【作者】 裘智勇；

【导师】 郭锡杰；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2008， 博士

【摘要】 家蚕浓核病毒是感染家蚕的四种主要病毒之一,主要通过食下感染,与其它浓核病毒不同,家蚕浓核病毒只感染家蚕中肠上皮组织的圆筒型细胞。患病蚕呈现空头,下痢,吐液等症状,是蚕桑生产上一种比较严重的病毒病。家蚕品种间对浓核病毒的感受性差异很大,一些品种对BmDNV具有完全的非感受性,而一些品种极易感染,家蚕对浓核病毒感受性差异的蛋白质分子机理目前尚不十分明确。本研究以对家蚕浓核病毒镇江株(Bombyx mori densovirus Zhenjiang Strain, BmDNV-ZJ)感受性和非感受性近等基因系蚕品种为材料,采用生物测定、地高辛标记的酶联免疫测定、蛋白质双向电泳和基质辅助质量飞行时间质谱技术,在蛋白水平上对家蚕感染浓核病毒相关组织进行了研究,研究结果将有助于进一步弄清家蚕对BmDNV的感受性分子机制,阐明家蚕浓核病毒的致病机理。通过生物测定和地高辛标记的酶联免疫吸附测定初步分析了家蚕对BmDNV-ZJ的感受性机制。试验表明:家蚕的消化液、血液中不存在对BmDNV-ZJ侵染性有影响的蛋白因子,感受性家蚕品种的中肠组织蛋白中可能存在对BmDNV-ZJ侵染性有影响的蛋白因子,而非感受性家蚕品种的中肠组织蛋白中不存在影响BmDNV-ZJ侵染性的蛋白因子,且BmDNV-ZJ与感受性家蚕品种中肠组织蛋白的结合具有组织特异性。通过蛋白质双向电泳和基质辅助质量飞行时间质谱技术对两个不同感受性家蚕品种的中肠组织蛋白进行了比较分析。2-DE电泳结果表明,两个品种间的中肠组织蛋白的点数、位置、点形等差异很小。MALDI-TOF MS鉴定的9个差异蛋白点中都有较强的肽质量指纹信号峰,其中感受性品种JS的中肠组织中有6个差异蛋白点,4个可能为氢离子转运ATP合酶β亚基1,氢离子转运ATP合酶β亚基2,组织蛋白酶D或3-羟酰辅酶A脱氢酶;抵抗性蚕品种NIL的中肠组织中有3个差异蛋白点, 2个可能是组织蛋白酶。通过对BmDNV-ZJ感受性和非感受性蚕品种血液和围食膜组织蛋白比较分析表明:两个蚕品种的血液和围食膜组织蛋白的差异很小,其凝胶图谱上的蛋白点数、位置、点形等都几乎一样。两品种的血液组织蛋白大多为一类中性略偏碱性的蛋白质,酸性和碱性极端的蛋白质很少;围食膜组织部分蛋白大多为一类中低分子量、偏中性的蛋白质。在血液组织蛋白中发现5个有显著差异的蛋白点:其中只在感受性蚕品种血液组织中发现的差异蛋白可能为酪蛋白激酶,另一个可能为新型组织蛋白;而在非感受性蚕品种血液中发现的特异蛋白点可能是类丝氨酸蛋白酶、线粒体延伸因子,而且其血淋巴蛋白的含量也极显著地高于感受性品种。从这些差异蛋白点可能的功能中可以看出,家蚕品种间对BmDNV-ZJ感受性差异可能与蚕品种间的血液组织蛋白差异无关。在围食膜组织蛋白中共发现4个有显著差异的蛋白点,其中在感受性蚕品种围食膜组织中,发现1个可能为新型组织蛋白的特异性蛋白点,其余3个蛋白点在含量上相互间存在显著差异。所发现的4个差异蛋白点是否与蚕品种对BmDNV-ZJ感受性差异有关尚待进一步研究。感受性蚕品种在BmDNV-ZJ侵染初期,中肠组织受BmDNV-ZJ诱导产生的蛋白可能为Serpin-2(调控蛋白酶活性和细胞凋亡)和Thiol peroxiredoxin(具有抗氧化、消除自由基等功能)。血液组织受诱导而得到特异性表达的蛋白可能是丝裂原活化蛋白激酶(调控细胞凋亡)或类抗氧化酶蛋白(具有抗氧化、消除自由基等功能)。表明感受性蚕品种在BmDNV-ZJ感染初期,其内生性免疫防御途径可能通过受侵染部位(中肠)和免疫防御组织(血液)分泌抗氧化蛋白等抗性蛋白因子和细胞凋亡等方式来阻止或延缓病毒的进一步侵染。感受性蚕品种在感染浓核病毒晚期,其中肠、血液组织蛋白量急剧下降,有近30%的蛋白点消失,其余蛋白点的含量大幅下调达50%以上。表明由于浓核病毒专一性地侵染中肠上皮组织,破坏了中肠的消化吸收功能,阻断了营养物质的输入,无法满足家蚕生长发育的需要,进而极大地减少了自身组织蛋白的合成。更多还原

【Abstract】 Bombyx mori Densovirus (BmDNV) is one of the four main viruses infecting silkworm. The BmDNV infects silkworm mainly through oral administration, and replicates only in the columnar cells of midgut epithelium, which is different from other densoviruses. The symptom of diseased silkworm includes body transparency, diarrhea, vomiting and so on. Thus the densonucleosis is a kind of severe viral diseases to the silkworm in sericultural production. The susceptibility to BmDNV differs according to the silkworm strains. Some strains are absolutely non-susceptible to BmDNV, while few are easily to be infected. The molecular mechanism of the silkworm susceptibility to BmDNV at protein level is unknown presently. In this paper, with two near isogenic silkworm strains, JS and NIL, respectively susceptible and non-susceptible to BmDNV Zhenjiang Strain (BmDNV-ZJ) as materials, proteins in the silkworm tissues related to the infection of BmDNV-ZJ were studied by bioassay, DIG-labeled ELISA, two dimensional electrophoresis (2-DE) and matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS).The results will be helpful for the understanding of the molecular mechanism of silkworm susceptibility to BmDNV and the pathogenesis of the silkworm densonucleosis.The purified virus was incubated with extract from different tissues of susceptible silkworm strain JS and non-susceptible silkworm strain NIL, then the infectivity of the virus was tested by bio-assay and the combination activity of the virus with proteins from the extract measured by DIG-labeled ELISA. The results indicated that there were no protein factors affecting the invasion of the virus in digestive juice and blood. Some protein factors enhancing the invasion probably exist in the midgut of JS but not in NIL. It is speculated that protein factor as virus receptor may exist in the midgut of JS, but not in the NIL. Meanwhile,the combination activity was tissue-specific to the midgut of JS.Proteins in the midgut of JS and NIL were analyzed comparatively by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed that there was less difference in the number, location and shape of the protein spots on the 2-DE image between the two strains. Protein peptide mass fingerprints (PMF) of all the 9 distinguished specific spots, six in JS and three in NIL , showed high intensity when analyzed by MALDI-TOF MS.Among the six specific spots in JS, four might be H+ transporting ATP synthase beta subunit isoforms 1 and 2, cathepsin D or 3-hydroxyacyl-CoA dehydrogenase, respectively. Of the three specific spots in NIL, two spots might be cathepsin.Comparative analysis of proteins in the hemolymph and peritrophic membrane (PM) between JS and NIL indicated that there was less difference in tissue proteins from hemalymph or PM between the two strains, the number, location and shape of the protain spots on the 2D image were very similar. The proteins from the blood of both the two silkworm strains were mainly neutral and slightly alkaline ones with very few extremely acid or extremely alkaline ones. The proteins from PM was mostly middle or low in molecular weight and neutral in isoelectric point value. Total five differentiated spots were distinguished on 2-D image of the hemolymph proteins. One spots distinguished in the susceptible strain might be casein kinase and another be an un-reported protein. Spots distinguished in the non-susceptible strain might be masquerade-like serine proteinase and mitochondrial elongation factor respectively. Moreover, the protein content in the hemolymph of non-susceptible strain was remarkably higher than that in the susceptible strain. According to the probable function of these differentiated spots, it is suggested that the susceptibility of silkworm strains against BmDNV-ZJ may be not related with the difference of their hemolymph proteins.Total four differentiated spots were detected in PM proteins. A specific spot distinguished in the PM of susceptible strain might be an un-reported protein. The protein content of each counterpart spots of the rest three is remarkably different between the susceptible and non-susceptible strains. It is not clear whether the four differentiated spots were related with the difference of susceptibility to BmDNV-ZJ among the silkworm strains.In the susceptible strains, serpin-2 (regulating protease activity and apoptosis) and thiol peroxiredoxin (anti-oxidation and eliminating free radicels) was detected in the midgut in the initial stage of infection by BmDNV-ZJ. They might be the products by the induction of the virus infection. The proteins specifically expressed in the hemolymph against the infection might be mitogen-activated protein kinase (regulating apoptosis) or peroxiredoxin-like protein (anti-oxidation and eliminating free radicels). According to the probable functions of these differentiated proteins, it is speculated that the silkworm of susceptible strain may prevent the BmDNV-ZJ infection from further development in the initial stage of infection through endogenous immune defense ways such as the apoptosis and the secretion of resistant protein factors in midgut (the infected position) and blood (the tissue of immune defense), like anti-oxidation proteins.In the advanced stage of infection by BmDNV-ZJ, the amount of proteins extracted from midgut and hemolymph of the silkworm of susceptible strain decreased rapidly. About 30% spots disappeared and the down-regulation of the rest spots was more than 50%. This implies that the protein synthesis is significantly reduced due to destruction of the digestion and absorption function of the midgut caused by the virus infection.更多还原