Main cis-acting element of shrimp white spot syndrome virus (WSSV) iel promoter and transcription factor combined with same and application

A cis-acting element, vitiligo technology, applied in applications, antiviral agents, gene therapy, etc., can solve the lack of understanding of virus-host interaction, invertebrates without a specific immune system, and the lack of effective prevention and treatment of viral diseases methods, etc.

Inactive Publication Date: 2011-09-07
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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Problems solved by technology

The complete sequencing of the genomes of three strains of WSSV in 2001-2002 opened the prelude to the study of WSSV molecular virology, but the analysis found that most of its open reading frames had no homology with the existing gene sequences in GenBank, so it is very difficult to Difficult to study gene expression and replication patterns of other viruses
[0004] Although some progress has been made in the molecular biology research of WSSV, the most important thing is to focus on the structural protein of the virus; there is no cell line suitable for the grow

Method used

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  • Main cis-acting element of shrimp white spot syndrome virus (WSSV) iel promoter and transcription factor combined with same and application
  • Main cis-acting element of shrimp white spot syndrome virus (WSSV) iel promoter and transcription factor combined with same and application
  • Main cis-acting element of shrimp white spot syndrome virus (WSSV) iel promoter and transcription factor combined with same and application

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Embodiment

[0025] 1. Materials and methods

[0026] 1.1 Reporter gene plasmid construction and its deletion and mutation analysis

[0027] Using the WSSV viral genome DNA as a template, a series of deletion and mutation analyzes were performed on the ie1 gene promoter from the 5' and 3' ends by using the primer sequence PCR method in Table 1. For the methods and strategies, see figure 1 , the position of the gene regulatory element in the mutant was deduced from the ie1 transcription start site (+1). Through a series of 5' and 3' deletion analysis, using different primer sequence combinations, a series of 5' and 3' promoter deletion sequences were generated. For mutation analysis of Sp1 binding site motif (TGTGGGCGGAGCA) and TATA box (GTGTATATAAGAGCC), site-directed mutagenesis ie1 promoter clones were obtained by performing PCR with mutation primers Mut1 and Mut2, and each PCR product was digested with Kpn I and Hind III , cloned into the phRG-B vector.

[0028] Table 1 is used to co...

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Abstract

The invention discloses a main cis-acting element of a shrimp white spot syndrome virus (WSSV) iel promoter and a transcription factor combined with the same and application. In the invention, by starting from transcriptional regulation of WSSV iel and carrying out structural and functional analysis on the promoter of the WSSV iel through deletion and mutation, a 12-bp DNA is found to be the maincis-acting element of the WSSV iel and is a crucial factor for the high expression of the iel. In the invention, a DNA affinity chromatography method is used for purifying a protein combined with a DNA segment from a nucleus protein Sf9, the protein is identified to be PHB2 (Poly-Beta-Hydroxybutyrate 2) through biological mass spectrometry, and the interaction between the protein and the DNA segment is proved to be specific by an electrophoretic mobility shift assay. Experiment results prove that PHB2 serves as a transcription factor and is specifically combined with a 12-bp DNA sequence of the iel promoter to start WSSV immediate early gene transcription so as to further regulate the replication of the WSSV, and can be used as an effective action target of medicaments for screening medicaments for resisting the shrimp WSSV.

Description

technical field [0001] The invention relates to a main cis-acting element of a viral gene promoter and a transcription factor combined with it, in particular to the main cis-acting element of a shrimp white spot syndrome virus (WSSV) ie1 promoter and a transcription factor combined with it. Their application, the invention belongs to the field of expression and regulation of shrimp white spot syndrome virus gene. Background technique [0002] Shrimp white spot syndrome (WSS) is the most serious infectious disease to the shrimp industry. The pathogen is white spot syndrome virus (WSSV). The disease first broke out in Taiwan, China in the 1990s, and then spread rapidly to the main shrimp farming areas throughout Asia, Europe and the United States. The virus has strong infectivity and replication ability, and the host mortality rate can reach 100%. It can infect various tissues of prawns and has a wide range of hosts. It can infect prawns, crayfish, crabs, and freshwater crust...

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Application Information

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IPC IPC(8): C12N15/113C12N15/12C07K14/435A61K48/00A61K38/17A61P31/20
Inventor 于力马国达
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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