2245 results about "Prokaryotic expression" patented technology

The present invention provides methods and compositions for improved expression and production of recombinant antibodies in prokaryotic expression systems. Particularly contemplated are prokaryotic expression and production of full length aglycosylated antibodies. The antibody products of the invention can be used in various aspects of biological research, diagnosis and medical treatment.

The first aspect of the invention relates to a bispecific antibody, which comprises a first functional domain for specific identification of phosphatidylinositol protein polysaccharide-3, a second domain for specific identification of human T cell antigen CD3, and a connection for connecting the functional domains. The second aspect of the invention relates to a nucleotide sequence encoding the above antibody. The third aspect of the invention relates to a carrier containing the above nucleotide sequence, and includes an expressive vector. The fourth aspect of the invention relates to a eukaryotic or prokaryotic expression system containing the above carrier. The fifth aspect of the invention relates to application of the above antibody to preparation of medicament for treating or preventing tumor.

The invention provides a method for knocking out a drug resistance gene mcr-1 through CRISPR-Cas9 in vitro and specialized cell penetrating peptide thereof. The method comprises the steps that a CRISPR-Cas9 system is constructed according to a target point sequence of an mcr-1 drug resistance gene sgRNA identification zone, a base sequence of the target point sequence is shown as SEQ NO.1, in the constructed CRISPR-Cas9 system, a T7 promoter is inserted into the portion in front of a start point of sgRNA transcription, a prokaryotic expression vector of Cas9 protein is constructed, regulation is conducted through the T7 promoter, the specialized cell penetrating peptide CPP2a is utilized for knocking out the mcr-1 drug resistance gene, a carrier pCas9-mcr is brought into a microbial cell, the microbial species can be gram negative bacteria, and the phenomenon that microbial mcr-a drug resistance gene is knocked out in vitro is achieved.

The invention relates to a method for recombinating and expressing human fibroblast growth factors, which comprises following steps: fusing an FGF-21 gene and a molecular chaperone Sumo sequence, building a new prokaryotic expression vector, transforming escherichia coli, thereby building engineering bacteria, and obtaining the FGF-21 through culturing the engineering bacteria and inducing expression. The method of the invention can promote soluble expression of protein, which is beneficial for folding recombined protein correctly and is convenient for separating and purifying the protein. Activity detection proves that the recombined protein which is obtained has comparatively high biological activity and the bioactivity is equivalent to the bioactivity of FGF21standard products.

The invention relates to an amino acid sequence of a recombinant human papillomavirus L1 capsid protein, a nucleotide sequence which encodes the amino acid sequence and a recombinant vector and an expression host which contain the nucleotide sequence. The invention further relates to an application of a HPV L1 protein which is composed of the amino acid sequence in the preparation of vaccines, drug combination and diagnostic antigens or antibodies. The invention allows the recombinant HPV L1 capsid protein which is expressed in a prokaryotic system to be dissolvable in water by the modification of the HPV L1 wild-type sequence, and an L1 pentamer which has the same immunogenicity and antigenicity with the VLP of HPV L1 is obtained by expression. The invention allows the industrial production of the HPV L1 capsid protein by utilizing the prokaryotic expression system to become a reality, compared with the currently used eukaryotic expression system, the invention has the advantages of more stable quality of the products, higher yield, low cost and convenient quality control, which has great economic benefits and social effects.

The present invention identified a recombinant synthetic collagen containing a triple helical backbone protein produced in a prokaryotic expression system where the protein contains at least one ‘inserted’ biologically active sequence(s).

The invention provides a molecule (Preproinsulin) of human proinsulin with a novel N-terminal expressed peptide sequence or analogs of the human proinsulin, a method for producing human insulin by using the molecule, and processes for building related expression vectors and engineering cells and expressing and purifying human proinsulin. The DNA sequence of the human proinsulin coded by the N-terminal expressed peptide sequence or the analogs of the human proinsulin is first introduced into a prokaryotic expression vector and then transferred into an escherichia coli to express the molecule in form of an inclusion body. The invention has the advantages that: the product has high expression amount and is easy to purify; the preparation method avoids the use of CNBr; and the process for processing the recombinant insulin is simple.

The invention discloses a monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and a method for detecting the nonstructural protein (NSP) antibody of a foot-and-mouth disease virus (FMDV) (FMD NSP B-ELISA); the kit comprises ELISA reaction plates, serum diluent, 25 times concentrated detergent, substrate solution, 100* concentrated ELISA detecting antibody, stop buffer, positive control serum and negative control serum; the ELISA reaction plates are two 96-pore high-affinity ELISA reaction plates, firstly 6* groups of amino acid monoclonal antibody or NSP 2C polyclonal antibody, and then FMDV 3ABC or 2C3AB NSP which is expressed by pronucleus and is provided with 6* groups of amino acid labels is captured through the monoclonal antibody or the polyclonal antibody; and compared with other similar kits, the method has higher coincidence rate and higher positive serum detection rate, and is applicable to detecting the serum of cattle, sheep, pigs and other susceptible animals.

The present invention provides methods and compositions for improved expression and production of recombinant antibodies in prokaryotic expression systems. Particularly contemplated are prokaryotic expression and production of full length aglycosylated antibodies. The antibody products of the invention can be used in various aspects of biological research, diagnosis and medical treatment.

The invention relates to the clone and the expression of alpha-cyclodextrin glycosyltransferase (alpha-CGTase), which belong to the field of enzyme gene engineering and enzyme engineering. The cgt gene expression method comprises the following steps: obtaining cgt gene SEQ ID NO:1 from total DNA of Peanibacillus macerans JFB05-01; selecting plasmid pET20b(+) as the expression vector of cgt gene; and selecting E.coli BL21(DE3) as the expression host to achieve high-efficiency extracellular expression of the cgt gene, wherein the cgt gene has 2,061 nucleotides and 687 coded amino acids; prokaryotic expression plasmid is constructed; and alpha-CGTase is expressed by transformed E.coli. Recombinase has cyclization activity and can transform starch and relevant matrix into cyclodextrin. The recombined alpha-CGTase has an optimal temperature of 40 to 45 DEG C and an optimal pH value of 5.5, and has higher thermal stability below 40 DEG C and poor thermal stability above 50 DEG C. The alpha-CGTase meets the requirement for industrial application such as food and medicines, and can be used for industrial production of alpha-cyclodextrin and beta-cyclodextrin.

The invention discloses a method to increase human papilloma virus L1 protein pronucleus expression productivity and also discloses a encode HPV L1 protein codon majorizing nucleic acid sequence from this method, which is characterized by the following: supplying expression carrier and host cell and HPVL1 protein poly body of nucleic acid sequence; disclosing appliance in preparing vaccine, drug compound and immunodiagnosis or antibody. The nucleic acid sequence expressing quantity possesses distinctive improvement, which decreases the preparing cost effectively.

The invention provides methods and compositions for improved expression and production of recombinant antibodies in host cell expression systems. In particular, prokaryotic expression and production of antibodies with modified hinge cysteine residues are provided. The invention further provides compositions, kits and articles of manufacture for practicing methods of the present invention.

The invention discloses a bacterial strain for producing farnesene and an application of the bacterial strain, belonging to the field of synthetic biology. The bacterial strain for producing the farnesene contains related genes for synthesizing the farnesene through a mevalonic acid pathway and codon optimization; and the sequence of a farnesene synthetic gene afs optimized by codons is as shown by SEQ ID NO.1. The bacterial strain can be used for producing the farnesene, and a seed solution of the bacterial strain is inoculated into a culture medium containing a carbon source to carry out prokaryotic expression to obtain the farnesene. The gene for synthesizing the farnesene is subjected to codon optimization or the farnesene is synthesized by using the mevalonic acid pathway to ensure that each protein is closer to a ratio of AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi:IspA:AFS=1:10:2:5:5:2:5:2:2, so that the production of the farnesene can be further promoted. By adopting the bacterial strain, the output of the farnesene is greatly improved to be more than 1g / L.

The invention discloses double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting an avian leukosis group specific antigen. The kit comprises an enzyme plate coated by a monoclonal antibody which is secreted by a hybridoma cell strain the preservation serial number of which is CGMCC (china general microbiological culture collection center) No.5961. The invention also discloses a double-antibody sandwich ELISA method which is established by utilizing the monoclonal antibody and is capable of rapidly and effectively detecting an ALV (avian leukosis virus). In the double-antibody sandwich ELISA method, the monoclonal antibody prepared by pronucleus expressive HLJ09mdj-1p27 albumen is utilized as a peridium antibody, and antibodies prepared by p27 are utilized as a detection antibody. According to the invention, the minimum detection amount of the p27 is 1.25 ng / ml, the method is not reacted with the common virus of birds, and the specificity is good. The method is utilized to detect egg white and an anus swab sample, and the coincidence rate is respectively 96.5% and 88.9% compared with a PCR (polymerase chain reaction) method; and the result proves that the method has the advantages of convenience, celerity, differentia, sensitivity and the like, and is useful for the detection and population purification of the ALV.

The invention relates to a competitive ELISA method based on a foot-and-mouth disease A type VP1 protein and its monoclonal antibody, also relates to a preparation method of the foot-and-mouth disease A type VP1 protein, and a preparation method of the monoclonal antibody of the foot-and-mouth disease A type VP1 protein, and belongs to the technical field of animal immunological detection. In the invention, a primer pair C1 and C2 and a primer pair E1 and E2 are amplified to obtain a gene sequence of the foot-and-mouth disease A type VP1 protein, the foot-and-mouth disease A type VP1 protein is obtained by constructing an expression plasmid, introducing the expression plasmid into a prokaryotic expression host and carrying out inducible purification, the foot-and-mouth disease A type VP1 protein monoclonal antibody is obtained by treating the foot-and-mouth disease A type VP1 protein as an antigen through a hybridomas technology, and the competitive ELISA method used for detecting a foot-and-mouth disease A type antibody is established based on the foot-and-mouth disease A type VP1 protein and its monoclonal antibody. The detection method has a strong specificity and a good stability, and can be used for detecting a foot-and-mouth disease A type serum antibody. By comparing a result obtained through the detection method with a liquid phase blocking ELISA kit, the coincidence rate is 95.8%.

Belonging to the field of biotechnologies, the invention discloses a competitive ELISA kit for detection of a peste-des-petits-ruminants virus antibody. The kit comprises a detection system composed of a coating antigen reaction solution and a monoclonal antibody reaction solution. The kit adopts prokaryotically expressed peste-des-petits-ruminants Nigeria 75 / 1 strain N protein as the coating antigen and employs a monoclonal antibody against N protein as the competitive antibody. The antibody against a peste-des-petits-ruminants virus in sheep serum is detected according to a competitive ELISA principle. The kit provided in the invention can rapidly and specifically detect the peste-des-petits-ruminants virus antibody in serum, and simultaneously has the advantages of large-scale production of monoclonal antibodies, good reaction specificity, high sensitivity, simple operation, low cost, stable, reliable and easily observable reaction results, thus being very suitable for import and export quarantine of sheep, food hygiene and screening of large batches of samples in livestock breeding farms, and being easy for large-scale popularization and application.

The invention discloses an E.tenella protein disulfide linkage isomerase gene EtPDI (Clone ID is BW1-E06,and the Genbank accession number of is EF552214). The gene is connected with a procaryon expression vector pGEX-4T-2; a procaryon expression recombination plasmid pGEX-4T-EtPDI is constructed and is expressed in a colibacillus system; and most of the expressed recombining protein exists in a soluble form. The recombining protein 4T-EtPDI is purified to carry out SPS-PAGE and is transferred to a PVDF film; and antiserum of E.tenella oocyst oral immunized chicken is used as first resistance and goat anti-chicken IgG is used as second resistance to carry out Western-blot analysis, thereby indicating that the gene has certain antigen. The gene is used for preparing an anti-chicken coccidiosis drug and an anti-chicken coccidiosis vaccine.

An anticancer vaccine of recombinant human MOC1-MBP fusion protein is disclosed, in which MBP is used as its adjuvant. The MBP gene and MUC1 gene are fused together. The MBP substituted for other fusion protein to induce CTL reaction. The pMAL-P2 is the carrier for effectively expressing maltose fusion protein. The serial repetitive sequence of MUC1 is inserted to downstream of malE gene.

The invention belongs to the field of molecular biology, and discloses a porcine circovirus type 2 recombinant cap protein and a subunit vaccine. The porcine circovirus type 2 cap protein expressed by recombinant Escherichia coli is obtained by steps of cloning a porcine circovirus type 2 cap protein in a nuclear localization signal area of which the N terminal is cut and which is rich in arginine into a prokaryotic expression vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into Escherichia coli BL21(DE3), and expressing by using the recombinant Escherichia coli BL21(DE3). Tests prove that the constructed recombinant strain expresses a foreign protein stably. When the subunit vaccine is prepared from the expressed recombinant protein, an antigen has high purity and safety, does not have pathogenicity on animals such as pigs and the like, and passes safety evaluation easily.

The invention discloses a large yellow croaker hepcidin antibacterial peptide and a preparation method thereof, relating to fish gene engineering in the biotechnology field, and providing a large yellow croaker hepcidin antibacterial peptide and a preparation method thereof. The preparation method of the large yellow croaker hepcidin antibacterial peptide comprises the following steps: cloning a large yellow croaker hepcidin antibacterial peptide gene; constructing a recombinant vector; converting a host cell; selecting positive clone for prokaryotic expression; separating and purifying an expression product; and obtaining the large yellow croaker hepcidin antibacterial peptide. During separation and purification, the yield of protein is improved, thus solving the problem of protein precipitation when the hepcidin antibacterial peptide expresses renaturation. The solution has simple preparation and low cost. The prepared large yellow croaker hepcidin antibacterial peptide has high broad-spectrum antimicrobial activity, can be used for scientific researches, can serve as feed additive for preventing and curing diseases of sea farming fishes and has wide application prospect.

The present invention generally relates to methods and compositions for expressing proteins or polypeptides in prokaryotic hosts using eukaryotic signal sequences.

The invention discloses a hybridoma cell line of a monoclonal antibody against African swine fever virus and the secreted monoclonal antibody thereof. The preparation method of the invention comprises the following steps: preparing a recombined P30 soluble antigen by prokaryotic expression; immunizing a BALB / c mouse; and finally fusing, screening and cloning by a hybridoma technology to obtain the hybridoma cell line which can stably secrete the monoclonal antibody against African swine fever virus P30 protein. The invention further discloses a method for preparing the monoclonal antibody with the cell line, an antibody purification method and a labeling method for horseradish peroxidase of the antibody. The monoclonal antibody can be used in detecting the African swine fever viral antibody in pig serum.

The invention discloses colibacillus and a method for performing soluble expression of transglutaminase proenzyme thereof. The method comprises the following steps: (1) designing PCR primer to perform PCR amplification according to the nucleotide sequence of transglutaminase proenzyme of streptomyces mobaraensis, cloning the related gene to prokaryotic expression vector pET22b (+),constructing expression vector pET22b-proMTG to conform to E.coli BL21 / proMTG of which strain preservation number is CCTCC M 208240; (2) performing soluble expression of transglutaminase proenzyme; (3) activating transglutaminase proenzyme; (4) performing high density fermentation; and (5) performing protein purification technology. In the method of the invention, the yield and specific activity of transglutaminase proenzyme can reach the level of that of transglutaminase proenzyme which is produced by firstly performing direct expression in colibacillus to form inclusion body and then performing denaturation and renaturation and the fermentation process is easier.

The invention discloses a bacterial strain for producing lycopene and an application of the bacterial strain, and belongs to the field of synthetic biology. The bacterial strain for producing lycopene contains a mevalonate pathway and a related gene which is synthetized by the lycopene optimized by a codon; and the synthetic gene sequence of the lycopene optimized by the codon is shown in SEQ ID NO.1-3. The bacterial strain can be used for producing the lycopene; and a seed solution of the bacterial strain is inoculated into a culture medium containing a carbon source to carry out prokaryotic expression, so as to obtain the lycopene. The gene synthetized by the lycopene is optimized by the codon or each protein of the mevalonate pathway is closer to A to B:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi=1:10:2:5:5:2:5; and production of the lycopene can be further promoted. By adopting the bacterial strain, the yield of the lycopene can be greatly improved to over 1g / L; and the fermentation cycle is shortened by over 50%.

The invention provides methods and compositions for improved expression and production of recombinant antibodies in host cell expression systems. In particular, prokaryotic expression and production of antibodies with modified hinge cysteine residues are provided. The invention further provides compositions, kits and articles of manufacture for practicing methods of the present invention.

The invention discloses a method for expression of a PCV 2 (Porcine circovirus type2) Cap protein by a pFast Bac Dual baculovirus. The method comprises the following steps of: amplifying a gene fragment of an encoded PCV 2 Cap protein with a His tag; connecting the gene fragment to a pFast Bac Dual plasmid so as to obtain a recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2; transforming Escherichia coli DH10Bac with the recombinant transfer plasmid, and carrying out blue-white selection to obtain a recombinant shuttle vector Bac-p10-ORF2-pH-ORF2; transfecting an insect cell with the recombinant shuttle vector so as to obtain a recombinant baculovirus Ac.Dual-Cap; and poisoning the insect cell with the recombinant baculovirus, performing cultivation, then collecting the insect cell, and purifying an expression product so as to obtain the recombinant Cap protein. The method disclosed in the invention solves the problem of low expression level of the Cap protein in eukaryotic cells. The recombinant Cap protein in the invention is designed with a His tag, thus being beneficial to the follow-up purification. And the recombinant Cap protein has biological activity superior to that of a Cap protein expressed by a prokaryotic expression system, thus being applicable to establishment of epidemiological diagnosis methods and reseach as well as development of PCV2 subunit vaccines.

The invention provides a Beta-mannose which has amino acid sequences shown in SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6 and SEQ ID: NO.8. Or substitution, depletion or addition of one or multiple amino acids is carried out to the amino acid sequences shown in the SEQ ID: NO.2, SEQ ID: NO.4, SEQ ID: NO.6 or SEQ ID: NO.8 to obtain amino acid sequences of Beta-mannose with the same activity. The invention also provides a gene for coding the Beta-mannose, a recombinant vector containing the gene and a host cell containing the recombinant vector. The invention further provides a preparation method of the Beta-mannose, including the culture of the host cell provided by the invention. The Beta-mannose provided by the invention has heat-resisting property and acid resistance and a prokaryotic expression system established by the invention can be utilized for production. Furthermore, six histidine tags can be utilized for purification.

The invention discloses a competitive ELISA (Enzyme-Linked Immuno Sorbent Assay)kit for detecting an antibody of an African swine fever virus and application thereof, belonging to the technical field of organisms. The kit is used for detecting an antibody of the African swine fever virus in pig serum by adopting prokaryotically expressed recombinant P54 protein as an envelope antigen according toa competitive ELISA principle. The envelope antigen in a 96 pore plate in the kit is prokaryotically expressed recombinant P54 protein and has favorable antigenicity. The enzyme-linked immuno kit provided by the invention comprises the P54 protein enveloped 96 pore plate, positive control, negative control, a horseradish peroxidase marked monoclonal antibody, a concentrated cleaning solution, serum diluent, a TMB substrate and a stopping solution. The kit can be used for screening samples in bulk, main reagents in the kit are provided in a working solution way, and the use is convenient.

The invention discloses a codon-optimized 7 beta-hydroxy steroid dehydrogenase gene, a recombination expression vector Pgex-6P-1-7 beta-HSDH containing an optimized gene and escherichia coli containing the recombination expression vector. Glutathione S-transferase (GST) fusion expression of the codon-optimized 7 beta-hydroxy steroid dehydrogenase gene and a prokaryotic expression vector Pgex-6P-1 in the escherichia coli is adopted, so that a large quantity of active target proteins with an integral structure can be generated in a short time; and a method for purifying the GST fusion expression protein is simple and quick, so that the active target proteins can be quickly obtained.

Provided are methods of generating a functional mammalian single chain MHC class I complex in prokaryotic expression systems and a host cell transformed with expression construct(s) capable of expressing a functional human single chain MHC class I complex capable of presenting specific antigenic peptides restricted to specific CTL clones.