47 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.

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.

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.

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.

Cloning vectors and methods useful for building multigenic and / or polycistronic constructs for delivery to cells and / or organisms are disclosed. The vectors are genetically engineered to comprise unique acceptor domains that independently enable the sequential or iterative addition of compliant DNA and / or RNA inserts. Insertion of a first nucleotide insert into an acceptor r domain destroys a complementary restriction site a 3'end of the first insert segment while the 3' restriction site is regenerated at the insertion point of the cloning vector to form a circular vector with a first insert. A subsequent cleavage at a 3' end of the first insert creates an insertion point for a second insert segment, and the iterative assembly of additional insert segments proceeds with each subsequent cleavage at a 3' end of a growing linear chain of a desired number of inserts.

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.

A non-viral vector comprising, from 5' to 3', a first coding region flanked by two transposon terminal inverted repeat sequences, an inducible promoter, a specific combination of genes whose expression is localized to the tumor microenvironment and whose function is to mediate CAR persistence and anti-tumor mechanisms to stimulate or enhance the patient's anti-tumor response; a second promoter and coding region expressing one or more artificial immune surveillance chimeric antigen receptors (AI-CAR), and a truncated CD20 or truncated EGFR safety target. The polycistronic mRNA consists of a specific combination of transiently expressed anti-tumor mechanisms designed to stimulate the patient's anti-tumor response, the same as the AI-CAR vector.

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.

The invention discloses a eukaryotic polycistron expression element obtained through machine learning guidance. The expression element can perform tandem expression on a plurality of genes in eukaryotes such as fungi, plants and the like, and realizes control of cooperative expression or inhibition of the plurality of genes through one promoter and one transcription terminator, so that the length of an exogenous multi-gene target DNA fragment is shortened, and the working efficiency is improved. The method has relatively high practical application value in the aspects of natural product mining, yield improvement, creation of non-natural compounds, improvement of disease and pest resistance and the like by utilizing synthetic biology.

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.

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.

The invention relates to the technical field of genetic engineering algae, in particular to a method for increasing the content of triglyceride-type eicosapentaenoic acid in marine globoid cells. The method comprises the following steps: constructing an expression vector comprising a delta 5 desaturase gene, an omega 3 desaturase gene and a delta 6 elongase gene; the expression vector adopts a Ribi bidirectional promoter, one end of the Ribi bidirectional promoter is connected with a delta 6 elongase gene, a delta 5 desaturase gene and an omega 3 desaturase gene are connected through a 2A peptide sequence, a polycistron is formed in a single open reading frame, and the polycistron is connected to the other end of the Ribi bidirectional promoter; the base sequence of the delta 6 elongase gene is as shown in SEQ ID NO. 1; the base sequence of the delta 5 desaturase gene is as shown in SEQ ID NO. 2; and the omega3 desaturase gene is as shown in SEQ ID NO. 3. The expression vector is transferred into marine nannochloropsis oculata chassis cells, so that the content of triglyceride eicosapentaenoic acid in the marine nannochloropsis oculata cells is increased.

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.

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.

Recombinant NK cells, particularly recombinant NK-92 cells express a chimeric antigen receptor (CAR) with an intracellular domain of FcεRIγ. It is noteworthy that the CAR construct with an intracellular domain of FcεRIγ has a significantly prolonged expression duration and significantly prolonged cytotoxicity over time. CAR can be expressed from RNA and DNA, preferably as a tricistronic construct that further encodes CD16 and cytokines to confer autocrine growth support. Advantageously, such constructs can also achieve high-level transfection and expression of recombinant proteins, and provide convenient selection markers to promote the rapid production of recombinant NK / NK-92 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.