CN109929894A - The preparation and activity identification method of one boar source second messenger molecule, 2 ' 3 '-cGAMP - Google Patents
The preparation and activity identification method of one boar source second messenger molecule, 2 ' 3 '-cGAMP Download PDFInfo
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Abstract
The invention discloses the preparations and activity identification method of 2 ' 3 '-cGAMP of a boar source second messenger molecule.The present invention is prepared for 2 ' 3 '-cGAMP synzyme pcGAS of Recombinant Swine source of solubility expression, then provides simple and quick, low in cost one kind, mild condition, the preparation method of yield height and 2 ' 3 '-cGAMP that can be mass-produced；A kind of method that can identify recombination pcGAS enzyme and its 2 ' 3 '-cGAMP biological activity of catalysate is provided simultaneously.2 ' 3 '-cGAMP prepared by the present invention is effective activator of hinge molecule STING in innate immunity signal path, can quickly activate the innate immunity with enhancing host；2 ' 3 '-cGAMP have the bioactivity such as wide antiviral and antitumor, Immune-enhancing effect and immunologic adjuvant again, have huge drug, immunopotentiator and immunologic adjuvant development prospect.
Description
Technical field
The present invention relates to biochemistries and gene engineering technology field, and in particular to a boar source second messenger molecule 2 '
The preparation and activity identification method of 3 '-cGAMP.
Background technique
2 ' 3 '-cGAMP are used as second messenger molecule, combinable and activate interferon gene stimulation molecule (stimulator
Of interferon genes, STING), then promote the phosphorylation of IRF3/7 and NF- κ B by activating TBK1 and IKKs
And enter nucleus, the expression of I type IFNs and inflammatory factor is induced, the innate immune reaction of host is finally caused.The study found that
2 ' 3 '-cGAMP not only pass through the expression of the I type IFNs and inflammatory factor of identification induction of STING, can also pass through cell gap junction
(gap junctions) is transferred quickly to adjacent cells from cell is generated, and quickly activates the STING of adjacent cells and induces I type
The secretion of IFN.In addition, 2 ' 3 '-cGAMP can also be packaged into new virion by togavirus, when new virion sense
The antiviral natural immune response of STING mediation can quickly be caused when contaminating new cell.Therefore, 2 ' 3 '-cGAMP can be used as
Potential antiviral drugs, anti-tumor drug, immunopotentiator and immunologic adjuvant carry out application and development.
2 ' 3 '-cGAMP are used as antiviral drugs, and the table of I type IFNs is induced by STING-TBK1-IRF3 signal path
It reaches, the antiviral natural of body is activated to be immunoreacted, then resist the locally and systemically infection of virus.In addition, 2 ' 3 '-cGAMP
Not only the apoptosis, karyorhexis and tumor tissues of tumour cell is induced to damage by STING, moreover it is possible to pass through STING-TBK1-IRF3
Signal path induces the release of I type IFNs and inflammatory factor, and then mediates the maturation and CD8 of DCs+The activation of T cell improves anti-
The activity of tumour medicine 5-FU；When 2 ' 3 '-cGAMP and PD-1 antibody, PD-L1 antibody or CTLA4 antibody are combined, can more have
The maturation and CD8 of the induction DCs of effect+The activation of T cell improves immune system to the lethality of tumour cell, and improves swollen
Oncocyte treats wider tumor type disease to the sensibility of chemotherapy.Further study show that 2 ' 3 '-cGAMP can also
High-caliber IgG1, IgG2a, IgG2c, IgA and cell factor IFN-γ and IL-2 are generated as immunopotentiator stimulation body,
And activation DCs, CD4+T cell, CD8+T cell, thus the infection of effectively prevention and treatment virus.Since 2 ' 3 '-cGAMP exist
Huge potential using value in terms of the anti-infective innate immunity of host and as novel antiviral, anti-tumor drug and exempt from
The great market application prospect of epidemic disease reinforcing agent, people show huge interest for the synthesis of 2 ' 3 '-cGAMP.With regard to existing
For technology, 2 ' 3 '-cGAMP are obtained at present and are mainly prepared by tissue purification and chemical synthesis process, but both methods is imitated
Rate is low, isolates and purifies difficulty, complex process and at high cost, limits its extensive use.Therefore seek one kind can it is simple and quick,
Low in cost, mild condition, yield height and the method that can be prepared on a large scale 2 ' 3 '-cGAMP are very for actual application
Important.
Summary of the invention
The object of the present invention is to provide the preparations and activity identification method of 2 ' 3 '-cGAMP of a boar source second messenger molecule.
The present invention provides a kind of method for preparing 2 ' 3 '-cGAMP of pig source second messenger molecule, include the following steps: with
ATP and GTP is substrate, under the stimulation of DNA, is passed through in vitro using pig source cyclic guanylic acid-adenosine acid enzyme as catalyst
Enzymatic reaction catalyzes and synthesizes 2 ' 3 '-cGAMP of pig source second messenger molecule.
The preparation method of the pig source cyclic guanylic acid-adenosine acid enzyme includes the following steps: that pig source cyclic guanosine will be encoded
Acid-adenosine acid enzyme channel genes host strain, obtains recombinant bacterium；The recombinant bacterium is cultivated, is obtained from recombinant bacterium described
Pig source cyclic guanylic acid-adenosine acid enzyme.
Coding pig source cyclic guanylic acid-adenosine acid enzyme gene is any one of following (b1)-(b4):
(b1) sequence 2 of sequence table DNA molecular shown in the nucleotide of 5 ' end 406-1488；
(b2) DNA molecular shown in sequence 2 in sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode pig source cyclic guanylic acid-adenosine
The DNA molecular of acid enzyme；
(b4) DNA sequence dna limited with (b1) or (b2) or (b3) has 90% or more homology and coding pig source cyclic guanosine
Acid-adenosine acid enzyme DNA molecular.
The stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
The host strain concretely Escherichia coli more specifically can be Escherichia coli Rosetta (DE3).
Coding pig source cyclic guanylic acid-adenosine acid enzyme gene can specifically be carried by the expression containing the gene
Body imports host strain.The expression vector is concretely by BamH I and the Hind III of prokaryotic expression carrier pET-28a-SUMO
Segment between site replaces with the expression load that the sequence 2 of the sequence table DNA sequence dna shown in 5 ' 406-1488, the ends obtains
Body.
Described " pig source cyclic guanylic acid-adenosine acid enzyme is obtained from recombinant bacterium " specifically may include step (1) and (2):
(1) it is crushed the recombinant bacterium thallus, is obtained containing pig source cyclic guanylic acid-adenosine acid enzyme crude extract；
(2) it separates pig source cyclic guanylic acid-adenosine acid enzyme in the crude extract and removes HIS6-SUMO fusion tag,
Obtain pig source cyclic guanylic acid-adenosine acid enzyme.
The method pair of affinity chromatography can be used in pig source cyclic guanylic acid-adenosine acid enzyme in the separation crude extract
Pig source cyclic guanylic acid-adenosine acid enzyme in the crude extract is purified, and Ni-NTA Ago-Gel parent specifically can be used
It is purified with column.
In the reaction system of the enzymatic reaction include following component: pig source cyclic guanylic acid-adenosine acid enzyme, HEPES,
MgCl2, ATP, GTP and HT-DNA.
The concentration of each component in the reaction system are as follows: pig source cyclic guanylic acid -5 μM of adenosine acid enzyme, HEPES
20mM、MgCl25mM, ATP 2mM, GTP 2mM and HT-DNA0.1mg/mL.
The reaction condition of the enzymatic reaction are as follows: 37 DEG C of incubation 2h.
After the enzymatic reaction, nuclease, 37 DEG C of incubation 30min are added into reaction system；Then reaction is produced
95 DEG C of incubation 10min of object, centrifuging and taking supernatant；Supernatant 0.5ml/10kDa ultra-filtration centrifuge tube is filtered, obtains 2 ' 3 '-
cGAMP。
The present invention goes back protection ring pig source guanylic acid-adenosine acid enzyme or its encoding gene in preparing 2 ' 3 '-cGAMP
Using；The pig source cyclic guanylic acid-adenosine acid enzyme is any one of following (a1)-(a4):
(a1) sequence 1 of sequence table amino acid sequence shown in N-terminal 136-495；
(a2) amino acid sequence shown in the sequence 1 of sequence table；
(a3) (a1) or (a2) is obtained by the substitution and/or deletion and/or addition of one or several amino acid residues
Amino acid sequence with the same function；
(a4) homology with (a1) or (a2) with 75% or 75% or more and amino acid sequence with the same function.
The encoding gene of the pig source cyclic guanylic acid-adenosine acid enzyme is any one of following (b1)-(b4):
(b1) sequence 2 of sequence table DNA molecular shown in the nucleotide of 5 ' end 406-1488；
(b2) DNA molecular shown in sequence 2 in sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode pig source cyclic guanylic acid-adenosine
The DNA molecular of acid enzyme；
(b4) DNA sequence dna limited with (b1) or (b2) or (b3) has 90% or more homology and coding pig source cyclic guanosine
Acid-adenosine acid enzyme DNA molecular.
The stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
The present invention also protects a kind of kit for being used to prepare 2 ' 3 '-cGAMP of pig source second messenger molecule, including pig source ring
Guanylic acid-adenosine acid enzyme, HEPES, MgCl2, ATP, GTP and HT-DNA.
The present invention also protects a kind of detection pig source cyclic guanylic acid-adenylate synthase activity and/or its product 2 ' 3 '-
The active method of cGAMP, includes the following steps:
(1) it using ATP and GTP as substrate, under the stimulation of DNA, is used as and is urged using pig source cyclic guanylic acid-adenosine acid enzyme
Agent carries out enzymatic reaction, obtains 2 ' 3 '-cGAMP of reaction product；
(2) 2 ' 3 '-cGAMP of reaction product of step (1) and reporter cell are co-cultured, by detecting cultivating system fluorescence
Intensity determines pig source cyclic guanylic acid-adenylate synthase activity and its product 2 ' 3 '-cGAMP activity；
The reporter cell reflected by uciferase activity in cell 2 ' 3 '-cGAMP stimulation after interferon regulation because
The activation situation of son, to reach detection pig source cyclic guanylic acid-adenosine acid enzyme and/or the activity of 2 ' 3 '-cGAMP of its product
Purpose.
It include following component: pig source cyclic guanosine in the reaction system of the enzymatic reaction in the step of the method (1)
Acid-adenosine acid enzyme, HEPES, MgCl2, ATP, GTP and HT-DNA.The concentration of each component in the reaction system are as follows: pig
Source cyclic guanylic acid -5 μM of adenosine acid enzyme, HEPES 20mM, MgCl25mM, ATP 2mM, GTP 2mM and HT-DNA0.1mg/
mL.The reaction condition of the enzymatic reaction are as follows: 37 DEG C of incubation 2h.After the enzymatic reaction, core is added into reaction system
Sour enzyme, 37 DEG C of incubation 30min；Then by 95 DEG C of incubation 10min of reaction product, centrifuging and taking supernatant；By supernatant 0.5ml/
The filtering of 10kDa ultra-filtration centrifuge tube, obtains 2 ' 3 '-cGAMP of reaction product.
In the step of the method (2), the reporter cell makes first before co-culturing with 2 ' 3 '-cGAMP of reaction product
It is cleaned once with permeabilized cells culture solution.The formula of the permeabilized cells culture solution is as shown in table 2.
The step of the method (2) specifically: after the reporter cell is cleaned once using permeabilized cells culture solution, by 1
2 ' 3 '-cGAMP of the reaction product of parts by volume is thin with the report after mixing with 2 × cell permeable membrane agent of 1 parts by volume
Born of the same parents co-culture (37 DEG C of co-cultivations 30min), using adding permeabilized cells culture solution after the cleaning once of permeabilized cells culture solution, 37
DEG C culture 20h.Then cultivating system supernatant is taken, luciferase substrate is added, fluorescent value is detected at 700nm wavelength, and calculate
Relative intensity of fluorescence (multiple), so that it is determined that the work of 2 ' 3 '-cGAMP of pig source cyclic guanylic acid-adenosine acid enzyme and/or its product
Property.The formula of 2 × cell permeable membrane agent is as shown in table 3.
The present invention also protects a kind of for detecting pig source cyclic guanylic acid-adenylate synthase activity and/or its product 2 ' 3 '-
CGAMP active agent box, including ATP, GTP, HT-DNA and reporter cell；The reporter cell passes through fluorescein enzyme activity in cell
Property come reflect 2 ' 3 '-cGAMP stimulation after interferon regulatory factor activation situation, thus reach detection ring pig source guanylic acid-gland
The active purpose of 2 ' 3 '-cGAMP of thuja acid synzyme and/or its product.
The kit further includes the permeabilized cells culture solution, the agent of 2 × cell permeable membrane and luciferase substrate (tool
Body can be invivogen company, France, and article No. is the product of rep-qlc1).
Any description above reporter cell concretely RAW264.7-LuciaTM(French invivogen is public for ISG cell
Department, article No. are the product of rawl-isg).
The molecular formula of 2 ' 3 '-cGAMP of pig source second messenger molecule of any description above is C20H22N10O13P2, No. CAS is
1441190-66-4, structural formula are as follows:
The preparation method of existing 2 ' 3 '-cGAMP is compared, important technical advantage of the invention is embodied in: (1) there is operation
The features such as simple and quick, low in cost, mild condition, yield are high and can be mass-produced；(2) pcGAS enzyme and catalysis are recombinated
2 ' 3 '-cGAMP of product has the characteristics that high activity and specificity；(3) this method can also identify that recombination pcGAS enzyme and its catalysis close
At the biological activity of 2 ' 3 '-cGAMP of product, it is convenient for subsequent applications.
The present invention prepares 2 ' 3 '-cGAMP synzyme of Recombinant Swine source of solubility expression using technique for gene engineering first
PcGAS, then using biochemical method using ATP and GTP as substrate, DNA stimulation under the biological enzyme synthesize high activity,
2 ' 3 '-cGAMP of specificity, provide simple and quick, low in cost, mild condition, yield it is high and can be mass-produced 2 '
The preparation method of 3 '-cGAMP；Recombination pcGAS enzyme and its catalysate 2 ' 3 '-cGAMP biology can be identified by providing one kind simultaneously
Learn active method.2 ' 3 '-cGAMP prepared by the present invention is that the effective of hinge molecule STING in innate immunity signal path swashs
Agent living, can quickly activate the innate immunity with enhancing host；2 ' 3 '-cGAMP have wide antiviral, anti-swollen again
The bioactivity such as tumor, Immune-enhancing effect and immunologic adjuvant have huge drug, immunopotentiator and immunologic adjuvant development prospect.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis for the recombination pcGAS enzyme that purifying obtains.
Fig. 2 is the SDS-PAGE electrophoresis for the recombination pcGAS enzyme that purifying obtains after cutting off His6-SUMO fusion tag albumen
Figure.
Fig. 3 is the active analysis result for recombinating pcGAS enzyme and 2 ' 3 '-cGAMP of enzymatic reaction product.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
Conventional biochemical reagent company is commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
2 ' 3 '-cGAMP: molecular formula: C20H22N10O13P2, No. CAS: 1441190-66-4, structural formula is as follows:
Escherichia coli Rosetta (DE3) competent cell: Beijing Hua Yue ocean Biotechnology Co., Ltd, article No.:
SJ00010。
Protease inhibitors: Roche, article No.: 04693132001；1 protease inhibitors is dissolved in 50mL solution.
Lysozyme: Amresco, article No.: 0663-5G, working concentration 10mg/ml.
Ni-NTA Ago-Gel affinity column: Webster Bo Hui chromatography Science and Technology Ltd., article No.: CS-A01-02.
PierceTMCoomassie (Bradford) protein quantification kit: Co., Ltd in silent your science and technology of winged generation of match,
Article No.: 23200.
HT-DNA: Sigma-Aldrich Shanghai trade Co., Ltd, article No.: D6898.
Nuclease Benzonase: Sigma-Aldrich Shanghai trade Co., Ltd, article No.: E1014-5KU.
RAW264.7-LuciaTMISG cell: French invivogen company, article No.: rawl-isg.
Luciferase substrate: French invivogen company, article No.: rep-qlc1.
The preparation of embodiment 1,2 ' 3 '-cGAMP synzyme pcGAS of Recombinant Swine source
The amino acid sequence of pcGAS enzyme (pig source cyclic guanylic acid-adenosine acid enzyme) as shown in the sequence 1 of sequence table,
In, it is the enzymatic activity catalyst structure domain from N-terminal 136-495.The encoding gene of pcGAS enzyme as shown in the sequence 2 of sequence table,
Wherein, from 5 ' 406-1488, end coding enzymatic activity catalyst structure domains.
1, by prokaryotic expression carrier pET-28a-SUMO's (Wuhan Miao Ling Biotechnology Co., Ltd, article No.: P0028)
Segment between the site BamH I and Hind III replaces with the sequence 2 of the sequence table DNA shown in 5 ' 406-1488, the ends
Molecule obtains recombinant expression carrier pET-28a-SUMO-pcGAS (sequence verification).
2, the recombinant expression carrier pET-28a-SUMO-pcGAS for obtaining step 1 imports Escherichia coli Rosetta (DE3)
Competent cell obtains recombinant bacterium.
3, the recombinant bacterium for obtaining step 2 is inoculated in LB liquid medium, and 37 DEG C, 180rpm cultivates to bacterium solution OD600nm
When for 0.6-0.8, it is added into cultivating system IPTG (concentration of the IPTG in cultivating system is 0.5mmol/L), 18 DEG C,
120rpm induce 16h, 4 DEG C after induction, 6000r/min be centrifuged 5min, collect bacterial sediment.
4,20mM Tris-Cl buffer (pH=7.5) washing step 3 for containing protease inhibitors using 50mL obtains
Bacterial sediment 3 times, then with containing lysozyme and protease inhibitors 4mL Tris-Cl be resuspended bacterial sediment, freeze repeatedly
Melt 3 times, using the broken cracking thallus of ultrasonic cell disruption instrument, (ultrasound is arranged: ultrasonic 5s, interval 5s, total 20min.).
5, after completing step 4,4 DEG C, 12000rpm/min centrifugation 30min collect supernatant.
6, the supernatant for obtaining step 5 passes through Ni-NTA Ago-Gel affinity column after 0.45 μm of filtering with microporous membrane
(to specifications operate) initial gross separation purifies to obtain pcGAS, and analyzing and identifying purifying pcGAS with SDS-PAGE, (purity is
90%), as a result as shown in Figure 1.
7, the pcGAS for taking step 6 to obtain is removed using SUMO protease ULP (Beijing Suo Laibao Science and Technology Ltd) digestion
HIS6-SUMO fusion tag albumen is removed, it is pure then (to operate) separation to specifications by Ni-NTA Ago-Gel affinity column
Change obtains recombination pcGAS enzyme.Recombination pcGAS enzyme (purity 95%) is analyzed and identified with SDS-PAGE, as a result as shown in Figure 2.
Endonuclease reaction system: recombination 1000 μ g, SUMO Protease Buffer of pcGAS enzyme, 20 μ L, SUMO protease, 2 μ
L, ddH2O is settled to 1000 μ L.Endonuclease reaction program: 4 DEG C of digestion 12h are stayed overnight.
8, using PierceTMCoomassie (Bradford) protein quantification kit (operating to specifications) measures weight
The concentration of group pcGAS enzyme is 10.75mg/ml.
Embodiment 2, with recombination 2 ' 3 '-cGAMP of pcGAS Enzyme catalyzed synthesis pig source
The pig source recombination pcGAS enzyme prepared with embodiment 1 is efficient in vitro, catalyzes and synthesizes pig source 2 ' 3 '-in specific manner
CGAMP, specific as follows:
1, enzymatic reaction system (as shown in table 1) is configured.Enzymatic reaction system configures on ice.
1 enzymatic reaction system of table
2, after completing step 1, then 50 μ L nuclease Benzonase, 37 DEG C of incubation 30min are added in 37 DEG C of incubation 2h.
3, after completing step 2, by 95 DEG C of incubation 10min of reaction product, then 16000g/min is centrifuged 10min, takes supernatant
Liquid.Supernatant is filtered with 0.5ml/10kDa ultra-filtration centrifuge tube (Merck-Millipore company, article No.: UFC501096), is obtained
To reaction product.
The extinction of the enzymatic reaction system of the reaction product and step 1 that are obtained with spectrophotometer in 260nm detecting step 3
Angle value, reaction yield 97.83%.
Reaction yield=reaction product OD260nm/ enzymatic reaction system OD260nm。
The activity identification of 2 ' 3 '-cGAMP of embodiment 3, pig source pcGAS and catalysate
RAW264.7-LuciaTMISG cell is that one kind is total to by ISG54 promoter and IFN-stimulated responsive element ISRE
With the reporter cell of driving luciferase gene (Lucia luciferase gene) expression.When 2 ' 3 '-cGAMP stimulation, quilt
RAW264.7-LuciaTMISG cell expression STING molecular recognition after have activated transcription factor IRF3 and IRF7, then with
ISRE is combined, the secretion of induced fluorescence element enzyme gene, and then passes through QUANTI-LucTMThe fluorescent value of luciferase is detected to reflect
The stimulating activity of the enzymatic activity of pcGAS and 2 ' 3 '-cGAMP.
1, by RAW264.7-LuciaTMIt is 5 × 10 that ISG cell, which adjusts density,5/ mL is inoculated in 96 orifice plates (every hole 100
μ L, cell number are 5 × 104/ mL), 37 DEG C of culture to cell densities reach 70%-80%.
2, after completing step 1,96 orifice plate is taken, culture solution is discarded, clean cell using 100 μ L permeabilized cells culture solutions
Once.The formula of permeabilized cells culture solution is as shown in table 2.
2 permeabilized cells culture formula of liquid of table
3, after completing step 2, following six groups of setting is operated (every group of 6 hole of repetition):
PcGAS (10 μM) group: add after enzymatic reaction product is mixed with the agent of 2 × cell permeable membrane according to volume ratio 1:1
Enter in hole (every 50 μ L of hole), liquid in hole is discarded after 37 DEG C of incubation 30min, adds permeabilized cells culture solution shown in 100 μ L tables 2
Careful cleaning cell is primary, adds 50 μ L permeabilized cells culture solutions, 37 DEG C of culture 20h.The enzymatic reaction product is to implement
The reaction product (concentration of pcGAS enzyme in the reaction system wherein, will be recombinated and be changed to 10 μM) obtained in example 2.
PcGAS (5 μM) group: it is added after enzymatic reaction product is mixed with the agent of 2 × cell permeable membrane according to volume ratio 1:1
In hole (every 50 μ L of hole), liquid in hole is discarded after 37 DEG C of incubation 30min, and it is small to add permeabilized cells culture solution shown in 100 μ L tables 2
Heart cleaning cell is primary, adds 50 μ L permeabilized cells culture solutions, 37 DEG C of culture 20h.The enzymatic reaction product is embodiment 2
The reaction product of acquisition.
2 ' 3 '-cGAMP (50ng/ml) group: by 2 ' 3 '-cGAMP of 100ng/ml (French invivogen company, article No.:
Tlrl-nacga23 (every 50 μ L of hole, working concentration are in adding hole after) mixing with the agent of 2 × cell-permeant according to volume ratio 1:1
50ng/ml), liquid in hole is discarded after 37 DEG C of incubation 30min, is added permeabilized cells culture solution shown in 100 μ L tables 2 and is carefully cleaned
Cell is primary, adds 50 μ L permeabilized cells culture solutions, 37 DEG C of culture 20h.
No ATP and GTP group: it is added after enzymatic reaction product is mixed with the agent of 2 × cell permeable membrane according to volume ratio 1:1
In hole (every 50 μ L of hole), liquid in hole is discarded after 37 DEG C of incubation 30min, and it is small to add permeabilized cells culture solution shown in 100 μ L tables 2
Heart cleaning cell is primary, adds 50 μ L permeabilized cells culture solutions, 37 DEG C of culture 20h.The enzymatic reaction product is embodiment 2
The reaction product (wherein, not adding ATP and GTP in reaction system) of acquisition.
No pcGAS group: adding hole after enzymatic reaction product is mixed with the agent of 2 × cell permeable membrane according to volume ratio 1:1
Interior (every 50 μ L of hole) discards liquid in hole after 37 DEG C of incubation 30min, it is careful to add permeabilized cells culture solution shown in 100 μ L tables 2
It is primary to clean cell, adds 50 μ L permeabilized cells culture solutions, 37 DEG C of culture 20h.The enzymatic reaction product is that embodiment 2 obtains
The reaction product (wherein, not adding recombination pcGAS enzyme in reaction system) obtained.
Control group: (every 50 μ L of hole) in 2 × cell permeable membrane agent adding hole is discarded in hole after 37 DEG C of incubation 30min
Liquid, adding permeabilized cells culture solution shown in 100 μ L tables 2, carefully to clean cell primary, adds 50 μ L permeabilized cells cultures
Liquid, 37 DEG C of culture 20h.
The formula of above-mentioned 2 × cell permeable membrane agent is as shown in table 3.
Table 32 × cell permeable membrane agent prescription
| Component | Concentration in the reaction system | Volume |
| 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 | It mends to 5ml |
4, after completing step 3,25 μ L supernatants are drawn into 96 hole micropore blanks, and 50 μ L luciferase substrates are added, stood
Fluorescent value is detected at 700nm wavelength using VICTORNivo multi-mode plate reader (PerkinElmer Co., Ltd), and is counted
It calculates relative intensity of fluorescence (multiple), so that it is determined that the biological activity of recombination pcGAS enzyme and its 2 ' 3 '-cGAMP of catalysate.
As a result see Fig. 3.The result shows that recombination pcGAS enzyme and its 2 ' 3 '-cGAMP of catalysate have high biological activity,
Can by the expression of STING-TBK1-IRF3/7 signal path induced strong luciferase reporter gene, and 2 ' 3 '-
The stimulating activity of cGAMP and the concentration of pcGAS are related.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
The preparation and activity identification method of 2 ' 3 '-cGAMP of<120>one boar source second messenger molecule
<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 (10)
1. a kind of method for preparing 2 ' 3 '-cGAMP of pig source second messenger molecule, includes the following steps: using ATP and GTP as substrate,
Under the stimulation of DNA, synthesized as catalyst by external enzymatic catalytic reaction using pig source cyclic guanylic acid-adenosine acid enzyme
2 ' 3 '-cGAMP of pig source second messenger molecule.
2. the method as described in claim 1, it is characterised in that: the pig source cyclic guanylic acid-adenosine acid enzyme is as follows
(a1) any one of-(a4):
(a1) sequence 1 of sequence table amino acid sequence shown in N-terminal 136-495；
(a2) amino acid sequence shown in the sequence 1 of sequence table；
(a3) tool for obtaining (a1) or (a2) by the substitution and/or deletion and/or addition of one or several amino acid residues
There is the amino acid sequence of identical function；
(a4) homology with (a1) or (a2) with 75% or 75% or more and amino acid sequence with the same function.
3. method according to claim 2, it is characterised in that: the preparation side of the pig source cyclic guanylic acid-adenosine acid enzyme
Method includes the following steps: that pig source cyclic guanylic acid-adenosine acid enzyme channel genes host strain will be encoded, and obtains recombinant bacterium；Training
The recombinant bacterium is supported, the pig source cyclic guanylic acid-adenosine acid enzyme is obtained from recombinant bacterium.
4. method as claimed in claim 3, it is characterised in that: the coding base of the pig source cyclic guanylic acid-adenosine acid enzyme
Because of any one of following (b1)-(b4):
(b1) sequence 2 of sequence table DNA molecular shown in the nucleotide of 5 ' end 406-1488；
(b2) DNA molecular shown in sequence 2 in sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode pig source cyclic guanylic acid-adenylate and close
At the DNA molecular of enzyme；
(b4) DNA sequence dna limited with (b1) or (b2) or (b3) has 90% or more homology and coding pig source cyclic guanylic acid-
The DNA molecular of adenosine acid enzyme.
5. the method as described in Claims 1-4, it is characterised in that: include such as the following group in the reaction system of the enzymatic reaction
Point: pig source cyclic guanylic acid-adenosine acid enzyme, HEPES, MgCl2, ATP, GTP and HT-DNA.
6. method as claimed in claim 5, it is characterised in that: the concentration of each component in the reaction system are as follows: pig source ring
Guanylic acid -5 μM of adenosine acid enzyme, HEPES20mM, MgCl25mM, ATP2mM, GTP2mM and HT-DNA0.1mg/mL.
7. pig source cyclic guanylic acid-adenosine acid enzyme or its encoding gene are preparing the application in 2 ' 3 '-cGAMP；The pig source
Cyclic guanylic acid-adenosine acid enzyme is any one of following (a1)-(a4):
(a1) sequence 1 of sequence table amino acid sequence shown in N-terminal 136-495；
(a2) amino acid sequence shown in the sequence 1 of sequence table；
(a3) tool for obtaining (a1) or (a2) by the substitution and/or deletion and/or addition of one or several amino acid residues
There is the amino acid sequence of identical function；
(a4) homology with (a1) or (a2) with 75% or 75% or more and amino acid sequence with the same function.
8. a kind of kit for being used to prepare 2 ' 3 '-cGAMP of pig source second messenger molecule, including pig source cyclic guanylic acid-adenylate
Synzyme, ATP, GTP and HT-DNA.
9. a kind of detection pig source cyclic guanylic acid-adenylate synthase activity and/or the active method of 2 ' 3 '-cGAMP of its product, packet
Include following steps:
(1) using ATP and GTP as substrate, under the stimulation of DNA, using pig source cyclic guanylic acid-adenosine acid enzyme as catalyst
Enzymatic reaction is carried out, 2 ' 3 '-cGAMP of reaction product is obtained；
(2) 2 ' 3 '-cGAMP of reaction product of step (1) and reporter cell are co-cultured, by detecting cultivating system fluorescence intensity
Determine pig source cyclic guanylic acid-adenylate synthase activity and its product 2 ' 3 '-cGAMP activity；
The reporter cell reflects interferon regulatory factor after 2 ' 3 '-cGAMP stimulation by uciferase activity in cell
Situation is activated, to reach detection pig source cyclic guanylic acid-adenosine acid enzyme and/or the active mesh of 2 ' 3 '-cGAMP of its product
's.
10. one kind is for detecting pig source cyclic guanylic acid-adenylate synthase activity and/or its 2 ' 3 '-cGAMP active agent of product
Box, including ATP, GTP, HT-DNA and reporter cell；The reporter cell reflects 2 ' 3 '-by uciferase activity in cell
CGAMP stimulation after interferon regulatory factor activation situation, thus reach detection pig source cyclic guanylic acid-adenosine acid enzyme and/
Or the active purpose of 2 ' 3 '-cGAMP of its product.
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