53results about How to "Optimize purification steps" patented technology

The present invention belongs to the field of biological engineering technology. The gene engineering bacteria is colibacillus DH5-alpha, BL21(DE3) or BLR(DE3) carrying the recombinant plasmid of human glicentin-1 gene, i. e., GLP-1 gene. The construction process of the gene engineering bacteria includes connecting serially DNA sequence containing human glicentin-1 gene to form polymer, constituting expression vector and converting to colibacillus to obtain efficiently expressing human glicentin-1 gene engineering bacteria. With the gene engineering bacteria and through a three-step process of liquid culture, purification to produce human glicentin-1 fusion protein and preparation of human glicentin-1, human glicentin-1 product may be produced. The present invention has the advantages of high expression amount, less purification steps, high yield and low production cost.

A method for producing Group 8 (VIII) metallocene or metallocene-like compounds employs a compound that includes a Cp′ anion, such as found, together with a counterion, in a cyclopentadienide or cyclopentadienide-like salt. In one embodiment, the method includes reacting a metal salt, a (Cp) compound, such as a substituted or unsubstituted cyclopentadiene or indene, and a ligand (L) to form an intermediate compound and reacting the intermediate compound with a Cp′ compound, eg., a cyclopentadienide or cyclopentadienide-like salt, where the metal salt can be is a ruthenium, an osmium or an iron halide or nitrate and L is an electron pair donor. Unsubstituted, mono-substituted as well as symmetrically or asymmetrically di- or multi-substituted metallocenes or metallocene-like compounds can be produced. In another embodiment, unsubstituted or symmetrically substituted metallocenes are formed by reacting MX2(PPh3)m with a Cp′ compound, where m=3 or 4. The method can be used to form precursors for chemical vapor deposition of thin films.

The present invention relates to a gene engineered bacteria, and its preparation method and uses, belongs to biological engineering field. The gene engineered bacteria is composed of plasmid and host bacteria in which host bacteria is any one of Ecoli DH5alpha,BL21(DE3) or Rosetta-gami(DE3), the recombinant plasmid is pET32a(+) containing gene GAD65. The construction method of engineered bacteria comprise: designing Nest-PCR upper-stream primer and down-stream primer, amplifying human GAD65 gene fragment using PCR from human pancreas cDNA library, transforming Ecoli via vector and obtaining highly effective expression recombinant human GAD65 gene engineered bacteria. The inventive engineered bacteria can be used to produce soluble active thioredoxin- human Glutamic Decarboxylase 65 fusion protein and active recombinant human GAD65 which has enzymatic activity and immunological activity and are easy to purify. The expression volume is high and cost is low .

The invention relates to a method for extracting taxol simply, conveniently and efficiently and belongs to a method for extracting taxol. The method comprises: crushing a taxus chinensis dry medicinal material into coarse powder, removing fat soluble impurities by a supercritical CO2 extraction process, and adding cellulase into medicinal residue for hydrolysis; and filtering, drying the medicinal residue, leaching in ethanol, filtering, recovering ethanol under reduced pressure till the residue gives off no alcohol smell, washing the residual with water, discarding washing solution, drying and obtaining taxol by chromatography. The method improves the extraction rate of taxol, uses a small volume of water, avoids using an organic solvent in a solvent extraction method, reduces column loading pressure and is more suitable for mass industrial production.

The invention discloses an immunoglobulin-binding protein comprising one or more mutated immunoglobulin-binding domains (monomers) of staphylococcal Protein A (E, D, A, B, C) or protein Z or a functional variant thereof, wherein in at least one of the one or more mutated monomers, the asparagine or histidine at the position corresponding to H18 of the B domain of Protein A or of Protein Z has been deleted or substituted with a first amino acid residue which is not proline or asparagine and wherein, if the amino acid residue at position 57 is proline and the amino acid residue at position 28 is asparagine, then the amino acid residue at the position corresponding to H18 of the B domain of protein A or of protein Z is not serine, threonine or lysine.

The invention relates to the technology of separation and purification, and aims at providing a method for separating and purifying antibiotic avilamycin for livestock. According to the method, organic solvent is adopted to extract mycelium containing avilamycin, then after crystallization, silica column chromatography, ODS column elution separation, recrystallization and leaching, the crystal is rinsed with pure water, and then drying is carried out to obtain pure products of avilamycin A and B. The pure products obtained by the method disclosed by the invention are respectively pure products of avilamycin A and avilamycin B instead of a pure product formed by mixing of various components of avilamycin A; the purifying step is simple and convenient and easy to operate; and the average yield is higher than 37.50%, the purity is higher than 98%, and the pure product can be taken as a reference substance in avilamycin measurement.

A method for producing fuel ethanol by multi-strains co-fermentation of straws comprises the following steps: (1) taking mixed liquor of glucose and xylose or hydrolysate of biomass (such as maize straws, wheat straws or rice straws) as a carbon source, and adding yeast powder and microelements to obtain a fermention culture medium; and (2) simultaneously inoculating a seed solution of glucose zymophyte and a seed solution of xylose zymophyte into the fermention culture medium according to the ratio of 1: 1-1: 3(v / v), and fermenting for 36-60 hours at the temperature of 25-38 DEG C. By the method, glucose can be efficiently transformed into ethanol, meanwhile, the xylose is transformed into ethanol to a maximum degree, the yield of glycerinum which is a by-product in fermentation broth is remarkably reduced, steps of extraction and purification are improved remarkably, and the production cost of cellulosic ethanol is reduced obviously.

The invention belongs to the technical field of bioengineering, in particular to a method for preparing and purifying 5-methylpyrazine-2-carboxylic acid by microbial fermentation. The technical scheme of the present invention uses 2,5-dimethylpyrazine as a raw material, uses Arthrobacter woluwensis HW-1 strain with xylene monooxygenase activity as a catalyst, and uses compressed air to bring in xylene vapor as a catalyst. inducer, and add raw materials in batches in due time, and ferment and transform for a period of time to obtain a fermentation broth containing 5-methylpyrazine-2-carboxylic acid. The invention makes up for the deficiencies in the process of chemically synthesizing 5-methylpyrazine-2-carboxylic acid, overcomes the defect of low conversion rate of shake flask fermentation, and provides a method for purifying 5-methylpyrazine-2-carboxylic acid in fermentation broth for the first time method.

The invention relates to sika deer antler thymosin beta10 recombinant protein, a preparation method and application of protein, belonging to the technical field of gene engineering. The method comprises the following steps: cloning deer antler thymosin beta10 genes, constructing a prokaryotic expression vector pET-28a-Tbeta10, performing inducible expression in host bacteria E.coli BL21 and separating and purifying the recombinant protein. The prokaryotic expression of the sika deer antler thymosin beta10 is attempted for the first time by utilizing a gene engineering technology, the prokaryotic expression vector is successfully constructed, high-efficiency expression of the recombinant protein is induced, and the purification step is simple and rapid. The in-vitro activity research proves that the sika deer antler thymosin beta10 recombinant protein has an effect of obviously promoting proliferation of osteoblasts M3T3.

A method for synthesising esters from alcohols by dehydrogenating coupling in the presence of a catalyst of formula 1 as well as to the use of catalysts of formula 1 for synthesising esters. The method according to the invention can be used in particular for the production of dihydrogen. The invention also relates to novel catalysts as well as to the uses thereof.

The invention belongs to the technical field of genetic engineering, and specifically discloses a genetically engineered bacterium for expressing solubility pig gamma-interferonPoIFN-gamma and construction method and application of the genetically engineered bacterium. The genetically engineered bacterium for expressing solubility pig gamma-interferonPoIFN-gamma is Escherichia coil BL21(DE3) carrying recombinant plasmid Pet-32a(+)-PoIFN-gamma and plasmid pTf16-ParaB-tig simultaneously, wherein the PoIFN-gamma gene sequence in the recombinant plasmid Pet-32a(+)-PoIFN-gamma is SEQ ID NO.4, and the plasmid pTf16-ParaB-tig can express the molecular chaperone tig. The method disclosed by the invention is capable of improving the solubility and output of the pig gamma-interferon, simple in process and low in cost, and has good industrial application value.

The invention discloses a VZV (varicella-zoster virus)glycoprotein E gene eukaryotic expression vector as well as a recombinant yeast strain and application thereof. The vector is a connector of alpha-gE fused gene and pPink-HC, and the alpha-gE fused gene is a gene complete sequence of VZV glycoprotein E and a fused gene alpha signal peptide. The glycoprotein E capable of successfully expressingthe VZV in an expression system of pichia pastoris discloses by the invention sets the foundation for the detection of the protein immunogenicity as well as the efficient expression of the protein inthe pichia pastoris as well as the research of the VZV vaccine.

The present invention is gene engineering bacteria with high efficiency expression of human alpha 1-thmulin and its construction and application, and belongs to the field of bioengineering technology. The gene engineering bacteria is colibacillus DH5-alpha, BL21(DE3) or BLR(DE3), and carry plasmid containing 1-16 alpha 1-thmulin genes. The plasmid promoter is IPTG induced promoter Lac, Tac of PT7, such as plasmid pET series, pGEX series, pQE series, etc. On DNA level, DNA sequences containing alpha 1-thmulin gene are connected serially to form serial body and constitute one series of expression vectors, which transform colibaccilus to obtain one series of gene engineering bacteria with high efficiency expression of human alpha 1-thmulin. The gene engineering bacteria may be used in preparing human alpha 1-thmulin samples. The present invention has high yield and low cost of human alpha 1-thmulin samples.

The invention discloses an expression method of VZV glycoprotein to pichia pastoris and application of the expression method. The expression method comprises the following steps: constructing an expression vector, screening a positive transformant, linearizing a yeast expression vector, preparing a yeast electrotransformation competence, identifying a recombinant, expressing recombinant yeast andidentifying target protein. The expression method disclosed by the invention can be used for successfully expressing glycoprotein E of varicella-varicella-zoster virus and the obtained protein has good immunogenicity; the pichia pastoris can be efficiently and stably expressed.

The invention relates to the fields of genetic engineering and veterinary biological pharmacy, and in particular discloses a subunit vaccine for porcine reproductive and respiratory syndrome as well as a preparation method and application of the subunit vaccine. The invention firstly provides a fusion protein, wherein an amino acid sequence of the fusion protein is as shown in SEQ ID No. 1; and then the invention provides the subunit vaccine for the porcine reproductive and respiratory syndrome, wherein the subunit vaccine comprises the fusion protein. The fusion protein in the subunit vaccinefor the porcine reproductive and respiratory syndrome is obtained by conducting fusion expression on an ORF5 full-length gene of a PRRSV NADC30-like strain, which is optimized by virtue of a codon, and a gene of pseudomonas aeruginosa, from which a Domain III structural Domain is removed, and by virtue of an insect baculovirus / insect cell expression system. The vaccine provided by the inventionis good in protective effect, capable of inducing effective humoral immunity and cellular immunity and is suitable for clinical prevention of the reproductive and respiratory syndrome.

The invention belongs to the field of biomedicine, and particularly relates to a novel polypeptide specifically bound with multiple tumor cells and application thereof. The amino acid sequence of thepolypeptide is selected from an amino acid sequence shown as one of SEQ ID NO. 1 to SEQ ID NO. 5. The polypeptide and bioactive fragments and derivatives thereof are applied to tumor diagnosis and treatment as molecular imaging probes and drug target heads. The polypeptide disclosed by the invention has the effects of specifically targeting various cancer cells, and is high in specificity and small in side effect, so that a series of tumor early diagnosis reagents and targeted treatment drugs are developed, and a new direction is opened up for designing and researching novel tumor targeted drugs.

The invention relates to a genetically engineered bacterium for efficiently expressing recombinant bacteriocin and a construction method and an application thereof. The genetically engineered bacterium is escherichia coli BL21(DE3) carrying a Pet-sumo-e50-52 recombinant expressing carrier. The genetically engineered bacterium is constructed by steps of cloning bacteriocin E50-52 genes with a nucleotide sequence shown in SEQ ID No.1 to fused expression plasmid pET-SUMO and then converting the competent cell of the escherichia coli BL21(DE3). The genetically engineered bacterium is expressed through induction of IPTG (Isopropyl-beta-d-Thiogalactoside) so that SUMO-E50-52 fuse protein is expressed in escherichia coli; the fuse protein is cut by a specific SUMO protease to obtain the recombinant bacteriocin E50-52. The recombinant bacteriocin provided by the invention has good antimicrobial activity and stability, hardly generates drug tolerance, and can be used as ideal antibacterial agents, antiviral drugs, feed additives, preservatives, killing agents and the like.

The invention discloses a method for efficiently expressing extracellular Serratia marcescens non-specific nuclease based on a Bacillus subtilis expression system. The method is characterized by comprising the following steps: taking Bacillus subtilis as a host expression system, and utilizing the extracellular secretion capacity of the Bacillus subtilis to realize the high-efficiency expression of the extracellular Serratia marcescens non-specific nuclease. The specific activity of the extracellular Serratia marcescens non-specific nuclease recombined and expressed by the invention is 3 timesas high as that of current commercial products. The Bacillus subtilis secretion expression system selected by the invention fundamentally overcomes the defects that endotoxin introduced by the self characteristics of other expression systems and the glycosylation modification affect enzyme activity or limit the application of the system, efficient secretion-type expression does not need to breakcells, the purification steps are simpler and more convenient, linear amplification can be realized, scale production is convenient, and the method is worthy of popularization.

The invention discloses a soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein as well as a preparation method and application of the soluble efficiently-expressed rChGM-CSF-IFNalpha fusionprotein. The preparation method comprises the following steps: (1) respectively extracting and amplifying chicken GM-CSF genes and chicken IFN-alpha genes; (2) linking the amplified chicken GM-CSF genes with the amplified chicken IFN-alpha genes, so as to obtain a fusion protein gene; (3) constructing a recombinant clone vector and a recombinant expression vector, and carrying out screening, so as to obtain gene engineering strains; (4) carrying out soluble efficient expression on a fusion protein; and (5) purifying the fusion protein. By virtue of ChGM-CSF-IFNalpha fusion expression, the antiviral activity of pure ChIFNalpha is improved, and the in-vivo drug half-life period of the pure ChIFNalpha is prolonged; and by virtue of the soluble efficient expression of a ChGM-CSF-IFNalpha pronucleus, the application cost problems of the chicken GM-CSF genes and the chicken IFN-alpha genes in the poultry breeding industry are effectively solved.

A purified multi-valent vaccine of influenza made of the kidney cells of primary vole is disclosed. Its preparing process includes such steps as preparing the viral culture base from the kidney cells of primary vole, culturing the current viruses of human or fowl's influenza, adaptive reproduction and passage to obtain influenza virus strains, culturing the said kidney cells infected by current virus to obtain the primary liquid of influenza virus, centrifugal separation, ultrafiltration, deactivating, ultra-speed centrifugal treating, and purifying by column chromatography.

The invention relates to a preparation method of a baloxavir marboxil intermediate compound. The preparation method includes: using 2-methyl-3-benzyloxy-4-oxo-4-hydropyrane as the initial raw materialand oxygen as the oxidizing agent to perform oxidizing reaction under the effect of a nitric oxide catalyst to generate 3-benzyloxy-4-oxo-4-hydropyrane-2-formaldehyde, and performing further oxidization to obtain the product 3-benzyloxy-4-oxo-4-hydropyrane-2-methyl carboxylate. The preparation method has the advantages that product purification steps are optimized, the raw materials of the methodis cheap and easy to obtain, and the method is simple in post-processing steps, capable of avoiding extra production cost, capable of greatly lowering production cost and product unit price, capableof favorably promoting the researches and development of various medicine intermediates using the product as the raw material and good in commercial value and industrial development potential.

The invention belongs to the technical field of analytical chemistry, and provides a fluorescent probe for detecting mitochondrial membrane potentials as well as a preparation method and application thereof. The fluorescent probe for detecting the mitochondrial membrane potentials has a structural formula shown as: , and can be prepared by allowing reaction between a reaction product of 4-fluorobenzaldehyde and piperazine with 1,2-dimethyl-quinoline iodate. The fluorescent probe for detecting the mitochondrial membrane potentials provided by the invention has the characteristics of being low in biological toxicity, excellent in membrane permeability, simple in synthesis method and convenient in a purification step; and the fluorescent probe can be applied to cell imaging for detecting, labeling or displaying mitochondrial membrane potential changes.

The invention discloses a pichia pastoris recombinant bacterium, a culture method and an application thereof, the prepared and cultured recombinant yeast can efficiently express GLP-1 analogues, and then the GLP-1 analogues are efficiently prepared.

The invention provides a preparation process of propofovir disoproxil fumarate, which is short in production period, simple to operate and suitable for industrial production, and the high-purity propofovir disoproxil fumarate is prepared by taking the propofovir disoproxil fumarate as a starting material through special phosphorus chiral atom synthesis, purification and separation. The purity of the prepared propofovir disoproxil fumarate is greater than 99.90%, the diastereoisomer is less than 0.15%, the content of other single impurities is less than 0.10%, and the method has high commercialscale production value.

The invention discloses an immunoglobulin-binding protein comprising one or more mutated immunoglobulin-binding domains (monomers) of staphylococcal Protein A (E, D, A, B, C) or protein Z or a functional variant thereof, wherein in at least one of the one or more mutated monomers, the asparagine or histidine at the position corresponding to H18 of the B domain of Protein A or of Protein Z has been deleted or substituted with a first amino acid residue which is not proline or asparagine and wherein, if the amino acid residue at position 57 is proline and the amino acid residue at position 28 is asparagine, then the amino acid residue at the position corresponding to H18 of the B domain of protein A or of protein Z is not serine, threonine or lysine.

The invention belongs to the technical field of organic synthesis, and particularly relates to a preparation method of 2-bromine-4-fluorobenzaldehyde. The method comprises the following steps that (1)4-fluorobenzaldehyde is dissolved in an acid solution to be prepared into 0.1 to 100mol / L of 4-fluorobenzaldehyde acid solution; (2) the temperature of the solution is raised to 30 to 100 DEG C; bromination reagents are added while stirring is performed; stirring reaction is performed for 1 to 24h; (3) the bromination reagents are added again; stirring reaction is performed for 24 to 72h; (4) after the reaction is finished, reaction liquid is poured into ice water; (5) water phases are extracted by alkane solvents; organic phases are merged and are washed; pressure reduction concentration isperformed to remove organic solvents; 2-bromine-4-fluorobenzaldehyde crude products are obtained; the crude products are refined; the target product of 2-bromine-4-fluorobenzaldehyde is obtained. Theraw materials are cheap and can be easily obtained; the used bromination reagents are environment-friendly water treatment agents; the purification method of the target product is simple; the industrial production is facilitated.