家蚕杆状病毒vp80, Bm21及家蚕SUMO基因研究
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摘要
家蚕核型多角体病毒(Bombyx mori nucloepolyhedrivirus，BmNPV)是杆状病毒的模式病毒之一，它的基因组全长128413bp，编码136个ORF，本文选择了一个BV和ODV都含有的结构蛋白基因Bmvp80基因，一个Group 1型杆状病毒特有的基因BmNPV ORF21基因进行了研究。同时研究了家蚕的一种新的修饰蛋白SUMO(small ubiquitin-like modifier)及其对BmNPV在BmN细胞中复制的影响。主要结论如下：
1、BmVP80功能研究
在大肠杆菌中表达了BmVP80部分片段，制备了多克隆抗体，免疫荧光定位发现BmVP80在细胞核内表达，利用ET重组系统构建了BmVP80敲除的BmBacmid，敲除后的病毒不能产生有侵染能力的BV，将完整的BmVP80异位补回后，敲除的病毒恢复了感染能力。透射电镜发现BmVP80敲除后核衣壳的数量明显下降。为了研究Bmvp80是否具有种属特异性，用在不同启动子启动下的Acvp80拯救BmVP80敲除的BmBacmid都告失败，表明Bmvp80有种属特异性。总的来说Bmvp80是BV产生和核衣壳成熟必需的，并且具有种属特异性。
2、BmNPV ORF21(Bm21)部分功能研究
Bm21是鳞翅目昆虫NPV特有的基因，在本实验中克隆和表达了该基因，RT-PCR分析了Bm21的转录时相，发现在12 h．i．开始转录，在BmN细胞中利用Bac-to-Bac表达系统间接定位发现Bm21表达产物在细胞核和细胞质中都有分布。利用ET系统构建了Bm21部分敲除的缺失病毒，转染细胞没有发现明显的细胞病变，表明Bm21的敲除影响了病毒的复制。
3、家蚕SUMO基因研究
BmSUMO编码一个含91个氨基酸的蛋白，序列分析发现它与SUMO2／3同源性较高(67％)，而与SUMO1只有51．7％的同源性。免疫荧光定位发现BmSUMO在整个细胞中都有分布，并且在细胞核内呈点状分布。Western blot实验表明在家蚕细胞内有许多蛋白是被SUMO修饰的。在极早期启动子IE-1启动下过量表达SUMO发现过量表达抑制了病毒感染后48小时内病毒的复制，在48小时后病毒的滴度恢复到了野生型水平。
The BMNPV genome was 128413 bp nucleotide long and contained 136 openreading frames (ORFs). In this study, Bmvp80 which is located at both BV and ODVof Bombyx mori nucleopolyhedrovirus (BmNPV), BmNPV orf21 (Bm21) which is theunique gene of group I NPVs, were characterized. At the same time, the characters ofSUMO gene of silkworm and the effect of over-expression of BmSUMO on BmNPVreplication in BmN cells were analyzed. The results were as following:
1. study of Bmvp80
Partial coding region of the Bmvp80 was cloned and expressed in E.coli BL21, andantiserum against BmVP80 was prepared. The sub-cellular localization of theBmVP80 proteins was investigated by immmunofluorescence. The fluorescence wasconcentrated within the nucleoplasm. A BmNPV Bacmid with the Bmvp80 genedisrupted was constructed using the ET-recombination system to investigate the roleof Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in singlecell infection phenotype, whereas a rescue BmBacmid restored BV titers to wild typelevels; however, the homologous gene Acvp80 from AcMNPV could not complementthe BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cellsinfected with BmNPV lacking functional Bmvp80 revealed that the number ofnucleocapsids was markedly lower. These results suggest that Bmvp80 is essential fornormal BV production and nucleocapsid maturation, and is functionally divergentbetween baculovirus species.
2. analysis of Bm21
The open reading frame 21 (Bm21) of BmNPV was one of the unique genes ofgroup I NPVs. In this study Bm21 was cloned and expressed. The RT-PCR analysis ofBm21 demonstrated that Bm21 transcribed as early as 12h p.i. and kept highly steadyat late phase. The Bm21 was located at both the nucleoplasm and the cytoplasm. ABmNPV Bacmid with the Bm21 gene disrupted was constructed using theET-recombination system, no obvious cytological change was observed in transfectedcells, which means the knockout of Bm21 may influence the replication of BmNPV.
3. analysis of BmSUMO
The SUMO of Bombyx mori (BmSUMO) contains 91 amino acids and the codingregion was interrupted by two introns. The BmSUMO was more similar to vertebrateSUMO-2/3, sharing 67% sequence identity with human sumo-2/3, while share only51.6% identity with human sumo-1. In BmN cells, many proteins were modified byBmSUMO peptide. Immunofluorescence microscopy demonstrated that theBmSUMO was present within the whole BmN cells and also aggregated as dots in thenucleus. The over-expression of BmSUMO under ie-1 promoter inhibited the growthof Bombyx mori nucleopolyhedrovirus (BmNPV) before the 48 p.i. of virusreplication cycle.
1、BmVP80功能研究
在大肠杆菌中表达了BmVP80部分片段，制备了多克隆抗体，免疫荧光定位发现BmVP80在细胞核内表达，利用ET重组系统构建了BmVP80敲除的BmBacmid，敲除后的病毒不能产生有侵染能力的BV，将完整的BmVP80异位补回后，敲除的病毒恢复了感染能力。透射电镜发现BmVP80敲除后核衣壳的数量明显下降。为了研究Bmvp80是否具有种属特异性，用在不同启动子启动下的Acvp80拯救BmVP80敲除的BmBacmid都告失败，表明Bmvp80有种属特异性。总的来说Bmvp80是BV产生和核衣壳成熟必需的，并且具有种属特异性。
2、BmNPV ORF21(Bm21)部分功能研究
Bm21是鳞翅目昆虫NPV特有的基因，在本实验中克隆和表达了该基因，RT-PCR分析了Bm21的转录时相，发现在12 h．i．开始转录，在BmN细胞中利用Bac-to-Bac表达系统间接定位发现Bm21表达产物在细胞核和细胞质中都有分布。利用ET系统构建了Bm21部分敲除的缺失病毒，转染细胞没有发现明显的细胞病变，表明Bm21的敲除影响了病毒的复制。
3、家蚕SUMO基因研究
BmSUMO编码一个含91个氨基酸的蛋白，序列分析发现它与SUMO2／3同源性较高(67％)，而与SUMO1只有51．7％的同源性。免疫荧光定位发现BmSUMO在整个细胞中都有分布，并且在细胞核内呈点状分布。Western blot实验表明在家蚕细胞内有许多蛋白是被SUMO修饰的。在极早期启动子IE-1启动下过量表达SUMO发现过量表达抑制了病毒感染后48小时内病毒的复制，在48小时后病毒的滴度恢复到了野生型水平。
The BMNPV genome was 128413 bp nucleotide long and contained 136 openreading frames (ORFs). In this study, Bmvp80 which is located at both BV and ODVof Bombyx mori nucleopolyhedrovirus (BmNPV), BmNPV orf21 (Bm21) which is theunique gene of group I NPVs, were characterized. At the same time, the characters ofSUMO gene of silkworm and the effect of over-expression of BmSUMO on BmNPVreplication in BmN cells were analyzed. The results were as following:
1. study of Bmvp80
Partial coding region of the Bmvp80 was cloned and expressed in E.coli BL21, andantiserum against BmVP80 was prepared. The sub-cellular localization of theBmVP80 proteins was investigated by immmunofluorescence. The fluorescence wasconcentrated within the nucleoplasm. A BmNPV Bacmid with the Bmvp80 genedisrupted was constructed using the ET-recombination system to investigate the roleof Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in singlecell infection phenotype, whereas a rescue BmBacmid restored BV titers to wild typelevels; however, the homologous gene Acvp80 from AcMNPV could not complementthe BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cellsinfected with BmNPV lacking functional Bmvp80 revealed that the number ofnucleocapsids was markedly lower. These results suggest that Bmvp80 is essential fornormal BV production and nucleocapsid maturation, and is functionally divergentbetween baculovirus species.
2. analysis of Bm21
The open reading frame 21 (Bm21) of BmNPV was one of the unique genes ofgroup I NPVs. In this study Bm21 was cloned and expressed. The RT-PCR analysis ofBm21 demonstrated that Bm21 transcribed as early as 12h p.i. and kept highly steadyat late phase. The Bm21 was located at both the nucleoplasm and the cytoplasm. ABmNPV Bacmid with the Bm21 gene disrupted was constructed using theET-recombination system, no obvious cytological change was observed in transfectedcells, which means the knockout of Bm21 may influence the replication of BmNPV.
3. analysis of BmSUMO
The SUMO of Bombyx mori (BmSUMO) contains 91 amino acids and the codingregion was interrupted by two introns. The BmSUMO was more similar to vertebrateSUMO-2/3, sharing 67% sequence identity with human sumo-2/3, while share only51.6% identity with human sumo-1. In BmN cells, many proteins were modified byBmSUMO peptide. Immunofluorescence microscopy demonstrated that theBmSUMO was present within the whole BmN cells and also aggregated as dots in thenucleus. The over-expression of BmSUMO under ie-1 promoter inhibited the growthof Bombyx mori nucleopolyhedrovirus (BmNPV) before the 48 p.i. of virusreplication cycle.
引文
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