36 results about "Cytidylic Acids" patented technology

The invention belongs to the technical field of biological medicine, and particularly discloses a composite vaccine adjuvant as well as a preparation method and application thereof. The composite vaccine adjuvant provided by the invention is an oil-in-water emulsion, and simultaneously contains squalene, polysorbate 80, sorbitan trioleate, polyinosinic acid (Poly I: C) and CpG oligodeoxynucleotide (CpG-ODN). The sequence-optimized CpG-ODN and Poly I: C are used in a specific emulsion system in a combined mode, a remarkable synergistic immune enhancement effect can be generated, the induced humoral immunity and cellular immunity response level is remarkably higher than the sum of the effects when all the components are independently used, and the composite adjuvant is good in stability, high in immunostimulatory activity and suitable for clinical application. The immune effect can be remarkably improved by combined application with various vaccine antigens. The invention also provides a preparation method and application of the composite vaccine adjuvant.

The utility model discloses an anion exchange resin column structure for synthesizing cytidine monophosphate, which comprises an exchange column body, a liquid inlet pipe and a regenerated liquid discharge pipe are arranged at the top of the exchange column body, and a liquid discharge pipe and a regenerated liquid filling pipe are arranged at the bottom of the exchange column body; the middle part of the exchange column body is divided into a plurality of compartments by filter plates, a static pressure chamber is reserved at the top, a confluence chamber is reserved at the top, and the compartments are filled with weakly alkaline organic acid anion exchange resin. Aiming at the impurity characteristics in the synthesis process of cytidine monophosphate, the utility model adopts a mode of feeding regeneration liquid from the bottom and discharging regeneration liquid from the top and countercurrent regeneration, and can ensure that the resin at the bottommost layer is in contact with the most fresh eluent, so that a good adsorption effect is kept in the next exchange, and the recovery rate is ensured. The resin is respectively arranged in the compartments in a manner of additionally arranging the partition plates, and although the resin can move in the compartments due to a suspended state in the exchange process, the partition plates can be maintained to be roughly layered and are not changed, so that the effect of countercurrent regeneration is ensured.

The present invention discloses a method and application for efficiently producing cytidylic acid, and belongs to the field of biotechnology. The present invention integrates the pyrimidine operon gene cluster pyr_Bs of Bacillus subtilis at the gene locus pta, reconstructs the UMP synthesis pathway in Escherichia coli, improves the metabolic flux of the 5'-cytidylic acid synthesis pathway, and realizes efficient production of 5'-cytidylic acid (5'-CMP). The yield of 60h fermentation in a 60L fermenter reaches 49g / L.

This invention relates to a zebrafish model, its establishment method, and its applications, relating to the biomedical field. The establishment method includes the following steps: injecting zebrafish with polyinosinic-polycytidylic acid (poly I:C), resuscitating, and culturing. This method can obtain a stable zebrafish model that can simulate an inflammatory state similar to that in humans following viral infection. This model can be used to assess whether antiviral drugs inhibit viral activity and the differences in efficacy among different antiviral drugs, thereby enabling preliminary screening of antiviral drugs.

The invention belongs to the technical field of bioengineering, and discloses a preparation method of an engineered exosome with a better treatment effect, and the preparation method comprises the following steps: S1, stimulating mesenchymal stem cells by using 10 [mu] g / mL of polyinosinic acid cytidine monophosphate for 1 h, and removing the polyinosinic acid cytidine monophosphate; s2, introducing a DMEM / F12 culture medium containing 10% of fetal calf serum into the mesenchymal stem cells subjected to stimulation treatment; s3, putting the mesenchymal stem cells into a cell incubator, culturing for 24 hours, and separating and purifying the exosome by adopting a conventional method; according to the invention, stem cells are pretreated by polyinosinic acid cytidine monophosphate, the stem cells are induced to secrete exosomes rich in therapeutic factors, and advanced preparation and purification technologies are combined, so that the engineered exosomes which are stable in quality and have a better therapeutic effect on osteoarthritis are successfully prepared.

The invention provides a method for simultaneously determining various flavor nucleotides in a livestock and poultry meat product and application, and the method comprises the following steps: freeze-drying the livestock and poultry meat product, and then preparing the freeze-dried livestock and poultry meat product into a test solution; measuring the contents of CMP (cytidylic acid), ATP (adenosine triphosphate), GMP (guanine monophosphate), IMP (inosinic acid), ADP (adenosine diphosphate), Hy (hypoxanthine), AMP (adenosine monophosphate) and IN (inosine) in the test solution by adopting HPLC (high performance liquid chromatography); in HPLC, a mobile phase A is a phosphate buffer solution with the volume concentration of 95%, a mobile phase B is methanol with the volume concentration of 5%, gradient elution is carried out, and the gradient elution condition is 0-11 min, 99% phosphate buffer solution and 1% methanol; when 12 min, 80% phosphate buffer solution and 20% methanol are added, and the conditions are maintained for 6-7 min; the phosphate buffer solution is heated to 99% within 2 minutes, and then the temperature is kept for 8-10 minutes.

The invention discloses a method for low-cost and large-batch production of high-purity 5 '-nucleotide, and belongs to the technical field of purification of biological products. Penicillium citrinum M843 with high yield of nuclease P1 is obtained through mutagenesis screening, and a nuclease P1 concentrated enzyme solution is obtained through fermentation, filtration and concentration; the method comprises the following steps: adding a 5 '-nucleotide solution into an RNA aqueous solution for enzymolysis to obtain a 5'-nucleotide solution, removing impurities, loading the 5 '-nucleotide solution to a chromatographic column filled with strongly basic styrene anion exchange resin, respectively collecting a 5'-cytidine acid eluent, a 5 '-adenosine acid eluent, a 5'-uridine acid eluent and a 5 '-guanosine acid eluent, carrying out nanofiltration desalination and concentration, carrying out activated carbon decoloration, carrying out soluble calcium compound dephosphorization and residual protein removal, and carrying out freeze drying to obtain the 5'-adenosine 5 '-monophosphate. According to the present invention, with the method, the wastewater discharge and the wastewater treatment are significantly reduced, the time and the production cost are saved, the high-purity nucleotide product can be prepared, and the good application prospect is provided.

The invention discloses a preparation method of a high-activity deoxycytidine monophosphate deaminase mutant, and relates to the technical field of bioengineering, and the preparation method comprises the following steps: 1, synthesizing a deoxycytidine monophosphate deaminase female parent and a mutant gene; 2, screening dominant mutants; 3, purifying deoxycytidine monophosphate deaminase and the mutants; and 4, determining the specific enzyme activity of the enzyme. A plurality of mutation sites are introduced through a site-specific mutagenesis technology to optimize the catalytic performance of the deoxycytidine monophosphate deaminase, and required mutation is introduced into a specific position of the enzyme, so that the accurate regulation and control of the catalytic performance of the enzyme are realized, the catalytic activity and the reaction rate of the enzyme are remarkably improved, and the efficiency of the whole biological catalysis process is further improved; the thermal stability, pH stability and the like of the enzyme are enhanced by a site-directed mutagenesis technology, so that the enzyme can maintain catalytic activity under wider conditions, the amino acid sequence of the enzyme is optimized, the enzyme can better adapt to specific substrates or reaction conditions, and the activity of the deoxycytidine monophosphate deaminase in a dCMP deamination reaction is improved.

The application discloses a transient transfection method for improving recombinant protein expression of CHO cells, and relates to the technical fields of eukaryotic cell gene transfection and animal cell culture. A cell suspension is obtained by mixing a CHO cell and a serum-free CHO cell basic culture medium; DNA and a transfection reagent are added into the cell suspension to mix a transfection culture, 5-azadeoxycytidine acid solution is added into the transfection culture at 0h-24h to form a transfection system and continue the transfection culture; the transfection system is added with a feeding culture medium at 1d-4d after the transfection culture to carry out suspension culture; the suspension culture is centrifuged at 7d to separate and harvest cell culture supernatant to complete recombinant protein expression. According to the application, 5-azadeoxycytidine acid is added into the transfection culture at a specific concentration, so that the expression of recombinant protein of the CHO cell is significantly improved during the transfection expression.

The present disclosure relates to the technical field of medicine, and in particular to a pharmaceutical composition, preparation method and use thereof. The pharmaceutical composition includes a first active ingredient, a second active ingredient, and a pharmaceutically acceptable carrier or excipient. The first active ingredient is a microbial agent, including one or more of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, and Salmonella paratyphi. The second active ingredient includes polyinosinic acid, polycytidylic acid, and vitamin. The pharmaceutical composition of the present disclosure pertains to an artificial active immunotherapy for tumors. It can “stimulate” the entire immune system, making the therapy of using bacteria to activate the human immune system to kill cancer cells highly stable and reliable. This composition can significantly save and prolong the lives of cancer patients while exhibiting extremely high safety, minimal toxic side effects, and low production costs.

This skin barrier function improving agent contains cytidylic acid or a precursor thereof as an active ingredient.

The invention discloses a method for chiral separation of tryptophan by using a capillary silica gel monolithic column modified by gold nanoflowers coated with cytidine monophosphate, and belongs to the field of pharmaceutical analysis. The method comprises the following steps: firstly synthesizing cytidine monophosphate coated gold nanoflower, using the cytidine monophosphate coated gold nanoflower as a chiral selector to prepare a capillary monolithic column modified by the cytidine monophosphate coated gold nanoflower, and separating chiral molecules under an electrophoresis condition. And the baseline separation of the tryptophan enantiomer can be realized. The separation method is simple to operate, and a novel high-selectivity chiral separation detection technology is established.

This invention provides a kit for the simultaneous detection of nine flavor nucleotides in livestock and poultry products. The flavor nucleotides are cytidine monophosphate, uridine monophosphate, guanylic acid, inosine monophosphate, adenosine monophosphate, hypoxanthine, inosine, adenosine diphosphate, and adenosine triphosphate. The kit includes: a high-concentration extraction reagent, a standard curve solution, a high-concentration phosphate buffer solution, pH adjustment solution A, and pH adjustment solution B. The high-concentration extraction reagent is perchloric acid; the standard curve solution is a mixed solution of the nine flavor nucleotides; the high-concentration phosphate buffer solution is a solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate; pH adjustment solution A is sodium hydroxide; and pH adjustment solution B is phosphate. This invention also provides a method for the simultaneous detection of the nine flavor nucleotides using the above kit. This invention enables the simultaneous determination of the content of nine flavor nucleotides in livestock and poultry products. The method is simple, rapid, accurate, and sensitive, and can be used for batch determination, showing good prospects for practical application.

The application discloses a kind of solid phase extraction-liquid quality connection simultaneously detecting multiple nucleotides in drinking water in the technical field of water pollution monitoring, first using solid phase extraction enrichment target material, nitrogen blow concentration, finally using the concentration of guanosine acid, adenosine acid, cytidine acid, uridine acid, deoxyguanosine acid, deoxyadenosine acid, deoxycytidine acid and deoxythymidine acid is determined using the multiple reaction monitoring mode of liquid chromatography tandem mass spectrometry, the present application combines solid phase extraction pretreatment technology with the detection technology of liquid chromatography tandem mass spectrometry, realizes the detection of trace nucleotide under complex matrix condition, the present application can realize the simultaneous determination of multiple nucleotides by one sample injection, with the advantages of high recovery rate, short detection time, many detection types and the like.

The invention relates to the field of enzymatic production of deoxyribonucleoside triphosphate, in particular to a method for enzymatic synthesis of deoxyribonucleoside triphosphate. According to the method, the salmon sperm DNA is subjected to enzymolysis through deoxyribonuclease to obtain dNMP, and the enzymolysis rate reaches 99.5% or above; and carrying out catalytic reaction on the dNMP obtained by enzymolysis by utilizing adenylate kinase, thymidylate kinase, cytidine monophosphate kinase, guanylate kinase, nucleoside diphosphate kinase and acetokinase to obtain corresponding dNTP, wherein the molar conversion rate is greater than 80%. The synthesis method is simple to operate, short in production period, easy to separate and purify and low in production cost, and has important industrial application prospects.

The invention discloses engineering escherichia coli for synthesizing sialyloyl-N-tetrasaccharide a as well as a construction method and application of the engineering escherichia coli, and belongs to the technical field of microbial metabolism engineering. Recombinant escherichia coli E.coli BL21 (DE3) delta lacZ delta wecB delta nagB delta ugD, delta recA:: Ptac-galE delta ISl86-1:: Ptac-lgtA, delta IS186-2:: Ptac-lgtA, delta IS186-4:: Ptac-lgtA and delta IS186-5:: Ptac-lgtA are taken as initial strains, 5 '-cytidine monophosphate-N-acetyl nerve amino acid pathway genes nenC, nenB and nenA and beta-1, 3-galactosyl transferase genes wbgO and alpha2, 3-galactosyl transferase genes wbgO are over-expressed by knocking out 5'-cytidine monophosphate-N-acetyl nerve amino acid The recombinant escherichia coli capable of stably synthesizing sialic acid lactyl-N-tetrasaccharide a is obtained by adding a 1, 3-sialyltransferase specific gene Pm0188 into the recombinant escherichia coli. By screening more efficient alpha 2, 3-sialyltransferase and optimizing a pathway gene expression combination, the yield of sialyloyl-N-tetrasaccharide a is increased, and the sialyloyl-N-tetrasaccharide a with the yields of 1.235 g / L and 4.85 g / L respectively is finally obtained under shake-flask culture and fed batch culture.

The present invention discloses a pharmaceutical composition, its manufacturing method, and its use, wherein the pharmaceutical composition comprises a first active ingredient, a second active ingredient, and a pharmaceutically acceptable carrier or excipient, the first active ingredient being a microbial agent containing one or more of Staphylococcus aureus, Bordetella pertussis, diphtheria toxoid, tetanus toxoid, Salmonella typhi, or Salmonella paratyphi, and the second active ingredient comprising polyinosinic acid, polycytidylic acid, and vitamins. The pharmaceutical composition is an artificial active immunotherapy for tumors, which, by "stimulating" the entire immune system, activates the human immune system using bacteria to kill cancer cells, making the treatment extremely stable and reliable, significantly saving and extending the lives of cancer patients, while simultaneously being extremely safe, having very few toxic side effects, and having low manufacturing costs.

The invention provides application of taurochenodeoxycholic acid and / or chenodeoxycholic acid and polyinosinic acid combined medicine in preparation of medicine for preventing and / or treating melanoma, and belongs to the field of biological medicine. It is found for the first time that taurochenodeoxycholic acid and / or chenodeoxycholic acid can influence the tumor immune microenvironment by regulating and controlling the antigen presentation function of cDC1, and the treatment effect of polyinosinic-cytidylic acid is improved. The melanoma model experiment directly shows that the combination of chenodeoxycholic acid and polyinosinic-cytidylic acid can better inhibit tumor growth, can effectively promote CD8 + T cell infiltration in tumors, has more cDC1 infiltration in tumor tissues, shows a stronger inhibition effect on tumors, and can be used for preparing drugs for treating tumors. The pharmaceutical composition plays a synergistic role in treatment of melanoma, and provides a new choice for clinical treatment of melanoma.

The invention relates to the technical field of biological models, in particular to a mouse model for simulating neurodevelopmental disorder, which comprises the following steps: injecting a polyinosine-polycytidylic acid compound into a mouse during pregnancy to simulate maternal immune activation; separating the mother and the baby after the mouse is delivered, so as to obtain the mouse model for simulating the neurodevelopmental disorder. Through the mouse model, the common influence of multiple environmental factors on nervous system development can be more accurately researched, and the mouse model is particularly suitable for researching a mechanism of long-term psychological and behavioral effects and is beneficial to developing new intervention strategies and treatment methods.

The invention relates to a preparation method of cytidine-5 '-diphosphate. The cytidine-5'-diphosphate comprises the following raw materials: 5 '-cytidine monophosphate, adenosine triphosphate and sodium hexametaphosphate. Immobilized enzymes: 5 '-cytidine monophosphate kinase and polyphosphate kinase; 5 '-cytidine monophosphate, adenosine triphosphate and sodium hexametaphosphate are used as raw materials, 5'-cytidine monophosphate kinase and polyphosphate kinase are subjected to a one-pot catalytic reaction, and cytidine-5 '-diphosphate is synthetized.The cytidine-5'-diphosphate prepared through the method has the advantages that the raw materials are easy to purchase and low in price, and generation of by-products is reduced.

The invention relates to a preparation method of uridine 5 '-disodium phosphate and uridine 5'-disodium phosphate. The uridine 5 '-disodium phosphate comprises the following raw materials: cytidine 5'-phosphoric acid; the catalytic enzyme is cytidine monophosphate deaminase; cytidine 5 '-phosphate is used as a raw material, an elastin tag is fused behind an initiation codon of cytidine deaminase to obtain cytidine deaminase with the elastin tag, the cytidine deaminase with the elastin tag is purified, and uridine 5'-disodium phosphate is synthesized through a one-step catalytic reaction. The uridine 5 '-disodium phosphate prepared by the preparation method disclosed by the invention does not contain a large amount of phosphate, magnesium ions and adenylic acid impurities, so that a large amount of sewage generated in the purification process is avoided.

According to the improved poly-ICLC compound provided by the invention, epsilon-polylysine is adopted to replace traditional poly-L-lysine, so that the cytotoxicity is remarkably reduced (ALT is reduced by 48.5%), and meanwhile, the excellent immune activation capability is kept (the tumor inhibition rate is increased by 9.5%). The compound can be stably preserved for more than 6 months at 4 DEG C, and is a safe and efficient cancer vaccine adjuvant.

The present disclosure relates to lipid nanoparticle (LNP) compositions including at least one ionizable cationic lipid and Polyinosinic:polycytidylic acid (poly(I:C)), and compositions and formulations incorporating such LNP compositions. The disclosure also provides methods of using these compositions to elicit an immune response, including for the prevention or treatment of infectious diseases.

The invention belongs to the technical field of biological medicines, and particularly discloses a DC cell-based tumor vaccine and a preparation method thereof, and the preparation method comprises the following steps: (1) separating peripheral blood to prepare a mononuclear cell suspension; (2) inoculating the mononuclear cell suspension into a first culture medium, culturing for 48-72 hours, then replacing 40-60% of the first culture medium every 48-72 hours, and continuing culturing to obtain a first cell sap; (3) preparing the tumor tissue into a cell lysate; and (4) adding the cell lysate, polyinosinic-cytidylic acid, TNF-alpha and alpha-ketoglutaric acid into the first cell sap, and carrying out co-culture to obtain the DC cell-based tumor vaccine. The DC cell-based tumor vaccine prepared by the method provided by the invention can specifically kill tumor cells.

The application discloses a method for enhancing 5'-cytidylic acid accumulation and application, and belongs to the field of genetic engineering and microbial engineering. The application knocks out ppnN, ushA, yrfG, yjjG, umpH and umpG genes, overexpresses 5'-CTP diphosphohydrolase gene nudG and orotidine-5'-phosphate ribosyltransferase gene pyrE, integrates expression of uridine phosphorylase mutant gene PyrH(R92G / D93G), further knocks out cytidine deaminase gene cdd and ribonucleoside-triphosphate reductase gene nrdD, and integrates expression of uridine-cytidine kinase gene udk, phosphoribosyl pyrophosphate synthetase gene prs, glucose-6-phosphate dehydrogenase gene zwf and 6-phosphogluconate dehydrogenase gene, so that the 5'-cytidylic acid accumulation capacity of Escherichia coli is improved. Under the condition that no antibiotic and inducer are added, the 5'-cytidylic acid yield of the recombinant bacteria reaches 39.6 g / L in a 60L fermenter.

The invention belongs to the field of gene engineering, and particularly relates to a polyphosphate kinase mutant and application thereof. According to the invention, modification and screening are carried out on the basis of amino acids of wild type polyphosphate kinase PPK (polyP-polyphosphate kinase [EC: 2.7.4. 1]) from Mycobacterium tuberculosis, and the polyphosphate kinase with high enzyme activity, high catalytic efficiency and high ATP regeneration efficiency is obtained. The amino acid sequence of the polyphosphate kinase mutant is as shown in SEQ ID NO.11, and the polyphosphate kinase mutant is obtained by mutating valine at the 267th site of wild type polyphosphate kinase into glutamic acid and simultaneously mutating threonine at the 369th site into proline. Uridine monophosphate and cytidine monophosphate are produced through catalysis of the polyphosphokinase, the conversion yield is larger than 95%, meanwhile, cyclic regeneration of ATP is efficiently achieved, the ATP adding amount is reduced by 97%-98%, the product concentration is increased, the production cost is reduced, the catalysis application potential is greatly improved, and the important industrial application prospect is achieved.

The present application relates to a kind of preparation, separation detection method and application of the impurity of sodium cytidine phosphate with structure I, comprising the following steps: 5'-cytidylic acid, choline phosphate and sodium tert-butanolate aqueous solution are mixed to be prepared into reaction solution, then catalyst is added to carry out reaction, the solution after reaction is collected, chromatographic column chromatography separation, crystallization drying, the impurity of sodium cytidine phosphate with structure I is obtained.The raw material of the preparation method of the present application is environmental protection, process is simple, yield is high, fills the technical blank of the preparation of the impurity of sodium cytidine phosphate with structure I, and the research on the preparation method is conducive to guiding the optimization of cytidine phosphate sodium synthesis process route and ensuring cytidine phosphate sodium safe drug use.