| Biological sciences & biotechnology |
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| Influence of BmNPV orf98 on DNA replication，transcription and virus package of Bombyx mori nucleopolyhedrovirus. |
| Shi Lili, Jiang Caiying, Yu Wei, Chen Chen, Jiang Lei, Gong Chengjian, Tong Fudan* |
| (Key Laboratory of Silkworm Bioreactor and Biological Medicine in Zhejiang, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China) |
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Abstract 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 λRed 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, TCID50) was determinated and virus proliferation curve was drawn, and total DNA was extracted using a eukaryotic DNA extraction kit. After DpnⅠ 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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Published: 20 November 2015
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BmNPV orf98对家蚕核型多角体杆状病毒复制、转录及包装的影响
为了研究家蚕核型多角体病毒(Bombyxmori nucleopolyhedrovirus,BmNPV)基因orf98 的功能,通过λRed重组系统定点敲除BmNPVorf98 基因,构建缺失型重组病毒Bm98-ko-Bacmid;以Bac-to-Bac系统补回BmNPVorf98 基因,构建补回型重组病毒Bm98-re-Bacmid;将野生型病毒(wtBacmid)、缺失型病毒(Bm98-ko-Bacmid)和补回型病毒(Bm98-re-Bacmid)分别转染家蚕细胞BmN.病毒滴度检测结果显示,Bm98-ko-Bacmid可形成侵染性的病毒粒子,但数量显著降低(P<0.05).透射电子显微镜观察发现,Bm98-ko-Bacmid只产生游离的杆状病毒粒子,数量明显减少,而wtBacmid和Bm98-re-Bacmid产生大量具有囊膜结构的成熟病毒粒子.荧光定量聚合酶链反应分析结果表明,BmNPVorf98 基因缺失对BmNPV病毒复制没有影响,而早期基因lef3、晚期基因vp39 和极晚期基因p10 的转录水平显著降低(P<0.05).综上所述,BmNPVorf98 基因对病毒复制是非必需的,但显著影响病毒的繁殖速度和包装(P<0.05);对病毒各个时期的基因转录也具有重要影响.
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