中国特有小型猪内源性反转录病毒的检测与特性分析
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摘要
由于人源供体器官的短缺,人们希望借助异种移植来获得大量的组织/器官移植物,猪被选定为最合适的异种移植供体。然而,猪内源性反转录病毒(Porcine endogenous retrovirus, PERV)在体外能够感染多种人源细胞的发现,引起了人们对异种移植病原安全性的广泛关注,即带有PERV的猪细胞/组织/器官一旦植入处于高度免疫抑制的病人体内,是否可能突破种间屏障造成人源大流行?国际上要求对人类异种移植建立远期评估的呼声越来越高,有关PERV生物学特性的研究及对PERV的病原安全性进行评价成为一个新的热点。
我国丰富的小型猪种资源有望为猪→人异种移植提供优良供体,目前已经开展异种移植的相关研究,人用转基因猪皮肤已经进入产业化阶段,但对PERV的研究尚未引起充分的重视。为了开发利用我国小型猪种资源,为异种器官移植受体提供安全可靠的供体器官,我们在前期研究的基础上,有必要深入开展PERV相关研究。一方面进一步了解我国小型猪种群中PERV的存在状况,为筛选PERV低拷贝猪奠定基础;另一方面研究我国小型猪内源性释放毒株的生物学特性,并探索嗜人性PERV感染人源细胞后产生的分子效应,为揭示PERV的潜在病理作用及进行异种移植的病原安全性评价打下基础。为此本研究主要进行了以下工作:
(1)中国特有小型猪内源性反转录病毒拷贝数的检测
设计并合成了检测PERV pol基因的引物,运用实时荧光定量PCR方法对中国特有小型猪基因组中整合的PERV拷贝数进行了系统检测,结果表明,五指山猪与巴马小型猪基因组中均可检测到pol基因的存在,五指山猪单细胞基因组中PERV的拷贝数为1.38±0.33至14.23±3.31之间,巴马小型猪单细胞基因组中PERV的拷贝数为2.96±0.76至58.46±3.50之间。统计学分析表明五指山猪与巴马小型猪单细胞基因组中PERV的拷贝数具有显著性差异,五指山猪基因组中整合的PERV拷贝数较低。
五指山猪基因组中PERV的基因序列负荷较低,因此有希望通过选择性育种获得PERV低拷贝猪,在此基础上则易于进一步筛选出无完整PERV前病毒的小型猪个体,也易于通过基因敲除去除PERV,从而为异种移植提供合适供体。此外,基于SYBR Green I的PERV拷贝数的定量PCR检测方法,与Southern Blot检测拷贝数的方法相比具有明显的优越性,简便易行,可在短时间内检测大量样品,费用低廉,利于对猪群进行大规模筛选,既可用于实验用猪的病原安全性评价,也可用于异种移植后PERV存在与表达状况的监测。
(2)五指山猪内源性反转录病毒前病毒全基因克隆与序列分析
应用PCR技术将五指山猪内源性反转录病毒前病毒全基因分为两段进行扩增,然后构建前病毒全基因克隆,运用生物信息学方法对其序列进行分析,并与Genbank中收录的其他PERV全基因序列进行比对,在此基础上还进行了进化分析,构建了基于Env氨基酸序列的进化树。结果表明,五指山猪来源的PERV前病毒全长为8899bp,含有完整的gag、pol、env的ORF,两端为5’LTR与3’LTR两个长末端重复序列。序列比对分析发现PERV-WZSP与其他来源的PERV毒株高度相似但仍具有一定的差异性。进化分析结果表明PERV-WZSP与其他A亚型毒株属于同一分支,这与PCR分型鉴定的结果也相一致。此外,我们还发现该株PERV前病毒的5’LTR中有多个U3重复盒的存在,这可能会导致PERV-WZSP在宿主细胞中复制能力的增强;3’LTR中含有PERV-C亚型中的特异性序列,这将可能导致部分的PERV-C序列与PERV-A序列发生重组。然而,我们还需要进行更深入的研究来对这种推测进行验证。
本研究有助于深入了解五指山猪内源性反转录病毒的分子生物学特性,并为下一步构建PERV感染性克隆奠定了良好基础。
(3)嗜人性PERV感染人源细胞模型的构建与鉴定
为了研究嗜人性PERV感染人源细胞后的分子效应并探索其可能的病理作用,我们首先构建并筛选具有较高反转录酶活性的PERV细胞感染模型。采用细胞共培养的方法,将五指山猪PBMC与HEK293细胞共培养24h后除去PBMC,建立五指山猪来源PERV的体外细胞感染模型。共培养后的HEK293细胞在形态、生长特性及折光性上与阴性对照HEK293细胞没有明显差异。
为证实共培养后的HEK293细胞感染了PERV,采用了PCR方法检测共培养后HEK293细胞中PERV结构基因gag、pol、env的存在,采用RT-PCR检测gag、pol、env的mRNA的表达,采用Western Blot方法鉴定Gag蛋白的表达,进一步采用反转录酶活性检测实验筛选具有较高反转录酶活性的嗜人性PERV的人源细胞,结果表明共培养后第49天左右时反转录酶活性较高,于是提取此时的细胞总蛋白,用于下一步蛋白质组学分析。(4)嗜人性PERV感染人源细胞后的蛋白质组学分析PERV感染人源细胞后,细胞并不产生任何形态学的改变或生长状态的改变,为了揭示嗜人性PERV感染人源细胞后究竟产生了怎样的分子效应,并探索其是否具有与其他γ群反转录病毒类似的致病性,我们运用比较蛋白质组学方法从全细胞水平上对嗜人性PERV感染人源细胞后的蛋白质表达的变化进行研究。
通过2-DE比较分析感染PERV的HEK293细胞与阴性对照HEK293细胞的总蛋白谱,采用高解析离子淌度质谱分析鉴定差异表达的蛋白,鉴定出10种差异表达蛋白,与对照相比,其中6个为上调蛋白,4个为下调蛋白。通过实时定量RT-PCR与Western Blot验证,证实差异表达结果可靠。这些差异蛋白参与信号转导、细胞凋亡、蛋白合成等生命过程,其中有数种蛋白与肿瘤的发生发展有关。该研究结果有望为深入研究嗜人性PERV与人源细胞的相互作用及PERV感染后的分子效应等提供一定的线索,也提示了PERV感染后可能会对细胞的生长、生理功能等造成影响,甚至存在潜在的致病性。本研究为研究PERV感染人源细胞后潜在的分子效应奠定了基础,对于异种移植中PERV的病毒安全性评价具有重要意义。
Xenotransplantation is considered to be a promising approach to alleviate the shortage of human donor organs. Pigs are selected as the most suitable animals. However, concerns regarding the microbial safety of xenotransplantation were raised when PERVs were shown to infect human cells in vitro. It is feared that xenotransplantation of porcine cells/tissue/organs to immunosupressed patients might cause cross-species infections of PERV and in a worst case scenario, the pandemic in the human community. It is necessary to establish long-term appraisal for the risk posed by PERV in xenotransplatation. Studies on PERV biology and potential for pathogenicity to increase our understanding of the risk in pig to human xenotransplantation have become a new research focus.
Chinese miniature pigs are the potential organ donors for pig-to-human xenotransplantation. Studies on xenotransplantation have been carried out and transgenic porcine skin for pig to human xenotransplantation is in industrialization. However, so far, in China, an adequate level of information on PERV from Chinese miniature pigs has not been available. To expore the resourses of Chinese miniature pigs and to provide safe organ donors for xenotransplantation, it’s essential to do further studies on PERV on the basis of our previous studies. In this study, we intended to estimate copy numbers of PERV integrated in the host genome. This will be helpful for screening donor pigs with few copies of PERV and be beneficial for pig to human xenotransplantation. Besides, we intended to do studies on the characteristics of PERV from Chinese miniature pigs and tried to learn the molecular effect of human-tropic PERV on human cells. These studies will be helpful for revealing PERV potential pathogenesis and for the safety appraisal in xenotransplatation. The studies include the following:
(1) Detection of copy numbers of porcine endogenous retrovirus from Chinese miniature
The oligonucleotide primers used for real-time amplification of PERV were designed from the polymerase (pol) gene. Real-time PCR was performed to detect the copy numbers of PERV integrated in the host genome.The copy numbers of PERV from WZSP genomic DNA was in the range of 1.38±0.33 to 14.23±3.31, while the copy numbers of PERV from BMP genomic DNA was in the range of 2.96±0.76 to 58.46±3.50 (Table 3). The statistical analysis showed significant differences between these two breeds (P<0.01; Wilcoxon 2-Sample Test). The copy numbers of PERV from WZSP genomic DNA was less than that from BMP genomic DNA.
The PERV sequence load was lower in the genome of WZSP. Thus WZSP with few copies of PERV could probably be screened by selecting breeding. Then miniature pigs without intact PERV proviruses could be screened and PERV might be knocked out. Thus the elimination of PERV could be achieved. Therefore, compared with other breeds, WZSP has the potential to be a favorable choice for xenotransplantation. In addition, compared with Southern Blot, the SYBR Green I-based real-time quantitative PCR assay described here provide a simple and rapid method for estimating copy numbers of PERV integrated in the genome and could be applied to do large-scale screening. It could be an excellent tool for screening donor pig and also be helpful for evidence of PERV transmission in clinical samples from human subjects treated with porcine xenotransplantation products.
(2) Cloning and characterization of full-length proviral DNA of PERV from WZSP
PERV provirus was amplified in two overlapping halves. 5’and 3’fragments were isolated, subcloned and fused to generate full-length proviral DNA using the unique restriction site within the overlap between 5’and 3’halves. Full-length nucleotide sequences of PERV-WZSP and other PERVs were aligned and phylogenetic tree was constructed from deduced amino acid sequences of env. The results indicated that the sequence of PERV-WZSP was 8,899 bp long. It had intact gag, pol, env ORFs flanking by two LTRs. Sequence alignment showed that PERV-WZSP had high similarities with PERV of other origins but differences still existed. Phylogenetic analysis indicated that PERV-WZSP belongs to the same branch with other PERV-A strains, which is consistent with the result of subtyping assay. In addition, the U3 region in 5’LTR contained three 39bp repeats which suggested that PERV of Chinese origin probably have potential capacity for increased replication in host cells. PERV-C specific sequence exited in 3’LTR of PERV-WZSP, thus suggested that partial PERV-C sequence may recombine with PERV-A sequence. Nevertheless, this implication need further study on characterization of LTR of PERV provirus from Chinese Wuzhishan miniature pigs inbred.
This study is helpful for further understanding the characteristics of PERV-WZSP and also important for our further study of constructing infectious clones of PERV-WZSP.
(3) Construction and identification of human cell model infected with human-tropic PERV
To study the molecular effect of human-tropic PERV on human cells and explore its potential pathogenicity, we constructed human cell model infected with human-tropic PERV and screened the cells with higher RT activity. PBMCs from WZSP and HEK293 cell line were co-cultured for 24h, then PBMCs were eliminated. There was no obvious difference in the appearance, growth characteristics and refraction between HEK293 cells post coculture and HEK293 cells as control. Several assays were done to demonstrate that HEK293 cells were infected by human-tropic PERV post coculture.
PCR was applied to detect the structural genes including gag, pol and env. RT-PCR was done to detect the expression of mRNA of gag, pol and env. Western Blot was done to detect Gag protein. Then RT activity assay was done to screen the infected cells with higher RT activity. The result indicated that RT activity reached the peak around the 49th day post coculture. Thus total protein of the cells around this day was prepared for the following analysis.
(4) Proteomic analysis of human cells infected with human-tropic PERV Human cells didn’t show any difference in cell morphology and growth after infection by PERV. In order to reveal the molecular effect of human-tropic PERV on human cells, and to explore the potential pathogenic effects of human-tropic PERV which may be similar with other C-type retroviruses, proteomic analysis was done to study the differences of protein profile after infection of human cells by human-tropic PERV.
The protein profiles of infected HEK293 cells and uninfected control were compared and analyzed by 2-DE. Ten differentially expressed proteins were identified by HDMS, including 6 upregulated proteins and 4 downregulated proteins. Real time RT-PCR and Western Blot were applied to confirm change of these proteins at the mRNA level and protein level, respectively. These differentially expressed proteins are closely related to signal transduction, cell apoptosis and protein synthesis. These identified proteins provide a number of clues and potential links to understanding the molecular effect of the infection by human-tropic PERV. This study will facilitate the appraisal of PERV in xenotransplantation.
我国丰富的小型猪种资源有望为猪→人异种移植提供优良供体,目前已经开展异种移植的相关研究,人用转基因猪皮肤已经进入产业化阶段,但对PERV的研究尚未引起充分的重视。为了开发利用我国小型猪种资源,为异种器官移植受体提供安全可靠的供体器官,我们在前期研究的基础上,有必要深入开展PERV相关研究。一方面进一步了解我国小型猪种群中PERV的存在状况,为筛选PERV低拷贝猪奠定基础;另一方面研究我国小型猪内源性释放毒株的生物学特性,并探索嗜人性PERV感染人源细胞后产生的分子效应,为揭示PERV的潜在病理作用及进行异种移植的病原安全性评价打下基础。为此本研究主要进行了以下工作:
(1)中国特有小型猪内源性反转录病毒拷贝数的检测
设计并合成了检测PERV pol基因的引物,运用实时荧光定量PCR方法对中国特有小型猪基因组中整合的PERV拷贝数进行了系统检测,结果表明,五指山猪与巴马小型猪基因组中均可检测到pol基因的存在,五指山猪单细胞基因组中PERV的拷贝数为1.38±0.33至14.23±3.31之间,巴马小型猪单细胞基因组中PERV的拷贝数为2.96±0.76至58.46±3.50之间。统计学分析表明五指山猪与巴马小型猪单细胞基因组中PERV的拷贝数具有显著性差异,五指山猪基因组中整合的PERV拷贝数较低。
五指山猪基因组中PERV的基因序列负荷较低,因此有希望通过选择性育种获得PERV低拷贝猪,在此基础上则易于进一步筛选出无完整PERV前病毒的小型猪个体,也易于通过基因敲除去除PERV,从而为异种移植提供合适供体。此外,基于SYBR Green I的PERV拷贝数的定量PCR检测方法,与Southern Blot检测拷贝数的方法相比具有明显的优越性,简便易行,可在短时间内检测大量样品,费用低廉,利于对猪群进行大规模筛选,既可用于实验用猪的病原安全性评价,也可用于异种移植后PERV存在与表达状况的监测。
(2)五指山猪内源性反转录病毒前病毒全基因克隆与序列分析
应用PCR技术将五指山猪内源性反转录病毒前病毒全基因分为两段进行扩增,然后构建前病毒全基因克隆,运用生物信息学方法对其序列进行分析,并与Genbank中收录的其他PERV全基因序列进行比对,在此基础上还进行了进化分析,构建了基于Env氨基酸序列的进化树。结果表明,五指山猪来源的PERV前病毒全长为8899bp,含有完整的gag、pol、env的ORF,两端为5’LTR与3’LTR两个长末端重复序列。序列比对分析发现PERV-WZSP与其他来源的PERV毒株高度相似但仍具有一定的差异性。进化分析结果表明PERV-WZSP与其他A亚型毒株属于同一分支,这与PCR分型鉴定的结果也相一致。此外,我们还发现该株PERV前病毒的5’LTR中有多个U3重复盒的存在,这可能会导致PERV-WZSP在宿主细胞中复制能力的增强;3’LTR中含有PERV-C亚型中的特异性序列,这将可能导致部分的PERV-C序列与PERV-A序列发生重组。然而,我们还需要进行更深入的研究来对这种推测进行验证。
本研究有助于深入了解五指山猪内源性反转录病毒的分子生物学特性,并为下一步构建PERV感染性克隆奠定了良好基础。
(3)嗜人性PERV感染人源细胞模型的构建与鉴定
为了研究嗜人性PERV感染人源细胞后的分子效应并探索其可能的病理作用,我们首先构建并筛选具有较高反转录酶活性的PERV细胞感染模型。采用细胞共培养的方法,将五指山猪PBMC与HEK293细胞共培养24h后除去PBMC,建立五指山猪来源PERV的体外细胞感染模型。共培养后的HEK293细胞在形态、生长特性及折光性上与阴性对照HEK293细胞没有明显差异。
为证实共培养后的HEK293细胞感染了PERV,采用了PCR方法检测共培养后HEK293细胞中PERV结构基因gag、pol、env的存在,采用RT-PCR检测gag、pol、env的mRNA的表达,采用Western Blot方法鉴定Gag蛋白的表达,进一步采用反转录酶活性检测实验筛选具有较高反转录酶活性的嗜人性PERV的人源细胞,结果表明共培养后第49天左右时反转录酶活性较高,于是提取此时的细胞总蛋白,用于下一步蛋白质组学分析。(4)嗜人性PERV感染人源细胞后的蛋白质组学分析PERV感染人源细胞后,细胞并不产生任何形态学的改变或生长状态的改变,为了揭示嗜人性PERV感染人源细胞后究竟产生了怎样的分子效应,并探索其是否具有与其他γ群反转录病毒类似的致病性,我们运用比较蛋白质组学方法从全细胞水平上对嗜人性PERV感染人源细胞后的蛋白质表达的变化进行研究。
通过2-DE比较分析感染PERV的HEK293细胞与阴性对照HEK293细胞的总蛋白谱,采用高解析离子淌度质谱分析鉴定差异表达的蛋白,鉴定出10种差异表达蛋白,与对照相比,其中6个为上调蛋白,4个为下调蛋白。通过实时定量RT-PCR与Western Blot验证,证实差异表达结果可靠。这些差异蛋白参与信号转导、细胞凋亡、蛋白合成等生命过程,其中有数种蛋白与肿瘤的发生发展有关。该研究结果有望为深入研究嗜人性PERV与人源细胞的相互作用及PERV感染后的分子效应等提供一定的线索,也提示了PERV感染后可能会对细胞的生长、生理功能等造成影响,甚至存在潜在的致病性。本研究为研究PERV感染人源细胞后潜在的分子效应奠定了基础,对于异种移植中PERV的病毒安全性评价具有重要意义。
Xenotransplantation is considered to be a promising approach to alleviate the shortage of human donor organs. Pigs are selected as the most suitable animals. However, concerns regarding the microbial safety of xenotransplantation were raised when PERVs were shown to infect human cells in vitro. It is feared that xenotransplantation of porcine cells/tissue/organs to immunosupressed patients might cause cross-species infections of PERV and in a worst case scenario, the pandemic in the human community. It is necessary to establish long-term appraisal for the risk posed by PERV in xenotransplatation. Studies on PERV biology and potential for pathogenicity to increase our understanding of the risk in pig to human xenotransplantation have become a new research focus.
Chinese miniature pigs are the potential organ donors for pig-to-human xenotransplantation. Studies on xenotransplantation have been carried out and transgenic porcine skin for pig to human xenotransplantation is in industrialization. However, so far, in China, an adequate level of information on PERV from Chinese miniature pigs has not been available. To expore the resourses of Chinese miniature pigs and to provide safe organ donors for xenotransplantation, it’s essential to do further studies on PERV on the basis of our previous studies. In this study, we intended to estimate copy numbers of PERV integrated in the host genome. This will be helpful for screening donor pigs with few copies of PERV and be beneficial for pig to human xenotransplantation. Besides, we intended to do studies on the characteristics of PERV from Chinese miniature pigs and tried to learn the molecular effect of human-tropic PERV on human cells. These studies will be helpful for revealing PERV potential pathogenesis and for the safety appraisal in xenotransplatation. The studies include the following:
(1) Detection of copy numbers of porcine endogenous retrovirus from Chinese miniature
The oligonucleotide primers used for real-time amplification of PERV were designed from the polymerase (pol) gene. Real-time PCR was performed to detect the copy numbers of PERV integrated in the host genome.The copy numbers of PERV from WZSP genomic DNA was in the range of 1.38±0.33 to 14.23±3.31, while the copy numbers of PERV from BMP genomic DNA was in the range of 2.96±0.76 to 58.46±3.50 (Table 3). The statistical analysis showed significant differences between these two breeds (P<0.01; Wilcoxon 2-Sample Test). The copy numbers of PERV from WZSP genomic DNA was less than that from BMP genomic DNA.
The PERV sequence load was lower in the genome of WZSP. Thus WZSP with few copies of PERV could probably be screened by selecting breeding. Then miniature pigs without intact PERV proviruses could be screened and PERV might be knocked out. Thus the elimination of PERV could be achieved. Therefore, compared with other breeds, WZSP has the potential to be a favorable choice for xenotransplantation. In addition, compared with Southern Blot, the SYBR Green I-based real-time quantitative PCR assay described here provide a simple and rapid method for estimating copy numbers of PERV integrated in the genome and could be applied to do large-scale screening. It could be an excellent tool for screening donor pig and also be helpful for evidence of PERV transmission in clinical samples from human subjects treated with porcine xenotransplantation products.
(2) Cloning and characterization of full-length proviral DNA of PERV from WZSP
PERV provirus was amplified in two overlapping halves. 5’and 3’fragments were isolated, subcloned and fused to generate full-length proviral DNA using the unique restriction site within the overlap between 5’and 3’halves. Full-length nucleotide sequences of PERV-WZSP and other PERVs were aligned and phylogenetic tree was constructed from deduced amino acid sequences of env. The results indicated that the sequence of PERV-WZSP was 8,899 bp long. It had intact gag, pol, env ORFs flanking by two LTRs. Sequence alignment showed that PERV-WZSP had high similarities with PERV of other origins but differences still existed. Phylogenetic analysis indicated that PERV-WZSP belongs to the same branch with other PERV-A strains, which is consistent with the result of subtyping assay. In addition, the U3 region in 5’LTR contained three 39bp repeats which suggested that PERV of Chinese origin probably have potential capacity for increased replication in host cells. PERV-C specific sequence exited in 3’LTR of PERV-WZSP, thus suggested that partial PERV-C sequence may recombine with PERV-A sequence. Nevertheless, this implication need further study on characterization of LTR of PERV provirus from Chinese Wuzhishan miniature pigs inbred.
This study is helpful for further understanding the characteristics of PERV-WZSP and also important for our further study of constructing infectious clones of PERV-WZSP.
(3) Construction and identification of human cell model infected with human-tropic PERV
To study the molecular effect of human-tropic PERV on human cells and explore its potential pathogenicity, we constructed human cell model infected with human-tropic PERV and screened the cells with higher RT activity. PBMCs from WZSP and HEK293 cell line were co-cultured for 24h, then PBMCs were eliminated. There was no obvious difference in the appearance, growth characteristics and refraction between HEK293 cells post coculture and HEK293 cells as control. Several assays were done to demonstrate that HEK293 cells were infected by human-tropic PERV post coculture.
PCR was applied to detect the structural genes including gag, pol and env. RT-PCR was done to detect the expression of mRNA of gag, pol and env. Western Blot was done to detect Gag protein. Then RT activity assay was done to screen the infected cells with higher RT activity. The result indicated that RT activity reached the peak around the 49th day post coculture. Thus total protein of the cells around this day was prepared for the following analysis.
(4) Proteomic analysis of human cells infected with human-tropic PERV Human cells didn’t show any difference in cell morphology and growth after infection by PERV. In order to reveal the molecular effect of human-tropic PERV on human cells, and to explore the potential pathogenic effects of human-tropic PERV which may be similar with other C-type retroviruses, proteomic analysis was done to study the differences of protein profile after infection of human cells by human-tropic PERV.
The protein profiles of infected HEK293 cells and uninfected control were compared and analyzed by 2-DE. Ten differentially expressed proteins were identified by HDMS, including 6 upregulated proteins and 4 downregulated proteins. Real time RT-PCR and Western Blot were applied to confirm change of these proteins at the mRNA level and protein level, respectively. These differentially expressed proteins are closely related to signal transduction, cell apoptosis and protein synthesis. These identified proteins provide a number of clues and potential links to understanding the molecular effect of the infection by human-tropic PERV. This study will facilitate the appraisal of PERV in xenotransplantation.
引文
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30. Tacke S, Specke V, Stephen O, et al. Porcine endogenous retroviruses: diagnostic assays and evidence for immunosuppressive properties. Transplant Proc. 2000, 32(5): 1166.
31. Tacke SJ, Kurth R, Denner J. Porcine endogenous retroviruses inhibit human immune cell function: risk for xenotransplantation? Virology. 2000, 268(1): 87-93.
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36.杨勇贤,马玉媛,章金刚.小型猪在异种移植中的应用前景.中国实验动物学报. 2008, 16(5): 368-371.
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