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Continuous c-di-AMP production method

A technology of c-di-amp and recombinant plasmids, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of few and unindustrialized applications, and achieve the effect of reducing production costs

Active Publication Date: 2015-09-30
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many research reports on the fusion expression of CBM and DAC and the biocatalytic synthesis of c-di-AMP, let alone industrial applications

Method used

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  • Continuous c-di-AMP production method

Examples

Experimental program
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Effect test

Embodiment 1

[0064] In this example, taking Bacillus subtilis DisA as an example, the DisA-CMB gene recombination and recombinant expression plasmid construction, DisA-CMB protein expression and purification are briefly introduced as follows.

[0065] (1) DisA gene modification, by means of genetic engineering, the DisA gene and the cbm gene are fused to construct a recombinant plasmid vector; the schematic diagram of the pCG-DisA recombinant plasmid vector construction is as follows figure 2 Shown; specifically include the following steps:

[0066] First, primers are designed according to the known DisA gene sequence, and the corresponding DisA gene sequence is obtained by PCR amplification or whole-gene artificial synthesis technology; specifically, according to the sequence of the DisA gene of Bacillus subtilis and the structural characteristics of the pCG plasmid, upstream addition XhoI on the upper side, two restriction sites of BamHI on the lower side, the designed upstream and down...

Embodiment 2

[0106] On the basis of Example 1, this example further explains the process of one-step purification and solidification of the CBM-DAC fusion protein, and the process of using the DisA protein to catalyze the synthesis of c-di-AMP and further purification, as follows.

[0107] (3) One-step purification and immobilization of CBM-DAC fusion protein; the specific steps are as follows:

[0108] First, the microcrystalline cellulose is preactivated and regenerated with phosphoric acid according to the method provided in the literature (Nat. Commun., 2014, 5, 3026); the preferred particle size of the microcrystalline cellulose is 10-100 μm. Specifically:

[0109] Weigh 100g microcrystalline cellulose (Aladdin, C104843-250g), dissolve in 0.3L deionized water, and mix well;

[0110] Add 5L of pre-cooled phosphoric acid, stir and mix evenly while adding, and clarification appears;

[0111] Place on ice for 1 hour, stir once every 10 minutes; then add 20L pre-cooled deionized water, s...

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Abstract

The present invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a method for continuous production of c-di-AMP by using immobilized DisA protein. The method comprises: 1, DisA gene transformation, wherein DisA gene and cbm gene are fused through gene engineering to construct a recombinant plasmid vector; 2, recombinant plasmid transformation and CBM-DAC fusion protein expression; 3, one-step DAC enzyme purification and immobilization; 4, continuous c-di-AMP production; and 5, reaction product separation concentration and purification. According to the present invention, the characteristic that the CBM domain is combined with cellulose and no hydrolysis exists is specifically utilized, the process is mature, the production cost is low, the environmental protection is provided, the types of the prepared DAC enzyme are diversified, the sources are wide, the concentration and the purity are high, the enzymatic reaction efficiency is high, and the good development and application value is provided.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to a method for continuously producing c-di-AMP by using immobilized DisA protein. Background technique [0002] Cyclic 3', 5'-diadenosine monophosphate (c-di-AMP) is a new type of second messenger discovered in bacteria and archaea in recent years. As a signal molecule widespread in prokaryotic microorganisms, it is an essential factor for cell growth, not only involved in cell wall synthesis, cell shape control, bacterial DNA damage repair, but also with bacterial resistance to antibiotics, pathogenic bacteria Disease-related; c-di-AMP can also stimulate eukaryotic host cells to produce immune responses, mediate the host to produce interferon, and enhance the body's non-specific immune defense capabilities. However, the current research on the mechanism of action of c-di-AMP is still in its infancy and has become a research hotspot ...

Claims

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Application Information

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IPC IPC(8): C12P19/28C12N15/62C12N11/12C12R1/19
Inventor 杨国宇张改平杨森孔江南张超韩莹倩郭豫杰
Owner HENAN AGRICULTURAL UNIVERSITY
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