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Method for preparing second messenger molecule 2 '3'-cGAMP through solid-phase multienzyme coupling

A messenger and coupling technology, which is applied in the field of solid-phase multi-enzyme coupling to prepare the second messenger molecule 2'3'-cGAMP, can solve the problems of high cost, complicated purification steps, expensive and other problems, and achieves cost reduction and simple steps. , The effect of process environmental protection

Pending Publication Date: 2022-07-26
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, enzymatic synthesis requires more expensive GTP and a large amount of double-stranded DNA, which increases the cost of raw materials and increases the cost of purification
The subsequent purification steps of chemical synthesis are cumbersome and not conducive to environmental protection
Due to the above reasons, the yield of 2'3'-cGAMP is low, and the cost is as high as 7000 yuan / mg, which is very expensive

Method used

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  • Method for preparing second messenger molecule 2 '3'-cGAMP through solid-phase multienzyme coupling
  • Method for preparing second messenger molecule 2 '3'-cGAMP through solid-phase multienzyme coupling
  • Method for preparing second messenger molecule 2 '3'-cGAMP through solid-phase multienzyme coupling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of recombinant plasmid

[0038] 1. The mouse-derived nucleoside transferase cGAS gene was synthesized by Beijing AuGCT DNA-SYN Biotechnology Company, and then the mouse-derived nucleoside transferase cGAS gene was used as a template for PCR amplification to obtain the tNmcGAS gene sequence,

[0039] PCR primer sequences are as follows:

[0040] Upstream primer: 5'-ACGGATCCCCGGACAAGCTAAAGAAGGTG-3' (containing BamHI restriction site), downstream primer: 5'-ACGCTCGAGTCAAAGCTTGTCAAAAATTGG-3' (containing XhoI restriction site);

[0041] PCR amplification program: pre-denaturation, 95°C for 5min; denaturation, 95°C for 30sec; annealing, 60°C for 40sec; extension, 72°C for 30sec (30 cycles); termination extension, 72°C for 10min; final incubation at 4°C;

[0042] Through the conventional recombinant plasmid construction method, the gene sequences of the vector plasmids pET28a-SUMO and tNmcGAS were digested with BamHI and XhoI, respectively, and then lig...

Embodiment 2

[0048] Example 2 Construction of recombinant bacteria

[0049] 1. Conversion

[0050] Take 100 μL of BL21-Cod onPlus(DE3)-RIL Escherichia coli competent cells and put them in a 1.5 mL small tube, put the small tube on ice quickly, so that it is buried in ice except the cap, and flick the tube after thawing wall to resuspend the cells. 1 μL, 100 ng / μL of pET28a-Sumo-mcGAS plasmid was added to the competent cells, after ice bathing for 30 min, heat shock at 42°C for 90 s, and ice bathing again for 2 min. Add 400 μL of anti-LB liquid medium, mix well and incubate at 37°C with shaking at 200 rpm for 40-60 min. Take 100 μL of the culture solution, spread it evenly on LB solid medium, and invert at 37° C. for 12-16 hours to obtain recombinant bacteria mcGAS.

[0051] According to the same method, using Escherichia coli BL21 (DE3) as competent cells, recombinant bacteria GMK and recombinant bacteria NDK were obtained.

[0052] 2. Positive clone verification

[0053] Single colon...

Embodiment 3

[0058] like figure 2 As shown, a preparation method of a second messenger molecule 2'3'-cGAMP, comprising the following steps:

[0059] (1) Take 1 L of the recombinant bacteria mcGAS supernatant obtained in Example 2 and fix it on 1.5 mL of Co-binding gel resin, and take 1 L of the recombinant bacteria GMK supernatant obtained in Example 2 and fix it on 5 mL of Co-binding gel resin On the gel resin, take 1L of the recombinant bacterial NDK supernatant obtained in Example 2 and fix it on 5mL of Co-binding gel resin, then use 20mM Tris pH7.5, 100mMNaCl, 5mM imidazole to remove impurities, and finally use 50mM Tris pH7 .5 cleaning to obtain cGAS immobilized resin, GMK immobilized resin and NDK immobilized resin;

[0060] (2) prepare 300mL reaction system, the molar concentration of each component in the reaction system is 50mM Tris-HCl (pH7.0), 10mM MgCl 2 , 8mM ATP (pH 7.0), 2mM GMP, 0.5μM GMK immobilization resin (0.5mL, containing GMK enzyme 3.9mg), 1μM NDK immobilization r...

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Abstract

The invention relates to a method for preparing a second messenger molecule 2 '3'-cGAMP through solid-phase multienzyme coupling. Comprising the following steps: constructing a recombinant bacterium mcGAS, a recombinant bacterium GMK and a recombinant bacterium NDK, preparing cGAS immobilized resin, GMK immobilized resin and NDK immobilized resin, and preparing a second messenger molecule 2 '3'-cGAMP through solid-phase multienzyme coupling. According to the invention, murine nucleoside transferase cGAS is adopted; the 2 '3'-cGAMP is efficiently synthesized through a multi-enzyme coupling reaction of staphylococcus aureus guanylate kinase GMK and escherichia coli nucleoside diphosphate kinase NDK, the yield reaches 179.8 mg, the total yield reaches 41.2%, only ATP and GMP are used as substrates, large-scale production of the 2 '3'-cGAMP is achieved without additionally adding exogenous DNA, and the cost of the 2 '3'-cGAMP is effectively reduced.

Description

technical field [0001] The invention relates to a method for preparing the second messenger molecule 2'3'-cGAMP by solid-phase multi-enzyme coupling, and belongs to the technical field of biochemistry and genetic engineering. Background technique [0002] 2'3'-cGAMP (2'3'-cyclic guanosine monophosphate-adenosinemonophosphate) is an important second messenger in mammalian innate immune pathways and is now widely used in various research fields, such as biology, pharmacy, and clinical research. Recent studies have shown that 2'3'-cGAMP is expected to be a vaccine adjuvant against viruses in humans, and even a new generation of immunotherapy drugs for cancer. [0003] The use of multi-enzyme coupling method to synthesize biological small molecules is a conventional technical solution, which has been widely used in the biosynthesis of amino acids, nucleic acids and other compounds. At the same time, immobilized enzymes have the advantages of being easy to separate from the reac...

Claims

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Application Information

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IPC IPC(8): C12P19/32C12N15/54C12N15/70C12R1/19
CPCC12P19/32C12N9/1241C12N9/1229C12Y207/04008C12Y207/04006C12N15/52C12N15/70
Inventor 朱德裕王晓丹张展
Owner SHANDONG UNIV
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