31 results about "Cistron" patented technology

Recombinant NK cells, and especially recombinant NK-92 cells express a chimeric antigen receptor (CAR) having an intracellular domain of FcεRIγ. Notably, CAR constructs with an intracellular domain of FcεRIγ had a substantially prolonged duration of expression and significantly extended cytotoxicity over time. The CAR may be expressed from RNA and DNA, preferably as a tricistronic construct that further encodes CD16 and a cytokine to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of the recombinant proteins and provide a convenient selection marker to facilitate rapid production of recombinant NK / NK-92 cells.

The invention discloses a pichia pastoris high-secretion strain screening system and a use method thereof. According to the system, pichia pastoris is used as a host, and the core structure of the system is as follows: an AOX1 promoter, an alpha-factor signal peptide, a target protein-P2A peptide, an alpha-factor signal peptide, a Gamilus fluorescent protein-G4S connecting peptide-Gcw21 anchoring sequence-AOX1 terminator. The use method comprises the steps of vector construction, conversion of enzyme digestion recovery products, clone strain selection and activation, fermentation, inducible expression and screening, and product collection to finally obtain the strain with high yield of target protein. Through integration of bicistronic design, P2A peptide self-cleavage and cell surface display technologies, efficient screening of target protein high-secretion type strains is realized.

This invention discloses a recombinant vector, recombinant bacteria, and fermentation method for high-yield GHK production, belonging to the field of biomedical technology. The recombinant expression vector of this application comprises a bicistronic expression cassette controlled by a T7 / lac heterozygous promoter, sequentially containing the encoding (GHK). n The vector encodes the first cistron of the polypeptide and the second cistron, where n is an integer from 2 to 200. When this vector is transformed into *E. coli* BL21, the resulting recombinant bacteria can simultaneously express (GHK) after IPTG induction. n Polypeptides and trypsinogen. During fermentation, trypsinogen is activated and enables the processing of (GHK). n In situ enzymatic cleavage of peptides efficiently releases GHK. This invention integrates the traditional multi-step process into a "one-step fermentation," simplifying the production process and reducing purification difficulty. Examples show that the GHK yield can reach up to 3.9 g / L, providing an efficient and convenient new strategy for the large-scale biomanufacturing of GHK.

The invention relates to a methanophilic genetically engineered bacterium for producing gamma-polyglutamic acid (gamma-PGA) as well as a construction method and application of the methanophilic genetically engineered bacterium, EQU2420670 gene is knocked out on a host bacterium genome, and meanwhile, an exogenous gamma-PGA synthetase encoding gene cluster pgsBCAE under the control of an inducible arabinose promoter (Pbad) is introduced at the site. The construction method comprises the following steps: obtaining a multi-cistron structure DNA fragment pgsBCAE for coding gamma-PGA synthetase through artificial gene synthesis in combination with a codon optimization strategy, and constructing the expression module Pbad-pgsBCAE under the control of a Pbad promoter. A suicide vector pK18mobsacB carrying a SacB sucrose reverse selection system is used for constructing a gene replacement vector, upstream and downstream homologous arms of an EQU2420670 gene on a genome of methanophilic bacteria M. buryatase 5GB1 are designed on two sides, an EQU2420670 site is replaced by a Pbad-pgsBCAE expression module, the methanophilic genetically engineered bacterium PGA is obtained, and the methanophilic genetically engineered bacterium PGA is applied to production of gamma-PGA and has important economic and social significance; the gamma-PGA produced by the engineering methanophilic bacteria by using methane as a carbon source and an energy source can be used as a cosmetic additive; the invention provides a novel low-carbon biosynthesis approach for commercial production and application of gamma-PGA, and has a good industrial application prospect.

Vectors and methods are disclosed for immortalizing mammalian cells by co-expression of v-raf and v-myc proteins. A replication-defective viral vector is used for improved safety. The vector comprises an optional marker gene, and is especially useful for producing an immortalized macrophage by a method that involves contacting the vector with a monocyte, proliferatively growing the monocyte, growing the monocytic cell on a solid surface, and then growing the monocytic cell on a porous surface. An immortalized macrophage is also disclosed.

The invention discloses a method for reducing the molecular weight of gamma-PGA based on a polycistron expression vector, which comprises the following steps: cloning a gamma-polyglutamic acid synthetase gene cluster pgsBCA of bacillus licheniformis into corynebacterium glutamicum F343 with high yield of glutamic acid in a polycistron expression mode for exogenous expression to obtain a recombinant strain pBCA; compared with a recombinant strain pBpCpA expressed by a monocistron, the recombinant strain pBCA carrying a polycistron expression system has the advantage that the molecular weight of gamma-PGA generated by the recombinant strain pBCA is reduced by 15.98%. Therefore, the polycistron expression system is an effective method for reducing the molecular weight of the gamma-PGA.

The invention discloses an Escherichia coli strain capable of bidirectionally regulating the copy number of CoIE1 type plasmids and application of the Escherichia coli strain. According to the method, escherichia coli Mach1-T1 is taken as an initial strain, a CRISPR / CAS9 gene editing technology is utilized, RNase E Q36R variant genes, rssB genes and pcnb genes are constructed into a polycistron expression cassette, and the polycistron expression cassette is placed under the control of an arabinose operon to replace endogenous pcnb genes of a genome. Each gene in the polycistron expression cassette is driven to translate by an independent ribosome binding site (RBS). By adjusting the concentration of the arabinose, continuous and bidirectional regulation and control of the copy number of the CoIE1 type plasmids from'inhibition 'to'overproduction' can be realized, and the method has important significance on industrial mass production of the CoIE1 type plasmids and diversified application of the CoIE1 type plasmids.

Multiple exogenous genes are expressed from a single transcript in algae and plants using engineered plant polycistronic loci.

The present disclosure relates to bicistronic polypeptide constructs for use in allogeneic gene therapy, such as CAR-T cell therapy. The bicistronic constructs comprise a first polynucleotide encoding a therapeutic molecule (e.g., CAR-T or an antibody) and a second polynucleotide encoding an immune surveillance masking molecule (ISMM). The ISMs comprise a human leukocyte antigen-E genetically fused to a non-functional form, such as a fragment, of a protein, such as beta-2 microglobulin or B2M, which is knocked out by insertion of the bicistronic construct. Also provided are vectors, cells (e.g., CAR-T cells) comprising the bicistronic constructs and methods of use. Kits and articles of manufacture are also provided. The present disclosure also provides four new insertion sites that can be used to insert an expression construct into the B2M gene.

The present application relates to the field of agricultural biotechnology, and in particular to an improved Pichia pastoris gene editing single plasmid and a method for improving Pichia pastoris gene editing efficiency thereof.According to the technical scheme of the present application, the polycistronic strategy is beneficial to reducing the size of the plasmid, thereby improving the transformation efficiency; by adjusting zeocin and rad52 the order, the expression amount of Rad52 is increased during gene editing to improve the homologous recombination efficiency, and the Rad52 plasmid form is expressed in Pichia pastoris. During gene editing, the plasmid is introduced into Pichia pastoris cells for expression, promotes the proteins of gene editing and homologous recombination, and improves the gene editing efficiency; after the gene editing is completed, the plasmid is eliminated, the expression level of Rad52 returns to the normal level to maintain the stability of the cells, thereby maintaining the stability of the cell performance.

This invention relates to a mouse model of Parkinson's disease and its applications. The construction method includes the following steps: 1) in mice... Snack An exogenous fragment containing the human SNCA G51D coding sequence was knocked into the region upstream of the gene stop codon via homologous recombination, causing the human SNCA G51D coding sequence to interact with the mouse endogenous gene. Snack The gene forms a bicistronic expression structure, and the coding sequence of the human SNCA G51D is shown in SEQ ID No:1; 2) Positive F0 generation mice are backcrossed with wild-type mice, and genotypes are identified and screened to obtain a stable SNCA G51D endogenous knock-in Parkinson's disease mouse model. The gene editing strategy of the Parkinson's disease mouse model preserves the mouse's... Snack Simultaneous endogenous expression of α-synuclein G51D in the gene can mimic two pathogenic forms, exhibiting high physiological relevance and genetic authenticity.

The invention discloses a method for constructing MSTN (myostatin) gene knockout porcine primary cells, which comprises the following steps: designing and constructing plasmids capable of efficiently knocking out MSTN genes by utilizing an efficient and simple gene modification technology TALEN (transcription activator-like effector nodules) technology; a gene editing target site is designed for a pig MSTN gene, the target site can be specifically recognized by Cas9 endonuclease, so that double-strand breakage is mediated, the gene is mutated in a non-homologous end connection mode under the action of a self-repairing system, then an sgRNA composition is designed, tRNA-sgRNA-sgRNA scaffold is used as a minimum repetitive unit, and the target site is specifically recognized by Cas9 endonuclease. The Cas9-CD163-MSTN polycistron vector is constructed by using a PTG / Cas9 strategy, so that the target MSTN gene is efficiently and accurately knocked out, and therefore, the Cas9-CD163-MSTN polycistron vector has a relatively good application prospect in pig germplasm improvement.

Provided herein arc polynucleotides comprising at least two coding sequences for expression of coding sequences of interest in a cell. Also provided are vectors, recombinant viral genomes, and recombinant virus compositions comprising said polynucleotides, as well as associated methods.

Provided herein are optimized 2A ribosome skipping elements and polynucleotide sequences encoding such optimized 2A ribosome skipping elements, as well as polycis-trans subsystems, constructs, vectors, and engineered cells that utilize the 2A ribosome skipping elements to express a plurality of polypeptides from a single transcript. Pharmaceutical compositions comprising the engineered vectors and / or cells for the treatment of disorders are also disclosed.

According to the bicistronic DNA vaccine for edwardsiella tarda disease of paralichthys olivaceus, provided by the invention, the bicistronic DNA vaccine can remarkably induce Th1 type immune bias, enhance immune response of fish cells and improve the resistance to intracellular bacterial infection, and has the advantages of being safe, efficient, easy to prepare, high in field popularization adaptability and the like. A fusion fragment of an immunologic adjuvant IL-12 is used in the bicistronic DNA vaccine, and the bicistronic DNA vaccine is constructed by connecting a nucleic acid fragment of a subunit 12p35 of which the coding amino acid sequence is SEQ ID NO: 1 and a nucleic acid fragment of a subunit 12p40 of which the coding amino acid sequence is SEQ ID NO: 3 through a flexible connecting peptide joint. According to the bicistronic DNA vaccine disclosed by the invention, the synergistic expression of the OmpC antigen and the IL-12 adjuvant is realized, the immunogenicity is enhanced, and the problem of insufficient immune effect of the traditional DNA vaccine is solved; and the vaccine has no obvious toxic or side effect on the fish body, does not depend on antibiotics, is green and environment-friendly, and has a good industrialization prospect.

The application discloses a method for increasing drought adaptability of plant osmotic regulation substance accumulation, and belongs to the field of plant genetic engineering and drought-resistant molecular breeding. First, a plant to be transformed is combined with 13C-glutamic acid and 15N-choline double isotope labels to quantize carbon and nitrogen flow redistribution of proline synthesis pathways and glycine betaine synthesis pathways in leaf cells and vascular bundle companion cells, and to identify rate-limiting enzyme nodes. Second, a multi-cistronic plant expression vector is constructed. A first expression box is driven by a leaf-specific promoter and a drought response cis-element composite promoter to drive P5CS. A second expression box is driven by a companion cell-specific promoter and a drought response cis-element composite promoter to drive BADH. Positive transformants are obtained through Agrobacterium mediation or gene gun transformation and molecular screening. The expression quantity of the positive transformants is increased by 5 to 50 times under stress and falls back to a baseline within 72 hours of rehydration. Root activity is significantly improved, and yield is preserved.

Provided herein are methods for detecting the expression of different bicistronic-expressed polypeptides, such as chimeric cytokine receptors, that have been engineered into a population of immune cells (e.g., a population of CAR T cells). In one aspect, the disclosure provides in vitro methods of detecting bicistronic expressed polypeptides in chimeric antigen receptor (CAR) T cells.

A recombinant NK cell, in particular a recombinant NK-92 cell, expresses a chimeric antigen receptor (CAR) having an intracellular domain of Fc [epsilon] RI [gamma]. Notably, the CAR constructs having the intracellular domain of Fc [epsilon] RI [gamma] have a significantly prolonged expression duration and a significantly prolonged cytotoxicity over time. The CARs can be expressed from RNA and DNA, preferably as tricistronic constructs that further encode CD16 and cytokines to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of recombinant proteins and provide convenient selection markers to promote rapid production of recombinant NK / NK-92 cells.

Described herein relates to novel methods for determining susceptibility for infection and / or disease (e.g., pregnancy complication and / or cancer), and / or predicting severity of infection and / or disease by measuring circulating Alu RNA by RT-PCR (e.g., circulating blood, serum, and / or plasma). Additionally, described herein relates to novel methods of treating infection and / or disease (e.g., pregnancy complication and / or cancer), via increasing immune response, optimizing vaccine delivery, via administering at least one Alu RNA into the subject.

The invention discloses a traceless induction method of eukaryote polycistron expression ability. The method disclosed by the invention comprises the following steps: introducing an expression cassette into a eukaryotic cell, or introducing a fragment except a promoter in the expression cassette into the downstream of a DNA fragment with promoter activity in the eukaryotic cell, so that the DNA fragment can drive the expression of a gene in the expression cassette to obtain a recombinant biological cell, the expression cassette contains a promoter, an upstream gene, a cistronic interregion and a downstream gene, and 2) carrying out induced culture on the recombinant biological cells by using the selection marker corresponding to the downstream gene to realize the induction of the eucaryon polycistronic expression competence. The method disclosed by the invention can be used for inducing the cells with the polycistronic expression competence in a short time.

The present disclosure provides multi ci str onic vaccines and method for producing and using the same in preventing infection or transmission, or reducing severity of disease caused by influenza and / or SARS-Cov-2 virus in a subject. Multicistronic vaccines of the disclosure can be administered via intramuscular, intranasal, or inhalation route. In one particular embodiments, the disclosure provides a recombinant adenovirus comprising at least two different extraneous oligonucleotides that are capable of stimulating an immune response in a subject. Each of the oligonucleotide independently comprises an oligonucleotide that encodes either influenza or SARS-Cov-2 virus antigens.

Provided herein are polynucleotides comprising at least two coding sequences for expression of coding sequences of interest in a cell and two transcriptional regulatory elements. The transcriptional regulatory elements may be tissue specific. Also provided are vectors, recombinant viral genomes, and recombinant virus compositions comprising said polynucleotides, as well as associated methods.

In variants, the method of generating a hypoimmunogenic cell can include: integrating an insertion sequence into a cell genome (e.g., into a Beta-2-Microglobulin (B2M) gene) and performing a cell selection. The method can optionally include: integrating a selection sequence into the cell genome, removing a selectable marker, editing an additional gene, and / or any other suitable steps. The hypoimmunogenic cell can include a genetically engineered sequence including: a Beta-2-Microglobulin:human leukocyte antigen (B2M:HLA) fusion gene, a multicistronic element, and a kill switch gene.

A ribonucleic acid comprising two non-natural pre-miRNA sequences, wherein each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein. The pre-miRNA sequences may target a different gene or target a different regions of the same gene. A deoxyribonucleic acid encoding the aforementioned ribonucleic acid. The deoxyribonucleic acid may further encode a protein such as a chimeric antigen receptor, a cytokine, a cell tag, and / or an immune checkpoint inhibitor. A vector comprising the aforementioned ribonucleoic acid or the aforementioned deoxyribonucleic acid. A method for modifying the expression of a gene in a cell, wherein the method comprises introducing the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the cell. A method for producing a genetically-engineered cell, wherein the method comprises introducing the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the cell. A genetically-modified cell comprising the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid. A composition comprising the aforementioned ribonucleic acid or the aforementioned deoxynbonucleic acid. A kit comprising the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid. A method of treating a disease or disorder in a subject, comprising administering the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the subject. A method of treating a disease or disorder in a subject, comprising administering the aforementioned cell to the subject. The use of the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid in the manufacture of a medicament for modifying the expression of a gene. The use of the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid in the manufacture of a medicament for the treatment of a disease or disorder in a subject.

The invention relates to the technical field of agricultural biology, in particular to an improved pichia pastoris gene editing single plasmid and a method for improving pichia pastoris gene editing efficiency. According to the technical scheme provided by the invention, the plasmid size can be reduced through the multi-cistronic strategy, so that the conversion efficiency is improved; by adjusting the sequence of zeocin and rad52, the expression quantity of Rad52 is increased during gene editing so as to improve the homologous recombination efficiency, and expression of the Rad52 plasmid form in pichia pastoris is realized. When gene editing is carried out, plasmids are introduced into pichia pastoris cells for expression, gene editing and homologous recombination are promoted, and the gene editing efficiency is improved; and after gene editing is completed, the plasmid is eliminated, and the Rad52 expression level is recovered to a normal level to maintain the cell stability, so that the cell performance stability is maintained.

Provided is a method for administering a single RNAi molecule or a combinatorial RNAi molecule via a polycistronic RNAi scaffold, where the polycistronic RNAi scaffold comprises one or more arms disposed on the polycistronic RNAi scaffold, wherein each of the one or more arms is an individual RNAi substrate, where at least one of the one or more arms comprise one or more of: a dicer independent loop sequence, a dicer independent loop-closing sequence, and a corresponding complement sequence. Further, at least one of the one or more arms may further comprise one or more of: an Ago2 cleavage sequence, a corresponding seed sequence, and a MID domain tight interaction.

The present disclosure provides compositions comprising IFNE and methods of using the same to treat cancer and / or enhancing responsiveness to immune checkpoint blockade therapy in a patient in need thereof. Also disclosed herein are compositions including tandem bicistronic expression cassettes, and methods of using the same to generate large genomic deletions and / or knock-in gene alterations.

Provided is a method for producing a microbial strain that activates a gene group and exhibits desired properties such as high yield and inhibition of by-products. In detail, the present application provides, in one aspect, a method for producing a strain having desired properties, the method comprising the steps of: 1) providing a plurality of constructs comprising a gene cluster containing a plurality of genes, said constructs being formed by monocistronic configuration of promoters; (2) carrying out combinatorial library on the promoter in the construct, and introducing the promoter into a target host cell; and 3) a step for screening a strain having desired properties from the host cell.

A nucleic acid molecule comprising: a first polycistronic cassette comprising: TFPI (EPI / LACI), EPCR (PROCR), and thrombomodulin (THBD); a second polycistronic cassette comprising: B2M HLA-E fusion pr

The present invention provides a polycistron self-amplifying RNA and a preparation method therefor. According to the present invention, based on the non-structural protein sequence and subgenomic promoter of the alphavirus-derived saRNA, a first target gene is connected behind a first subgenomic promoter which is partially overlapped with nsP1234, and then subgenomic promoters and other target gene frames are repeatedly connected, achieving the expression of a plurality of target genes, and achieving self-amplification of the plurality of target genes.