【作者】 王永杰；

【导师】 陈克平； 杨胜利；

【作者基本信息】 江苏大学 ， 食品科学， 2007， 博士

【摘要】 浓核病毒无囊膜,直径18～26nm,基因组大小约为4.0～6.5kb。该病毒内含有正负链单分子（ssDNA）,或为正链,或为互补的负链。浓核病毒具有高度的宿主特异性,并且在通常情况下能引起宿主的死亡。目前浓核病毒最为关注的是可作为生物杀虫剂和表达载体表达外源蛋白。家蚕作为经济昆虫的重要性,自上世纪80年代人们先后从日本（BmDNV-1、BmDNV-2、BmDNV-5）、中国镇江（BmDNV-3）和印度（BmDNV-4）的家蚕中分离到了不同株系DNVs。其中BmDNV-3和BmDNV-2（Yamanashi isolate）都拥有双分子基因组（VD1,VD2）,并且感染相同的宿主,但在感受性、血清学上有差异,在宿主感染部位上也存在着不同。本论文中以BmDNV-3作为研究对象,对其基因组的结构和功能进行研究。主要结果如下:1.浓核病毒BmDNV-3基因组中含有两种没有同源性的单链线形DNA分子（VD1,VD2）,这两种核酸分子以单链（+VD1,-VD1;+VD2,-VD2）线型方式被分开包装在各自的衣壳蛋白中。该病毒DNAs在高盐状态下被分离、纯化。用T4酶（300ngDNA/10U）补平抽提过程中复性为双链的病毒DNAs,然后用HindⅢ进行酶切,酶切后的片段克隆到经过HindⅢ和SmaⅠ平端酶切的pUC119载体上,完成了对该病毒的全基因组克隆和序列测定。序列装配的结果显示:VD1全基因组序列长为6543个核苷酸,末端拥有224个核苷酸末端反向重复序列;VD2全基因组序列长为6022个核苷酸,末端拥有524个核苷酸末端反向重复序列。GenBank序列登录号:[DQ017268;DQ017269]。2.生物信息学分析显示:VD1基因组正链含有3个大的开放阅读框（ORF1～3）,ORF1和ORF2可能编码非结构蛋白,因为它们含有细小病毒保守区域序列NTP-binding和DNA解旋酶结合域,而ORF3可能编码结构蛋白,负链含有1个大的开放阅读框（ORF4）,由于它与假定的DNA聚合酶有一定的同源性,因此推测可能编码非结构蛋白。对VD1 ORF4的1115氨基酸序列与其它病毒DNA聚合酶基因氨基酸序列比对,发现N端含有3个外切酶的保守区域与原核和真核生物3′→5′外切酶相关,5个保守区域涉及到DNA聚合酶的合成功能,这8个保守区域显示VD1 ORF4编码DNA聚合酶;VD2基因组正链含有1个大的开放阅读框,负链含有1个小的开放阅读框。根据同源性比较推测该基因组负链上开放阅读框（ORF2）可能编码非结构蛋白,而正链上开放阅读框（ORF1）可能编码病毒的结构蛋白。3.比较BmDNV-3和BmDNV-2（Yamanashi isolate）基因组全序列,两者VD1的同源性为98.4%,VD2同源性达97.7%,并且有228个碱基的替代、11个碱基的删除和2个碱基插入。比较这两个不同株系病毒的开放阅读框和末端反向重复序列,突变的热点集中在结构蛋白（VD1 ORF3,VD2 ORF1）,VD1 ORF4和末端反向重复序列上。本研究中的结论为更好地理解家蚕浓核病毒不同株系差异性及其可能的原因,也为研究家蚕浓核病毒进化提供了有益的线索。4.在对VD1基因转录组织结构研究中,Northern杂交显示VD1的左边正链上有1.1kb非结构蛋白和1.5kb结构蛋白这两个转录本,右边的负链上有一个3.3kb非结构蛋白转录本。3′和5′RACE序列测定结果显示1.1kb非结构蛋白开始于nt290,结束于nt 1437。ORF1和ORF2完全位于其中,因此有可能通过核糖体扫描机制在不同的阅读框上翻译两种蛋白NS1和NS2。1.5kb结构蛋白开始于nt 1423,结束于nt 2931。编码结构蛋白的转录本（ORF3）和编码非结构蛋白的转录本（ORF4）在图距50处拥有10个核苷酸的3′端的共同序列。该病毒的转录方式与已报道的其它浓核病毒存在着较大差异。5.该病毒感染的家蚕中肠组织经匀浆、40%（W/W）CsCl₂梯度离心和定量PCR,结果显示VD1和VD2两者的浮力密度接近相等,约为1.29 g/cm³。该病毒的浮力密度比其它浓核病毒明显小,再次说明该浓核病毒的特殊性。从不同梯度等份样品中VD1、VD2的起始拷贝数判断,得出它们可能位于各自不同的病毒衣壳蛋白中。另外,SDS-PAGE电泳分析结果显示BmDNV-3病毒的衣壳蛋白的分子量为48kDa、55kDa、72kDa和97kDa的4条明显条带,其中53kDa为主要组分。6.在原核表达载体中构建了pET28a（+）-VD1 ORF4 3′端部分,然后转化到大肠杆菌BL21中。通过IPTG诱导表达后,SDS-PAGE显示在62 kDa左右有一特异条带,与预测的蛋白质分子量大小一致。另外,还在杆状病毒表达系统中,构建了重组转座载体pF_ASTB_ACHTB-VD1 ORF4 3′端部分。更多还原

【Abstract】 Densoviruses（DNVs） are small no-enveloped particles particles of 18～26 nm in diameter and their genomes consist of 4～6.5 kb single-stranded linear DNA encapsidated as plus and minus strands in separate virions.DNVs tend to be highly host-specific and cause death of the host in most cases.The current interest in the molecular biology of these viruses is fostered by their potential as modified virus pesticide,and by their use as vectors for the expression of foreign proteins.Due to important of the silkworm industry,many isolates were obtained from silkworm,they were termed as BmDNV-1（Ina isolate）,BmDNV-2（Saku isolate and Yamanashi isolate）,BmDNV-5,BmDNV-3（China isolate） and BmDNV-4（Kenchu insolate）. BmDNV-3 and BmDNV-2（Yamanashi isolate） both have bipartite genomes（VD1,VD2） and have the same host species,but their virulence and the symptoms of infection are different.In present studies,we reported the characterizations and potential function of Bombyx mori densonucleosis virus type 3.The results are shown as follows.1.The genome of BmDNV-3 contains two kinds different single-stranded linear DNA molecules（VD1,VD2）,and each of them is encapsidated respectively in the form of single-stranded liner DNA（+VD1,-VD1,+VD2,-VD2） in equal percentage.Viral DNA was extracted from the purified viral particles under high salt concentrations,the viral DNA hybridized upon extraction was blunted using T4 DNA polymerase（10 U each with 300 ng of DNA）,Restriction fragments digested with HindⅢwere cloned into HindⅢ/SmaⅠ-digested pUC119,and the complete nucleotide sequence was determined.Sequence analysis showed VD1 genome consisted of 6543 nts including inverted terminal repeats（ITRs） of 224 nts,and VD2 genome consisted of 6022 nts including ITRs of 524 nts.The GenBank accession numbers are DQ017268 and DQ017269,respectively.2.Computer analysis showed that the ’plus’ strand of the genome contained three large open reading frames（ORF1 to 3）,ORF1 and ORF2 most likely encoded non-structural proteins（NS） since they contained conserved replication initiator motifs and NTP-binding and helicase domains shared by all parvoviruses,and ORF3 encoded viral capsid proteins（VP）.The ’minus’ strand in the right-half had one open reading frame（ORF4）,it most likely encoded NS since it contains sequences conserved among a putative DNA polymerases.Analysis of the predicted DNA polymerase sequence using neighbor-joining and protein parsimony algorithms indicated the predicted 1115-residue polypeptide contain five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3′to 5′exonuclease activity.In VD2,one major open reading frame（ORF1） in the plus strand and one minor ORF2 in the complementary strand were identified. Computer analysis suggested the minus strand ORF2 most likely encode the major nonstructural protein,while the plus stand ORF1 most likely encodes the major structural protein.3.Comparison of the complete genome sequence between BmDNV-3 and BmDNV-2（Yamanashi isolate） showed an identity of 98.4%in VD1 and 97.7%in VD2, with a total number of 228 bp substitutions,11 bp deletions and 2 bp insertions found in BmDNV-3.Comparative polymorphisms of ORFs and ITRs regions of the two virus genomes showed that there were three highly variable regions（VD1 ORF3,VD2 ORF1, VD1ORF4 and ITRs）.These results make us further understand the strain heterogeneity and the possible reasons for variation of the virulence in this virus,and provide clues for their evolution.4.The transcripts of the virus were mapped to examine the transcription strategy of VD1.Northern blotting revealed one NS genes of 1.1 kb and another VP transcript of 1.5 kb in the left-half ’plus’ strand,and one transcript about 3.3 kb of ’minus’ strand in the right-half.Sequencing of 3′and 5′ends products of the 1.1 kb NS transcript demonstrated that it started nt 290 and ended at nt 1437.ORF1 and ORF2 were contained completely within this NS cassette but translated from a different reading frame,allowing leaky scanning translation of NS1 and NS2.The 1.5 kb VP transcript was found to start nt 1423 and ended at nt 2931.The VP（ORF3） and NS（ORF4） transcripts overlapped for 10 nts at the 3′ends（antisense RNAs） at 50 m.u.These results indicated that this genus transcription and translation strategies are radically different from that of other reported densonucleosis viruses.5.The midguts was homogenized and isopycnic centrifuged in 40%（W/W） cesium chloride（CsCl）,then each fraction of the gradient was submitted in parallel to quantitative PCR,the result showed the buoyant density of VD₁ and VD₂ were all approximately 1.29 g/cm³.The buoyant density of BmDNV-3 is smaller than that of other DNVs.Judging from copies of VD1 and VD2 in different gradients,we think they are probably in different capsids.In addition,the relative quantity of each type of viral protein was estimated after separation on SDS-polyacrylamide gel,four structural proteins with molecular weights of 48 kDa,55 kDa,72 kDa and 97 kDa were found, and 55 kDa was predominance.6.The prokaryotic expression plasmid pET28a（+）-VD1 ORF4 3′fragment was constructed,and then was transformed into BL21.The gene was expressed in the formation of inclusion bodies induced by IPTG.The expression products were with the weight of 62 kDa.In addition,the eukaryotic expression plasmid pF_ASTB_ACHTB-VD1 ORF4 3′fragment was constructed.更多还原