75results about How to "High expression yield" patented technology

The invention discloses recombinant proserum / growth hormone fusion protein, a preparation and purification method of the recombinant fusion protein, and the use of the recombinant fusion protein to preparation of medicines for treating children dwarfism. The amino acid sequence of the recombinant proserum / growth hormone fusion protein is SEQID NO.1, and the nucleotide sequence of the recombinant proserum / growth hormone fusion protein is SEQID NO.2. According to a preparation technology of the recombinant proserum / growth hormone fusion protein disclosed by the invention, yeast engineering bacteria are constructed and expressed, so that high-density expression recombinant fusion protein is obtained; and through a purification technology, the recombinant proserum / growth hormone fusion proteinwhich can be used clinically is obtained. The recombinant proserum / growth hormone fusion protein obtained by the preparation and purification method adopts a creative medicine structure for treatingthe children dwarfism, has long residual action that administration can be performed once every two weeks, is more suitable for children medication demands, and has more excellent treatment effects, less administration frequency and lower production cost.

The invention provides a coding sequence of recombined human plasminogen Kringle 5 (hk5) suitable for yeast expression, a high-effective method for preparing hk5, establishment of relevant engineering cell, expression and purification of rhk5. The optimalized human plasminogen Kringle 5 (hk5) gene is quite suitable for yeast expression (especially multi-copy expression) and it is characterized by high expression, high stability and high secretion. The invention is characterized in that it can prepare pure rhk5 with high-effective, simple and low-cost method.

The invention provides an anti-chicken infectious bursal disease (IBD) recombinant protein subunit vaccine. The vaccine is a fusion protein having high immunogenicity of Salmonella typhimurium flagellin and an infectious bursal disease virus (VP2). The above flagellin + VP2 fusion protein is obtained through the expression of a recombinant baculovirus containing a flagellin + VP2 gene by utilizing a Bac-to-Bac baculovirus expression system. The recombinant baculovirus obtained through the system has a short period, and the expressed flagellin + VP2 fusion protein has high immune protection force.

The invention provides a bovine viral diarrhea virus E0 protein amino acid and a preparation method thereof. The amino acid sequence of the bovine viral diarrhea virus E0 protein amino acid is represented by SEQ ID NO:3. The preparation method comprises the following steps:S1, obtaining a BVDV NADL whole genome sequence from NCBI GenBank, and finding out a BVDV E0 gene sequence from the BVDV NADLwhole genome sequence; S2, optimizing and synthesizing the E0 gene sequence obtained in step S1; and S3, selecting a prokaryotic expression vector pMal-c5X with an MBP label, connecting the E0 gene with the pMal-c5X, inserting an E0 fragment into the pMal-c5X, converting the connected expression vector pMAL-c5x-E0 into an expression host bacterium BL21 (DE3), inducing the expression of the obtained recombinant protein, and separating and purifying the target protein. The target gene E0 is optimized, and is subjected to fusion expression with the MBP tag protein to finally achieve the efficientsoluble expression of the E0 protein, so the bovine viral diarrhea virus E0 protein amino acid has the advantages of high expression yield, good solubility, and convenience in expression protein purification; and the solubility of the Escherichia coli expression protein can be improved during the fusion expression of the foreign protein and the MBP so as to provide convenience for the purification of the expression protein.

The invention provides a recombinant protein subunit vaccine for resisting a porcine circovirus serotype 2. The recombinant protein subunit vaccine is a fused protein of a salmonella typhosa flagellin with relatively high immunogenicity and a porcine circovirus serotype 2 Cap protein. Manually coded Flagellin-ORF2 (Open Reading Frame) and Flagellin-delta ORF2 genes are fused and cloned in a pFastBac expression vector, and recombined and cloned together with ORF2 and deltaORF2 (pFastBac vector) for transfecting Sf9 cell, four fused proteins are expressed by using a baculovirus system and identified by using immunofluorescence assay (IFA) and Western-blot. A period of recombining baculovirus by the system is short, and the expressed flagellin+Cap fused protein is high in immune protection force.

The invention relates to a production method of recombination human antibody, in particular to a production method of recombination human anti rabies virus antibody, which is characterized in that the recombination antibody is neutralizing antibody specially combined with rabies virus, which is determined by the specific gene sequence in hypervariable region (CDRs) of antibody heavy chain and light chain variable region and can be expressed in procaryotic cell and eukaryotic cell. The CDR area, part or all gene of the antibody can generate the antibody in any expression system of procaryotic cell and eukaryotic cell, to prevent and treat rabies clinically.

The present invention discloses preparation and application of a hepatitis C virus (HCV) recombinant protein, specifically discloses a separated HCV antigen peptide, which is derived from an E2 protein of HCV virus; the antigenic peptide can bind to an anti-HCV antibody; and the HCV is a Con1 strain of HCV 1b genotype. The invention provides a composition containing the above antigen peptide and a preparation method thereof. The HCV antigen peptide of the invention can effectively and specifically combine with anti-E2 antibody in serum of an infected patient in a broad-spectrum way, and can be used to develop a diagnostic kit for the detection of HCV infection.

The invention relates to a human NT-proBNP preparation capable of stable preservation, a preparation method thereof, and a method for high-yield recombinant expression of human NT-proBNP; particularly, an escherichia coli expression system is used to produce a recombinant human B-type natriuretic peptide precursor (NT-proBNP), and the preparation process and conditions are optimized to obtain a preparation process for high-yield expression of human NT-proBNP; and the human NT-proBNP preparation capable of stable preservation is obtained by adding a stabilizing protective agent. The natural human NT-proBNP preparation prepared by adding the stabilizing protective agent in the invention can exist stably under conditions of -20 DEG C and -80 DEG C, and the biological activity is not decreased. A stable raw material source is provided for the preparation of NT-proBNP as a clinical diagnosis reagent standard substance and an antibody.

The invention provides truncated biologically active ADAMTS polypeptides, particularly those with hyalectenase activity, and more particularly those with aggrecanase activity, that exhibit greater stability and homogeneity and higher expression yields than their full-length counterparts. The invention also provides nucleic acid molecules encoding such truncated biologically active ADAMTS polypeptides and methods for producing the truncated biologically active ADAMTS polypeptides. In addition, the invention provides methods for identifying compounds capable of modulating biologically active ADAMTS polypeptides, particularly those compounds that inhibit aggrecanase activity.

The invention belongs to the technical field of biology, and particularly relates to a corynebacterium and colibacillus double expression vector with high copy capability. The nucleotide sequence of an expression plasmid is shown as SEQ ID NO:9. Through artificial mutagenesis on a nucleotide C in a 1786-th site in a corynebacterium replicon regulation position of a pXMJ19 carrier, a corynebacterium / colibacillus dual-purpose vector pXMJ19C1786T plasmid with the copy capability being about 12.5 times higher than that of a pXMJ19 plasmid is successfully obtained. The copy number of the pXMJ19 plasmid in the corynebacterium is about 20; and the copy number of the pXMJ19C1786T plasmid in the corynebacterium is 251. Meanwhile, the invention also discloses a building method of the expression plasmid.

The invention provides a method for preparing grass carp reovirus (GCRV) gene engineering vaccines. The method comprises the following steps of: selecting recombinant baculoviruses (vAcGCRV-VP5 / VP7) of sf9 insect cell proliferated recombinant GCRV outer capsid proteins VP5 and VP7; separating recombinant protein components by freezing, thawing and cracking recombinant virus infected sf9 cells and adopting sucrose lining centrifuging method, and thus obtaining a purified in vitro expression VP5 and VP7 protein complex; and performing fish protection experiments on the purified VP5 and VP7 proteins to prove that the VP5 and VP7 proteins have good immune protection effect. The GCRV gene engineering vaccines prepared by the method not only have good stability and immunogenicity, but also have good immune protection effect on grass carp fries; the immune protection rate of the GCRV gene engineering vaccines reaches 90 percent; and the method is suitable for large-scale production and application of the vaccines.

The invention provides a method for increasing human C5 monoclonal antibody expression yield through targeted screening of a foreign gene double-integrated locus. The method comprises the following steps: (1) inserting a light chain encoding gene and a heavy chain encoding gene of a human complement C5 antibody into two eukaryotic expression vectors respectively; (2) acquiring a recombined light chain encoding plasmid and a recombined heavy chain encoding plasmid, and co-transfecting the light chain encoding plasmid and heavy chain encoding plasmid to Chinese hamster ovary (CHO)DG44 cells; (3)screening a light chain encoding gene and a heavy chain encoding gene with a FISH technology, and locating on the cells of a chromosome 1q1 and a chromosome 1q13 by double integration for cloning on.By adopting the method, 1q1 and 1q13 regions obtained by screening support strong transcription of the light chain encoding gene and the heavy chain encoding gene, efficient and stable expression ofthe human C5 monoclonal antibody in the CH0-DG44 cells can be realized, and the product can be used for treating various diseases caused by rise of the C5 level.

The invention discloses a codon-optimized gene for encoding a precursor protein of a type-3 wild polio virus coat and a gene for encoding 3CD protein of type-1 attenuated polio virus. The genes are transferred into yeast cells and can be efficiently and spontaneously assembled into VLP (virus-like particles) in the cells through coexpression. The invention further discloses a macromolecule with immunogenicity. The macromolecule is mainly prepared from the genes in the yeast cells by expressing. The invention also discloses an application and composition of the macromolecule with immunogenicity.

The invention discloses a pyruvate oxidase gene (AvPyOD) derived from viridian aerococcus, recombinant expression plasmid constructed by the gene and an engineering strain of escherichia coli gene transformed by the recombinant expression plasmid. The AvPyOD gene has a nucleotide sequence illustrated in SEQ ID NO: 1 and is a brand-new gene. After the recombinant expression plasmid that contains the gene AvPyOD transforms into the escherichia coli gene, the engineering stain is obtained, the enzyme production ability of the engineering stain is greatly improved, thus being suitable for large-scale industrialized production of the pyruvate oxidase.

The invention relates to taenia multiceps protective antigen enolase TmENO recombinant protein with immunizing protection and a prepared method and application thereof. Amino acid sequence of the recombinant protein is GenBank. Amino acid sequence of ORF sequence encoding of accession number is SEQ No.5. Gene sequence of encoding gene sequence according to escherichia coli prefered codons to optimize is SEQ No.6. The recombinant protein can obviously reduce the number of cysts parasitized in sheep brains, and immunizing protection is achieved. Related experiments show that high title antibodies can be produced after animals are immunized by the recombinant protein, and the stronger immunizing protection can be created to taenia multiceps egg attack infection. The TmENO recombinant protein is a candidate target of coenurosis vaccine development and can be used for immunoprophylaxis of the coenurosis.

The invention discloses a process which uses pGAP as a promoter and Pichia Yeast GS115 as an engineering bacterium to perform fermentation expression on an intestinal trefoil factor (ITF) in a fermentation tank. The method comprises the following steps: the recombinant Pichia Yeast expression vector pGAPZ alpha A-ITF is firstly constructed and then recombined in Escherichia coli Top10 to amplify, Escherichia coli plasmid is extracted and linearized to be transformed in Saccharomyce GS115, a high copy transformant is screened according to the zeocin resistance gene carried by the vector to perform constitutive expression, a high expression pilot test engineering bacterium is screened and fermented in a culture medium for 2 days to perform secretory expression on the ITF; and the target protein ITF of which the purity is more than 95% is obtained through purification treatment, wherein the output is about 200mg / L and the yield is about 50%. The intestinal trefoil factor recombinant expression vector has low production cost, short fermentation period and simple purification method; methanol induction is not adopted, the protein expression level is high; and a good foundation is laid for the large-scale industrial production of ITF.

The invention discloses a construction method of a gene knockout CHO cell line and application of the gene knockout CHO cell line in the expression of therapeutic recombinant proteins. The construction method comprises the following steps: imputing a knockout vector in a CHO cell, and then performing the screening to obtain the knockout plasmid mediated CYLD expression-deficient cell line. The nucleotide sequence SEQ ID NO:1 in the knockout cell can be replaced with SEQ ID NO: 2, NO: 3, NO: 4, NO: 5 or NO: 6; and the transfected cell can be the CHO-K1 cell or the CHO cell for stably expressingan antibody. The construction method has important application value for increasing the expression yield of the therapeutic recombinant proteins.

The invention provides a multi-functional tumor-targeted nanometer preparation and a construction method and application in treating tumors thereof. By means of the nanometer preparation, the tumors can be treated by using the RNA interference technology cooperating with the photothermal therapy, the RNA interference technology and the photothermal therapy cooperatively inhabit the growth of the tumors, and a better tumor treatment effect is achieved. The nanometer preparation comprises effector molecules, a nanometer material and targeting molecules, wherein the effector molecules comprise nucleic acid molecules for RNA interference; a photothermal effect is formed by the nanometer material; the targeting molecules are used for delivering the particular cells which the nanometer preparation arrives at. The nanometer preparation process is mild in condition, the cost is low, and the nanometer preparation is good in biocompatibility, targeting ability, siRNA loading and unloading capacity, serum protection effect and tumor-suppression effect. The industrialization implementating prospect of the nanometer preparation is wide, and the corresponding treatment method is a new potentialtumor treatment thought.

The invention discloses application of yeast upstream activation element in filamentous fungi, and particularly relates to application of an upstream activation element UAS coming from brewer's yeast and shown as SEQ ID NO.1 in increasing expression amount of target protein. The upstream activation element UAS is preferably cloned to an upstream of a promoter or a downstream position of terminator while positions are not limited to the upstream and the downstream position. After the upstream activation element coming from the brewer's yeast is fused with the promoter, transformation level of a downstream coding protein sequence corresponding to the upstream activation element can be improved, so that the expression amount of the target protein is increased.

The invention discloses an expression system for expressing an HAS-Vmip-II fusion protein, and the expression system is produced by the transformation of plasmid pPICZaA-HSA-vMIP-II into Pichia pastoris. The invention also discloses a construction method of the expression system, comprising the following steps of: extracting recombinant plasmid from escherichia coli containing the plasmid pPICZaA-HSA-vMIP-II, linearizing the recombinant plasmid, transforming Pichia pastoris competent cells by electrotransformation, followed by resistance screening, performing the PCR identification to obtain positive clones, carrying out an inducible expression by the use of a positive clone, followed by the SDS-PAGE analysis of the expression product and Western-Blot identification. According to the invention, the half life of vMIP-II in blood plasma is greatly prolonged without the loss of vMIP-II functions. Therefore, the HAS-vMIP-II fusion protein will reduce the administration frequency and dosage, and exert a drug effect similar to that of vMIP-II.

The invention discloses a method for secretory expression of littin signal peptide-mediated IBV N protein, that is firstly, IBV N gene HBM-N fusing honeybee melittin signal peptide is obtained, then a recombination transfer vector pFast-HBM-N and recombination bacmid rHBM-N are constructed, and finally, the recombination bacmid transfers Sf9 insect cells for secretory expression of IBV N protein. According to the method, the honeybee melittin signal peptide is introduced before IBV N protein, N protein efficient secretory expression is realized, the obtained IBV N protein has excellent biological activity, hydrolysis of endogenous protease to target protein is reduced with the method, purification is facilitated, the foundation is laid for further study of N protein biological functions, development of novel diagnostic antigen, development of novel vaccine and the like, and at the same time, a new thought is provided for expression of other structural protein of the IBV.

The invention provides a SUMO-Drosomycin fusion protein and a preparation method thereof, and a preparation method of an antifungal peptide Drosomycin. When expressed in an Escherichia coli expressionsystem, the protein can overcome the problem that most of the target proteins are easily folded in the system, and avoids the formation of non-functional proteins. The protein can be correctly folded, has high antibacterial activity, does not produce drug resistance, and has broad application prospects in preparing antibacterial drugs, sterile foods, and cosmetics, and has high market value.

The invention relates to a method for producing American cockroach allergen protein Pera 9, in particular to a method of producing the American cockroach allergen protein Pera 9 in a baculovirus-insect expression system, and belongs to the technical field of gene engineering. The method produces the American cockroach allergen Pera 9 protein by the baculovirus-insect expression system so as to avoid the problem that a Pera 9 allergen structure is influenced in different expression systems (such as bacteria and yeast). The method provides an affinity chromatography method to solve the problem that the purification process of natural Pera 9 allergen is complicated. The activity of the Pera 9 protein obtained by the baculovirus-insect expression system is as 2.1 times as that of Pera 9 protein obtained by Escherichia coli.

The invention discloses bacillus subtilis capable of efficiently expressing Fe<3+>-dependent food-grade acid urease, and belongs to the technical field of biological engineering. Ni-dependent urease has potential safety hazards due to Ni and is a non-food enzyme, and Fe is essential trace elements of a human body and is beneficial for the human body. Accordingly, food-grade efficient expression of the Fe<3+>-dependent food-grade acid urease is successfully achieved in bacillus subtilis 168 (pTTB) through a food-grade bacillus subtilis expression system, and the enzyme activity of the Fe<3+>-dependent food-grade acid urease reaches 5.8 U / mL and reaches 1.3 U / mL when EC is taken as a substrate; compared with an original bacillus licheniformis strain, the expression yield of the food-grade urease is greatly increased, and a foundation is laid for industrialized application of the food-grade acid urease.

The invention provides a nano antibody for specifically recognizing vibrio parahaemolyticus, which comprises a coding gene of the nano antibody, and also provides a vector and a host cell containing a nucleotide sequence for coding the nano antibody. The amino acid sequence of the nano antibody is shown as SEQ ID NO: 1, and the nano antibody has good specificity and binding capacity to vibrio parahaemolyticus. The nano antibody for specifically recognizing the vibrio parahaemolyticus, provided by the invention, is good in specificity, strong in stability and high in expression yield, is not combined with other non-vibrio parahaemolyticus, and can specifically recognize the vibrio parahaemolyticus.

The invention discloses a dysoxia-resistant lactase yeast strain with a preservation number of the dysoxia-resistant lactase yeast strain is CCTCC NO:M2016252. The invention further discloses a construction method of the dysoxia-resistant lactase yeast strain, wherein the construction method comprises the following steps: sequentially transferring expression vectors in vitreoscilla hemoglobin cells and lactase secretory expression vectors into pichia pastoris; screening a strain with high lactase activity to obtain the dysoxia-resistant lactase yeast strain. The strain disclosed by the invention can be used for preparing recombinant lactase protein by high-density fermentation; a utilization rate, on oxygen gas, of cells is increased by synthesizing hemoglobin (VHB) in cells, and growth, under an oxygen-lean condition, of the cells is promoted, so that the expression yield of extracellular recombinant lactase is increased.

The invention belongs to the field of biotechnology, and relates to an application of a soluble urokinase receptor mutant suPARH47C / N259C (suPARcc) in eukaryotic extracellular protein expression, specifically increasing expression level of exogenous protein by means of fusion expression, and further utilizing high affinity of suPARcc to a ligand ATF, and utilizing an ATF affinity column to directly and efficiently capture a target protein from a large-volume fermentation broth. According to the application, suPARcc and a conventional protein enzyme digestion site are integrated into a eukaryotic expression system vector to obtain a suPARcc gene fusion expression vector, a target gene to be expressed and the suPARcc gene are fused in a correct reading frame to construct a fusion expressionrecombinant plasmid, the fusion expression recombinant plasmid is transfected and screened for stable cell lines, a fusion protein containing the tag and the target protein is obtained through induction, secretion and expression in eukaryotic cells, and the fusion tag can be finally removed by a conventional protease.

The invention relates to a method for expressing an antibacterial peptide apidaecin by using a bombyx mori cell and for preparing the antibacterial peptide apidaecin. The method comprises the following steps: chemically synthesizing AP2 gene with an enterokinase enzyme cutting site, constructing a recombinant plasmid pFast-His-AP2, converting a BmDH10Bac competent cell by using the recombinant plasmid, extracting recombinant BmBacmid gene, transfecting the recombinant BmBacmid gene to a BmN culture cell, culturing to obtain a recombinant virus, and continuously infecting the BmN culture cell by using the recombinant virus to obtain an AP2 fusion protein with His fusion; and carrying out eluting purification on the AP2 fusion protein by using His affinity chromatography, concentrating, and carrying out enzyme cutting to obtain a crude AP2 product. Compared with the prior art, the method using a baculovirus expression system to express the antibacterial peptide apidaecin in the bombyx mori BmN culture cell has the advantages of effective increase of the expression output, effective expression of the expressed product due to various natural protein protection agents in the bombyx mori cell, stability of the gene expressed product, and improvement of the expression safety.

The invention provides a method for preparing grass carp reovirus (GCRV) gene engineering vaccines. The method comprises the following steps of: selecting recombinant baculoviruses (vAcGCRV-VP5 / VP7) of sf9 insect cell proliferated recombinant GCRV outer capsid proteins VP5 and VP7; separating recombinant protein components by freezing, thawing and cracking recombinant virus infected sf9 cells and adopting sucrose lining centrifuging method, and thus obtaining a purified in vitro expression VP5 and VP7 protein complex; and performing fish protection experiments on the purified VP5 and VP7 proteins to prove that the VP5 and VP7 proteins have good immune protection effect. The GCRV gene engineering vaccines prepared by the method not only have good stability and immunogenicity, but also have good immune protection effect on grass carp fries; the immune protection rate of the GCRV gene engineering vaccines reaches 90 percent; and the method is suitable for large-scale production and application of the vaccines.