo预测及功能注释。结果测定FPV282E4全基因组为308829bp,GC含量约为28%,预测出313个编码序列(Coding sequence, CDS),并与FPVUS株、CNPV ATCC株进行核酸平等共线性分析,通过功能注释表明FPV282E4的基因具有核营酸代谢、运输、转录、复制、重组、修复及翻译后修饰、伴侣蛋白和信号转导功能。在此基础上,采用LC-ESI-MS/MS对鸡痘病毒282E4株进行蛋白质全谱分析,鉴定出76个蛋白,对其进行功能注释,结果与基因组序列相互印证,从而为重组鸡痘病毒载体构建及感染与致病机制研究奠定理论基础。
基于FPV282E4全基因组序列测定数据,针对本实验室前期在鸡痘病毒载体构建中遇到的重组效率较低、筛选周期长等问题,本研究优化FPV282E4株5个非必需CDS区作为候选同源重组臂,通过插入含有PE/L早／晚期启动子和终止信号的三基因表达盒,进行重组鸡痘病毒载体的构建,分别以红、绿、蓝三种荧光蛋白基因作为报告基因,进行病毒重组及外源基因表达活性的验证。荧光蛋白表达检测结果表明,三个表达盒均可独立表达外源基因；通过对rFPV进行形态学、生物特性检测,结果成功构建出3个rFPV,且基因缺失未影响重组鸡痘病毒的形态及其复制,3株重组鸡痘病毒无差异。
在成功获得表达载体的基础上,以本实验室前期构建的包含HIV-1复合多表位(MEGNp24)、CpG基序和CTB基因(CCMp24)的免疫原为基础,构建表达HIV-1复合多表位的rFPV-CCMP24,通过荧光噬斑筛选纯化rFPV-CCMP24。采用PCR、RT-PCR、Western Blot方法进行重组病毒及外源基因表达的鉴定。成功获得rFPV-CCMp24,重组病毒复制效率及病毒滴度与野生株FPV一致,重组病毒能够在体外表达外源基因且具有反应原性。然后开展了rFPV-CCMp24的小鼠免疫试验,分析其免疫原性,同时结合前期构建的重组痘苗病毒rddVTT-CCMp24进行联合免疫研究,通过检测特异性HIV-1抗体、淋巴细胞刺激指数、淋巴细胞分泌IFNy以及流式细胞术检测CD3+CD4+、CD3+CD8+等指标,综合分析免疫效果,探寻有效的病毒载体为基础的免疫策略。结果显示,rFPV-CCMp24单独免疫及与rddVTT-CCMp24联合免疫,均能诱导细胞免疫及体液免疫。本研究为研制有效鸡痘病毒活载体疫苗及多载体疫苗联合免疫的临床前实验研究奠定了基础。
Fowlpox virus (FPV) is a representative type of the avipoxvirus genus of the Poxviridae. It could only infect poultry and lead to disease. Fowlpox virus can not infect humans and other mammals, but it can express exogenous DNA effectively, and it replicates in the cytoplasm of infected cell, the study about fowlpox as vector become the current hot spot. In order to design a safer and more effective FPV expression vector system, an in-depth understanding of FPV virulence, pathogenic mechanism, host range and immune escape is needed. In this study, the FPV282E4was constructed as vector to express HIV-1multi-epitope to do background analyze of genome and protein composition, construction of neotype expression vector, vaccine screening of recombinant fowlpox virus and experimental immune research.
In this study, Illumina high-throughput sequencing technologies were used to sequence the whole genome of FPV282E4strains. The sequences data were assembled, predicted and annotated using the software for de novo prediction and functional annotation after assembly the sequencing data. The results showed that the whole genome of FPV282E4was308826bp, the GC%content was around28%, and313Coding sequence (CDS) were predicted and carry out the equality synteny analysis of the nucleic acid of FPV282E4, FWPV and CNPV. Analysis of functional annotation indicates that FPV has the function of nucleotide metabolism, transport, transcription, replication, recombination and repair, and modification after translation, partner and signal transduction which lay a theoretical foundation for the further study of the fowlpox virus. Based on the results, we did full spectrum proteomic analysis of FPV282E4by LC/ESI-MS/MS, and we have identified76proteins and proceeded functional annotation which showed a results that were similar to the whole genome sequencing. The results provided basis research for FPV infection and pathogenic mechanism.
Based on the FPV282E4whole-genome sequencing data and the problems such as low recombinant efficiency and long screen cycle that occurred during construction of fowlpox virus, we have chosen5CDSs zone of recombinant non-essential genes as homologous recombination arm to construct the fowlpox virus vector through inserting three gene-expression-box which contains PE/L early/late promoter and termination signal; and then made red, green and blue fluorescent protein to be the report gene to validate to expression activity of recombinant virus and exogenous gene. The result of fluorescent protein expression showed that three express boxes can express exogenous genes independently; and through the detection of morphology and biological characteristics, we have constructed three rFPV successfully of which the gene deletion has no impact on the morphous and replication of recombinant fowlpox virus and there was no difference among of three rFPV.
Base on the well abtain of expression vector, and the construction of immunogen of HIV-1multi-epitope(MEGNp24), CpG motif and CTB genes (CCMp24), we got an rFPV-CCMp24through plaques screening and purification, and then, the recombinant virus was identified by PCR, RT-PCR and WB methods. Results showed that the rFPV-CCMp24was successful constructed, and replication efficiency of recombinant virus was concordance with wild stain FPV, it could express exogenous genes in vitro and possess reactionogenicity. Then we proceeded combined immunization in mice with the rFPV-CCMp24and the recombinant Fowlpox virus rddVTT-CCMp24to analyze the immunogenicity, then comprehensively analyze the immune effect through detecting the specific HIV-1antibodies, lymphocyte stimulation indices, lymphoid cells secrete IFN gamma and CD3+/CD8+, CD4+/CD8+to retrieval effective immunization strategy based on virus vector. The results showed that immunization in mice with rFPV-CCMp24only and with combination of rFPV-CCMp24and rddVTT-CCMp24would show the advantage in induction of cellular and humoral immunity. This study lays the foundation for the further study of rFPV live vector vaccine development and the pre-clinical experiment.
本研究首先采用高通量Illumina测序技术对中国弱毒疫苗株FPV282E4株进行全基因组测序,通过对测序数据组装后利用基因预测软件进行基因de novo预测及功能注释。结果测定FPV282E4全基因组为308829bp,GC含量约为28%,预测出313个编码序列(Coding sequence, CDS),并与FPVUS株、CNPV ATCC株进行核酸平等共线性分析,通过功能注释表明FPV282E4的基因具有核营酸代谢、运输、转录、复制、重组、修复及翻译后修饰、伴侣蛋白和信号转导功能。在此基础上,采用LC-ESI-MS/MS对鸡痘病毒282E4株进行蛋白质全谱分析,鉴定出76个蛋白,对其进行功能注释,结果与基因组序列相互印证,从而为重组鸡痘病毒载体构建及感染与致病机制研究奠定理论基础。
基于FPV282E4全基因组序列测定数据,针对本实验室前期在鸡痘病毒载体构建中遇到的重组效率较低、筛选周期长等问题,本研究优化FPV282E4株5个非必需CDS区作为候选同源重组臂,通过插入含有PE/L早／晚期启动子和终止信号的三基因表达盒,进行重组鸡痘病毒载体的构建,分别以红、绿、蓝三种荧光蛋白基因作为报告基因,进行病毒重组及外源基因表达活性的验证。荧光蛋白表达检测结果表明,三个表达盒均可独立表达外源基因；通过对rFPV进行形态学、生物特性检测,结果成功构建出3个rFPV,且基因缺失未影响重组鸡痘病毒的形态及其复制,3株重组鸡痘病毒无差异。
在成功获得表达载体的基础上,以本实验室前期构建的包含HIV-1复合多表位(MEGNp24)、CpG基序和CTB基因(CCMp24)的免疫原为基础,构建表达HIV-1复合多表位的rFPV-CCMP24,通过荧光噬斑筛选纯化rFPV-CCMP24。采用PCR、RT-PCR、Western Blot方法进行重组病毒及外源基因表达的鉴定。成功获得rFPV-CCMp24,重组病毒复制效率及病毒滴度与野生株FPV一致,重组病毒能够在体外表达外源基因且具有反应原性。然后开展了rFPV-CCMp24的小鼠免疫试验,分析其免疫原性,同时结合前期构建的重组痘苗病毒rddVTT-CCMp24进行联合免疫研究,通过检测特异性HIV-1抗体、淋巴细胞刺激指数、淋巴细胞分泌IFNy以及流式细胞术检测CD3+CD4+、CD3+CD8+等指标,综合分析免疫效果,探寻有效的病毒载体为基础的免疫策略。结果显示,rFPV-CCMp24单独免疫及与rddVTT-CCMp24联合免疫,均能诱导细胞免疫及体液免疫。本研究为研制有效鸡痘病毒活载体疫苗及多载体疫苗联合免疫的临床前实验研究奠定了基础。
Fowlpox virus (FPV) is a representative type of the avipoxvirus genus of the Poxviridae. It could only infect poultry and lead to disease. Fowlpox virus can not infect humans and other mammals, but it can express exogenous DNA effectively, and it replicates in the cytoplasm of infected cell, the study about fowlpox as vector become the current hot spot. In order to design a safer and more effective FPV expression vector system, an in-depth understanding of FPV virulence, pathogenic mechanism, host range and immune escape is needed. In this study, the FPV282E4was constructed as vector to express HIV-1multi-epitope to do background analyze of genome and protein composition, construction of neotype expression vector, vaccine screening of recombinant fowlpox virus and experimental immune research.
In this study, Illumina high-throughput sequencing technologies were used to sequence the whole genome of FPV282E4strains. The sequences data were assembled, predicted and annotated using the software for de novo prediction and functional annotation after assembly the sequencing data. The results showed that the whole genome of FPV282E4was308826bp, the GC%content was around28%, and313Coding sequence (CDS) were predicted and carry out the equality synteny analysis of the nucleic acid of FPV282E4, FWPV and CNPV. Analysis of functional annotation indicates that FPV has the function of nucleotide metabolism, transport, transcription, replication, recombination and repair, and modification after translation, partner and signal transduction which lay a theoretical foundation for the further study of the fowlpox virus. Based on the results, we did full spectrum proteomic analysis of FPV282E4by LC/ESI-MS/MS, and we have identified76proteins and proceeded functional annotation which showed a results that were similar to the whole genome sequencing. The results provided basis research for FPV infection and pathogenic mechanism.
Based on the FPV282E4whole-genome sequencing data and the problems such as low recombinant efficiency and long screen cycle that occurred during construction of fowlpox virus, we have chosen5CDSs zone of recombinant non-essential genes as homologous recombination arm to construct the fowlpox virus vector through inserting three gene-expression-box which contains PE/L early/late promoter and termination signal; and then made red, green and blue fluorescent protein to be the report gene to validate to expression activity of recombinant virus and exogenous gene. The result of fluorescent protein expression showed that three express boxes can express exogenous genes independently; and through the detection of morphology and biological characteristics, we have constructed three rFPV successfully of which the gene deletion has no impact on the morphous and replication of recombinant fowlpox virus and there was no difference among of three rFPV.
Base on the well abtain of expression vector, and the construction of immunogen of HIV-1multi-epitope(MEGNp24), CpG motif and CTB genes (CCMp24), we got an rFPV-CCMp24through plaques screening and purification, and then, the recombinant virus was identified by PCR, RT-PCR and WB methods. Results showed that the rFPV-CCMp24was successful constructed, and replication efficiency of recombinant virus was concordance with wild stain FPV, it could express exogenous genes in vitro and possess reactionogenicity. Then we proceeded combined immunization in mice with the rFPV-CCMp24and the recombinant Fowlpox virus rddVTT-CCMp24to analyze the immunogenicity, then comprehensively analyze the immune effect through detecting the specific HIV-1antibodies, lymphocyte stimulation indices, lymphoid cells secrete IFN gamma and CD3+/CD8+, CD4+/CD8+to retrieval effective immunization strategy based on virus vector. The results showed that immunization in mice with rFPV-CCMp24only and with combination of rFPV-CCMp24and rddVTT-CCMp24would show the advantage in induction of cellular and humoral immunity. This study lays the foundation for the further study of rFPV live vector vaccine development and the pre-clinical experiment.
引文
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