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Preparation and activity identification method of a pig-derived second messenger molecule 2′3′-cgamp

An active, porcine-derived technology, applied in the field of preparation and activity identification of porcine-derived second messenger molecule 2'3'-cGAMP, can solve the problems of limited wide application, difficult separation and purification, complicated process, etc. Low cost, broad antiviral effect

Active Publication Date: 2021-06-01
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as the existing technology is concerned, 2′3′-cGAMP is currently obtained mainly through tissue purification and chemical synthesis methods, but these two methods are inefficient, difficult to separate and purify, complicated processes and high costs, which limit their wide application

Method used

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  • Preparation and activity identification method of a pig-derived second messenger molecule 2′3′-cgamp
  • Preparation and activity identification method of a pig-derived second messenger molecule 2′3′-cgamp
  • Preparation and activity identification method of a pig-derived second messenger molecule 2′3′-cgamp

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Preparation of recombinant pig-derived 2′3′-cGAMP synthetase pcGAS

[0065] The amino acid sequence of the pcGAS enzyme (porcine cyclic guanylate-adenylate synthetase) is shown in Sequence 1 of the Sequence Listing, wherein the active catalytic domain of the enzyme is at positions 136-495 from the N-terminus. The coding gene of the pcGAS enzyme is shown in Sequence 2 of the Sequence Listing, wherein the enzyme activity catalytic domain is encoded from the 406-1488 position at the 5' end.

[0066] 1. Replace the fragment between the BamH I and Hind III sites of the prokaryotic expression vector pET-28a-SUMO (Wuhan Miaoling Biotechnology Co., Ltd., article number: P0028) with sequence 2 from the 5' end 406- The DNA molecule indicated at position 1488 was used to obtain the recombinant expression vector pET-28a-SUMO-pcGAS (sequencing verification).

[0067] 2. Introduce the recombinant expression vector pET-28a-SUMO-pcGAS obtained in step 1 into Escherichia col...

Embodiment 2

[0075] Example 2. Using recombinant pcGAS enzyme to catalyze the synthesis of pig-derived 2'3'-cGAMP

[0076] Using the pig-derived recombinant pcGAS enzyme prepared in Example 1 to efficiently and specifically catalyze the synthesis of pig-derived 2′3′-cGAMP in vitro, the details are as follows:

[0077] 1. Configure the enzymatic reaction system (as shown in Table 1). The enzymatic reaction system was configured on ice.

[0078] Table 1 Enzymatic reaction system

[0079]

[0080] 2. After completing step 1, incubate at 37°C for 2 hours, then add 50 μL of nuclease Benzonase, and incubate at 37°C for 30 minutes.

[0081] 3. After completing step 2, incubate the reaction product at 95°C for 10 minutes, then centrifuge at 16000g / min for 10 minutes, and take the supernatant. The supernatant was filtered with a 0.5ml / 10kDa ultrafiltration centrifuge tube (Merck-Millipore, catalog number: UFC501096) to obtain the reaction product.

[0082] The absorbance value of the reaction ...

Embodiment 3

[0084] Example 3: Activity identification of pig-derived pcGAS and catalytic product 2′3′-cGAMP

[0085] RAW264.7-Lucia TM ISG cells are a reporter cell in which the expression of luciferase gene (Lucia luciferase gene) is jointly driven by the ISG54 promoter and the interferon stimulation response element ISRE. When stimulated by 2′3′-cGAMP, by RAW264.7-Lucia TM The STING molecule expressed by ISG cells recognizes and activates the transcription factors IRF3 and IRF7, and then combines with ISRE to induce the secretion of luciferase gene, and then through QUANTI-Luc TM The fluorescence value of luciferase was detected to reflect the enzymatic activity of pcGAS and the stimulating activity of 2′3′-cGAMP.

[0086] 1. Connect RAW264.7-Lucia TM ISG cells were adjusted to a density of 5 × 10 5 / mL, seeded in a 96-well plate (100 μL per well, the number of cells is 5×10 4 / mL), cultured at 37°C until the cell density reached 70%-80%.

[0087] 2. After completing step 1, ta...

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Abstract

The invention discloses a method for preparing and identifying the activity of the porcine second messenger molecule 2'3'-cGAMP. The present invention prepares soluble expressed recombinant porcine 2'3'-cGAMP synthetase pcGAS, and then provides a simple, rapid, low-cost, mild condition, high yield and large-scale production of 2'3'-cGAMP The preparation method; meanwhile, a method capable of identifying the biological activity of the recombinant pcGAS enzyme and its catalyzed product 2'3'-cGAMP is provided. The 2'3'-cGAMP prepared by the present invention is an effective activator of the key molecule STING in the natural immune signaling pathway, which can quickly activate and enhance the natural immune response of the host; Biological activities such as tumors, immune enhancement and immune adjuvants have great prospects for the development of drugs, immune enhancers and immune adjuvants.

Description

technical field [0001] The invention relates to the technical fields of biochemistry and genetic engineering, in particular to a method for preparing and identifying the activity of the pig-derived second messenger molecule 2'3'-cGAMP. Background technique [0002] 2′3′-cGAMP, as the second messenger molecule, can bind and activate the stimulus of interferon genes (STING), and then promote the phosphorylation of IRF3 / 7 and NF-κB and enter the nucleus by activating TBK1 and IKKs , induce the expression of type Ⅰ IFNs and inflammatory factors, and finally cause the host's natural immune response. Studies have found that 2′3′-cGAMP not only induces the expression of type I IFNs and inflammatory factors through the recognition of STING, but also rapidly transfers from producing cells to adjacent cells through gap junctions, rapidly activating STING in adjacent cells and Induces the secretion of type I IFN. In addition, 2′3′-cGAMP can also be packaged into new virions by envelo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/32C12N9/12C12N15/54C12N15/70C12Q1/48
Inventor 景志忠何小兵龙朝琳陈国华贾怀杰房永祥
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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