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Construction method and application of engineering bacteria capable of highly-producing CpG-ISS

A construction method and technology of engineering bacteria, which are applied in the directions of fungi, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve the problems of low production efficiency and high production cost of CpGISS, and achieve high production costs and simple and feasible construction methods. , the effect of increasing gene expression

Pending Publication Date: 2018-12-07
浙江皇冠科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The construction method of engineering bacteria is simple and feasible, and the constructed engineering bacteria grow and reproduce rapidly, are not pathogenic, do not produce toxins, are safe and reliable, and can maintain the activity and stability of CpG-ISS products, effectively solving the problem of high production cost of CpG ISS, The problem of low production efficiency

Method used

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  • Construction method and application of engineering bacteria capable of highly-producing CpG-ISS
  • Construction method and application of engineering bacteria capable of highly-producing CpG-ISS

Examples

Experimental program
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Effect test

Embodiment 1

[0026] A method for constructing high-yielding CpG ISS engineering bacteria, based on 10 CpG-ISSs that have better immune activity to aquaculture fish and shrimp, 10 CpG-ISSs are connected in series and artificially synthesized, and the homologous enzyme digestion technology is used to The tandem sequences were cloned five times in the pYES2 vector to construct a plasmid vector pYES2-90CpG with multiple copies of the CpG motif (90 copies), and introduced into Saccharomyces cerevisiae INVSc1 strain by electroporation.

[0027] The specific steps of the construction method are:

[0028] Step 1: Take 25 CpG ISSs that have immune activation effects in different animals such as humans, mice, and fish as templates, and use macrophage respiratory burst, phagocytosis, lysozyme, phenoloxidase, and serum bactericidal activity as indicators , to detect its immune activation effect in aquatic animals, and finally screen out 10 most suitable CpG ISSs for aquaculture animals, and reserve th...

Embodiment 2

[0040]A method for constructing high-yield CpG ISS engineering bacteria, the specific steps of which are:

[0041] Step 1: Take 25 CpG ISSs that have immune activation effects in different animals such as humans, mice, and fish as templates, and use macrophage respiratory burst, phagocytosis, lysozyme, phenoloxidase, and serum bactericidal activity as indicators , to detect its immune activation effect in aquatic animals, and finally screen out 10 most suitable CpG ISSs for aquaculture animals, and reserve them;

[0042] Step 2: Connect the 10 nucleotide sequences screened in step 1 in sequence, and introduce an Xho I restriction site at the 3' end to form a DNA fragment containing the 18CpG motif, and then mix it with annealing buffer and sterile water , incubate in a hot water bath at 90°C for 5 minutes, cool naturally to room temperature after incubation, and then clone the DNA fragment into the pMD18-T vector, and the cloned plasmid is called pMD18-T / 18CpG;

[0043] Step ...

Embodiment 3

[0053] The method for engineering bacteria to produce CpG ISS, the further optimization steps are:

[0054] 1) Activate the engineered bacteria and inoculate them into YPD medium and fermentation medium for shake flask culture of primary and secondary species, and inoculate the culture of secondary species into fermenters for fed-batch culture to maximize activation For the expression of the CpG ISS gene fragment, the thalline is collected after the cultivation of the yeast engineered bacterium is completed, washed with distilled water and then used for subsequent use;

[0055] 2) Add STET buffer solution to the washed yeast cells, stir magnetically for 30 minutes under the condition of 1000r / min, then add freshly prepared lysozyme to a final concentration of 100μg / mL, stir well and place in a 38°C refrigerator In a water bath for 25 minutes, then add cell lysate, heat in a boiling water bath for 15 minutes, rapidly cool to room temperature, then centrifuge at 12,000 r / min for...

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Abstract

The invention discloses a construction method and an application of engineering bacteria capable of highly-producing CpG-ISS. The construction method includes: on the basis of ten CpG-ISSes, which have excellent immunoactivity on aqua-cultured fish and shrimps, mutually linking the ten CpG-ISSes in series and performing artificial synthesis; repeatedly cloning the in-series sequence in a pYES2 carrier for five times through isocaudamer digestion technology, thus forming a plasmid vector pYES2-90CpG of poly-copied CpG motif; through electro-transformation, introducing the product into saccharomyces cerevisiae strain to obtain engineered saccharomyces cerevisiae, finally screening out the engineering bacteria capable of highly-producing CpG-ISS. The method is simple and available, wherein the constructed engineering bacteria can be rapidly reproduced, are safe and reliable and are high in yield and productivity of the CpG-ISS. The engineering bacteria also can maintain the activity and stability of the CpG-ISS product and can be applied to prevention and control on aquatic animal diseases.

Description

technical field [0001] The invention relates to the technical field of construction of engineering bacteria, in particular to a construction method and application of high-yield CpG ISS engineering bacteria. Background technique [0002] In recent years, my country's livestock, poultry and aquaculture industry has developed rapidly, and its total output ranks first in the world in the same industry. It not only meets the needs of the domestic people, but also is the main agricultural product for export. However, with the rapid development of the breeding industry, there has also been a very serious problem, that is, focusing on the increase of production, but ignoring the quality of the product, the meat quality and taste of the product have declined, and the safety cannot be guaranteed. have a serious negative impact on the vast number of consumers. Analysis of the reasons for the decline in the quality of aquaculture products is mainly due to the three major factors of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19A61K36/064A61P37/04
CPCA61K36/064A61P37/04C12N1/18C12N15/81
Inventor 胡向东项黎新叶茂
Owner 浙江皇冠科技有限公司
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