【作者】 孙成群；

【导师】 孔宪刚；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2004， 博士

【摘要】 慢病毒如同其它逆转录病毒，可以将前病毒基因组持久地整合到感染细胞的染色体上并且在感染的细胞和子代细胞中表达病毒基因，可以作为将目的外源基因引入细胞的载体。由于慢病毒除了能够感染分裂状态的细胞外还具有感染非分裂细胞的能力，因而，慢病毒载体备受关注。以HIV-1为基础的慢病毒基因转移载体是目前研究最为透彻的慢病毒载体。尽管已经在增加HIV载体的生物安全性和减少产生具备复制能力的病毒方面做了很多改进，但要应用于人类临床实验仍然存在较大的安全隐患。因此，开发非灵长类慢病毒基因转移载体系统成为当前研究的热点之一。 马传染性贫血病毒(Equine Infectius Anemia Virus，EIAV)是一种非灵长类慢病毒，而且是基因组结构最简单的慢病毒。中国EIAV驴白细胞弱毒疫苗株(EIAV-DLA)是目前唯一成功开发并大规模应用的慢病毒疫苗。本研究的目的在于利用我国在马传染性贫血病毒(EIAV)研究上的成果，在EIAV驴白细胞弱毒疫苗株的感染性克隆(pOK8266)的基础上，构建有中国特色的EIAV基因转移系统，为基因治疗、疫苗的开发和马传染性贫血病毒或其它相关的病毒的致病机制和免疫机制的研究提供一个新的研究手段。 本实验利用反向遗传操作技术，以EIAV驴白细胞弱毒疫苗的感染性克隆质粒(pOK8266)为基础，用CMV立即早期启动子替换了EIAV-5’LTR-U3区，以及载体中加入内部启动子、乙型肝炎病毒转录后调节元件(HPRE)和中央聚嘌呤序列(cPPT)／Rev反应元件(RRE)对载体进行修饰，构建了系列复制缺陷性EIAV载体质粒。同时将EIAV基因片段插入不同真核表达载体中构建了囊膜缺失的EIAV gag／pol表达质粒。而且克隆水泡性口炎病毒囊膜糖蛋白(VSV-G)基因并构建了不同的提供囊膜伪型的VSV-G的真核表达载体质粒，建立了EIAV三质粒基因转移载体系统。 实验结果表明用CMV立即早期启动子替换了EIAV-5’LTR-U3区的复制缺陷型EIAV载体转染或制备的载体病毒转导293T细胞能够有效地表达报告基因-增强绿色荧光蛋白(EGFP)。具有内部启动子载体转染后EGFP的表达明显高于无内部启动子的载体，说明增加一个内部启动子能够提高转基因的表达。反向插入HPRE的载体转染后未见有EGFP的表达，表明反向插入HPRE可以抑制EGFP的表达，而这种抑制作用可以被cPPT／RRE所纠正。正向插入HPRE的载体转染后未见对EGFP的表达有明显的抑制作用，说明HPRE的作用是方向特异性的。载体中增加中央聚嘌呤序列(cPPT)和Rev反应元件(RRE)等cis作用元件能够明显地增加报告基因的表达。构建的VSV-G表达载体在VSV-G基因前增加一个内含子能够明显地增加VSV-G的表达，而EIAVgag／pol表达载体却得出相反的结果，说明内含子可以增加VSV-G表达载体的蛋白表达，但是不能增加EIAVgag／pol载体的蛋白表达。更多还原

【Abstract】 Lentiviruses, like other retroviruses, with the abilities to integrate efficiently their genome into the chromosomal DNA of host cells and be stably replicated and transmitted to all of the progeny of these cells offering the potential for long-term expression, can be as gene transfer vectors to introduce the foreign target gene into host cells. In contrast to typical vectors derived from oncoretroviruses, which can only infect dividing cells, lentivirus-based vectors have received more considerable attention for gene delivery because lentiviruses are also able to infect nondividing, terminally differentiated target cells. Gene transfer systems based on human immunodeficiency virus type 1 (H1V-1) are by far the most developed lentiviral systems. Although HIV-1 vectors have been much more modified to maximize their biosafety and to minimize the potential for production of replication-competent viruses, the potential safety risks still limit HIV-1 vectors for human clinical applications. Such concerns are leading to the development of non-primate lentiviral vectors.Equine infectious anemia virus (EIAV) is a non-primate and the genetically simplest lentivirus. Chinese equine infectious anemia virus donkey leukocyte attenuated strain (E1AV-DLA) is a unique lentiviral vaccine strain used widely for prevention and control of equine infectious anemia (EIA) in China up to now. This study is to construct EIAV gene transfer vector systems based on an infectious molecular clone, pOK.8266, derived from the ElAV-DLA and to offer a new approach for gene therapy, development of vaccines, and viral pathogenic and immunological mechanisms of the EIAV and other related viruses.A series of replication-deficient EIAV gene transfer vector plasmids, envelope gene-deleted gag/pol expression plasmids and VSV-G gene expression plasmids were constructed and the gene transfer vector systems of these three plasmids were established by reverse genetic system. The results showed that the 293T cells could efficiently express the enhanced green fluorescent protein (EGFP) after transient transfection or transduction of the replication-deficient EIAV gene transfer vectors replaced EIAV-5’LTR-U3 region with human cytomegalovirus immediate-early promoter (CMV). The internal promoter and the "central polypurine tract (cPPT)/Rev responsive element (RRE) inserted into the vectors might increase expression of EGFP. The function of hepatitis B virus posttranscriptional regulatory element (HPRE) for EGFP expression is orientation-specific, HPRE inserted in reverse orientation could inhibit the EGFP expression, but this could be counteracted by cPPT/ RRE. An intron located between the CMV promoter and the VSV-G gene in VSV-G expression vector might increase the VSV-G expression, but the intron located between the CMV promoter and the EIAV gene in gag/pol expression vector was not for gag/pol expression.更多还原