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Baculovirus-mediated gene transfer systems in silkworm larvae using constitutive host promoters
Jae Man Lee,Jian Xu,Hiroaki Mon,TakumiMitsudome,Atsushi Masuda,Kaito Yoshimura,Kazuhiro Iiyama,Yuuka Chieda,Takahiro Kusakabe 한국응용곤충학회 2014 Journal of Asia-Pacific Entomology Vol.17 No.1
Baculoviruses serve as efficient viral vectors for gene delivery into vertebrate and invertebrate cells. The identificationand characterization of the functional promoters in different baculovirus-infected hosts are essentialfor the efficient gene expression. To establish a baculovirus-mediated gene transfer system in the silkworm,Bombyxmori, we investigated the activities of silkworm-derived TCTP, ACTIN3, and HSC70-4 promoters deliveredby AcNPV or BmNPV in various tissues of silkworm. In many of the tested silkworm tissues, the BmHSC70-4promoter exhibited a higher transcription activity than those of BmTCTP or BmACTIN3 promoters when deliveredby AcNPV, which is reported to be incapable of replicating in silkworms. In contrast, the BmACTIN3 promoter wasfound to be the strongest promoterswhen delivered by BmNPV. The present results indicate that the BmHSC70-4promoter is potentially useful for the stable gene expression by the non-replicating AcNPV vector for gene functionanalysis in the silkworm.
Masato Hino,Takuji Kawanami,Jian Xu,Daisuke Morokuma,Kazuma Hirata,Mami Yamashita,Noriko Karasaki,Tuneyuki Tatsuke,Hiroaki Mon,Kazuhiro Iiyama,Noriho Kamiya,Yutaka Banno,Takahiro Kusakabe,이재민 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.2
Interleukin 2 (IL-2) is a pharmacologically vital cytokine secreted mainly by activated CD4+ T lymphocyte. Recombinant human IL-2 (rhIL-2) protein has already been globally applied as an immune-therapeutic reagent for various diseases. Therefore, there is great interest in developing an active form of rhIL-2 in very large amounts and with excellent purity for clinical use. In this study, we successfully mass-expressed and purified N- or C-terminal tandem tag-fused rhIL-2 in a baculovirus expression vector system (BEVS) using silkworm larvae as factories. We confirmed that the intrinsic instability of hIL-2 causes the loss and low recovery of N-tagged rhIL-2 and that C-tagged rhIL-2 ismore suitable for mass production. Furthermore, the activity of purified rhIL-2s was further validated by a cell proliferation assay of a human natural killer cell line, and both rhIL-2 proteins produced in the silkworm-BEVS exhibited comparable activity to that of the commercial E.coli-derived rhIL-2. Taken together, our results and strategies could contribute greatly to the mass production of active rhIL-2.