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Novel Infection System of Recombinant BmBDV DNA into BmN Cells of Silkworm, Bombyx mori

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Abstract

Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous single-stranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.

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Acknowledgments

Authors greatly acknowledge the financial support of the National Natural Science Foundation of China (31272500), State Key Laboratory of Silkworm Genome Biology (SKLSGB201200011), the National Basic Research Program of China (973 Program, 2012CB114600), the Specialized Research Fund for the Doctoral Program of Higher Education (20113201130002), and a project funded by the Priority Academic Program of Development of Jiangsu Higher Education Institutions. Thanks to Prof. Xijie Guo for providing plasmid pGEM-VD1, and to Prof. Keping Chen and Prof. Xiaofeng Wu for providing antibodies.

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    Authors and Affiliations

    1. College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

      Rui Guo

    2. School of Biology and Basic Medical Science, Soochow University, Suzhou, 215123, China

      Rui Guo, Guangli Cao, Yuexiong Zhu, Dhiraj Kumar, Renyu Xue, Yahong Lu, Xiaolong Hu & Chengliang Gong

    3. National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, People’s Republic of China

      Guangli Cao, Renyu Xue, Xiaolong Hu & Chengliang Gong

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    1. Rui Guo
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    Correspondence to Chengliang Gong.

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    Rui Guo and Guangli Cao have contributed equally to this work.

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    284_2016_1102_MOESM1_ESM.docx

    Supplementary material 1 (DOCX 40 kb) Fig. S1 Construction steps of recombinant baculovirus BmBac-VD1. VD1 genome excised from the pGEM-VD1 with restriction endonucleases SphI and EcoRI was subcloned into pFastBac™ Dual to obtain the donor plasmid pFastBac™ Dual-VD1, then the constructed plasmid was transformed into E. coli DH10 containing B. mori nucleopolyhedrovirus bacmid and helper plasmid. The transformations were screened on a LB agar medium containing kanamycin (50 µg/ml), gentamycin (7 µg/ml),tetracycline (10 µg/ml), X-Gal (100 µg/ml) and IPTG (40 µg/ml) to generate the recombinant Bmbacmid-VD1 according to the protocol of Bac-to-Bac baculovirus expression system

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    Guo, R., Cao, G., Zhu, Y. et al. Novel Infection System of Recombinant BmBDV DNA into BmN Cells of Silkworm, Bombyx mori . Curr Microbiol 73, 587–594 (2016). https://doi.org/10.1007/s00284-016-1102-0

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