1002 results about "Expression cassette" patented technology

To provide a medicine for treating a disease in a mammal.SOLUTION: Provided is a medicine for treating a disease in a mammal, comprising an expression cassette that is a first expression cassette comprising a chimeric gene operably linked to a first expression control element, the chimeric gene comprising a first portion comprising a minigene having an alternatively spliced exon and a second portion encoding a first RNA encoding a protein, the first portion being 5' to the second portion, the second portion lacking an initiation codon or start codon, the alternatively spliced exon comprising a translation initiation regulatory sequence, expression of the protein being controlled by inclusion of the alternatively spliced exon, and the medicine being used in combination with a small molecule splicing modifier that modulates splicing of the alternatively spliced exon.SELECTED DRAWING: Figure 1

The invention belongs to the field of gene engineering and biological treatment, and provides a hypoxia specific gene expression cassette, gram-negative bacteria containing the expression cassette and application of the gram-negative bacteria in preparation of tumor drugs. According to the invention, the forward hypoxia promoter and the reverse hyperxia promoter are reasonably designed, and appropriate survival essential genes are matched, so that the hypoxia specific gene expression cassette which is more efficient and safer is obtained. Gram-negative bacteria modified by the hypoxia specific gene expression cassette designed by the invention are low in overall mutation rate, and in a mouse in-vivo tumor model, more bacteria are distributed in tumors and the clearing speed in normal organs is higher.

The invention discloses an aspergillus niger recombinant bacterium AnCT-XynB-K-agdA and application thereof, and belongs to the technical field of microbiology and bioengineering. According to the invention, Aspergillus niger AnCat is taken as an expression host, firstly, an acid protease regulatory factor prtT is knocked out to obtain a defective strain, and then different chaperonins are subjected to fusion expression by optimizing integration sites of a laccase lcc9 expression cassette, so that the high-expression laccase chaperonin is obtained. According to the method, an expression vector pC3-5 'agdA-PcitA-XynB-L-lcc9-hph-3' agdA is constructed, a recombinant bacterium AnCT-XynB-K-agdA is further obtained, the enzyme activity of the extracellular laccase subjected to shake flask fermentation reaches 1821.2 U / L, and finally, the recombinant efficient expression of the laccase Lcc9 in aspergillus niger is realized.

The invention discloses a watermelon transgenic screening expression cassette and application thereof, and belongs to the technical field of biology. According to the invention, an expression cassette of a TagRFP gene driven by a d35s promoter is constructed on a pCambia1300 vector, and a recombinant vector is obtained. A recombinant vector is introduced into watermelon cells by utilizing an agrobacterium-mediated genetic transformation technology, and a red fluorescent protein TagRFP gene is integrated into a watermelon genome, so that transgenic watermelons can be screened and identified by taking red fluorescence as an identifier in the whole growth period of watermelon genetic transformation. Compared with a traditional screening method, the screening method has the advantages that the screening process is greatly simplified, and the transgenic watermelon and the non-transgenic watermelon are clearly distinguished, so that the genetic transformation efficiency of the watermelon is remarkably improved.

The present invention provides an ATP phosphoribosyltransferase mutant and its application in histidine production. The mutant is obtained by performing point mutations at positions 84 and / or 205 on the amino acid sequence of the wild-type ATP phosphoribosyltransferase derived from Acinetobacter baumannii. The mutant, the nucleic acid molecule encoding the mutant, the expression cassette containing the nucleic acid molecule, the recombinant vector or the recombinant microorganism are used for the production of L-histidine, and the yield of L-histidine is significantly increased, laying a good foundation for the large-scale industrial production of L-histidine.

The invention belongs to the technical field of genetic engineering, and particularly relates to an agrobacterium rhizogenes-mediated visualized genetic transformation method for cucurbitaceae plants, which comprises the step of transfecting the cucurbitaceae plants by using agrobacterium rhizogenes containing recombinant plasmids, and the recombinant plasmids are expression cassettes containing Ruby reporter genes. A visual agrobacterium rhizogenes-mediated transformation system of the cucurbitaceae plants is established by utilizing the RUBY gene, professional equipment or chemical treatment is not needed, red hairy roots can be visually screened to identify and induce the positive plants, and the method is simple, rapid and efficient and can be used for identifying and inducing the positive plants of the cucurbitaceae plants such as bottle gourds and the like. And the gene is transferred into an exogenous gene or is subjected to gene editing on the cucurbitaceae plant, so that the gene has important significance.

The invention relates to the field of plant genetic engineering, and discloses application of a ZmWOX5b gene in improvement of corn genetic transformation efficiency. By separating the ZmWOX5b gene and constructing a recombinant vector, an expression cassette or a recombinant bacterium containing the gene, genetic transformation is carried out on corn inbred lines B73, B104, Zheng 58, FSJ115 and Xiang 249 by utilizing an agrobacterium-mediated method, and a research result shows that the gene can remarkably improve the transformation efficiency. Compared with a traditional method, the method has the advantages that the efficiency of easily-transformed genotypes is effectively improved, meanwhile, the transformation effect of difficultly-transformed maize inbred lines is greatly improved, and the limitation of receptor genotypes in maize genetic transformation is effectively broken through. The achievement obviously widens the application range of corn genetic transformation, and provides a new tool for efficiently developing gene function research and application of high yield, high quality, disease and pest resistance, stress resistance and the like of corn.

Reported herein is, inter alia, a method for producing a trivalent bispecific antibody, the method comprising the steps of culturing a mammalian cell comprising a deoxyribonucleic acid encoding the trivalent bispecific antibody, and recovering the trivalent bispecific antibody from the cell or culture medium, wherein the deoxyribonucleic acid encoding the trivalent bispecific antibody is stably integrated into the genome of the mammalian cell and comprises in the 5' to 3' direction a first expression cassette encoding a first heavy chain, a second expression cassette encoding the first heavy chain, a third expression cassette encoding a first light chain, a fourth expression cassette encoding the first light chain, a fifth expression cassette encoding the second heavy chain, a sixth expression cassette encoding the first light chain or the second heavy chain or the second light chain, and a seventh expression cassette encoding the second light chain, wherein the first heavy chain The chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, a peptide linker, a second heavy chain variable domain and a CL domain, the second heavy chain comprises, from N-terminus to C-terminus, a first heavy chain variable domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain, the first light chain comprises, from N-terminus to C-terminus, a first light chain variable domain and a CH1 domain, and the second light chain comprises, from N-terminus to C-terminus, a second light chain variable domain and a CL domain, wherein the second heavy chain variable domain and the first light chain variable domain form a first binding site, and the first heavy chain variable domain and the second light chain variable domain form a second binding site.

The invention provides a structure, a sequence and a construction method of a plant virus gene editing vector and application of the plant virus gene editing vector in plant gene editing. The method is characterized in that a TRV2 genome of a tobacco embrittlement virus (TRV) is used for carrying and expressing a small Cas gene AsCas12f and gRNA of the small Cas gene AsCas12f. Preferably, the AsCas12f and the gRNA of the AsCas12f are subjected to tandem expression by utilizing the TRV2 to form an expression cassette structure of the AsCas12f-tRNA-gRNA, the expression cassette substitutes a part of 2b gene sequence of the TRV2 on a TRV2 genome to form a plant gene editing vector pTRV2-Cas12f-gRNA based on the TRV virus, and long-time stable expression and gene editing can be performed after wild type nicotiana benthamiana is inoculated. The invention also provides a method for carrying out gene editing in a wild type plant by utilizing the plant virus.

The invention provides a sphingomonas paucimobilis CRISPRi / dCas12a gene regulation system as well as a construction method and application thereof, and particularly belongs to the technical field of gene engineering. The gene regulation and control system disclosed by the invention comprises a pBBR1-dCas12a-crRNA plasmid, wherein the plasmid comprises a pBBR1 replicon, a dCas12a gene, a crRNA expression cassette and a chloramphenicol resistance gene; the crRNA expression cassette comprises a Ptac promoter, two DR fragments and an rrnB T1 terminator, and is characterized in that the crRNA expression cassette comprises a Ptac promoter, two DR fragments and an rrnB T1 terminator. The system disclosed by the invention can stably express in sphingomonas paucimobilis and inhibit the transcription of genes so as to reduce the expression of specific genes without modifying the DNA (Deoxyribose Nucleic Acid) sequence of the specific genes, has lower toxicity to S.paucimobilis, is high in host growth speed and can realize the efficient expression of dcas12a.

The invention belongs to the technical field of gene engineering, and particularly relates to a strain for efficiently expressing beta-lactoglobulin as well as a construction method and application of the strain. By utilizing the beta-lactoglobulin expression vector disclosed by the invention, beta-lactoglobulin is guided to be secreted out of a host cell by an exogenous signal peptide, and efficient secretory expression of the beta-lactoglobulin in the host cell is realized by integrating a multi-copy expression cassette through a genome and co-expressing ATP enzyme Ssa4 participating in protein folding and coping with pressure; the amino terminal and the carboxyl terminal of the secreted and expressed beta-lactoglobulin do not contain redundant amino acids, and do not contain animal-derived components. Meanwhile, the invention provides a construction method and a fermentation culture method of the engineering strain, compared with a single-copy genetically engineered strain, the expression quantity of the genetically engineered strain in shake-flask fermentation is increased by 2.47 times, and the genetically engineered strain has a good industrial application prospect.

The invention provides a system and a method for screening Cas protein mutants capable of identifying different PAM sequences, and belongs to the technical field of bioengineering. The system comprises a Cas protein mutant screening vector and an sgRNA expression vector, the Cas protein mutant screening vector comprises the following expression elements: a Cas protein coding gene, a plasmid replicon and a first resistance screening tag expression cassette; the sgRNA expression vector comprises the following expression elements: a plasmid replicon, a second resistance screening tag expression cassette, a third resistance screening tag expression cassette and an sgRNA expression cassette. According to the system and the method, aiming at the problem that the efficiency of identifying PAM sequences except NGG by wild type spCas9 protein is low, an active Cas9 mutant capable of identifying PAM sites except NGG is screened out, and the application field and the editing efficiency of a CRISPR gene editing system can be greatly expanded.

The present invention provides a novel therapeutic drug for treating a Prom1-associated retinal disease. Specifically, the present invention provides an optimized Prom1 gene expression cassette, an rAAV viral vector, and a gene therapy drug. The drug of the present invention can specifically express the PROM1 protein in the retinal photoreceptor layer, and is suitable for the clinical treatment of a retinal disease associated with Prom1 gene mutation.

The present invention relates to a nucleic acid sequence derived from a regulatory region of a human Lgr5 gene and having promoter activity in retina non-long-process cells. The invention also relates to an expression cassette or vector comprising the promoter, a viral particle or cell comprising the expression cassette or vector, and a pharmaceutical composition comprising the expression cassette or vector and the viral particle or cell. The invention also relates to the use of the expression cassette, vector, viral particle, cell or pharmaceutical composition in the treatment of eye diseases, especially diseases associated with retinal ganglion cell degeneration.

The present invention relates to an exogenous target gene expression cassette for delivering an exogenous target gene to the retina, in particular AIPL1 to retinal pigment epithelial cells and photoreceptor cells, comprising an IRBP enhancer sequence, a rhodopsin kinase (RK) promoter sequence and a CAG intron sequence, which are operatively linked, and an exogenous target gene. The present invention also relates to a recombinant adeno-associated viral vector comprising a viral capsid comprising a capsid protein or a capsid protein variant and a viral vector genome comprising an expression cassette encoding for specifically expressing an exogenous target gene in retinal pigment epithelial cells and photoreceptor cells. The recombinant adeno-associated virus vector can be used for relieving or treating retinal degenerative eye diseases by intravitreal administration or subretinal administration.

The invention relates to the field of plant breeding, and particularly provides a method for fixing rice heterosis by using an OsZFPP gene. The method comprises the following steps: firstly, constructing an OsZFPP1 gene or OsZFPP2 gene expression cassette driven by an OsECA1 promoter; then, an expression box of rice with three target points of OsPAIR1, OsREC8 and OsOSD1 being knocked out of CRISPR / Cas9 is constructed; integrating the expression cassette into the same vector to transform hybrid rice; screening three-gene homozygous mutation and OsZFPP positive plants, and identifying diploid cloned offspring through flow cytometry and genome sequencing. According to the method, the rice OsZFPP gene is combined with MiMe to provide a novel method for fixing the heterosis of the rice through apomixis, the heterosis of the rice can be successfully fixed through the method, an apomixis system with the high maturing rate can be obtained, and a novel solution is provided for fixing the heterosis of the rice through the apomixis.

The invention provides a salt-tolerant gene, namely a PrABC1 gene and application thereof, and also provides a vector containing the gene, an expression cassette, a recombinant cell, a transgenic plant and a method for producing the transgenic plant. Specifically, the gene has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The plant growth regulator can be used for improving the tolerance of plants to a salt stress environment, relieving the adverse effects of a high-salt environment on the germination rate and root length of the plants, reducing the water loss rate of the plants under the high-salt stress condition and reducing the reduction of the chlorophyll content.

The invention relates to a high-yield trans-aconitic acid engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of gene engineering. In the scheme of the invention, the inventor introduces mitochondrial localization signal peptide to be combined with the aconitic acid isomerase to form a gene element, and an expression cassette containing the gene element is introduced into the strain by adopting a genetic engineering technical means, so that the aconitic acid isomerase expressed in the constructed recombinant strain can be localized into mitochondria, and the expression efficiency of the recombinant strain is improved. Metabolic pathways related to trans-aconitic acid synthesis are located in the same zone, so that the capacity of fermenting and producing trans-aconitic acid by the recombinant strain is improved. The technical scheme disclosed by the invention not only has a good application prospect and production economic value, but also provides a referential new thought for transformation of strains related to other fermentation processes.

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises an AAV 5' inverted terminal repeat (ITR), an expression cassette comprising engineered nucleic acid sequence encoding human Tafazzin and operably linked thereto regulatory sequences, and an AAV 3' ITR. Also provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer, and a method of treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene.

The invention provides a preparation method, application and the like of large intestine expression recombinant A-type botulinum toxin (BONT / A). The recombinant BONT / A nucleic acid expression cassette comprises a tag, a restriction enzyme cutting site and a BONT / A nucleic acid sequence, and an exogenous protease recognition site is not introduced between a BONT / A light chain and a BONT / A heavy chain. The invention also provides a recombinant vector containing the nucleic acid expression cassette, a recombinant bacterium, a coded and expressed protein and the like, the prepared recombinant BONT / A does not introduce exogenous amino acid or only introduces two amino acids at one position, and the consistency of the recombinant BONT / A with the natural A-type botulinum toxin is ensured to the greatest extent. Through novel molecular design and preparation process, the recombinant reBONT / A with higher toxicity and low immunogenicity is efficiently obtained, and wide industrial application is facilitated.

The present subject matter relates to recombinant lentiviral transfer plasmids for producing lentiviral vectors with increased vector titer and performance. These transfer plasmids are constructed so that a central polypurine tract (cPPT) is centered between the lentiviral LTRs and include a 6x stop codon sequence block downstream of the 3' LTR. When the transfer plasmids comprise a globin expression cassette, the lentiviral vectors produced therefrom are useful for treating hemoglobinopathies by gene therapy. Transduced cells, pharmaceutical compositions and methods of treating or ameliorating hemoglobinopathies with these lentiviral vectors are also provided. Additional transfer plasmids, cells, and compositions are provided that are useful for delivering any gene of interest to a cell, as well as plasmids for production of lentiviral vectors for treating other erythroid-specific diseases or disorders such as anemia and cancer.

The invention relates to a photoreceptor cell specific bidirectional promoter and application thereof. Specifically, the invention relates to a novel photoreceptor cell specific bidirectional promoter, an expression cassette containing the bidirectional promoter, a vector, a viral particle, a cell and a pharmaceutical composition, and application of the bidirectional promoter to treatment of eye diseases, such as retinal diseases.

This invention provides vectors (e.g., lentiviral vectors) and expression cassettes for incorporation into vectors for expressing an alpha-globin (α-globin) gene. In certain embodiments method of treatment using such vectors are provided.

The invention provides a virus RNAi inhibitor screening vector as well as a construction method and application thereof, and belongs to the technical field of viruses. The virus RNAi suppressor screening vector comprises a green fluorescent protein coding gene expression cassette and a shRNA expression cassette targeting a green fluorescent protein coding gene, the nucleotide sequence of the shRNA is as shown in SEQ ID NO: 2 or SEQ ID NO: 3. According to the invention, the green fluorescent protein coding gene and the shRNA are cloned into the same expression vector, so that the consistency of transfection efficiency is ensured. The invention further provides a screening method of the virus RNAi suppressor, the virus RNAi suppressor screening vector and the recombinant vector for expressing the virus RNAi suppressor to be screened are co-transfected into cells, and the screening result is judged according to the green fluorescence intensity. According to the technical scheme, operation is easy and convenient, the screening result is accurate and reliable, and wide application and popularization value is achieved.

The invention discloses an application of an ALKBH9B gene in promotion of plant callus formation, belongs to the technical field of gene engineering, and provides an application of the ALKBH9B gene in promotion of plant callus formation. The invention also provides an application of an expression cassette, a vector or a recombinant bacterium containing the ALKBH9B gene in promoting formation of poplar leaf calluses. The ALKBH9B gene is knocked out, the expression quantity of the ALKBH9B gene is reduced, it is found that when the gene editing strain with the knocked-out gene is cultured on a callus induction culture medium for 5 days, callus tissue starts to be formed, the callus forming speed is high, and the net weight of the callus and the callus generation rate are remarkably higher than those of a wild strain. The result shows that ALKBH9B has an inhibition effect in the process of inducing the callus by the leaf, and the deletion of ALKBH9B can promote the formation of the callus.

96 New PCT-Patent Application based on EP 24 186 635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 Abstract The present invention relates to a linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising one or more Tus protein (Tus) binding site(s) at the 5'-terminus, one or more Lac repressor protein (LacI) binding site(s) at the 3'-terminus, and a segment comprising a DNA sequence of interest (DOI) between said Tus binding site(s) and said LacI binding site(s). The present invention further relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from the linear dsDNA molecule of the invention, and to an expression DNA cassette comprising a promoter, an RBS, a GOI encoding a POI, and one or more LacI binding site(s), wherein said expression cassette does not comprise a terminator sequence between the 3´ end of said GOI and said one or more LacI binding site(s). The present invention also relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from said expression cassette. The present invention further relates to method of protecting a linear deoxyribonucleic acid (DNA) molecule from exonuclease degradation by adding one or more Tus binding site(s) at the first terminus of the DNA molecule and adding one or more LacI binding site(s) at the other terminus of the DNA molecule. The present invention further relates to a method of synthesizing a protein of interest (POI) in a cell- free protein synthesis (CFPS) reaction mixture by using the (ds)DNA and / or RNA molecules of the invention, and also to a cell-free biological system or CFPS reaction mixture comprising the (ds)DNA, expression cassette and / or RNA molecules of the invention.

The invention discloses a method for improving the solubility and thermal stability of sweet protein, and belongs to the technical field of biosynthesis, the method comprises the following steps: constructing a gene tandem recombinant plasmid containing a monellin x-3C-sfGFP-3C-monellin y expression cassette, x is greater than or equal to 1, y is greater than or equal to 1, 5 is greater than or equal to x + y is greater than or equal to 3, a 3C protease recognition sequence is also inserted between adjacent copies of monellin, and each monellin is connected with a purification tag; the gene tandem recombinant plasmid is transformed into escherichia coli and inducible expression is carried out, thalli are split and purified to obtain fusion protein, 3C protease is used for enzyme digestion, and the target protein is obtained after re-purification. The total protein yield and the solubility proportion are remarkably improved by adopting a series construction mode of plasmids, meanwhile, the stability of monellin is effectively improved, the purification step is simpler and more convenient, and the method is suitable for industrial production.

The invention discloses a novel single-cell CRISPR screening library. The invention provides a double-gRNA expression cassette, a double-gRNA expression cassette vector for single-cell CRISPR (clustered regularly interspaced short palindromic repeats) screening and a construction method of a single-cell CRISPR screening library. According to the CRISPR screening library constructed by the method, the single-cell CRISPR screening range can be expanded from a protein coding gene to ncRNA, and the ncRNA can be knocked out.

The invention belongs to the technical field of synthetic biology genome editing, and relates to a chassis strain for gene cluster in-vivo traceless editing, a construction method and a gene cluster traceless editing method.The chassis strain is based on escherichia coli BL21 (DE3) and integrates a CRISPR-Cas9 system and a homologous recombination system (lambda Red) of a lambda bacteriophage source; and then knocking in an inducible sgRNA expression cassette (sgT) of a targeted editing vector to construct the vector. The strain has an efficient editing capability aiming at exogenous DNA, traceless editing of a target biosynthetic gene cluster is realized, and editing plasmids can be quickly eliminated, so that iterative editing is facilitated. The method is simple, convenient and rapid to operate and high in practicability, and provides powerful technical support for metabolic engineering and synthetic biology breeding transformation of streptomyces industrial strains, activation of synthetic gene clusters of recessive natural products and excavation of novel natural products.

The present invention relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. The present invention provides an expression cassette, which sequentially includes: the rop gene, the replicon ori, and the resistance gene; the rop gene, the replicon ori, and the resistance gene are connected by a linking fragment. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of relatively low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences of more than 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently and rapidly obtain the DNA template for in vitro transcription of the target mRNA.