CN106318997A

CN106318997A - Efficient preparation and purification method of thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP

Info

Publication number: CN106318997A
Application number: CN201510383060.0A
Authority: CN
Inventors: 张跃茹; 向道凤; 谭相石
Original assignee: Liaocheng City Run Bio Pharmaceutical Technology Co Ltd
Current assignee: Liaocheng City Run Bio Pharmaceutical Technology Co Ltd
Priority date: 2015-07-03
Filing date: 2015-07-03
Publication date: 2017-01-11

Abstract

The invention belongs to the field of bioengineering technology, and in particular discloses a method for efficiently preparing and purifying thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP by virtue of bio-enzyme catalysis. According to the method disclosed by the invention, firstly, a biological enzyme, namely cyclic di-nucleotide cGAMP synthetase (cGAS), is subjected to mass production by virtue of genetic engineering technology so as to subsequently synthesize adenosine (vernine) 5'-([alpha]-thio-/seleno-phosphoric acid) triphosphate, and then efficient and specific thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP is synthesized by conducting catalysis with the biological enzyme cGAS; therefore, the method for preparing the thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP is provided, and the method is large in scale, high in yield, low in cost, mild in conditions and is rapid and convenient. The thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP prepared by the invention, as an effective activator for STING in a human natural immune signaling pathway cGAS-STING-IRF3, is capable of activating and enhancing the natural autoimmune system; the thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP has relatively broad bio-activities of resisting viruses, resisting tumors and the like; and the thio- (seleno-) phosphoric acid cyclic di-nucleotide cGAMP has a good drug development prospect.

Description

Sulfur (selenium) is for phosphoric acid ring dinucleotide cGAMP Efficiently preparation, purification process

Technical field

The invention belongs to technical field of bioengineering, be specially that one uses catalyzed by biological enzyme efficiently to prepare, purification sulfur (selenium) is for the method for phosphoric acid ring dinucleotide cGAMP.

Background technology

The nucleic acid of antibacterial and virus can cause host immune response, and host starts immunoreation by the nucleic acid sensing producing pathogen.Recent studies have shown that cyclo GMP-AMP synzyme (cGAS) is the cytoplasmic DNA sensor of a kind of key.CGAS is activated by double-stranded DNA, catalyzes and synthesizes a kind of non-classical ring dinucleotide 2 ', 3 '-cGAMP.CGAMP is combined promotion to interferon beta and the sensing of other cytokines as second message,second messenger by the receptor protein STING on endoplasmic reticulum.Model about STING regulation signal conduction shows that the combination of receptor can cause the conformation change of STING, causes convening and activating of protein kinase TBK1 in Signaling complex.Transcription factor IRF3 entering signal complex the most therewith by TBK1 phosphorylation, the IRF3 of phosphorylation forms oligomer and is transported in nucleus, starts the expression of interferon beta.Interferon beta can regulate and control the expression more than 200 kinds of interferon-stimulated genes, they can down-regulation protein matter synthesis, promote cell growth arrest and inducing cell apoptosis, thus the state forming a kind of antiviral controls the propagation of virus.

Research in the model of STING mediation signal conduction shows, the combination of cGAMP can cause STING that allosteric effect occurs, thus cause enrichment and the activation of protein kinase TBK1 in Signaling complex, and then cause transcription factor interferon regulatory factor 3(IRF3) phosphorylation, the IRF3 of phosphorylation can occur oligomerization and be transported in nucleus, it can induce the expression of interferon-β (IFN-β) in nucleus, and the expression of IFN-β cytokine gene interferon-induced to kind more than 200 has regulation effect, thus by the expression of albumen in downward tumor cell, growth of tumour cell is stagnated and inducing apoptosis of tumour cell is to embody its antineoplastic function.IFN-β has been applied to the clinical treatment of leukemia, multiple sclerosis etc. by FDA approval, and cGAMP is as the activator of STING, strengthens human body innate immunity, the up-regulated expression of induction I-type interferon, and then activates T cell.Therefore, cGAMP has the huge potential quality being applied to antitumor clinical treatment.Two nearest researchs show that the cytoplasmic DNA sensing depending on STING path can regulate the innate immunity identification function for immunogenic cancer cell, and the immunological effect that can rely on by activating I type interferon becomes the complementary therapy of radiation treatment.Dendritic cell is the internal topmost class antigen-presenting cell presenting tumor associated antigen, and when dendritic cell is exposed to danger or inflammatory signals, they have high susceptibility to this task, it is possible to cause the cross activation of CD8+ T cell to react.I type interferon (comprising interferon-ALPHA and interferon beta) is as a class well-known immune activation factor family, it is possible to the cross immunity of Dendritic Cells Induced is produced maximally effective activation.Recent internal and in vitro study all shows that the interferon-induced dendritic cell cross-immune reaction for tumor specific antigen of I type is the mechanism of a kind of key of tumour immunity monitoring, and can promote the tumor-killing effect of CD8+ T cell.Recently, we have discovered that sulfur (selenium) shows the antitumor more higher than cGAMP, antiviral activity for phosphoric acid ring dinucleotide cGAMP.Therefore, sulfur (selenium) has potential significant application value for phosphoric acid ring dinucleotide cGAMP at antitumor, antiviral drugs development field.

Summary of the invention

1. prepared by the scale of Mus source cGAMP synzyme cGAS

2, using the Mus source ring dinucleotide cGMP-AMP synzyme cGAS catalysis of restructuring, scale prepares sulfur (selenium) for phosphoric acid ring dinucleotide cGAMP.

3, sulfur (selenium) is for phosphoric acid ring dinucleotide cGAMP high efficiency separation and purification method.

Accompanying drawing explanation

Figure 1 It is that sulfur (selenium) is for phosphoric acid ring dinucleotide cGAMP Molecular structure

Detailed description of the invention

Embodiment 1 : Mus source cGAMP Synzyme cGAS Efficient preparation

( 1 )Mus source ring dinucleotide cGAMP synzyme cGAS gene is bought from ATCC company of the U.S., by genetic engineering means by its successful clone to pET-28 (a) carrier being purchased from Novagen company, Mus cGAS gene N-end is with SMT3 fusion tag, containing 6 histidine Ni-NTA post affinity tag on SMT3 label protein.Gene sequencing result shows that we successfully construct Mus source ring dinucleotide cGAMP synzyme cGAS gene expression plasmid.

( 2 )Inoculation: (be purchased from Novagen company) with e. coli bl21 (DE3) as Host Strains, convert, choose speckle in LB culture medium culturing.The LB culture fluid that shake flask culture concentration is 25 g/L for inoculation.Shaking flask is placed in full temperature control shaking table, and 240 rpm shaken cultivation are overnight.The OD600 measured value of seed is between 2-4.Inoculum concentration is the 2%-5% of working volume.

( 3 )Fermentor cultivation E. coli expressing protein:

Desk-top fermentation cylinder is used to cultivate 20 L tank bodies and the Rushton stirring paddle of E. coli feeding culture.Control and regulation speed of agitator, temperature, pH, dissolved oxygen, foam/liquid level, three peristaltic pumps and ventilations etc..It is provided that supply control by a thermal mass gas flow meters (TMFC).Initial incubation based component is: sodium dihydrogen phosphate (NaH2PO4,3.5 g/L), disodium hydrogen phosphate (Na2HPO4,5.0 g/L), ammonium sulfate ((NH4) 2SO4,5.0 g/L), yeast extract (5.0 g/L), defoamer.After high temperature sterilize cooling, add: magnesium sulfate (25% solution, ultimate density is 4 ml/L), glucose, (10g/L, it is usually added into the solution of 50%), K-12 trace meter (ultimate density is 1 ml/L), thiamine (addition determines according to Stock concentrations, and ultimate density is 2.2 mg/L), calcium chloride dihydrate (addition determines according to Stock concentrations, and ultimate density is 0.15g/L).[K-12 Trace Metal solution, including: sodium chloride (NaCl, 5 g/L), zinc sulphate heptahydrate (ZnSO4-7H2O, 1 g/L), manganese chloride tetrahydrate (MnCl2-4H2O, 4 g/L), Iron trichloride hexahydrate (FeCl3-6H2O, 4.75 g/L), copper sulphate pentahydrate (CuSO2-5H2O, 0.4 g/L), boric acid (H3BO3,0.575 g/L), Sodium Molybdate Dihydrate (NaMoO4-2H2O, 0.5 g/L), 6N sulphuric acid (H2SO4, ~ 12.5 ml/L).】

Control pre-set parameter: fermentation the operation time be 22 hours, control temperature be 37 ° of C, pH be 7.0, DO is 30%.Initial speed of agitator is 200rpm, is controlled rotating speed by DO association thereafter.After initial incubation 5 hours, proceeding by feeding culture, after fermenting 5 hours, along with exhausting of carbon source, pH value terminates in 7.1, and BioCommand Automatic Program opens feed supplement pump.DO and pH is also accurately controlled.The dry cell weight cultivating E. coli acquisition is 25 g/L.

( 4 ) Mus sourceCGAS pheron purification

Somatic cells is dissolved in 50mM Tris.HCl(pH 7.5), use cell crushing instrument smudge cells, protein-contg supernatant is obtained after high speed centrifugation, then it being purchased from Qiagen company with Ni-NTA() affinity column initial gross separation purifies cGAS albumen, with SDS-Page analyzing proteins purity (85%), and then remove SMT3 fusion tag albumen with SUMO protease (being purchased from Qiagen company) enzyme action, then it is isolated and purified to carry out second time Ni-NTA affinity column.Finally use HiLoad^TMSuperdex 75 gel column is further purified, and obtains 95% purity cGAS pheron, preserves and-80 degree ultralow temperature refrigerators after lyophilization.

Embodiment 2: Adenosine ( Guanosine )5 ’ - The preparation of α sulfur generation (seleno) tricresyl phosphate phosphoric acid

Adenosine (guanosine) 5 '-α sulfur generation (seleno) tricresyl phosphate phosphoric acid according to literature method synthesis (Caton-Williams Julianne etc., Science China, Chemistry, 2012, 55(1), 80-89; Boyle Nicholas A., Nucleosides, Nucleotides and Nucleic Acids, 2005, 24, 1651-1664.)。

Embodiment 3: Use the Mus source ring dinucleotide of restructuring cGMP-AMP Synzyme cGAS Catalysis , Prepare sulfur ( Selenium ) For phosphoric acid ring dinucleotide cGAMP

Use the Mus source restructuring cGAS enzyme catalysis of preparation, prepare sulfur (selenium) the most in specific manner for phosphoric acid ring dinucleotide cGAMP.Reaction system is 20 liters of biological enzyme reactors, reaction system (10 mM) Han Mus source cGAS, adenosine 5 '-α sulfur generation (seleno) tricresyl phosphate disodic alkaliine (10 mM), GTP [or guanosine 5 '-α sulfur generation (seleno) tricresyl phosphate disodic alkaliine] (10 mM), MgCl2 (10 mM) DNA (0.2mg/ml), NaCl (100 mM), reaction temperature 37 degree, 8 hours time.With spectrophotometer in 260nm detection product and reactant, reaction yield more than 95%.

4 , sulfur ( Selenium ) For phosphoric acid ring dinucleotide cGAMP Scale high efficiency separation and purification method

Isolated and purified with 100 milliliters of bulk ionic exchange columns (being purchased from GE company), gradient elution sulfur (selenium), for phosphoric acid ring dinucleotide cGAMP, obtains the purity sulfur (selenium) higher than 95% for phosphoric acid ring dinucleotide cGAMP.About 250 grams of sulfur (selenium) are obtained for phosphoric acid ring dinucleotide cGAMP lyophilizing white powder samples after-50 degree freezer dryer lyophilizations.

Claims

1. the efficient large-scale preparation method of sulfur generation (seleno) phosphoric acid ring dinucleotide cGAMP.

2. the high efficiency preparation method of Mus source cGAMP synzyme cGAS.

3. using ring dinucleotide cGAMP synzyme cGAS catalysis, the method for sulfur generation (seleno) phosphoric acid ring dinucleotide cGAMP is prepared in scale.

4. sulfur generation (seleno) phosphoric acid ring dinucleotide cGAMP high efficiency separation and purification method.