Universal strong promoter among species derived from white spot syndrome virus and application thereof

A technology of white spot syndrome and strong promoter, which is applied in the field of promoters, can solve the problems of not widely used, high experimental cost, and poor versatility, and achieve the effects of strong activity, improved efficiency, and strong versatility

Inactive Publication Date: 2010-09-01
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there are powerful promoters in several expression systems commonly used at present, which can ensure the high-efficiency expression of genes, but because the promoters carried by the vectors are species-specific, these expression vectors are universal among different expression systems Not strong, so when it is necessary to express foreign genes in different species, people need to replace the appropriate expression vector for the target gene through experimental steps such as gene amplification, enzyme digestion, and ligation, which is time-consuming and laborious
Although some companies have developed some methods based on DNA recombination technology (such a

Method used

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  • Universal strong promoter among species derived from white spot syndrome virus and application thereof
  • Universal strong promoter among species derived from white spot syndrome virus and application thereof
  • Universal strong promoter among species derived from white spot syndrome virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of Very Early Transcription Samples of White Spot Syndrome Virus

[0038] In order to screen very early genes, it is first necessary to prepare very early transcription samples of white spot syndrome virus. Select one of the hosts of white spot syndrome virus, Procambarus clarkii as the experimental material, and extract the hemolymph of Procambarus clarkii without the virus after being detected: first use a 2ml disposable syringe to draw anticoagulant (0.8% sodium citrate , 0.05% citric acid, 1.87% glucose, 0.42% sodium chloride, pH5.0), blood was collected from the heart of Procambarus clarkii, and the volume of hemolymph collected could not exceed the volume of anticoagulant. Immediately after blood collection, invert and mix well. After pulling out the needle, inject the mixture of blood cells and anticoagulant in the needle tube into a sterile centrifuge tube. After collecting an appropriate amount of hemolymph, it was centrifuged at room tem...

Embodiment 2

[0039] Example 2 Chip Screening of Very Early Genes

[0040] Chip preparation:

[0041]According to the sequence of the white spot syndrome virus (Chinese strain) genome (GenBank no.AF332093), CapitalBio Co., Ltd. (CapitalBio Company) was entrusted to apply computer analysis to design primers, and use about 1kb as a basic unit to amplify gene fragments by PCR , the probes needed for the preparation of the whole genome chip of white spot syndrome virus were obtained. Using a gene chip spotting machine to spot the probes on the glass slide, the whole genome chip of white spot syndrome virus was prepared, and the positive and negative control genes were also included on the chip (see reference 6, Fang Li, Mingyuan Li, Wei Ke , Yong chang Ji, Xiaofang Bian, Xiumin Yan. Identification of the immediate-early genes of white spot syndrome virus. Virology. 2009 Mar 1; 385(1): 267-274).

[0042] Chip hybridization:

[0043] The RNA samples of the very early gene screening group and t...

Embodiment 3

[0049] Example 3 RT-PCR verification whether WSV249 is a very early gene of white spot syndrome virus

[0050] In order to further test whether WSV249 is the very early gene of white spot syndrome virus, 4 groups of samples were prepared:

[0051] 1. Negative samples treated with cycloheximide: Procambarus clarkii cells were treated with cycloheximide and were not infected with white spot syndrome virus;

[0052] 2. Infected samples treated with cycloheximide: the cells of Procambarus clarkii were treated with cycloheximide, and then the cells of Procambarus clarkii infected by white spot syndrome virus were added in the presence of cycloheximide;

[0053] 3. Normal cell samples: Procambarus clarkii cells without cycloheximide and without infection with white spot syndrome virus;

[0054] 4. Normal infected cell sample: Procambarus clarkii cells infected with white spot syndrome virus were added without adding cycloheximide.

[0055] Among them, the treatment concentration o...

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Abstract

The invention relates to a universal strong promoter among species derived from white spot syndrome virus and an application thereof. The invention provides the universal strong promoter among the species derived from the white spot syndrome virus, two primers of the strong promoter, an expression vector of the strong promoter and recombinant host cells of the expression vector. The promoter is an immediate early gene promoter of the white spot syndrome virus and can high-efficiently start the expression of downstream genes in insect cells, mammalian cells and the white spot syndrome virus host cells (including cells of crayfish and shrimp). The recombinant host cells are obtained by transfection of the expression vector containing the universal strong promoter among the species derived from the white spot syndrome virus in the host cells, and the recombinant host cells can express target protein genes controlled by the universal strong promoter among the species derived from the white spot syndrome virus.

Description

technical field [0001] The present invention relates to a promoter, in particular to a strong interspecies general promoter derived from white spot syndrome virus, and an expression vector containing the above promoter, which is used for expressing foreign genes in cells of different species. Background technique [0002] In the field of genetic engineering, the recombinant expression of target genes is of great significance both in theoretical research and practical application. To express the target gene in the host cell, it must be cloned into a genetic engineering vector with various elements required for gene expression, which is called an expression vector. Gene expression systems can be divided into prokaryotic expression systems and eukaryotic expression systems. [0003] Escherichia coli is currently the most widely used prokaryotic expression system, which can express and purify recombinant proteins conveniently and efficiently. However, when the target gene is a...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/63C12N5/10
Inventor 李钫柯韡林梵宇鄢秀敏杨丰
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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