CN109929894B - Preparation and activity identification method of porcine second messenger molecule 2 '3' -cGAMP - Google Patents

Abstract

The invention discloses a preparation method and an activity identification method of a porcine second messenger molecule 2 '3' -cGAMP. The invention prepares the soluble expression recombinant porcine 2 '3' -cGAMP synthetase pcGAS, and then provides a simple and rapid preparation method of 2 '3' -cGAMP with low cost, mild condition, high yield and large-scale production; also provided is a method for identifying the biological activity of the recombinant pcGAS enzyme and the catalytic product 2 '3' -cGAMP thereof. The 2 '3' -cGAMP prepared by the invention is an effective activator of the central knob molecule STING of a natural immune signal pathway, and can quickly activate and enhance the natural immune response of a host; the 2 '3' -cGAMP has wide biological activities of antivirus, antitumor, immune enhancement, immune adjuvant and the like, and has huge development prospects of medicines, immune enhancers and immune adjuvants.

Description

Preparation and activity identification method of porcine second messenger molecule 2 '3' -cGAMP

Technical Field

The invention relates to the technical field of biochemistry and genetic engineering, in particular to a preparation method and an activity identification method of a porcine second messenger molecule 2 '3' -cGAMP.

Background

2 '3' -cGAMP is used as a second messenger molecule, can combine and activate interferon gene stimulating molecules (STING), promotes the phosphorylation of IRF3/7 and NF-kB and enters the nucleus by activating TBK1 and IKKs, induces the expression of I type IFNs and inflammatory factors, and finally causes the natural immune response of a host. It was found that 2 '3' -cGAMP induces the expression of type I IFNs and inflammatory factors not only through the recognition of STING but also through the rapid transfer of gap junctions (gap junctions) from producer cells to neighboring cells, rapidly activating the STING of neighboring cells to induce the secretion of type I IFN. In addition, 2 '3' -cGAMP can be packaged by enveloped viruses into new virions, and can rapidly elicit STING-mediated antiviral innate immune responses when new virions infect new cells. Therefore, the 2 '3' -cGAMP can be applied and developed as a potential antiviral drug, an antitumor drug, an immunopotentiator and an immunologic adjuvant.

2 '3' -cGAMP is used as an antiviral drug, induces the expression of I type IFNs through a STING-TBK1-IRF3 signal channel, activates the antiviral natural immune response of the organism, and then resists the local and systemic infection of viruses. In addition, 2 '3' -cGAMP not only induces the apoptosis, nuclear lysis and tumor tissue damage of tumor cells through STING, but also induces the release of I-type IFNs and inflammatory factors through STING-TBK1-IRF3 signaling pathway, thereby mediating the maturation of DCs and CD8⁺Activation of T cells, and improvement of the activity of the anti-tumor drug 5-FU; when 2 '3' -cGAMP is used in combination with PD-1 antibody, PD-L1 antibody or CTLA4 antibody, DCs maturation and CD8 can be induced more effectively⁺Activation of T cells, increased lethality of the immune system to tumor cells, and increased sensitivity of tumor cells to chemotherapy, and treatment of a wider range of tumor-type diseases. Further research shows that the 2 '3' -cGAMP can also be used as an immunopotentiator to stimulate the body to produce high-level IgG1, IgG2a, IgG2c, IgA and cytokines IFN-gamma and IL-2, and activate DCs, CD4⁺T cell, CD8⁺T cells, thereby effectively preventing and treating viral infection. Because of the huge potential application value of the 2 '3' -cGAMP in the aspect of host anti-infection natural immune response and the huge market application prospect of the 2 '3' -cGAMP as a novel antiviral and antitumor drug and an immunopotentiator,there is great interest in the synthesis of 2 '3' -cGAMP. In terms of the prior art, the currently obtained 2 '3' -cGAMP is mainly prepared by tissue purification and chemical synthesis methods, but the two methods have low efficiency, difficult separation and purification, complex process and high cost, and limit the wide application of the two methods. Therefore, it is important for practical applications to find a method for preparing 2 '3' -cGAMP in a simple and rapid manner, at low cost, under mild conditions, in high yield, and in a large scale.

Disclosure of Invention

The invention aims to provide a preparation method and an activity identification method of a porcine second messenger molecule 2 '3' -cGAMP.

The invention provides a method for preparing a porcine second messenger molecule 2 '3' -cGAMP, which comprises the following steps: ATP and GTP are taken as substrates, and pig-derived cyclic guanosine monophosphate-adenylate synthetase is taken as a catalyst to catalyze and synthesize pig-derived second messenger molecule 2 '3' -cGAMP through in vitro enzymatic reaction under the stimulation of DNA.

The preparation method of the pig-derived cyclic guanosine monophosphate-adenylate synthetase comprises the following steps: introducing a gene for coding the pig-derived cyclic guanosine monophosphate-adenylate synthetase into a host bacterium to obtain a recombinant bacterium; culturing the recombinant strain to obtain the porcine cyclic guanosine monophosphate-adenylate synthetase from the recombinant strain.

The gene for encoding the pig-derived cyclic guanosine monophosphate-adenylate synthetase is any one of the following genes (b1) - (b 4):

(b1) DNA molecule shown by 406-position 1488 nucleotide from 5' end of sequence 2 in the sequence table;

(b2) DNA molecule shown in sequence 2 in the sequence table;

(b3) a DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (b1) or (b2) and which encodes a cyclic guanosine-adenylate synthetase of porcine origin;

(b4) a DNA molecule which has more than 90% homology with the DNA sequence defined in (b1) or (b2) or (b3) and encodes a cyclic guanosine-adenosine synthetase derived from swine.

The stringent conditions can be hybridization and membrane washing with a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ in DNA or RNA hybridization experiments.

The host bacterium may specifically be E.coli, more specifically E.coli Rosetta (DE 3).

The gene for coding the pig-derived cyclic guanosine monophosphate-adenylic acid synthetase can be specifically introduced into a host bacterium through an expression vector containing the gene. The expression vector can be obtained by replacing the fragment between the BamH I site and the Hind III site of the prokaryotic expression vector pET-28a-SUMO with a DNA sequence shown in the 406-1488 site from the 5' end of the sequence 2 in the sequence table.

The method for obtaining the porcine cyclic guanylic acid-adenylic acid synthetase from the recombinant bacteria specifically comprises the following steps (1) and (2):

(1) crushing the recombinant bacteria to obtain a crude extract containing the porcine cyclic guanylic acid-adenylic acid synthetase;

(2) separating the pig-derived cyclic guanosine monophosphate-adenylate synthetase in the crude extract and removing the HIS6-SUMO fusion tag to obtain the pig-derived cyclic guanosine monophosphate-adenylate synthetase.

The separation of the cyclic guanosine monophosphate-adenylate synthetase in the crude extract can be carried out by purifying the cyclic guanosine monophosphate-adenylate synthetase in the crude extract by adopting an affinity chromatography method, and specifically can be carried out by adopting a Ni-NTA agarose gel affinity column.

The reaction system of the enzymatic reaction comprises the following components: pig-derived cyclic guanosine monophosphate-adenylate synthetase, HEPES and MgCl₂ATP, GTP and HT-DNA.

The concentration of each component in the reaction system is as follows: pig-derived cyclic guanosine monophosphate-adenylate synthetase 5 mu M, HEPES 20mM and MgCl₂5mM, ATP 2mM, GTP 2mM and HT-DNA0.1mg/mL.

The reaction conditions of the enzymatic reaction are as follows: incubate at 37 ℃ for 2 h.

After the enzymatic reaction is finished, adding nuclease into the reaction system, and incubating for 30min at 37 ℃; then incubating the reaction product at 95 ℃ for 10min, centrifuging and taking supernatant; the supernatant was filtered through 0.5ml/10kDa ultrafiltration centrifuge tubes to give 2 '3' -cGAMP.

The invention also protects the application of the ring-derived guanylic acid-adenylate synthetase or the coding gene thereof in the preparation of 2 '3' -cGAMP; the pig-derived cyclic guanosine monophosphate-adenylate synthetase is any one of the following (a1) - (a 4):

(a1) the amino acid sequence shown in the No. 136-495 bit of the sequence 1 in the sequence table from the N terminal;

(a2) an amino acid sequence shown in a sequence 1 in a sequence table;

(a3) amino acid sequences with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) or (a 2);

(a4) and (b) an amino acid sequence which has 75% or more homology with (a1) or (a2) and has the same function.

The encoding gene of the pig-derived cyclic guanosine monophosphate-adenylate synthetase is any one of the following genes (b1) - (b 4):

(b1) DNA molecule shown by 406-position 1488 nucleotide from 5' end of sequence 2 in the sequence table;

(b2) DNA molecule shown in sequence 2 in the sequence table;

(b3) a DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (b1) or (b2) and which encodes a cyclic guanosine-adenylate synthetase of porcine origin;

(b4) a DNA molecule which has more than 90% homology with the DNA sequence defined in (b1) or (b2) or (b3) and encodes a cyclic guanosine-adenosine synthetase derived from swine.

The stringent conditions can be hybridization and membrane washing with a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ in DNA or RNA hybridization experiments.

The invention also provides a kit for preparing the porcine second messenger molecule 2 '3' -cGAMP, which comprises porcine cyclic guanylic acid-adenylic acid synthetase, HEPES, MgCl₂ATP, GTP and HT-DNA.

The invention also provides a method for detecting the activity of the pig-derived cyclic guanosine monophosphate-adenylate synthetase and/or the activity of the product 2 '3' -cGAMP thereof, which comprises the following steps:

(1) using ATP and GTP as substrates, and adopting pig-derived cyclic guanosine monophosphate-adenylate synthetase as a catalyst to carry out enzymatic reaction under the stimulation of DNA to obtain a reaction product 2 '3' -cGAMP;

(2) co-culturing the reaction product 2 '3' -cGAMP in the step (1) and a report cell, and determining the activity of the pig-derived cyclic guanosine monophosphate-adenylate synthetase and the activity of the product 2 '3' -cGAMP by detecting the fluorescence intensity of a culture system;

the reporter cell reflects the activation condition of the interferon regulatory factor after 2 '3' -cGAMP stimulation through luciferase activity in the cell, thereby achieving the purpose of detecting the activity of the porcine cyclic guanosine monophosphate-adenosine synthetase and/or the product 2 '3' -cGAMP thereof.

In step (1) of the method, the reaction system of the enzymatic reaction comprises the following components: pig-derived cyclic guanosine monophosphate-adenylate synthetase, HEPES and MgCl₂ATP, GTP and HT-DNA. The concentration of each component in the reaction system is as follows: pig-derived cyclic guanosine monophosphate-adenylate synthetase 5 mu M, HEPES 20mM and MgCl₂5mM, ATP 2mM, GTP 2mM and HT-DNA0.1mg/mL. The reaction conditions of the enzymatic reaction are as follows: incubate at 37 ℃ for 2 h. After the enzymatic reaction is finished, adding nuclease into the reaction system, and incubating for 30min at 37 ℃; then incubating the reaction product at 95 ℃ for 10min, centrifuging and taking supernatant; the supernatant was filtered through a 0.5ml/10kDa ultrafiltration tube to give the reaction product 2 '3' -cGAMP.

In step (2) of the method, the reporter cells were first washed once with permeabilized cell culture fluid before co-culturing with the reaction product 2 '3' -cGAMP. The formulation of the permeabilized cell culture medium is shown in table 2.

The step (2) of the method is specifically as follows: after the reporter cell is washed once by using a permeabilized cell culture solution, 1 volume part of the reaction product 2 '3' -cGAMP and 1 volume part of 2 multiplied by cell membrane penetrating agent are mixed and then co-cultured with the reporter cell (co-culture at 37 ℃ for 30min), and after the reporter cell is washed once by using the permeabilized cell culture solution, the permeabilized cell culture solution is added and cultured for 20h at 37 ℃. Then, the culture system supernatant was taken, added with a luciferase substrate, and the fluorescence value was detected at a wavelength of 700nm, and the relative fluorescence intensity (fold) was calculated, thereby determining the activity of the porcine-derived cyclic guanosine-adenylate synthetase and/or the product thereof, 2 '3' -cGAMP. The formulation of the 2 × cell membrane permeabilizing agent is shown in table 3.

The invention also provides a kit for detecting the activity of the pig-derived cyclic guanosine monophosphate-adenylate synthetase and/or the activity of the product 2 '3' -cGAMP, which comprises ATP, GTP, HT-DNA and a report cell; the reporter cell reflects the activation condition of the interferon regulatory factor after 2 '3' -cGAMP stimulation through luciferase activity in the cell, thereby achieving the purpose of detecting the activity of the cyclic pig guanylate-adenylate synthetase and/or the product 2 '3' -cGAMP thereof.

The kit also comprises the permeabilized cell culture solution, a2 x cell permeabilized agent and a luciferase substrate (specifically, a product with the product number of rep-qlc1 from French invivogen).

Any one of the above reporter cells may specifically be RAW264.7-Lucia^TMISG cells (product of Rawl-ISG, Invivogen, France).

The molecular formula of any one of the porcine second messenger molecules 2 '3' -cGAMP is C₂₀H₂₂N₁₀O₁₃P₂CAS number 1441190-66-4, structural formula as follows:

compared with the existing preparation method of 2 '3' -cGAMP, the main technical advantages of the invention are as follows: (1) the method has the characteristics of simple and quick operation, low cost, mild condition, high yield, large-scale production and the like; (2) the recombinant pcGAS enzyme and the catalytic product 2 '3' -cGAMP have the characteristics of high activity and specificity; (3) the method can also identify the biological activity of the recombinant pcGAS enzyme and the 2 '3' -cGAMP catalytic synthesis product thereof, and is convenient for subsequent application.

The invention utilizes the genetic engineering technology to firstly prepare the soluble expressed recombinant porcine 2 '3' -cGAMP synthetase pcGAS, then utilizes the biochemical method to take ATP and GTP as substrates, and catalyzes and synthesizes the 2 '3' -cGAMP with high activity and specificity under the stimulation of DNA by the biological enzyme, thereby providing the simple and rapid preparation method of the 2 '3' -cGAMP with low cost, mild condition, high yield and large-scale production; also provided is a method for identifying the biological activity of the recombinant pcGAS enzyme and the catalytic product 2 '3' -cGAMP thereof. The 2 '3' -cGAMP prepared by the invention is an effective activator of the central knob molecule STING of a natural immune signal pathway, and can quickly activate and enhance the natural immune response of a host; the 2 '3' -cGAMP has wide biological activities of antivirus, antitumor, immune enhancement, immune adjuvant and the like, and has huge development prospects of medicines, immune enhancers and immune adjuvants.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of purified recombinant pcGAS enzyme.

FIG. 2 is an SDS-PAGE of the recombinant pcGAS enzyme purified after removal of the His6-SUMO fusion tag protein.

FIG. 3 is an assay of the activity of recombinant pcGAS enzyme and the enzymatic reaction product 2 '3' -cGAMP.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

2 '3' -cGAMP: the molecular formula is as follows: c₂₀H₂₂N₁₀O₁₃P₂CAS number: 1441190-66-4, the structural formula is as follows:

coli Rosetta (DE3) competent cells: beijing Huayuyo Biotech Co., Ltd., Cat No.: SJ 00010.

Protease inhibitors: roche, cat number: 04693132001, respectively; 1 tablet of protease inhibitor was dissolved in 50mL of the solution.

Lysozyme: amresco, cat No.: 0663-5G, working concentration 10 mg/ml.

Ni-NTA agarose gel affinity column: wechbo hui chromatography technologies ltd, cat #: CS-A01-02.

Pierce^TMCoomassie (bradford) protein quantification kit: sammer Feishale science and technology, China Co., Ltd., Cat No.: 23200.

HT-DNA: sigma aldrich shanghai trade ltd, cat #: D6898.

nuclease Benzonase: sigma aldrich shanghai trade ltd, cat #: e1014-5 KU.

RAW264.7-Lucia^TMISG cells: french invivogen, cat No.: rawl-isg.

Luciferase substrate: french invivogen, cat No.: rep-qlc 1.

Example 1 preparation of recombinant porcine 2 '3' -cGAMP synthetase pcGAS

The amino acid sequence of pcGAS enzyme (pig-derived cyclic guanosine monophosphate-adenylate synthetase) is shown as sequence 1 in the sequence table, wherein the 495 th site from the N end 136-495 th site is the catalytic domain of the enzyme activity. The coding gene of the pcGAS enzyme is shown as a sequence 2 in the sequence table, wherein the 406-th 1488 site of the 5' end codes the catalytic domain of the enzyme activity.

1. The fragment between the BamH I and Hind III sites of the prokaryotic expression vector pET-28a-SUMO (Wuhan vast Ling Biotech Co., Ltd., product number: P0028) is replaced by a DNA molecule shown in the 406 th 1488 th site from the 5' end of the sequence 2 of the sequence table to obtain the recombinant expression vector pET-28a-SUMO-pcGAS (sequencing verification has been carried out).

2. And (2) introducing the recombinant expression vector pET-28a-SUMO-pcGAS obtained in the step (1) into escherichia coli Rosetta (DE3) competent cells to obtain recombinant bacteria.

3. Inoculating the recombinant bacteria obtained in the step 2 into an LB liquid culture medium, and culturing at 37 ℃ and 180rpm until the OD of the bacterial liquid_600nmWhen the concentration is 0.6-0.8, adding IPTG (concentration of IPTG in the culture system is 0.5mmol/L), inducing at 18 deg.C and 120rpm for 16h, centrifuging at 4 deg.C and 6000r/min for 5min after induction, and collecting thallus precipitate.

4. The cell pellet obtained in step 3 was washed 3 times with 50mL of 20mM Tris-Cl buffer (pH 7.5) containing protease inhibitor, then resuspended with 4mL of Tris-Cl containing lysozyme and protease inhibitor, freeze-thaw repeated 3 times, and disrupted by ultrasonic cell disruption apparatus (ultrasonic setting: 5s, 5s pause, 20min total).

5. After completion of step 4, the mixture was centrifuged at 12000rpm/min at 4 ℃ for 30min, and the supernatant was collected.

6. The supernatant obtained in step 5 was filtered through a 0.45 μm microfiltration membrane and then preliminarily separated and purified by a Ni-NTA agarose gel affinity column (according to the instructions) to obtain pcGAS, which was identified by SDS-PAGE analysis as purified pcGAS (purity: 90%), and the results are shown in FIG. 1.

7. Taking the pcGAS obtained in the step 6, carrying out enzyme digestion by SUMO protease ULP (Beijing Sorley technologies, Ltd.) to remove HIS6-SUMO fusion tag protein, and then separating and purifying by a Ni-NTA agarose gel affinity column (operating according to the instruction) to obtain the recombinant pcGAS enzyme. The recombinant pcGAS enzyme (95% pure) was identified by SDS-PAGE analysis and the results are shown in FIG. 2.

An enzyme digestion reaction system: recombinant pcGAS enzyme 1000. mu.g, SUMO Protease Buffer 20. mu.L, SUMO Protease 2. mu.L, ddH₂O is metered to 1000. mu.L. And (3) enzyme digestion reaction program: the digestion was carried out at 4 ℃ for 12h or overnight.

8. Use of Pierce^TMThe Coomassie (Bradford) protein quantification kit (according to the instructions) determined a concentration of 10.75mg/ml of recombinant pcGAS enzyme.

Example 2 enzymatic Synthesis of porcine 2 '3' -cGAMP Using recombinant pcGAS

The porcine recombinant pcGAS enzyme prepared in the example 1 is used for catalyzing and synthesizing the porcine 2 '3' -cGAMP in vitro efficiently and specifically as follows:

1. an enzymatic reaction system was configured (as shown in table 1). The enzymatic reaction system was set up on ice.

TABLE 1 enzymatic reaction System

2. After completion of step 1, incubation was carried out at 37 ℃ for 2h, followed by addition of 50. mu.L nuclease Benzonase and incubation at 37 ℃ for 30 min.

3. After step 2 was completed, the reaction product was incubated at 95 ℃ for 10min, and then centrifuged at 16000g/min for 10min to obtain a supernatant. The supernatant was filtered through a 0.5ml/10kDa ultrafiltration centrifuge tube (Merck-Millipore, cat # UFC501096) to obtain a reaction product.

The absorbance values of the reaction product obtained in step 3 and the enzymatic reaction system of step 1 were measured at 260nm using a spectrophotometer, and the reaction yield was 97.83%.

Reaction yield-reaction product OD_260nmEnzymatic reaction System OD260_nm。

Example 3 Activity characterization of porcine pcGAS and catalytic product 2 '3' -cGAMP

RAW264.7-Lucia^TMISG cells are reporter cells in which the ISG54 promoter and ISRE, an interferon-stimulated response element, co-drive the expression of a luciferase gene (Lucia luciferase gene). When 2 '3' -cGAMP is stimulated, RAW264.7-Lucia^TMThe transcription factors IRF3 and IRF7 are activated after recognition by STING molecule expressed by ISG cells, then combined with ISRE to induce the secretion of luciferase gene, and further processed by QUANTI-Luc^TMLuciferase fluorescence was measured to reflect pcGAS enzymatic activity and 2 '3' -cGAMP stimulatory activity.

1. Mixing RAW264.7-Lucia^TMISG cells were adjusted to a density of 5X 10⁵Perml, seeded in 96-well plates (100. mu.L per well, cell number 5X 10⁴/mL) at 37 ℃ until the cell density reaches 70-80%.

2. After completion of step 1, the 96-well plate was removed, the culture medium was discarded, and cells were washed once with 100 μ L of permeabilized cell culture medium. The formulation of the permeabilized cell culture medium is shown in Table 2.

TABLE 2 formula of permeabilized cell culture solution

3. After completion of step 2, the following six groups were set up for operation (6 replicates per group):

pcGAS (10. mu.M) group: mixing the enzymatic reaction product with 2 Xcell cell membrane permeable agent according to the volume ratio of 1:1, adding into the hole (50. mu.L per hole), incubating at 37 ℃ for 30min, removing the liquid in the hole, adding 100. mu.L of the permeable cell culture solution shown in Table 2, carefully cleaning the cells once, adding 50. mu.L of the permeable cell culture solution, and culturing at 37 ℃ for 20 h. The enzymatic reaction product was the reaction product obtained in example 2 (wherein the concentration of the recombinant pcGAS enzyme in the reaction system was changed to 10. mu.M).

pcGAS (5. mu.M) group: mixing the enzymatic reaction product with 2 Xcell cell membrane permeable agent according to the volume ratio of 1:1, adding into the hole (50. mu.L per hole), incubating at 37 ℃ for 30min, removing the liquid in the hole, adding 100. mu.L of the permeable cell culture solution shown in Table 2, carefully cleaning the cells once, adding 50. mu.L of the permeable cell culture solution, and culturing at 37 ℃ for 20 h. The enzymatic reaction product is the reaction product obtained in example 2.

Group 2 '3' -cGAMP (50 ng/ml): 100ng/ml of 2 '3' -cGAMP (French invivogen, cat # tlrl-nacga23) and 2 Xcell permeant were mixed at a volume ratio of 1:1 and added to wells (50. mu.L/well, working concentration 50ng/ml), and after incubation at 37 ℃ for 30min, the liquid in the wells was discarded, 100. mu.L of the permeabilized cell culture shown in Table 2 was added and the cells were washed once carefully, and 50. mu.L of the permeabilized cell culture was added and cultured at 37 ℃ for 20 hours.

ATP and GTP free group: mixing the enzymatic reaction product with 2 Xcell cell membrane permeable agent according to the volume ratio of 1:1, adding into the hole (50. mu.L per hole), incubating at 37 ℃ for 30min, removing the liquid in the hole, adding 100. mu.L of the permeable cell culture solution shown in Table 2, carefully cleaning the cells once, adding 50. mu.L of the permeable cell culture solution, and culturing at 37 ℃ for 20 h. The enzymatic reaction product was the reaction product obtained in example 2 (wherein ATP and GTP were not added to the reaction system).

pcGAS-free group: mixing the enzymatic reaction product with 2 Xcell cell membrane permeable agent according to the volume ratio of 1:1, adding into the hole (50. mu.L per hole), incubating at 37 ℃ for 30min, removing the liquid in the hole, adding 100. mu.L of the permeable cell culture solution shown in Table 2, carefully cleaning the cells once, adding 50. mu.L of the permeable cell culture solution, and culturing at 37 ℃ for 20 h. The enzymatic reaction product was the reaction product obtained in example 2 (wherein no recombinant pcGAS enzyme was added to the reaction system).

Control group: 2 Xcell membrane permeabilizing agent is added into the holes (50. mu.L of each hole), the cells are incubated at 37 ℃ for 30min, then the liquid in the holes is discarded, 100. mu.L of permeabilized cell culture solution shown in Table 2 is added, the cells are carefully cleaned once, 50. mu.L of permeabilized cell culture solution is added, and the cells are cultured at 37 ℃ for 20 h.

The formulation of the 2 × cell membrane permeabilizing agent is shown in table 3.

TABLE 32X cell permeant formulations

Components	Concentration in the reaction System	Volume of
			HEPES(pH7.5)	100mM	500μL
MgCl2	6mM	30μL
			KCl	200mM	1ml
DTT	0.2mM	10μL
			Sucrose	170mM	850μL
BSA	7.5％	267μL
			ATP	2mM	100μL
GTP	0.2mM	10μL
			Digitonin (Digitonin)	20μg/mL	20μL
DEPCH2O		Make up to 5ml

4. After completion of step 3, 25. mu.L of the supernatant was pipetted into a 96-well microwell plate and 50. mu.L of luciferase substrate was added, and the fluorescence value was immediately detected at a wavelength of 700nm using a VICTORNivo multimode plate reader (Perkin Elmer Co., Ltd.) and the relative fluorescence intensity (fold) was calculated, thereby determining the biological activity of the recombinant pcGAS enzyme and its catalytic product, 2 '3' -cGAMP.

The results are shown in FIG. 3. The results show that the recombinant pcGAS enzyme and the catalytic product 2 '3' -cGAMP thereof have high biological activity, the expression of luciferase reporter gene can be strongly induced through the STING-TBK1-IRF3/7 signal pathway, and the stimulation activity of the 2 '3' -cGAMP is related to the concentration of pcGAS.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> preparation and activity identification method of pig-derived second messenger molecule 2 '3' -cGAMP

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 495

<212> PRT

<213> pig (Sus scrofa)

<400> 1

Met Ala Ala Arg Arg Gly Lys Ser Thr Arg Thr Ala Ser Glu Val Gly

1 5 10 15

Ala Ala Gly Pro Arg Ala Ser Ala Arg Ser Val Asn Gly Ala Pro Thr

20 25 30

Val Pro Glu Ala Ala Arg Pro Gly Ala Arg Arg Asn Gly Pro Ser Arg

35 40 45

Ala Ser Gly Cys Arg Arg Glu Lys Ser Gly Pro Asp Pro Arg Glu Lys

50 55 60

Pro Gln Val Arg Thr Arg Thr Ala Arg Ala Glu Asp Gln Ala Glu Gly

65 70 75 80

Pro Ser Ala Pro Ser Glu Arg Val Glu Pro Pro Ser Ala Gln Gly Ala

85 90 95

Ser Leu Leu Arg Ala Gly Ser Cys Arg Ala Arg Glu Ala Arg Ser Ala

100 105 110

Arg Glu Leu Arg Pro Gln Ala Gly Ala Thr Glu Leu Ala Ala Pro Ala

115 120 125

Arg Met Glu Ala Pro Pro Gly Ala Trp Lys Leu Gln Thr Val Leu Glu

130 135 140

Lys Val Arg Leu Ser Arg His Glu Ile Ser Glu Ala Ala Glu Val Val

145 150 155 160

Asn Trp Val Val Glu His Leu Leu Arg Arg Leu Gln Gly Gly Glu Ser

165 170 175

Glu Phe Lys Gly Val Ala Leu Leu Arg Thr Gly Ser Tyr Tyr Glu Arg

180 185 190

Val Lys Ile Ser Ala Pro Asn Glu Phe Asp Val Met Phe Lys Leu Glu

195 200 205

Val Pro Arg Ile Gln Leu Glu Glu Tyr Cys Asn Ser Gly Ala His Tyr

210 215 220

Phe Val Lys Phe Lys Arg Asn Pro Gly Gly Asn Pro Leu Glu Gln Phe

225 230 235 240

Leu Glu Lys Glu Ile Leu Ser Ala Ser Lys Met Leu Ser Lys Phe Arg

245 250 255

Lys Ile Ile Lys Glu Glu Ile Lys Asn Ile Glu Gly Val Thr Val Glu

260 265 270

Arg Lys Arg Arg Gly Ser Pro Ala Val Thr Leu Leu Ile Ser Lys Pro

275 280 285

Lys Glu Ile Ser Val Asp Ile Ile Leu Ala Leu Glu Ser Lys Ser Ser

290 295 300

Trp Pro Ala Ser Thr Gln Lys Gly Leu Pro Ile Ser Gln Trp Leu Gly

305 310 315 320

Ala Lys Val Lys Asn Asn Leu Lys Arg Gln Pro Phe Tyr Leu Val Pro

325 330 335

Lys His Ala Lys Glu Gly Ser Gly Phe Gln Glu Glu Thr Trp Arg Leu

340 345 350

Ser Phe Ser His Ile Glu Lys Asp Ile Leu Lys Asn His Gly Gln Ser

355 360 365

Lys Thr Cys Cys Glu Ile Asp Gly Val Lys Cys Cys Arg Lys Glu Cys

370 375 380

Leu Lys Leu Met Lys Tyr Leu Leu Glu Gln Leu Lys Lys Lys Phe Gly

385 390 395 400

Asn Arg Arg Glu Leu Ala Lys Phe Cys Ser Tyr His Val Lys Thr Ala

405 410 415

Phe Phe His Val Cys Thr Gln Asp Pro His Asp Asn Gln Trp His Leu

420 425 430

Lys Asn Leu Glu Cys Cys Phe Asp Asn Cys Val Ala Tyr Phe Leu Gln

435 440 445

Cys Leu Lys Thr Glu Gln Leu Ala Asn Tyr Phe Ile Pro Gly Val Asn

450 455 460

Leu Phe Ser Arg Asp Leu Ile Asp Lys Pro Ser Lys Glu Phe Leu Ser

465 470 475 480

Lys Gln Ile Glu Tyr Glu Arg Asn Asn Gly Phe Pro Val Phe Trp

485 490 495

<210> 2

<211> 1488

<212> DNA

<213> pig (Sus scrofa)

<400> 2

atggcggccc ggcggggaaa gtccacgcgg acagcttcag aggtgggagc cgctggcccc 60

agggcctcag cgcggagtgt aaatggcgct ccaacggtgc ccgaggccgc ccggcctggc 120

gcacggagga acggcccctc cagggcgtcg ggatgccgac gggagaagag cggcccggac 180

ccccgggaga agccgcaggt acgcacgagg acggcccgcg ccgaggacca ggcggagggc 240

ccatcggctc cctccgaaag ggtagagcct ccttcggccc agggggcttc ccttctcagg 300

gctggatctt gccgcgcgag agaggcgcgc tcggcgcggg aactgagacc ccaggccggg 360

gccacagagc tcgcagcccc cgcgcggatg gaggcacccc ccggcgcctg gaagctccag 420

acggtgctgg agaaggtgag gctgagccgc cacgaaatct cagaagcggc ggaggtggtg 480

aactgggtcg tggagcacct gctgcggagg ctgcagggcg gagagtccga gttcaaaggc 540

gttgccctgc tgcgcaccgg gagctactat gagcgagtga agatttctgc tcccaatgaa 600

tttgatgtta tgttcaaact ggaagttccc cgaattcagc tagaggaata ttgcaacagt 660

ggtgctcatt attttgtcaa gttcaaaaga aatcctggag gaaatcctct ggaacagttc 720

ttagaaaagg aaatattatc agcttctaag atgctgtcca agtttaggaa aattattaag 780

gaagaaatta aaaatataga aggtgtcact gtggagagga agaggagagg gagccctgca 840

gtaacacttc tgattagcaa acctaaagaa atatctgtgg atataatcct ggctttggaa 900

tctaaaagca gctggcctgc tagcacccag aaaggcctgc ccatcagtca gtggcttgga 960

gcaaaagtta aaaacaatct gaaacgacag ccattttacc tggtacccaa gcatgccaag 1020

gaaggaagtg gttttcaaga agaaacatgg aggctttcct tctctcacat tgaaaaggac 1080

attttgaaaa atcatggaca gtctaaaaca tgctgtgaaa ttgatggagt gaaatgttgc 1140

aggaaagagt gtttaaaact aatgaaatac cttttagaac aactgaaaaa aaagtttgga 1200

aaccgaaggg aactggctaa gttctgttct tatcacgtga aaactgcctt ctttcacgtc 1260

tgtacccagg accctcatga caatcagtgg cacctcaaga accttgagtg ctgctttgat 1320

aactgtgtgg catattttct gcagtgcctc aagacagaac aactggccaa ttatttcatt 1380

cctggagtca atctgttctc tcgagaccta attgacaaac caagtaaaga atttctgtca 1440

aagcaaattg aatatgaaag aaacaatgga tttccagttt tttggtga 1488

Claims (1)

1. A method for detecting the activity of pig cyclic guanosine monophosphate synthetase and/or the activity of 2 '3' -cGAMP of a product thereof, which comprises the following steps:

(1) using ATP and GTP as substrates, and adopting pig-derived cyclic guanosine monophosphate-adenylate synthetase as a catalyst to carry out enzymatic reaction under the stimulation of DNA to obtain a reaction product 2 '3' -cGAMP;

(2) co-culturing the reaction product 2 '3' -cGAMP in the step (1) and a report cell, and determining the activity of the pig-derived cyclic guanosine monophosphate-adenylate synthetase and the activity of the product 2 '3' -cGAMP by detecting the fluorescence intensity of a culture system;

the reporter cell reflects the activation condition of the interferon regulatory factor after 2 '3' -cGAMP stimulation through luciferase activity in the cell, thereby achieving the purpose of detecting the activity of the porcine cyclic guanosine monophosphate-adenosine synthetase and/or the product 2 '3' -cGAMP thereof;

the step (2) is as follows: before the report cell is co-cultured with the reaction product 2 '3' -cGAMP, firstly, washing the report cell once by using a permeabilized cell culture solution, mixing 1 volume part of the reaction product 2 '3' -cGAMP with 1 volume part of 2 multiplied cell membrane penetrating agent, then, co-culturing the mixture with the report cell at 37 ℃ for 30min, washing the report cell once by using the permeabilized cell culture solution, then, adding the permeabilized cell culture solution, and culturing the mixture for 20h at 37 ℃; then taking culture system supernatant, adding luciferase substrate, detecting fluorescence value at 700nm wavelength, and calculating relative fluorescence intensity, thereby determining activity of pig-derived cyclic guanosine-adenylate synthetase and/or product 2 '3' -cGAMP thereof;

the permeabilized cell culture solution consists of FBS, HEPES, L-glutamine, Zeolin and DMEM, wherein the concentration of FBS is 10%, the pH value of HEPES is 7.5, the concentration is 25 mM, the concentration of L-glutamine is 2mM, and the concentration of Zeolin is 200 mu g/mL;

the 2 × cell membrane-penetrating agent is prepared from HEPES, MgCl2, KCl, DTT, sucrose, BSA, ATP, GTP, Digitonin and DEPC H₂O, wherein HEPES has a pH of 7.5 and a concentration of 100 mM, MgCl₂Has a concentration of 6 mM, KCl concentration of 200 mM, DTT concentration of 0.2 mM, sucrose concentration of 170 mM, BSA content of 7.5%, ATP concentration of 2mM, GTP concentration of 0.2 mM; the porcine cyclic guanylic acid-adenylic acid synthetase is (a1) or (a 2):

(a1) the amino acid sequence shown in the No. 136-495 bit of the sequence 1 in the sequence table from the N terminal;

(a2) an amino acid sequence shown in a sequence 1 in a sequence table.

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