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

Active Publication Date: 2018-12-14
SOUTHWEST UNIV
View PDF6 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no better induction system in insect genetic engineering research, so it is of great significance to construct disease-inducible promoters in disease resistance breeding and gene therapy

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Bombyx mori nuclear polyhydrosis virus (BmNPV) inducible 39K promoter, and recombinant vector and application thereof
  • Bombyx mori nuclear polyhydrosis virus (BmNPV) inducible 39K promoter, and recombinant vector and application thereof
  • Bombyx mori nuclear polyhydrosis virus (BmNPV) inducible 39K promoter, and recombinant vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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'...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a BmNPV inducible 39K promoter, and a recombinant vector and application thereof. The promoter uses the optimal sequence of a promoter of the BmNPV delayed early gene 39K as aparental promoter; the optimal sequence is gradually truncated and analyzed to reduce the length of the promoter, and a recombinant promoter is constructed and still has strong BmNPV induced promoteractivity; and it is verified that the immediate early gene IE-1 protein of BmNPV can transcribe and bind to the 39K (-310 to -355) region so as to induce the expression of the 39K promoter. Accordingto the invention, the region is used for constructing different synthetic inducible promoters, and the inducible expression activity of 39K and other different promoters can be efficiently improved;and the synthetic inducible promoter are applicable to molecular biological theory analysis such as gene function analysis, the application research on the expression system of BmNPV and the improvement of Bombyx mori varieties via the genetic engineering technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Bombyx mori nucleopolyhedrosis virus inducible 39k promoter, and also relates to a recombinant vector containing the promoter and its application. Background technique [0002] Silkworm is a model insect of Lepidoptera with important economic value. The silk industry has made important contributions to the world's economic, cultural and social development. Silkworm and silkworm nuclear polyhedrosis virus, as bioreactors for high-efficiency expression of foreign proteins, have important application value in the field of bioengineering. The low expression activity and non-specificity of natural promoters have certain limitations in the application of bioengineering. In the previous research work, the inventor's research group invented the BmNPV 39K inducible promoter (ZL201010231957.9) and the modified enhanced En39K (ZL201310641709.5). The promoter has BmNPV-induced start-up activi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/866A01K67/033
CPCA01K67/0335A01K2227/706C12N15/113C12N15/86C12N2710/14143
Inventor 潘敏慧董战旗鲁成陈鹏曹明亚李海清蒋亚明胡志刚
Owner SOUTHWEST UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap