系统性红斑狼疮患者新的内源性逆转录病毒 NP9基因表达及其蛋白功能预测

目的研究新发现的人内源性逆转录病毒 NP9基因在系统性红斑狼疮(systemic lupus erythematosus, SLE)患者体内表达及其蛋白功能预测.方法逆转录PCR、T-A克隆技术和DNA序列测定分离、克隆和分析 NP9基因, 检测40例SLE患者和48名正常对照的 NP9基因表达, 借助NCBI BLAST系列分析工具及相应的基因分析软件预测其蛋白功能.结果 SLE患者组逆转录病毒 NP9基因表达阳性率为77.5%(31/40),明显高于正常对照的8.3%(4/48), P<0.01. NP9基因编码产物由74个氨基酸组成,含有多个细胞核内分布信号,其等电点(pI)为9.59,与SLE患者体内高表达的某些细胞因子具有较高同源性.结论 SLE患者存在逆转录病毒 NP9基因特异性转录, NP9表达可能与SLE发生发展相关.     

异种移植领域猪内源性逆转录病毒的研究进展

供体器官来源不足严重阻碍和困扰着器官移植手术的推广和应用，随着免疫学、基因工程与克隆技术的迅速发展，转基因猪的出现，异种移植有望解决器官短缺的问题，但猪内源性逆转录病毒种间传播的可能性必须进一步深入研究，本文就近期相关研究作一综述。     

人内源性逆转录病毒的生物学功能及与疾病的关系

人内源性逆转录病毒(human endogenousretroviruses,HERV)是逆转录病毒在几百万年前感染人类并整合到人类基因组中,以孟德尔方式遗传至今的残余物.其在人体内数量众多,并且每个家族都存在多拷贝.HERV各家族基因结构基本相同,但许多功能都不明确,过去大多数研究人员将内源性逆转录病毒视为垃圾DNA.但随着研究的深入,人们发现HERV与人类的进化关系密切,是哺乳动物生殖所必需的,并且影响哺乳动物胎盘发育,是妊娠所不可或缺的基因.同时和胎盘共同构建了一个防止微生物感染胎儿的屏障.除了以上生理功能外,HERV还参与人体多种自身免疫性疾病和肿瘤的发生和发展过程.     

异种移植领域猪内源性逆转录病毒的研究进展

供体器官来源不足严重阻碍和困扰着器官移植手术的推广和应用,随着免疫学、基因工程与克隆技术的迅速发展,转基因猪的出现,异种移植有望解决器官短缺的问题,但猪内源性逆转录病毒种间传播的可能性必须进一步深入研究,本文就近期相关研究作一综述.     

猪内源性逆转录病毒体外感染人胚肾细胞系HEK-293的初步研究

目的对猪内源性逆转录病毒(PERV)体外感染人胚肾细胞系HEK-293的能力进行检测。方法建立体外感染人胚肾细胞系HEK-293的方法,用免疫电镜的方法观察PERV粒子,聚合酶链反应(PCR)、逆转录聚合酶链反应(RT—PCR)检测与PERV共培养24h后HEK-293细胞基因组中PERV前病毒基因的整合、表达并对PERV亚型进行鉴定,激光共聚焦显微镜检测PERV—gag蛋白的表达并进行定量分析。结果电镜下可观察到PERV的圆形病毒粒子;与PERV共培养24h后HEK-293细胞基因组中检测到PERV前病毒DNA,且mRNA也有效表达,PERV亚型为PERV—A,B型;激光共聚焦显微镜观察到PERV—gag蛋白在感染细胞HEK-293的胞质表达,但表达量下降。结论PERV具有体外感染人胚肾细胞系HEK-293的能力,病毒蛋白可以有效表达,因此在猪到人的异种器官移植研究中对PERV可能引起的人兽共患病进行安全性评价是极有必要的。     

猪内源性逆转录病毒在异种移植中的感染风险

猪作为异种移植供体可有效解决人类同种移植供体短缺的问题,但猪内源性逆转录病毒具有潜在的感染风险,严重影响了猪器官、组织、细胞在临床中的应用。因此,在异种移植应用于临床前,阐明猪内源性逆转录病毒的感染风险性至关重要。     

壳聚糖纳米纤维支架对猪肝细胞分泌猪内源性逆转录病毒的影响

探讨壳聚糖纳米纤维支架对肝细胞的PERV分泌量以及其感染性的影响。将新鲜分离的猪肝细胞接种于壳聚糖纳米纤维支架或普通条件下连续培养7 d,分为实验组(Nano组)和对照组(Hep组)。每天收集培养液检测逆转录酶(RT)活性,并通过RT-PCR和实时定量PCR测定PERV RNA水平。通过western blot检测细胞裂解液中PERV gag 30蛋白含量。细胞培养液体外孵育HEK293细胞以测定其感染性。结果表明:实时定量PCR、RT活性以及western blot具有相似的变化趋势。在体外培养10 h和2 d出现PERV分泌高峰然后逐渐下降。除第6 dPERV RNA水平和第5 d蛋白水平实验组明显高于对照组外,其余均无明显差异。体外感染实验无HEK293细胞感染。壳聚糖纳米纤维支架会延长猪肝细胞的PERV分泌时间但对分泌量以及体外感染性并无明显影响,可安全用于生物人工肝。     

携带hIL-2cDNA的逆转录病毒载体的构建及其在人肝癌细胞中的特异表达研究

将人白细胞介素２（ｈＩＬ-２）ｃＤＮＡ克隆到逆转录病毒载体ＭＮＳＭ的ＰＬ位点　，分别构建了转录受ＳＶ４０早期启动子和人甲胎蛋白增强子调控的重组逆转录病毒载体ＭＮＳＩ和ＭＮＳＩＡ。用脂质体转染法将ＭＮＳＩ和ＭＮＳＩＡ分别转导ＰＡ３１７包装细胞，测质粒转染率为（５～２０）×１０~(－３)克隆／μｇＤＮＡ·１０~６细胞，病毒感染率为（５．４～４５０）×１０~４ＣＦＵ／ｍｌ。重组病毒在４μｇ／ｍｌ ｐｏｌｙｂｒｅｎｅ存在条件下感染人肝癌细胞、肾癌细胞和黑色素瘤细胞，Ｎｅｏ~Ｒ克隆经Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证明ｈＩＬ－２ｃＤＮＡ转入人肿瘤细胞并整合，　Ｒ　ＮＡ斑点杂交及ＩＬ－２活性表达分析证明，人甲胎蛋白增强子可促进异源启动子启动ｈＩＬ－２ｃＤ－ＮＡ在合成甲胎蛋白的人肝癌细胞中高效特异转录和表达。该研究对肝癌特异性免疫增强基因治疗有重要意义。     

Beneficial Role of Human Endogenous Retroviruses: Facts and Hypotheses

Human endogenous retroviruses (HERVs) have recently been suggested as mediators of normal biological processes such as cellular differentiation and regulation of gene expression. Moreover, a direct role for HERVs in pathogenesis and the development of disease is now better appreciated. Elucidation of the mechanisms regulating HERV biology should provide information about fundamental cellular activities and the pathogenesis of multifactorial diseases such as cancer and autoimmune disease. The importance of understanding the roles of HERVs is underscored by the recently obtained insight that activation of endogenous retroviruses poses potential risks following xenotransplantation and in gene therapy using retroviral vectors. Furthermore, HERV-encoded superantigens have recently been implicated as causes of autoimmune disease. This review discusses the established and possible biological roles of HERVs, and proposes hypotheses concerning their involvement as mediators of fundamental cellular responses. We propose that the evolutionary persistence of endogenous retroviruses in the genomes of eukaryotic cells reflects their indispensability in important normal functions in specialized cellular environments. HERVs can also be potentially hazardous through their involvement in the development of disease. In addition, the creation of new retroviruses can occur through recombination, between different HERVs and between HERVs and exogenous retroviruses.     

Human Endogenous Retroviral Expression by Leukocytes

Budding endogenous retroviral particles were identified on the plasma membrane of a high percentage of neutrophils in four widely varied pathological specimens.     

The Evolution,Distribution and Diversity of Endogenous Retroviruses

The retroviral capacity for integration into the host genome can give rise to endogenous retroviruses (ERVs): retroviral sequences that are transmitted vertically as part of the host germ line, within which they may continue to replicate and evolve. ERVs represent both a unique archive of ancient viral sequence information and a dynamic component of host genomes. As such they hold great potential as informative markers for studies of both virus evolution and host genome evolution. Numerous novel ERVs have been described in recent years, particularly as genome sequencing projects have advanced. This review discusses the evolution of ERV lineages, considering the processes by which ERV distribution and diversity is generated. The diversity of ERVs isolated so far is summarised in terms of both their distribution across host taxa, and their relationships to recognised retroviral genera. Finally the relevance of ERVs to studies of genome evolution, host disease and viral ecology is considered, and recent findings discussed.     

Reliable Classification and Recombination Analysis of Porcine Endogenous Retroviruses

Prevention of cross-species infection with porcine endogenous retroviruses (PERV) is crucial for xenotransplantation. Previous studies described the potential risk of infection for the PERV 1 subfamilies A, B and C. Replication competent PERV 1 proviruses designated to a particular subfamily and hybrid viruses originating from retroviral recombination events between the subfamilies were observed. Future pig genome sequencing projects will reveal multiple novel PERV proviruses from additional breeds and animals. Evaluation of these viral genomes has to be carried out to assess the potential risk of retroviral cross-species infection. In this study, we tested common sequence comparison methods for the classification of PERV sequences and the detection of hybrid clones. The examination of the polymorphic nucleotide positions was found to be the most suitable procedure. We describe a fast and simple method using bioinformatic software tools which can also be applied to analogous analyses of other viral genomes.