79 results about "High level expression" patented technology

The invention provides a construction method and application of a high-efficiency expression system based on a Seali forest virus replicon. The method comprises the following steps: constructing a saRNA system for expressing a reporter gene; constructing to obtain replicase providing plasmids in the taRNA system; constructing to obtain an antigen providing plasmid in the taRNA system; the method comprises the following steps: respectively linearizing pSFV-CMV-T7-X, pSFV-CMV-T7-TR-X and pSFV-CMV-T7-replicase, and then carrying out in vitro transcription to prepare mRNA (Messenger Ribonucleic Acid); transfecting the prepared mRNA into a host eukaryotic cell for expression according to a certain proportion to obtain an antigen protein; and detecting the antigen protein to evaluate the expression efficiency of the saRNA and taRNA system based on the SFV replicon. According to the self-amplification DNA and RNA vaccine based on the virus replicon, the development period can be shortened, and the vaccine antigen can be expressed at a high level.

The process comprises providing a recombinant vaccine construct, wherein said construct comprises genetically modified Lactobacillus plantarum NC8 as a live vector; modifying the genetic structure of said Lactobacillus plantarum NC8 to express conserved Salmonella antigens, including PagN, SopE2, and FliC, anchored by a ptrk 892 backbone with a constitutive promoter, phosphoglycerate mutase (PGM); incorporating Signal Lp_2145 and cAM12 Anchor sequences into said genetic construct to enhance surface expression of recombinant proteins on Lactobacillus plantarum NC8; administering said recombinant vaccine orally to poultry, leveraging the probiotic properties of Lactobacillus plantarum NC8 for effective colonization of the poultry gastrointestinal tract; inducing a prolonged and intensified immune response by ensuring sustained high-level expression of target antigens through the utilization of the robust constitutive promoter, phosphoglycerate mutase (PGM); and optimizing immunogenicity through the surface expression of recombinant proteins on Lactobacillus plantarum NC8, fostering a robust and precisely targeted immune response.

The invention belongs to the technical field of genetic engineering and microbial fermentation, particularly relates to a recombinant strain capable of efficiently producing microrhin Mccj25, and further discloses a method for producing microrhin Mccj25 through fermentation of the recombinant strain and application of the recombinant strain. A recombinant strain capable of producing Mccj25 at high yield is constructed on the basis of a genetic engineering means, an Escherichia coli F-750 recombinant strain capable of producing Mccj25 at high yield is preferably obtained by modifying an mcjABCD expression element and a chassis strain, and fermentation experiment determination shows that the recombinant strain can realize high-level expression of Mccj25, and the expression quantity reaches 6 g / L or above.

The invention relates to the technical field of genetic engineering, in particular to recombinant human collagen as well as a preparation method and application thereof. The recombinant human collagen provided by the invention is a polypeptide containing an amino acid sequence such as SEQ ID No.1. The recombinant human collagen disclosed by the invention is expressed at a high level outside cells, the culture is easy to amplify, the target protein can be produced at high yield by utilizing a high-density fermentation mode, secretory expression is beneficial to separation and purification of an expression product and reduction of production cost, and the recombinant human collagen has a certain post-translational modification function, such as glycosylation and the like, on the expressed foreign protein.

Provided is a gene for efficiently expressing hyaluronic acid hydrolase. A nucleotide sequence of the gene is represented by SEQ ID NO: 4. By truncating a segment of a signal peptide sequence at the N-terminus of a full-length hyaluronic acid hydrolase gene, the Pichia pastoris engineering strain of high level expression genetic engineering hyaluronic acid hydrolase is constructed. The enzyme activity of the hyaluronic acid hydrolase in a fermentation broth obtained by high-density fermentation using the constructed Pichia pastoris engineering strain is up to 4.7×105 U / mL.

The invention discloses bacillus subtilis for recombinant expression of a hermetia illucens HIAMP0438 gene and a construction method of the bacillus subtilis, and belongs to the technical field of gene engineering. The hermetia illucens recombinant protein used in the invention is obtained by expression of bacillus subtilis WB800N transferred into a pHT43-HIAMP0438 high-efficiency expression vector, and the pHT43-HIAMP0438 high-efficiency expression vector is obtained by a homologous recombination technology, so that high-level expression of the HIAMP0438 gene is realized, the HIAMP0438 gene can be efficiently secreted out of cells, the preparation process of the recombinant protein is greatly simplified, and the production cost is reduced. According to the present invention, the recombinant protein can significantly inhibit the growth of Escherichia coli, Salmonella and Listeria monocytogenes, and the recombinant protein can be used as the animal health product, and can be used for preventing and treating pig and chicken intestinal diarrhea caused by Escherichia coli and Salmonella and food-borne digestive tract diseases caused by Listeria monocytogenes.

The invention discloses a method for improving the efficiency of a baculovirus expression system. According to the invention, actin is overexpressed in insect BmN cells, so that the expression of exogenous genes is improved. The principle is as follows: after overexpression of actin, the recruitment efficiency of virus nucleation and late gene polymerase is improved, so that the replication and transcription of recombinant virus containing exogenous genes are promoted. According to the invention, the problem of low protein expression quantity by using the baculovirus is solved, ultrahigh-level expression of the protein is promoted, and the efficiency of a baculovirus expression system is improved.

The present invention relates to a method for constructing a host bacterium with high expression of human serum albumin, which method comprises: S100, designing an optimized gene encoding human serum albumin; S200, constructing a recombinant expression vector; and S300, transforming the recombinant expression vector into a Pichia pastoris host bacterium. The optimization design of a full-length sequence of a target gene is performed at the molecular level to obtain a target gene with the most suitable expression in Pichia pastoris; at the cellular level, the latest international Pichia pastoris expression system host bacterium CBS7435 is introduced for the gene expression of human serum albumin; and Pichia pastoris transformants harboring high-copy-number exogenous genes are selected by means of using a G418 resistance gene for the construction of an engineering bacterium with high expression of recombinant human serum albumin.

The present invention relates to an N-terminal extension sequence for enhancing the expression of a recombinant therapeutic peptide. The present invention also relates to a method for expressing a recombinant therapeutic peptide at a high level using the N-terminal extension sequence. The present invention also provides nucleic acids, vectors and recombinant host cells for efficiently producing biologically active proteins, such as liratide.

Provided are a translation regulatory element, an expression vector, a pharmaceutical composition, a vaccine composition, a construction method, a method for expressing a target protein, and the use thereof. The translation regulatory element is selected from recombinant IRES sequences having sequences as shown in SEQ ID NOs: 8-11, and / or one or more segments of recombinant IRES sequences complementary to the sequences as shown in SEQ ID NOs: 8-11; and can show high translation efficiency when being used for regulating gene expression, thereby realizing high-level expression of a target gene.

The application discloses a recombinant pseudorabies virus strain expressing classical swine fever virus E2 protein and application, and relates to the technical field of biology.The recombinant E2 protein provided by the application successfully realizes high-level expression of the E2 protein by removing the transmembrane region of the E2 protein and using the 18aa signal peptide of the E2 protein itself and a pig albumin signal peptide.The recombinant PRV expressing the classical swine fever virus (CSFV) E2 protein can induce E2 antibodies and cellular immunity after immunization of mice.The recombinant pseudorabies virus strain provided by the application can be used as a bivalent vaccine for preventing CSFV and PRV infection.

The invention discloses a sugar-inducible promoter and application thereof in prevention and treatment of monochamus alternatus, and relates to the technical field of genetic engineering and biological prevention and treatment. Based on the technical background of transgenic symbiotic bacteria in prevention and treatment of monochamus alternatus, the scheme has the advantages that the transgenic symbiotic bacteria and wild strains have a competitive relationship in ecological niche; under high-level expression driven by a constitutive promoter, the transgenic symbiotic bacteria may be too greedy in resource utilization, so that the transgenic symbiotic bacteria conflict with wild strains and even are eliminated by the wild strains; according to the scheme, expression of Cry3Aa-T protein is regulated and controlled by using the sugar-induced promoter so as to reduce the cost of fitness of exogenous genes carried by serratia marcescens, and a hidden and effective method is provided for preventing and controlling propagation of vector insect monochamus alternatus.

Methods to treat cancer in a subject comprising administering to the subject a therapeutically effective amount of T-cells of the subject having increased IRF4 polypeptide expression compared to a control are disclosed. Also disclosed are methods of increasing tumor reactivity of a T-cell by increasing IRF4 polypeptide expression, and methods to predict the likelihood that a subject having cancer will respond therapeutically to administered T-cells having increased IRF4 polypeptide expression. Also disclosed are compositions comprising a T-cell and a viral vector encoding an IRF4 polypeptide. The compositions are methods are useful for treating numerous cancers in which higher level expression of IRF4 in T-cells would be beneficial. In some embodiments, activated tumor-specific T-cells having increased IRF4 expression have greater infiltration in tumors and enhanced local immunological responses.

The present disclosure is related generally to systems and methods for high level expression of recombinant proteins from baculovirus in insect cells. In particular, the methods and systems described herein allow for high levels of baculovirus production in insect cells and / or high levels of protein production in insect cells using a chemically-defined, yeast lysate-free insect cell medium. The disclosure also relates to compositions and kits for culturing, transfecting, and / or producing recombinant protein in insect cells.

The invention provides a mammal protein efficient expression system based on a prokaryotic two-component signal transduction system (TCS, two-component system) and application of the mammal protein efficient expression system, and the mammal protein efficient expression system comprises the TCS capable of efficiently recruiting transcription factors and a carrier capable of being induced by the TCS to express a target gene. By transfecting mammalian cells with the system, a protein of interest can be produced in the cells or extracellular proteins can be produced in a secreted form. Based on the mammal protein high-efficiency expression system provided by the invention, stable and high-level expression of the target protein can be realized. The invention provides a new method for production of therapeutic proteins and antibodies, provides key technical support for preparation of mammalian protein samples in the research fields of structural biology and the like, and has important scientific significance for development of life science, diagnosis and treatment of diseases and research and development of medicines.

This invention relates to a wholly recombinant four factor hemostatic complex concentrate (4F-PCC) composition, human liver cell-produced product. Methods of producing a hemostatic composition using a hepatocyte cell line and a CRISPR / Cas9 gene activation multiplexing, which allows for simultaneous and high level expression of the FII, FVII, FIX, and FX clotting factors. The resultant product is a recombinant resuscitation solution to aid in mitigation of coagulopathy, reversal of coagulopathic states, and fluid resuscitation.

The invention relates to the technical field of biology, in particular to a method for synthesizing silphinene in saccharomyces cerevisiae by sesquiterpene cyclase SilpS. According to the invention, a novel sesquiterpene synthase SilpS is obtained through genome mining, a recombinant plasmid expressed by a SilpS gene is constructed, a SilpS gene expression cassette is transferred to a saccharomyces cerevisiae chromosome, and the obtained saccharomyces cerevisiae recombinant strain can realize high-level expression of silphinene; the yield of silphinene in the recombinant strain is 139.2 + / -2.2 mg / L, and the recombinant strain has a wide application prospect.

The invention discloses a recombinant PBase transposase, a PiggyBac vector system and a gene recombinant cell constructed by the recombinant PBase transposase and the PiggyBac vector system. The invention provides a recombinant PBase transposase. The amino acid of at least one of the 81st site, the 114 site, the 337th site, the 500th site and the 553th site of a wild type PBase transposase is mutated. According to the invention, the sequence of wild type PBase transposase is improved, codon optimization is carried out according to CHO cell preference, and then the codon optimization is constructed on an expression vector to obtain helper plasmids of a PiggyBac vector system; and connecting a target gene between two terminal inverted repetitive sequences of the expression vector to obtain the transposon plasmid of the PiggyBac vector system. A PiggyBac vector system is transfected into CHO cells, a stably transfected cell strain is constructed in a pressurized screening mode, and cells with high-level expression are obtained after the stably transfected cell strain is used for only 3-5 weeks.

The invention discloses an application of PbGAB2 in prevention and control of rape clubroot, a G protein coupled receptor PbGAB2 with high expression level in each stage is screened out from a plasmodiophora brassicae ZJ-1 transcriptome, an RNAi vector of the gene is constructed, a corresponding transgenic plant is obtained, and the application of PbGAB2 in prevention and control of rape clubroot is realized. A disease resistance test of plasmodiophora brassicae shows that the PbGAB2-RNAi transgenic line disclosed by the invention shows excellent disease resistance phenotype, and the G protein coupled receptor PbGAB2 of the plasmodiophora brassicae can be used as a gene resource for resisting clubroot.

The present application relates to the field of microorganisms and genetic engineering, and specifically discloses an Escherichia coli engineering bacterium for efficiently producing plastic depolymerase and application thereof. The present application uses Escherichia coli as a host, and through synergistic optimization of multiple key dimensions such as signal peptide, promoter, solubility-promoting tag and co-expression of molecular chaperones, a multi-dimensional engineering system for efficiently expressing plastic depolymerase is constructed. The construction method provided by the present application includes a rational design and a screening strategy for combination and assembly of the multiple elements. The engineering bacterium can realize extracellular high-level expression of plastic depolymerase during induced fermentation, and the enzyme preparation prepared can be directly used for catalyzing efficient depolymerization of polyester plastics. The present application provides an Escherichia coli cell factory with stable high yield for industrialization and low-cost production of plastic depolymerase, and has important industrial application prospects for promoting large-scale biological degradation and resource recycling of waste plastics.

The invention discloses a transgenic soybean event AS-1 with high expression of alpha-amylase and a detection method and application of the transgenic soybean event AS-1. The transgenic soybean event AS-1 can express the alpha-amylase at a high level and has very high amylase activity, and the amylase activity reaches up to 62.22 thousand U / g and is far higher than that of commercial enzymes produced through microbial fermentation; and the method has no obvious adverse effect on growth, development and agronomic traits of plants. The transgenic soybean event can be used as a bioreactor for large-scale production of alpha-amylase, and has a remarkable cost advantage and a wide application prospect. The transgenic soybean can be used as an enzyme preparation, and the production cost is lower than that of microbial fermentation.

The invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof in order to overcome the practical application problem of a genetic engineering method in the process of producing the bovine lactoferrin peptide at present. According to the invention, bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein are connected in series to form bovine lactoferrin peptide fusion protein, a pichia pastoris recombinant strain is constructed, and high-level expression of bovine lactoferrin peptide is realized. According to the method, positive charges of bovine lactoferrin are neutralized on the basis of calmodulin, toxicity of antibacterial peptide to a host is weakened, the tandem enhanced green fluorescent protein can emit green fluorescence under 488 nm exciting light, and real-time analysis of the expression level of bovine lactoferrin fusion protein is achieved. According to the method, by adopting a fusion expression technology, the toxicity to a host when the antibacterial peptide is produced by a genetic engineering method is overcome, so that the expression quantity is increased, and a foundation is laid for high-density fermentation production of the bovine lactoferrin peptide.

The invention discloses an EpCAM gene high expression vector based on a lentiviral vector and a cell strain thereof, and belongs to the technical field of gene engineering and biology. According to the vector, an EpCAM gene expression structure is connected between multiple cloning sites Xba1 and Nhe1 of a vector pCHD-CMV-MCS-EF1-RFP-T2A-puro, high-level expression is carried out under the driving of a CMV (cytomegalovirus) promoter, and a recombinant plasmid pCDH-OE-EpCAM is obtained. And transforming the recombinant plasmid into escherichia coli DH5alpha to obtain the recombinant microbial cell. The recombinant plasmid is respectively transfected with colorectal cancer cell strains HT-29 and HT-115 through a Lipo2000 transfection reagent, and then monoclonal cells are screened, so that transgenic cells are obtained. The vector for overexpression of the human EpCAM gene and the colorectal cancer cell strain for overexpression of the EpCAM gene are constructed, and a very important foundation is laid for molecular mechanism research of generation and development of the EpCAM gene and colorectal cancer.

The invention provides an application of high-expression MTA1 in targeted glioma stem cells (GSC) in treatment of glioblastoma (GBM). According to the invention, the high-level expression of MTA1 protein molecules by the GSC in the GBM is proposed for the first time, the high-expression MTA1 is positively correlated with the poor prognosis of a GBM patient, and the dryness, cell proliferation and balling ability of the GSC are also promoted. The MTA1 highly expressed in the targeted GSC can inhibit the malignant progress of GBM. The invention provides a new target and an inhibitor for targeted therapy of GBM.

A broadly protective Shigella-ETEC vaccine includes components to cover multiple Shigella species and serotypes as well as multiple different colonization factors of ETEC. ETEC colonization factors CS2 and CS3 are among the most prevalent CFs found on ETEC isolates associated with diarrhea. These two factors are important components of a vaccine that can confer broad protection against ETEC. High level expression of these heterologous antigens in Shigella live vectors results in stronger immune responses. This specification describes the genetic engineering of DNA sequences encoding upstream sequences, including promoter and ribosome binding sites, that results in increased expression of CS2 and CS3 in Shigella live vectors and that leads to stronger immune responses when used as a vaccine in animal models.

The present invention discloses a recombinant human type 17 collagen mussel mucin polypeptide, its preparation method and use, and belongs to the field of genetic engineering technology. The present invention recombines a fragment of human type 17 collagen with the histidine and 3,4-dihydroxyphenylalanine-rich regions of mussel mucin, and the obtained recombinant protein has higher cell adhesion activity. The amino acid sequence of the polypeptide is shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4. The recombinant human type 17 collagen mussel mucin polypeptide of the present invention can be expressed at a high level in Escherichia coli as a host, its preparation method is simple, easy to scale up production, and can be widely used in industries such as medicine and cosmetics.

The invention discloses a xylanase mutant and application thereof. The amino acid sequence of the xylanase mutant comprises SEQ ID NO: 7. The xylanase mutant MUT-1 provided by the invention has higher specific enzyme activity compared with a parent P1, the specific enzyme activity of the xylanase mutant MUT-1 is improved by 41% compared with that of the parent P1, and the xylanase mutant MUT-1 also has good acid resistance and heat resistance. The invention also provides an expression vector and a host cell of the xylanase mutant, high-level expression of xylanase can be realized by using engineering bacteria, and the production cost is greatly reduced.

The present invention belongs to the field of genetic engineering technology, and specifically relates to a method for improving the expression level of recombinant proteins in CHO cells. The method comprises the following steps: (1) combining a target gene recombinant vector with a gene containing cell cycle regulation-related genes; p18 and / or genes that regulate cell growth aFGF The co-transgenic recombinant vector is co-transfected into CHO cells; the gene p18 The sequence of is shown in SEQ ID NO: 1; the gene aFGF The sequence is shown in SEQ ID NO:2; (2) co-transfecting the co-transgenic recombinant vector and the target gene recombinant vector into CHO cells, culturing and screening to obtain a CHO cell expression system; (3) culturing the transient and stable transfected CHO cell lines in suspension serum-free culture to obtain the target protein. The method of the present invention can significantly improve the expression level of the target gene in CHO cells and achieve transient and stable high-level expression of the target protein in CHO cells.

The invention discloses an application of a targeted ABC transporter PbABCB25 in prevention and control of clubroot of rape. ABC transporter protein ABCB25 (abc transporter b family meber 25, ABCB25) with high-level expression in each stage is screened out from a plasmodiophora brassicae ZJ-1 transcriptome, then an RNAi carrier of the ABC transporter protein ABCB25 is constructed through an HIGS technology, the RNAi carrier is transformed into wild type arabidopsis thaliana, then the resistance of transgenic arabidopsis thaliana to clubroot is tested, the result shows that PbABCB25 has remarkable resistance to plasmodiophora brassicae, and the PbABCB25 can be used for preparing the plasmodiophora brassicae. The result shows that the ABCB25 in the plasmodiophora brassicae can be used as a gene resource for resisting the clubroot of the cruciferae.

The present invention relates to the technical fields of fermentation engineering and genetic engineering, and particularly relates to a method for synthesizing lactic acid monomer using sucrose as a raw material. Through the genetic recombination technology of site-directed gene replacement, the promoter sequence of the sucrose metabolic operon in the production strain is corrected, enabling it to maintain a high level of expression throughout the production process of lactic acid monomer, demonstrating an obvious ability to highly catabolize sucrose to synthesize lactic acid monomers with high optical purity and high chemical purity (D-lactic acid or L-lactic acid). For example, strains DSAPW5 and LSAPW5 are fermented for 28-30 h under the condition of 25-50 °C, and the fermentation levels of D-lactic acid and L-lactic acid can reach 158.58 g / L and 159.65 g / L respectively, and the optical purity can reach 99.94% and 99.95%. The present invention can be applied to the industrial production of lactic acid monomer using sucrose or sucrose-containing raw materials.