39 results about "Apramycin" patented technology

The invention discloses a method for preparing apramycin through fermentation. The method comprises the following steps: performing slant culture, culturing a seed liquid, and performing fermentation culture. The method is simple to operate, and due to replenishment of corn steep liquor in the fermentation culture, the fermentation unit of apramycin is greatly increased. The fermentation unit in apramycin prepared by the method can be up to 8400U / mL.

The invention discloses a method for simultaneously analyzing various aminoglycoside compound residues in agricultural and animal products. The method comprises the following steps: weighing a sample;adding an ammonium acetate buffering solution; after carrying out oscillation and extraction, carrying out centrifuging; separating an extracting solution obtained by centrifuging and regulating to asuitable range by utilizing a PH (Potential of Hydrogen) meter; purifying by adopting dispersed solid-phase extraction and a solid-phase extraction small column. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), electrospray ionization (ESI) and multi-reaction monitoring (MRM) scanning modes are adopted. Compared with other similar methods, the method disclosed by the invention has the advantages of simplicity in operation and high sample flux; eight types of aminoglycoside compounds including streptomycin, dihydrostreptomycin, spectinomycin, gentamicin, apramycin, kanamycin, amikacin and neomycin are detected at the same time; the detection efficiency is greatly improved, and rapid qualitative and quantitative detection can be rapidly carried out; the detection limit can completely meet domestic and overseas laws and regulations and limitation requirements, and powerful support is provided for food safety guarantees and import and export of the agricultural and animal products of China.

The invention discloses a milbemycin positive regulation gene milR and an overexpressed genetic engineering bacterium, a preparation method and application thereof. The sequence of the milbemycin positive regulation gene milR is shown as SEQ ID NO.1, an overexpression plasmid containing the gene and a promoter sequence is established and converts an escherichia coli host cell, conjugational transfer is conducted on the obtained converter and streptomyces bingchengensis, a zygote having apramycin resistance is picked, namely the genetic engineering bacterium having high milbemycin yield can be obtained, and the milbemycin can be produced by fermenting the genetic engineering bacterium. The yield of milbemycin A3 / A4 is improved, the production cost is reduced, and the economic benefit is improved.

The invention relates to a method for detecting aminoglycosides in milk, which comprises the following steps: (1) extracting; (2) preparing a standard working solution; (3) performing determination bya high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), wherein the aminoglycosides are paromomycin, tobramycin, gentamicin, kanamycin, amikacin, apramycin, streptomycin, dihydrostreptomycin, neomycin, spectinomycin and hygromycin B. An MIP solid phase extraction column is uses in the step of extracting, thereby simplifying the pretreatment step, reducing the consumption of reagents and the time uses for the pretreatment; a SiELC Obelisc R chromatographic column is used for the separation of 11 kinds of aminoglycosides; and the mobile phase does not require heptafluorobutyric acid and does not contaminate the mass spectrometry.

The invention relates to a tylosin gene engineering strain. The construction method for the tylosin gene engineering strain comprises the following steps: cloning tylosin through polymerase chain reaction (PCR) to synthesize crucial rate-limiting enzyme gene TylF and Vitreoscilla hemoglobin VHb gene, constructing a recombinant plasmid by using an E. coli-streptomyces shuttle vector, importing the recombinant plasmid into Escherichia coli ET12567 / pUZ8002, performing conjugal transfer culture on the Escherichia coli ET12567 / pUZ8002 and streptomyces fradiae spores, converting streptomyces, screening a converter through apramycin resistance, further confirming the converter by using PCR, and finally obtaining the gene engineering strain. The tylosin gene engineering strain can be directly used in the industrial production of tylosin; and compared with the tylosin gene engineering strain used in the conventional industrial production, the tylosin gene engineering strain has the advantages that the yield of the tylosin can be obviously improved to be 17,000-18,000u / ml and the content of a component A in the tylosin is effectively improved to be 94-95 percent.

The invention discloses an apramycin liposome. The apramycin liposome is a composition with a spherical or ellipsoidal double-film structure, and is formed by encapsulating apramycin with a liposome film. A preparation method comprises the following steps: 1) dissolving the apramycin and the liposome film with ethanol to obtain a mixed solution, and dissolving the mixed solution into supercritical CO2 to obtain a supercritical solution, wherein the mass ratio of the apramycin to the liposome film is (1 : 1) to (1 : 30); the liposome film is phosphatidylcholine, or a mixture of phosphatidylcholine and cholesterol; 2) dissolving a stabilizer into an aqueous medium to obtain a stabilizer solution; 3) spraying the supercritical solution to the stabilizer solution at an expansion pressure of 15-30MPa and a pre-expansion temperature of 323-343K, dispersing and precipitating to obtain an apramycin liposome suspension.

An oil-in-water type compound apramycin nano-emulsion belongs to the technical field of medicines, and is composed of the compositions in percent by weight: 1%-18% of apramycin, 1%-20% of rheum officinale extractive, 15%-40% of a surfactant, 1%-25% of an auxiliary surfactant, 1%-20% of oil and 20%-80% of distilled water. The compound apramycin bacteriostatic nano-emulsion is relatively narrow in emulsion particle size, transparent in system, good in stability, relatively low in surface tension, extremely good in fluidity and convenient to administrate. Because of the compounding of rheum officinale extractive and apramycin, the bacteriostatic effect is relatively good, and because of the nano-emulsion form, apramycin is relatively easy to absorb by bodies, and the usage amount and the usage frequency of the medicine can be reduced.

The invention relates to a new streptomyces secretion expression plasmid and application thereof. The new streptomyces secretionexpression plasmid is particularly characterized by being cloned with a colon bacillus replication original region ori, a streptomyces conjugational-transfer essential region oriT, a penbritin resistance gene bla, an apramycin resistance gene aac (3) IV, a promoter ermE*p, a lidamycin prosthetic-group protein gene promoter cagAp, a lidamycin prosthetic-group protein gene mutation type signal peptide SPcagA (CTG) and multiple cloning sites by taking a minimum replicon of a pSGL1 plasmid naturally existing in streptomyces globisporus C-1027 as a framework. The new streptomyces secretory expression plasmid can be used for efficiently secreting and expressing various heterologous proteins in streptomyces.

The invention relates to a preparation method and a use method of a colloidal gold test paper strip for fast detection of apramycin drug residues. The preparation method comprises orderly pasting a sample pad, a gold marking pad, a cellulose nitrate membrane and a sample absorption pad on the backing of a test paper. The surface of the cellulose nitrate membrane is coated with a detection antigen strip as a detection line and with an anti-second specie animal protein IgG strip as a reference line. The apramycin-detection colloidal gold test paper strip for fast detection has strong singularity, can realize semi-quantitative detection, can be used at an environment temperature of 4-40 DEG C, is suitable for fast detection of apramycin residues of animal-derived foods by individual farmers, food hygiene quality control department and customhouse. The apramycin-detection colloidal gold test paper strip has strong singularity and high sensitivity, can realize semi-quantitative detection and can be operated simply and conveniently.

The invention relates to ptsG gene knocked out recombination bacterial efficiently expressing human-like collagen protein, a construction method thereof, and protein expression. In the prior art, when escherichia coli is adopted to carry out fermentation production of human-like collagen protein, acetic acid accumulation can affect bacterial growth and protein expression, and a collagen protein yield can be reduced with the existing acetic acid byproduct reduction method. According to the present invention, escherichia coli BL21 with a preservation number of CGMCC No.0743 is adopted as starting bacterial, Red homologous recombination is adopted, and apramycin resistance gene is adopted to replace ptsG gene on the escherichia coli genome, and FLP incision enzyme expressed by plasmid pCP20 is adopted to eliminate the apramycin resistance gene to obtain the ptsG gene knocked out escherichia coli engineering bacterial CGMCC No.7331. According to the present invention, a gene engineering tool is adopted to transform an escherichia coli glucose absorption way so as to reduce acetic acid accumulation, improve human-like collagen accumulation, reduce glucose consumption by 9-28%, and improve human-like collagen protein yield by 20-30%.

The invention belongs to the field of biotechnology and relates to preparation of animal sperm low-temperature and freeze preservation diluents, in particular to a horse sperm low-temperature and freeze preservation liquid. The sperm low-temperature and freeze preservation diluent is prepared by combining sodium bicarbonate, hydroxyethylpiperazine ethane sulfonic acid, trisodium citrate dihydrate, potassium citrate monohydrate, sodium hydroxide, lactose, glucose, inositol, gentamicin, neomycin, apramycin, colistin, amphotericin, sodium pyrophosphate, sodium caseinate, whey protein, an antioxidant and deionized water. The horse sperm low-temperature and freeze preservation diluent can effectively increase the survival rate of sperms obtained after unfreezing of low-temperature and freezing of horse seminal fluid and the hybridization conception rate. Compared with a conventional method, the method is easy and stable to implement and especially remarkable in technical effect.

The present invention provides a method for overexpressing the zwf2 gene to increase the yield of daptomycin and its application, and clones a key enzyme 6-phosphate glucose dehydrogenase gene (zwf2) in the primary metabolic pentose phosphate pathway, using Escherichia coli-Streptomyces Shuttle plasmid expression vector construction of recombinant plasmids. The recombinant plasmid was introduced into Escherichia coli ET12567, ET12567 was co-cultured with Streptomyces roseospora, and Streptomyces roseospora was transformed by the combined transfer method, and then the transformants were screened by apramycin resistance, and the genetically engineered bacteria were further verified by PCR , thereby constructing the genetically engineered strain HP-2. The Streptomyces roseospora genetically engineered bacterium HP-2 constructed by the present invention has an 18% higher output than the starting strain, and the output of daptomycin is as high as 700mg / L, which can be directly used in the industrialized production of daptomycin, which can improve Yield reduces cost.

The invention discloses a compound antibiotic composition for preventing and curing piglet bacterial diarrhea and a preparation technology of a particulate premix thereof. Active components of the composition are Amoxicillin and apramycin with effective curative dose, and the dosage form is a particulate premix. With the organic combination of Amoxicillin and apramycin, the antibiosis effect of a single agent is greatly utilized, synergetic antibacterial advantages of the combination of the two drugs are displayed, and the combination of the two drugs widens antimicrobial spectrum, enhances antibiosis effectiveness and alleviates the generation speed of bacterial resistance. The compound antibiotic composition is mainly used to prevent and cure piglet bacterial diarrhea in clinic and simultaneously can be used to prevent and cure mixed infection caused by animal gram-positive bacteria and gram negative bacteria. The preparation technology is simple and easy to realize industrialization, requires low cost, is safe and efficient, and has high popularization and application values.

The invention relates to derivatives of apramycin-based aminoglycoside antibacterial drugs modified in positions C5 and / or C6 and O5 and / or O6. The modifications impart favourable properties regarding increased selectivity and retention of activity in the presence of resistance determinants of the AAC(3) class. The invention further relates to said compounds for use in the therapy of bacterial infection by systemic administration, especially in instances where the infection is caused by a pathogen comprising a resistance determinant of the AAC(3) class, in particular AAC(3)-IV.

The invention belongs to the technical field of organic-inorganic composite materials, and particularly relates to a slow-release bactericide suitable for a hospital environment and a preparation method of the slow-release bactericide. The slow-release bactericide suitable for the hospital environment includes apramycin, dodecylbenzene sulfonate and benzene sulfonate intercalated hydrotalcite. The preparation method comprises the following steps: firstly preparing a hydrotalcite precursor, then intercalating dodecylbenzene sulfonate and benzene sulfonate into the hydrotalcite precursor according to a certain proportion to obviously enlarge the interlayer spacing of hydrotalcite, then mixing the hydrotalcite with an apramycin aqueous solution, and diffusing apramycin into the interlayer of the hydrotalcite to obtain apramycin intercalated hydrotalcite, namely, the slow-release bactericide. According to the invention, the effect of adjusting the apramycin slow release rate can be achieved by adjusting the ratio of dodecylbenzene sulfonate radicals to benzene sulfonate radicals in layers.

The invention provides a magnetic immunochemiluminescence detection kit for apramycin. The magnetic immunochemiluminescence detection kit comprises an enzyme labeled antigen, an enzyme labeled antigendilution solution, a magnetically labeled antibody, a magnetically labeled antibody dilution solution, apramycin series standard solutions, a chemiluminescent substrate solution A, a chemiluminescentsubstrate solution B, a redissolution solution and a washing solution, wherein the enzyme labeled antigen is horseradish peroxidase-labeled apramycin hapten, and the magnetically labeled antibody isimmunomagnetic bead labeled apramycin monoclonal antibody. The invention further discloses a method for detecting the residual amount of apramycin in milk by using a chemiluminescence detector supporting the kit. According to the present invention, the method has advantages of high sensitivity, high specificity and short detection time in the detection of apramycin.

An apramycin containing supplement feeding stuff for poultry and is used for the prophylaxis and / or the treatment of histomoniasis.

The invention discloses a high-yielding kanamycin B genetically engineered bacterium and its construction and application. In the present invention, the apramycin biosynthetic gene in Streptomyces darker aprD3‑D4 In-frame knockout followed by further knockout of the carbamyltransferase gene tobZ , to obtain a production strain that accumulated a large amount of kanamycin B Streptomyces tenebrarius 314 (△ aprD3‑D4 +△ tobZ ). The bacterial strain of the present invention has high yield, good quality, single component and stable genetics, can be used for large-scale production of kanamycin B, and solves the long-standing need to separate a small amount of kanamycin A from the raffinate in the production process of kanamycin A The predicament of kanamycin B has realized the unique advantages of direct fermentation production of kanamycin B, achieved the purpose of clean production with low cost, low energy consumption and no secondary pollution, and created a new method for preparing kanamycin B. The method solves the problem of shortage of raw materials for the synthesis of arbekacin and dibekacin, and has a good application prospect.

The invention discloses a detection method for the logarithmic growth phase of an actinomycete strain producing apramycin, and relates to the technical field of microbial fermentation seed preparation. The detection method comprises the following steps of: S1, strain preservation: preparing a batch of actinomycete seed liquid culture medium and fermentation culture medium, subpackaging the liquidculture medium and the fermentation culture medium into seed shake flasks, setting three parallel experiments, and subpackaging the fermentation culture medium into fermentation shake flasks; and S2,seed culture medium: inoculating the preserved strain into the seed liquid culture medium in the step S1, taking the culture medium which is not inoculated with the strain as a blank control, and carrying out culture under the same condition and synchronizing with seed tank culture. According to the detection method for the logarithmic growth phase of the actinomycete strain producing apramycin, the original measurement technology is combined together and is combined with production experience, so that the defect of a single technology is overcome, the measurement result is more accurate, thetheory and the actual production experience are combined together, the laboratory and the production are tightly combined, and excellent seeds meeting the conditions are provided for production.

The invention provides a magnetic immunochemiluminescence detection kit for apramycin. The magnetic immunochemiluminescence detection kit comprises an enzyme labeled antigen, an enzyme labeled antigendilution solution, a magnetically labeled antibody, a magnetically labeled antibody dilution solution, apramycin series standard solutions, a chemiluminescent substrate solution A, a chemiluminescentsubstrate solution B, a redissolution solution and a washing solution, wherein the enzyme labeled antigen is horseradish peroxidase-labeled apramycin hapten, and the magnetically labeled antibody isimmunomagnetic bead labeled apramycin monoclonal antibody. The invention further discloses a method for detecting the residual amount of apramycin in milk by using a chemiluminescence detector supporting the kit. According to the present invention, the method has advantages of high sensitivity, high specificity and short detection time in the detection of apramycin.

The invention discloses a detection method for residual apramycin in chicken and a test strip. The detection method comprises the following steps: S1, first buying a live chicken with a normal body shape and a healthy growth condition separately from an individual farmer, a livestock farm and a vegetable market, then killing the chickens, taking out 1 to 2 kg of chicken tissues, placing the fetched chicken tissues respectively in different volumetric flasks, respectively marking as A1, A2 and A3, taking out an apramycin solution as a reference, marking as A4 and S2, and respectively taking out 1g to 5g of chicken tissues from A1, A2 and A3. The detection method adopted by the invention is simple in operation, short in detection time and capable of performing the detection at any time, and can judge reasons causing the residual apramycin in the chicken and an amount of the residual apramycin in the chicken to improve a raising method, so that chicken become healthier, the harm to a human body can be avoided, the loss can also be reduced, and the cost can be decreased.

The invention relates to ptsG gene knocked out recombination bacterial efficiently expressing human-like collagen protein, a construction method thereof, and protein expression. In the prior art, when escherichia coli is adopted to carry out fermentation production of human-like collagen protein, acetic acid accumulation can affect bacterial growth and protein expression, and a collagen protein yield can be reduced with the existing acetic acid byproduct reduction method. According to the present invention, escherichia coli BL21 with a preservation number of CGMCC No.0743 is adopted as starting bacterial, Red homologous recombination is adopted, and apramycin resistance gene is adopted to replace ptsG gene on the escherichia coli genome, and FLP incision enzyme expressed by plasmid pCP20 is adopted to eliminate the apramycin resistance gene to obtain the ptsG gene knocked out escherichia coli engineering bacterial CGMCC No.7331. According to the present invention, a gene engineering tool is adopted to transform an escherichia coli glucose absorption way so as to reduce acetic acid accumulation, improve human-like collagen accumulation, reduce glucose consumption by 9-28%, and improve human-like collagen protein yield by 20-30%.

The invention relates to a preparation method of crystallized apramycin sulfate, which comprises the steps of: with an apramycin concentrated solution as a raw material, blending a feed solution, salifying and crystallizing, drying and the like. The concentration of the feed solution is regulated to a certain range, filtrate is added in alcohol quantitatively under the stirring, the crystallizingtemperature is controlled, therefore, most of impurities in the feed solution are removed, and the crystallized apramycin sulfate with good graininess and purer component is obtained.

The invention discloses an apramycin liposome. The apramycin liposome is a composition with a spherical or ellipsoidal double-film structure, and is formed by encapsulating apramycin with a liposome film. A preparation method comprises the following steps: 1) dissolving the apramycin and the liposome film with ethanol to obtain a mixed solution, and dissolving the mixed solution into supercritical CO2 to obtain a supercritical solution, wherein the mass ratio of the apramycin to the liposome film is (1 : 1) to (1 : 30); the liposome film is phosphatidylcholine, or a mixture of phosphatidylcholine and cholesterol; 2) dissolving a stabilizer into an aqueous medium to obtain a stabilizer solution; 3) spraying the supercritical solution to the stabilizer solution at an expansion pressure of 15-30MPa and a pre-expansion temperature of 323-343K, dispersing and precipitating to obtain an apramycin liposome suspension.

The invention discloses a preparation method of an apramycin industrial production strain. Comprising the following steps: selecting single colonies from a flat plate to a shake flask, culturing a seed solution in the shake flask, preparing a frozen seed tube for the seed solution, performing slant activation on the seed solution in the frozen seed tube, preserving mature slant spores in a milk frozen tube, counting viable bacteria, preparing a spores solution in a tank, and inoculating the spores in the tank. According to the preparation method of the apramycin industrial production strain, the phenomenon that the fermentation level is unstable due to frequent slope replacement in a traditional method can be avoided. The preparation method is easy and convenient to operate, the amount of spore liquid fed into the tank each time is constant, and then the stability of the seed quality of the seed tank is guaranteed.

The invention provides a construction method and application of streptomyces roseosporus gene engineering bacteria. The construction method comprises the following steps of: cloning three key genes ORF53-55 for coding ATP-binding transport protein at the downstream part of a non-ribosomal protein synthase (NRPS) gene in a daptomycin synthetic gene cluster, and constructing recombinant plasmids by using an Escherichia coli-streptomyces shuttle plasmid expression vector; leading the recombinant plasmids into Escherichia coli ET12567; culturing the ET12567 together with the streptomyces roseosporus; converting the streptomyces roseosporus by a combining and transferring method; then, screening converters through the resistance of apramycin; and making a further verification on the gene engineering bacteria through polymerase chain reaction (PCR). The streptomyces roseosporus gene engineering bacteria constructed by the invention can be directly applied to the industrialized production of daptomycin, the yield can be improved, and the cost can be lowered.

The invention provides a milbemycin synthetic positive-regulation gene kelR, encoding protein and genetically engineered bacterium and a preparation method and application thereof, and belongs to thefield of microorganism pharmacy. According to the milbemycin synthetic positive-regulation gene kelR, the encoding protein and the genetically engineered bacterium and the preparation method and the application thereof, an overexpression vector containing the milbemycin synthetic positive-regulation gene and an upstream promoter sequence is constructed and transformed into escherichia coli, an obtained transformant and streptomyces bingchenggensis are subjected to intergeneric conjugational transferring, zygotes with apramycin resistance are picked, and then the kelR overexpression geneticallyengineered bacteria are obtained. The milbemycin synthetic positive-regulation gene kelR, the encoding protein and the genetically engineered bacterium and the preparation method and the applicationthereof are suitable for milbemycin biosynthesis.

The invention discloses a fermentation medium and application thereof. The fermentation medium comprises: (1) 7% of a carbon source, the carbon source comprising soybean oil and one or more of glucose, maltodextrin and corn flour; (2) 5.5% of a nitrogen source, wherein the nitrogen source is selected from one or more of cold bean powder, peanut powder, dolphin peptone and yeast powder YP600; and (3) 0.9-1.1% of inorganic salt which is selected from one or more of ammonium chloride, magnesium sulfate, manganese chloride, zinc sulfate, ferrous sulfate and calcium carbonate; and the balance is water, and the percent is the mass volume percent g / 100mL of the fermentation culture medium. The fermentation medium can improve the fermentation unit of apramycin from 5.3 g / L in the prior art to at least 6.25 g / L, and the fermentation unit is improved by 18%.

Oil-in-water compound apramycin nanoemulsion belongs to the field of medical technology, and its weight percentage is composed of: apramycin 1%-18%, rhubarb extract 1%-20%, and surfactant 15%-40% , Co-surfactant 1% to 25%, oil 1% to 20%, distilled water 20% to 80%. The compound apramycin bacteriostatic nanoemulsion of the present invention has a narrow emulsion particle size distribution, a transparent system, good stability, low surface tension, good fluidity and convenient administration. The combination of rhubarb extract and apramycin has a better antibacterial effect, and it is made into a nanoemulsion, which is easier to be absorbed by the body and can reduce the amount and frequency of use of drugs.

The invention discloses a seed culture medium for improving apramycin fermentation unit and application thereof. The seed culture medium comprises: (1) 3% of a carbon source, wherein the carbon source comprises glucose and corn starch; (2) 1.4-3.9% of a nitrogen source which comprises peanut powder and also comprises one or more of yeast extract, peptone, hot pressed bean flour and / or yeast powder YP200; and (3) 0.1-0.28% of inorganic salt, the inorganic salt comprises calcium carbonate, and the balance is water, and the mass volume percentage of the inorganic salt in the fermentation medium is g / 100mL. The seed culture medium can improve the phenomenon that hyphae are easy to break in the culture process, so that the shape of the hyphae becomes thick, strong and continuous, and the fermentation yield is increased from 3.02 g / L to at least 5.42 g / L.