15 results about "Cistron" patented technology

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.

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.

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.

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.

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.

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.

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.

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.

The invention discloses a recombinant vector for high yield of GHK, recombinant bacteria and a fermentation method, and belongs to the technical field of biological medicines. The recombinant expression vector comprises a bicistronic expression cassette controlled by a T7 / lac hybrid promoter, the bicistronic expression cassette sequentially comprises a first cistronic for coding (GHK) n polypeptide and a second cistronic for coding trypsinogen, and n is an integer from 2 to 200. The vector is transferred into Escherichia coli BL21, and the obtained recombinant bacterium can synchronously express the GHK n polypeptide and trypsinogen after being induced by IPTG (isopropyl-beta-d-thiogalactoside). In the fermentation process, trypsinogen is activated and in-situ enzyme digestion of the GHK n polypeptide is realized, so that the GHK is efficiently released. According to the method, a traditional multi-step process is integrated into one-step fermentation, the production process is simplified, the purification difficulty is reduced, the highest GHK yield can reach 3.9 g / L according to the embodiment, and an efficient and convenient new strategy is provided for large-scale biological manufacturing of GHK.