【作者】 肖仕全；

【导师】 鲁成； 潘敏慧；

【作者基本信息】 西南农业大学 ， 细胞生物学， 2004， 硕士

【摘要】 家蚕浓核病毒(中国株)引起的家蚕浓核病是我国蚕业生产上的重要病害，严重影响蚕丝的产量和质量，削弱了国际竞争力。家蚕类浓核病毒(DNV-like)是一株从西南农业大学家蚕基因库分离到的家蚕类浓核病毒。本论文对其病原性和病理学进行了研究，并对它的分子生物学特性进行了初步研究。 1．病原性研究：用不同浓度梯度的DNV-like和BmDNV-2，分别对不同品种的家蚕攻毒，逐日统计累计死亡率。结果表明，DNV-like属于慢性致死性病毒，从接种的第12天开始死亡。在病原性上与BmDNV-2有一定的差异：DNV-like的致病力远大于BmDNV-2；而且它们对不同家蚕品种的敏感性也不一样。 2．病理学研究：常规HE染色和组织荧光原性杂交显示，家蚕感染该病毒以后，首先寄生在家蚕中肠的圆筒形细胞，并表现为核的极度膨大并破裂，最大核为正常核的3倍以上；荧光原位杂交结果表明，该病毒在圆筒形细胞核内增殖，随着感染加深，邻近的杯形细胞也会受到感染。从这一点上看，与BmDNV-2类似。但蚕体其它组织也表现出阳性信号，比如脂肪体、肌纤维。这与BmDNV-2在感染部位上有一定的差异。病蚕中肠组织超微切片也显示出该病毒首先在核内增生，然后核仁发生病变，产生病毒发生基质，继而形成病毒粒子并广泛分布于细胞核中。 3．病毒DNA的研究：对DNV-like抽提的DNA进行电泳，发现只有一条大约6.5kb的带。明显不同于BmDNV-2的两条带(6.6kb和6.1kb)，也大于BmDNV-1的一条带(5.0kb)。 综合以上因素，可以初步认为该病毒是一种新型家蚕浓核病毒，但要对它准确分类，还需要很多数据，故暂命名为DNV-like。 昆虫细胞培养是是伴随着昆虫病毒研究而发展起来的，同时也是动物细胞培养工程中的一个重要方面，广泛应用于医学、农业及生物学的各个领域。为了满足学科研究和发展的多方面需要，人们还在不断致力于新的昆虫细胞系的建立。因此，建立和筛选有效支持病毒增殖和外源基因高水平表达的昆虫细胞系受到了特别关注。 为了进一步研究DNV-like以及其它家蚕病毒的致病机理，建立高效的病毒离体复制系统，更为了向细胞工程研究提供理想的材料，本研究还尝试建立高繁殖力、对病毒敏感的家蚕细胞系。对多个品种家蚕的中晚期胚胎使用胰酶半消化法建立原代初级培养，在长达11个月的分化、淘汰及筛选过程中，先后出现了多种组织，其中以网状组织、管状组织、脂肪体组织以及根瘤状组织为主。其中以杂交种的活力最大，组织更替快，生长也比较快。最后从杂交种得到的根瘤状组织中获得了高繁殖力的细胞群体，这时细胞种类复杂，繁殖力也不稳定。经过3个月的继续筛选，获得了繁殖力稳定、细胞形态比较均一的细胞系。命名为西农蚕胚1-.-..~~.翻~挂.~~图划号细胞系，英文名段扣由west Agricuhanl University Bom如morL LI，简写为S认叭UI.B州E。该细胞系以梭形和近圆形细胞为主，杂以少童多突起和巨型细胞.细胞系的倍增时间在28℃时为57.6小时，染色体呈短杆状或顺粒状，弥散型着丝粒，染色体数目异倍化，具有典型鳞翅目昆虫细胞系染色体特征。本细胞系对同钾的多角体病毒丑加NPv特别敏感，远远大于本研究室现保存细胞系，是研究家蚕杆状病毒及其表达载体的理想材料.更多还原

【Abstract】 Great losses in sericulture have been caused by silkworm disease, among which ,Bombyx mori. densovirus (BmDNV) is one of die main causes. DNV-like is a DNV strain from silkworm gene bank of Southwest Agricultural University, hi order to characterize the properties of this virus and prevent the breakout of silkworm disease, studies on its pathogeny, pathology and nucleic acid have been made.The study on DNV-Like’s pathogeny in silkworm had been done by dealing with different concentration solution of BmDNV-2 and DNV-like respectively. The results demonstrated that DNV-Like’s pathogeny was similar to BmDNV-2. But the virulence of DNV-like was much higher than that of BmDNV-2, and different varieties of silkworm had different sensitivity to them.Histopathology study indicated that the first autoeciousness position was cylindrical cell of midgut. And the nucleus of cylindrical cell extremely inflated, even broke. In Situ Hybridization Histochemistry (ISHH) also demonstrated that DNV-like first multiplied in nucleus of cylindrical cells of midgut, it could also infect the goblet cells, the fat body cells ,even the muscle cells.The result of electrophoresis of DNA extracted from DNV-like has only one band, approximately 6.5kb in length, which is different from that of BmDNV-2 with two bands of approximately 6.6kb and 6.1kb, and is also different from that of BmDNV-1 with only one band approximately 5.0kb.According to the results above, a conclusion can be drawn that DNV-like is a new kind of densoviruses, but lots of data is still needed to determine its accurate classification, v lIn order to make further study on the nosogenesis of DNV-like and other silkworm viruses and establish an efficient replicate system in vitro, a silkworm cell line which would be reproductive and susceptible to silkworm viruses have been tried to establish. Silkworm embryo in blastokinesis was chosen as the materials of primary culture. The embryos were minced by scissors and then digested by trypsin. By this method, cells and tissues could be subsisted in vitro, thus could be subcultured. The differentiation and types of cells and tissue were observed. The types of tissues were as follow: epithelioid tissue, fibroblast tissue, net tissue, glandiform tube -like tissue and fat -like tissue. After a long time of differentiation and filteration, a reproductive cell group consisting of several types of cells was gamed, but had an unstable reproducibility. After a 3-months- successive selection, a cell line was established. This cell line was named as Southwest Agricultural University Bombyx moriEmbryo 1, abbreviated as SWAUl-BmE. The cell line is mainly composed of two cell types: shuttle and spheroid. Also a few protuberant and huge cells were found in culture. The cell population doubling time is 57.6h at 28℃. The chromosome of SWAUl-BmE cells show typical characteristics of lepidoteran insect cell lines that chromosomes of metaphase have no apparent centromere, and are short pole-like and pellet-like. The longest chromosome is about 4 um. The chromosomes are heteroploidy. The infection of BmNPV on the different bisect tines showed that SWAUl-BmE was by far the more sensitive to BmNPV. So it would be a good material for silkworm baculovirus expression system.更多还原