Bombyx mori nuclear polyhydrosis virus (BmNPV) inducible 39K promoter, and recombinant vector and application thereof
A nuclear polyhedron and promoter technology, applied in virus/phage, application, virus and other directions, to achieve good application prospects, eliminate disease spread, and improve the effect of induction activity
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Embodiment 1、39
[0036] Example 1. Structural and functional analysis of the 39K promoter
[0037] To generate a more optimized virus-inducible specific promoter, the 39K promoter core region was gradually removed using a truncation and mutation strategy, and the changes in 39K promoter activity were analyzed. Firefly luciferase (FLUC) controlled by the promoter and sea cucumber luciferase (RLUC) controlled by the promoter of the control plasmid IE1 (SEQ ID No.7) were co-transfected into BmN-SWU1 cells, and then the cells were infected with BmNPV, Cells were collected 72 hours after infection, lysed, and centrifuged to take the supernatant. The PPL and PRL of the lysed supernatant were measured with a dual-luciferase reporter gene detection kit, and the relative luciferase activity was calculated to investigate the effect of transfected BmN-SWU1 cells on BmNPV infection. Post 39K promoter activity. Promoter activity was identified by detecting changes in relative luciferase FLUC / RLUC ( figur...
Embodiment 2
[0040] Example 2, construction of artificially inducible 39K promoter
[0041] Based on the deletion analysis of the 39K promoter, the regions contributing to the activity of the 39K promoter were roughly identified. At the same time, the key regulatory elements of the promoter core region were analyzed by the promoter prediction program. The results of online analysis showed that the 39K promoter contained core components, such as two enhancer components CGTGCGC, six CAAT sites, two transcriptional inhibitors TGAC, two cis-regulated pro-CCAT and two TATA boxes ( Figure 5 ). Combining the positions of the core elements and key regulatory regions of the 39K promoter, three artificially inducible promoters were first constructed, including p39K-1 (-573~-273 and +1~+62 fragments) (SEQ IDNo.2), p39K- 5 (-573 to -273 and +1 to +134) (SEQ ID No. 4), p39K-9 (-773 to -273 and +1 to +134) (SEQ ID No. 5). The activity of P39K-1, P39K-5 and P39K-9 promoter reached 87.24%, 75.94% and ...
Embodiment 3
[0042] Example 3, Identification of the transcriptional regulatory protein of the inducible promoter 39K
[0043] Expression of silkworm nuclear polyhedrosis virus genes is regulated by a cascade, and each subsequent gene expression is dependent on the previous one. Bombyx mori nuclear polyhedrosis virus 39K gene is a delayed early expression gene. In order to determine the transcriptional binding protein of the 39K promoter, first download the whole genome sequence of BmNPV (NC001962.1) from the National Center for Biotechnology Information (NCBI), design and synthesize five immediate early genes (IE-0, IE-1, IE -2, PE38 and ME53) protein primers and add corresponding restriction enzyme sites (underlined sequences in the following primer sequences) at the two sections of the primers:
[0044] IE0(EcoR I)-F: 5'-c ggaatt catgataagaaccagcagtc-3' (SEQ ID No. 11);
[0045] IE0 (Not I)-R: 5'-ataagaat gcggccg ctttatacgatgtcctgca-3' (SEQ ID No. 12);
[0046] IE1 (EcoR I)-F: 5'...
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