Influence of BmNPV orf98 on DNA replication, transcription and virus package of Bombyx mori nucleopolyhedrovirus
Baculoviruses have been considered as the powerful vectors to express the exogenous gene. And the representative vectors in baculovirus expression vector system is Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). The AcMNPV expressi...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2015-11-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2015.01.191 |
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| Summary: | Baculoviruses have been considered as the powerful vectors to express the exogenous gene. And the representative vectors in baculovirus expression vector system is Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). The AcMNPV expression system has been widely applied in American and European countries. However, the BmNPV expression reaches a higher level over other systems, because BmNPV can infect silkworm larva or pupa. Moreover, silkworm is pretty normal in China, with lower cost and mature breeding technology, thus it is really popular to use the silkworm as a “biofactory” to produce recombinant protein. The BmNPV genome sequenced in 1999 was 128 413 nucleotides long with a G + C content of 40% and contained about 136 open reading frames (ORFs) encoding predicted proteins of over 60 amino acids.The gene of BmNPV orf98 is found in all Group Ⅰ and the most of Group Ⅱ genomes. It is not a highly conserved gene, as the deletion of this gene in BmNPV, it has no apparent effect on infectivity. The function of BmNPV orf98 has not been reported until now. In order to study the specific function of BmNPV orf98 gene, a BmNPV orf98 knockout bacmid by λ Red recombination was constructed, naming Bm98-ko-Bacmid. Additionally, Bm98-re-Bacmid was constructed by Bac-to-Bac system. BmN cells were infected with three kinds of virus DNA from wild-type bacmid (wtBacmid), Bm98-ko-Bacmid and Bm98-re-Bacmid, and the cells were respectively collected in 12 h, 24 h, 48 h and 72 h phases, then virus titer (50% tissue culture infective dose, TCID<sub>50</sub>) was determinated and virus proliferation curve was drawn, and total DNA was extracted using a eukaryotic DNA extraction kit. After Dpn Ⅰ enzyme digestion overnight, the effects of lacking BmNPV orf98 gene on virus replication and transcription were analyzed by fluorescence quantitative polymerase chain reaction (PCR).The results showed that the knockout bacmid was able to produce viral progeny after transfecting the DNA of Bm98 -ko-Bacmid into BmN cells, but the number of viral progeny reduced significantly (P<0.05); meanwhile, the virus infection level of repair was recovered which was similar with that of wild-type virus, indicating that the virus infection level in the incidence of BmN cells could be delayed after the deletion of BmNPV orf98. Assembly of virus was observed by transmission electron microscope in 48 h phase. The results indicated that, after 48 h of transfecting host cell, wtBacmid and Bm98-re-Bacmid viruses produced a large number of virus-packed capsule membrane except the BmNPV orf98 knockout virus. The BmNPV orf98 deletion didn't show significantly effects on viral DNA replication, suggesting that it was not essential for viral replication; however, the transcription of early gene lef3, late gene vp39 and very late gene p10 decreased obviously (P<0.05), because of lack of BmNPV orf98 gene.All the above results show that BmNPV orf98 gene is non-essential for viral replication, but it can significantly affect viral progeny and assembly, and lack of the gene will lead to significant decline of the transcription level of early gene lef3, late gene vp39 and very late gene p10. |
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| ISSN: | 1008-9209 2097-5155 |