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中国巴马小型猪内源性反转录病毒的检测 总被引:2,自引:0,他引:2
目的 :对我国特有巴马小型猪内源性反转录病毒 ( porcine endogenous retrovirus,PERV)的存在与 m RNA的表达情况进行检测 ,了解巴马小型猪内源性反转录病毒的携带情况。方法 :根据以往建立的 PCR、RT-PCR检测方法 ,对来自于巴马小型猪外周血淋巴细胞的 DNA和RNA样品进行 PERV核心蛋白基因 ( gag)、多聚酶基因 ( pol)及囊膜基因 ( env)的存在与表达进行检测 ;同时 ,根据目前通用的 env基因分型方法检测 PERV env-A、env-B、env-C的存在与表达。结果 :在 1 2个被检的 DNA样品中均检出了PERV特异性 DNA的存在 ;同样 ,在 1 2个被检的 RNA样品中均有 PERV特异性 RNA的表达 ,且所表达的 PERV均为 A型和 B型 ;其中有9个 DNA样品检测出 PERV-C型的存在 ,所有样品中均未检出 C型 PERV的表达。结论 :检测结果表明 1 2个被检巴马小型猪基因组中存在着内源性反转录病毒序列 ,且能以 m RNA的形式表达 ,这一结果为我国特有小型猪的开发、利用及其病毒安全性评价奠定了基础。 相似文献
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利用CDNA末端快速扩增技术(RACE)成功扩增了广西巴马小型猪近交系连续4代次及封闭群的猪内源性反转录病毒(PERV)3UTR,共获得8条序列(GenBank登录号分别为:GQ475493、GQ475494、GQ475495、GQ475496、GQ475497、GQ475498、GQ475499、GQ475500),分为638、749bp 2种不同的类型,都属于PERV-A亚型。以PERV-3UTR构建遗传进化树,PERV-A,B,C分群清晰,3UTR可作为PERV亚型区分的另一基因序列。对调控元件定位分析,推测具有749bp类型3UTR的PERV病毒转录水平更高。在所获得序列的209~227bp位置,发现了"CTTGAAACTT"10个碱基的1.9个重复。模拟RNA二级结构,广西巴马小型猪PERV-3UTR 2种类型无论从空间构型、自由能,还是茎环数量都存在明显的不同。这些结果为全面评价广西巴马小型猪来源的PERV病原安全性提供了参考。 相似文献
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根据猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因和β-肌动素(β-actin)基因核酸序列设计并筛选特异性引物6对,利用PCR方法对五指山小型猪PERV gag、pol和env基因存在情况进行系统检测;利用半定量RT-PCR方法对五指山小型猪PERV gag、pol和env基因在心、肝、脾、肺、肾、脑、肌肉7个组织中的表达情况进行系统检测与分析.结果显示,在202个被检样品中,gag、pol基因和envB基因亚型均存在,envA基因亚型仅3个样品未发现,envC基因亚型共检出34个,检出率为16.8%.所测试的7个组织均表达gag、pol和env基因序列,但envC仅在1头个体的组织中表达.各组织gagmuRNA丰度以肾脏为最高,其次为肺、心、肝、脾,脑和肌肉组织的丰度最低.各组织pol mRNA丰度以肾脏为最高,其次为脾、肺、肝、心,肌肉和脑组织的丰度最低.各组织envA、envBmRNA丰度信号强度分布与gag、pol信号类似,亦以肾脏为最高,脑和肌肉组织的丰度最低.各组织envC mRNA的信号强度明显低于envA、envB.以上结果表明,在五指山小型猪中存在PERV,且在各组织中的表达量有差异. 相似文献
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根据猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因和β-肌动素(β-actin)基因核酸序列设计并筛选特异性引物6对,利用PCR方法对五指山小型猪PERVgag、pol和env基因存在情况进行系统检测;利用半定量RT-PCR方法对五指山小型猪PERVgag、pol和env基因在心、肝、脾、肺、肾、脑、肌肉7个组织中的表达情况进行系统检测与分析。结果显示,在202个被检样品中,gag、pol基因和envB基因亚型均存在,envA基因亚型仅3个样品未发现,envC基因亚型共检出34个,检出率为16.8%。所测试的7个组织均表达gag、pol和env基因序列,但envC仅在1头个体的组织中表达。各组织gag mRNA丰度以肾脏为最高,其次为肺、心、肝、脾,脑和肌肉组织的丰度最低。各组织pol mRNA丰度以肾脏为最高,其次为脾、肺、肝、心,肌肉和脑组织的丰度最低。各组织envA、envB mRNA丰度信号强度分布与gag、pol信号类似,亦以肾脏为最高,脑和肌肉组织的丰度最低。各组织envC mRNA的信号强度明显低于envA、envB。以上结果表明,在五指山小型猪中存在PERV,且在各组织中的表达量有差异。 相似文献
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聚合酶链反应检测4种猪源细胞系中的内源性反转录病毒 总被引:5,自引:0,他引:5
为了建立检测猪内源性反转录病毒(porcine endogenous retrovirus,PERV)的特异性方法,根据巳发表的PERV的序列,设计并合成了针对PERV核心蛋白(gag)、多聚酶(pol)、囊膜蛋白(env)基因的3对引物,预期扩增片段分别为361、150、265bp。应用PCR技术检测了PERV在4株猪源细胞中的整合情况。结果表明,在所有被检细胞的基因组中均存在有PERV的前病毒序列,应用RT-PCR检测上述4株猪源细胞中PERV特异性MRNA的表达,结果均为阳性。试验还对建立的上述2种方法的特异性进行了探讨,结果表明试验建立的PERV检测法具有较高的特异性。该方法的建立为进一步研究PERV奠定了基础,还可对异种移植动物模型及异种移植受体进行病原安全性监测。 相似文献
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首先将巴马小型猪(BM)、巴马香猪(BMXZ)外周血淋巴细胞分别与G418抗性HEK293细胞(G418^R293)共培养,通过G418加压筛选,除去共培养体系中巴马小型猪、巴马香猪外周血淋巴细胞,然后应用PCR及RT-PCR的方法对所制备的感染细胞模型进行系统鉴定。结果经6周共培养及3周加压筛选后,细胞的形态、生长速度及折光性均未见明显变化;PCR及RT-PCR鉴定表明,筛选后的共培养体系中已没有巴马小型猪、巴马香猪外周血淋巴细胞的存在;PERV特异性检测方法检测显示,该细胞模型的DNA中已有PERV的整合且有PERV特异性mRNA的表达。从而证实了猪外周血淋巴细胞来源的PERV在体外也能够感染人源细胞系,为研究PERV的生物学特性及病原安全性的评价搭建了技术平台。 相似文献
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作者介绍了国内外猪内源性反转录病毒(PERV)研究进展,简述了中国不同品种小型猪PERV存在情况、PERV前病毒全基因克隆与序列分析、细胞水平鉴定方法、感染HEK293细胞研究平台的创建、猪A3F对PERV的抑制作用研究,以及从分子遗传学上揭示不同品系PERV特异性等创新性研究成果,分析了五指山小型猪近交系具有PERV基因拷贝少且没有PERV-C等特异性,以及展望培育中国PERV阴性猪新品系方法等研究,以期为人类异种器官移植和生物医用材料产品安全性研发,解决小型猪PERV疾病传播危险性提供科学对策和新的方向。 相似文献
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为了解猪内源性反转录病毒(Porcine endogenous retrovirus, PERV)在海南地方猪种五指山猪和海南猪基因组中的存在状况。试验利用猪内源性反转录病毒特异性引物对这两种猪的基因组进行PCR检测。结果显示:PERV普遍存在于海南地方猪基因组中,其中在五指山猪中PERV-A、PERV-B、PERV-C存在率分别是100%、100%、20.69%;在海南猪中PERV-A、PERV-B、PERV-C存在率分别是100%、100%、25.49%。该研究为海南地方猪的开发、利用及其病毒安全性评价奠定了基础。 相似文献
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Wu J Ma Y Lv M Yang Y Guo Y Yu X Tian K Zhang J 《Comparative immunology, microbiology and infectious diseases》2008,31(4):367-371
We conducted a large-scale survey on the existence and expression status of porcine endogenous retrovirus (PERV) in seven breeds of Chinese miniature pigs. Genotyping of PERV was examined by PCR using type-specific primers according to the env genotyping method. The presence and expression status of viral gag, pol and env genes were further analyzed in Wuzhishan pigs (WZSP) and Bama minipigs (BMP). The results showed that PERV existed in all 348 genomic DNA samples. The genotype distribution was subtype A-74.43%, subtype B-95.40% and subtype C-30.46%. No expression of subtype C was found in WZSP and BMP. This research obtained an adequate level of information on the molecular epidemiology of PERV in China. The results indicated that it is possible to monitor pig herds for individuals with the lowest PERV prevalence, especially lacking PERV-C. 相似文献
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The paper is a brief introduction of porcine endogenous retrovirus (PERV) innovative research results in the world and also expounds the passing PERV existence situation on different varieties of miniature pig, analyzes the PERV-virus gene cloning and sequence, appraises method on cell level, create the platform of infection HEK293 cells research, study on pigs A3F inhibition of PERV, and reveal the innovative research results on the specific molecular genetics in the different strain of PERV, analyzes the advantages of Wuzhishan miniature pig inbred line such as low gene copy of PERV,and there is no passing PERV-C specificity and as well as looking forward to cultivate the methods for new strain of PERV negative pigs. It will provide a scientific counter measure and new perspective to solve the spread of disease risk of miniature pig PERV and product safety for human xenotransplantation and biomedical materials of research and development. 相似文献
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Hyoung-Joon Moon Seong-Jun Park Hye-Kwon Kim Soo-Kyung Ann Semi Rho Hyun-Ok Keum Bong-Kyun Park 《Journal of veterinary science (Suw?n-si, Korea)》2010,11(3):269-271
The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes. 相似文献
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Expression of porcine endogenous retroviruses (PERVs) in melanomas of Munich miniature swine (MMS) Troll 总被引:1,自引:0,他引:1
Dieckhoff B Puhlmann J Büscher K Hafner-Marx A Herbach N Bannert N Büttner M Wanke R Kurth R Denner J 《Veterinary microbiology》2007,123(1-3):53-68
Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear. 相似文献
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猪内源性逆转录病毒对异种组织器官移植影响的研究进展 总被引:1,自引:0,他引:1
猪内源性逆转录病毒(Porcine en-dogenous retrovirus,PERV)是新发现的对人细胞具有感染性的且存在于正常猪体内的病毒,在异种组织器官移植过程中可能向人传播,直接威胁到接受移植者的健康甚至生命,这是异种组织器官移植首要克服的难题。由于PERV近年来才受到广泛关注,所以人们对PERV的分类,对人类和其他动物的危害性及病原的检测均不能完全确定。文章旨在提供上述几方面的研究现状,以期为PERV的深入研究奠定基础。PERV能影响移植受体的生殖,抑制受体免疫体系;对其他物种的细胞或活体都可能产生体外和体内感染性;目前有多种PERV检测方法,为受体和供体的病毒监控提供了依据。 相似文献
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Phillips ND La T Adams PJ Harland BL Fenwick SG Hampson DJ 《Veterinary microbiology》2009,134(3-4):294-299
Feral pigs are recognized as being a potential reservoir of pathogenic microorganisms that can infect domestic pigs and other species. The aim of this study was to investigate whether feral pigs in Western Australia were colonized by the pathogenic enteric bacteria Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli. A total of 222 feral pigs from three study-populations were sampled. DNA was extracted from faeces or colonic contents and subjected to a previously described multiplex PCR for the three pathogenic bacterial species. A subset of 61 samples was cultured for Brachyspira species. A total of 42 (18.9%) of the 222 samples were PCR positive for L. intracellularis, 18 (8.1%) for B. hyodysenteriae and 1 (0.45%) for B. pilosicoli. Four samples were positive for both L. intracellularis and B. hyodysenteriae. Samples positive for the latter two pathogens were found in pigs from all three study-sites. A strongly haemolytic B. hyodysenteriae isolate was recovered from one of the 61 cultured samples. Comparison of a 1250-base pair region of the 16S rRNA gene amplified from DNA extracted from the isolate and five of the B. hyodysenteriae PCR positive faecal samples helped confirm these as being from B. hyodysenteriae. This is the first time that B. hyodysenteriae has been detected in feral pigs. As these animals range over considerable distances, they present a potential source of B. hyodysenteriae for any domesticated pigs with which they may come into contact. 相似文献