29 results about "Gene replacement" patented technology

The invention provides a recombinant herpes simplex virus and a herpes simplex virus modification method. The herpes simplex virus modification method comprises the step of replacing an ICP4 protein coding gene in a herpes simplex virus genome containing an infected cell protein 4 (ICP4) gene with an hTERTp-fluorescent protein expression cassette. The expression cassette comprises an hTERTp promoter and a fluorescent protein coding sequence controlled by the hTERTp promoter, and the transcription direction is opposite to that of an ICP4 promoter in a genome. The hTERTp-fluorescent protein expression cassette in the recombinant virus obtained by the method disclosed by the invention can be normally expressed in response to telomerase activity. Therefore, the recombinant virus is capable of expressing a fluorescent protein, such as mBaoJin, in a cell having human telomerase activity. Cells infected by the virus can be identified through fluorescence signals, and the higher the telomerase activity is, the stronger the fluorescence intensity is. The virus has wide application value in research of tumor action mechanisms and stem cell action mechanisms, health assessment, screening of tumor drugs, research and development of diagnostic reagents and establishment of animal models.

The invention relates to the field of biology, and particularly provides a replication-deficient influenza virus vaccine skeleton which comprises a PA gene, an HA gene, an NP gene, an NA gene, an M gene, an NS gene and a PB2 gene of an influenza virus. The PB2 gene is replaced into a PB1 gene coding region of a vaccine skeleton; the open reading frame of the original PB2 gene can be replaced by an exogenous gene, so that the exogenous gene is inserted into the vaccine skeleton and expressed. The skeleton has the advantages that the capacity is large, the antigen can be efficiently and stably expressed, the passage is stable, and the like. Meanwhile, the invention also discloses a replication-defective influenza virus as well as a preparation method and application thereof.

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.

This invention discloses the application of the NCgl0214 protein and its biomaterials in the production of L-isoleucine, belonging to the field of biotechnology. The technical problem solved by this invention is how to increase the yield of L-isoleucine. The sequence of the protein disclosed in this invention is SEQ ID No. 4 or SEQ ID No. 2. Replacing the coding gene of the protein shown in SEQ ID No. 2 in the starting organism cell with the coding gene of the protein shown in SEQ ID No. 4, or increasing the content or activity of the protein shown in SEQ ID No. 2 or SEQ ID No. 4 in the starting organism cell, can both increase the yield of L-isoleucine.

The invention discloses expansin recombinant aspergillus niger suitable for liquid fermentation as well as a construction method and application thereof, and belongs to the technical field of recombinant microorganisms. The technical problem of efficiently degrading cellulose is solved. The invention provides a recombinant aspergillus niger, and the recombinant aspergillus niger is obtained by taking aspergillus niger TH-2-D as a starting strain and replacing a saccharifying enzyme gene glaA as shown in SEQ ID NO.31 with a penicillium oxalicum expansin Swo gene as shown in SEQ ID NO.28. The invention also provides a preparation method of the recombinant aspergillus niger. The strain is subjected to liquid state fermentation, fermentation liquor is suitable for fiber puffing application, the cellulose degradation efficiency can be improved when the strain is used in cooperation with cellulase, linked starch can be effectively removed when the strain is used in cooperation with corn wet grinding enzyme, and the starch yield is increased.

The present invention features a dual vector system for disrupting and replacing a target gene comprising a mutation (e.g., dominant, recessive mutation). Embodiments of the invention may also provide compositions comprising the dual vector system, and methods of using the dual vector system, including but not limited to methods of modifying the genome of a cell, methods of genomic editing, and methods of treating cells or a subject suffering from a genetic disease comprising a mutation.

The present application discloses a kind of Magnaporthe grisea and Xanthomonas oryzae resistance related protein derived from rice and its coding gene and application.The present application provides a kind of method for improving the disease resistance of plant: the gene of plant coding OsAspRS protein is replaced by the gene of mutant protein.The present application also protects the application of osasprs mutant in the preparation of plant with improved disease resistance.osasprs mutant is a kind of spot-like mutant plant controlled by single recessive nuclear gene, which is screened by EMS on Huanghuazhan plant.Furthermore, the present application successfully locates and clones AspRS gene, and the mutation of the gene site can cause spot-like phenotype on rice leaf, and further improve the broad-spectrum disease resistance of rice.The present application is helpful to improve the disease resistance of crop, provides gene resources and technical support for cultivating new rice varieties with broad-spectrum resistance, and has important significance and application value for crop disease resistance breeding work.

This invention discloses a genetically engineered bacterium producing high levels of L-homoserine, its construction method, and its applications, belonging to the field of biotechnology. The genetically engineered bacterium uses *Escherichia coli* W3110 as the substrate cell. By knocking out the metA, lysA, thrBC, pflB, ldhA, poxB, adhE, iclR, sthA, tdcC, lacI, and ptsG genes, and replacing the promoters of ppc, aspC, and gdhA genes with the inducible strong promoter Ptrc, and by performing site-directed mutagenesis on the thrA and asd genes, and simultaneously overexpressing the thrA*, pnhsd, asd, asd*, and rhtA genes using free plasmids, a recombinant strain HOM20 was constructed. This strain, fermented in a 5 L fermenter for 48 h, achieved an L-homoserine yield of 135.4 g / L and a sugar-acid conversion rate of 44%, significantly superior to existing technologies. This invention achieves directional carbon flow allocation, cofactor balance, feedback inhibition removal, and enhanced product efflux through multi-gene synergistic modification, and has broad prospects for industrial application.

The present disclosure relates in part to gene replacement for the treatment of cardiomyopathy. In embodiments, the present invention provides compositions and methods of use for the treatment of ALPK3 cardiomyopathy. In embodiments, the present invention provides a composition comprising a nucleic acid encoding all or a portion of an ALPK3 protein.

The invention provides modified brucella as well as a preparation method and application thereof and a vaccine, and relates to the technical field of biology. The Omp25 gene of the modified brucella is replaced by an IFN (interferon)-gamma gene. The vaccine containing the modified Brucella is short in bacterium discharging time when being used for immunizing animals, the cellular immune effect can be improved, and prevention and control of Brucella are facilitated.

The application discloses a protein mutant coded by NCgl0216 and application of biological material of the protein mutant in production of L-isoleucine, and belongs to the technical field of biotechnology.The application solves the technical problem of how to improve the yield of L-isoleucine.The sequence of the disclosed protein is SEQ ID No.4 or SEQ ID No.2.The coding gene of the protein shown in SEQ ID No.2 in the original biological cell is replaced by the coding gene of the protein shown in SEQ ID No.4, or the content or activity of the protein shown in SEQ ID No.2 or SEQ ID No.4 in the original biological cell is improved, so that the yield of L-isoleucine can be improved.

The present invention features a dual vector system for disrupting and replacing a target gene comprising a mutation (e.g., dominant, recessive mutation). Embodiments of the invention may also provide compositions comprising the dual vector system, and methods of using the dual vector system, including but not limited to methods of modifying the genome of a cell, methods of genomic editing, and methods of treating cells or a subject suffering from a genetic disease comprising a mutation.

The invention relates to the technical field of biology, in particular to an engineering strain of zymomonas mobilis for high-yield acetoin and application of the engineering strain. The engineering strain is characterized in that Zymomonas mobilis ZM4 delta pdc-BDO is taken as an original strain, a ZMO0038 gene is replaced by an NADH oxidase gene noxE, a 2, 3-butanediol dehydrogenase gene bdh is knocked out, and potential reductase genes outside two conventional knowledge systems, namely ZMO0318 and ZMO1576, are further knocked out, so that the engineering strain capable of producing acetoin at high yield is obtained. When the strain is fermented in typical non-grain biomass raw materials with complex components, about 80% of production capacity can still be maintained, and the strain shows good potential for industrial production by utilizing renewable lignocellulose biomass.

The application discloses a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Sindbis virus G protein, and is a self-replicating recombinant oncolytic virus VSV-SING which is obtained by replacing the G protein coding gene of a wild type VSV with the G protein coding gene of a Sindbis virus through reverse genetic manipulation technology, and has non-neurotoxicity and high oncolytic activity.

The application provides a preparation method of rice with resistance to rice blast. By inhibiting the expression of a DHD6 gene in rice or constructing a DHD6 gene-deficient plant, a rice plant with the property of resistance to rice blast can be obtained. The CDS sequence of the DHD6 gene is shown as SEQ ID NO:1, or the sequence of the protein encoded by the DHD6 gene is shown as SEQ ID NO:2. The rice plant with improved resistance to rice blast can be identified by detecting the loss of function of the DHD6 gene. The application also provides a mutated DHD6 gene, the CDS sequence of the mutated DHD6 gene is shown as SEQ ID NO:3, or the sequence of the protein encoded by the mutated DHD6 gene is shown as SEQ ID NO:4. By replacing the wild-type DHD6 gene with the mutated gene through genetic engineering, a rice plant with resistance to rice blast can be constructed.

Compositions and methods discussed herein provide for treatment or prevention of a disease or disorder, or its symptoms, using a modified herpes simplex virus (mHSV) vector comprising at least two transgenes, wherein at least a first transgene encoding a gene-editing system, gene-deletion system, or bridge-editing system is expressed to knock out or delete an endogenous target gene and at least a second transgene encoding a corrected copy of the endogenous targeted gene, wherein the gene product of the second transgene replaces the gene product of the endogenous target gene.

The invention discloses an anti-tumor enhanced neurotoxicity-free VSV vector recombinant oncolytic virus, which is characterized in that a G protein coding gene of a wild type VSV is replaced by a G protein coding gene of a Luabout virus through a reverse genetic manipulation technology, and a gene for coding an anti-tumor enhancing factor IFN-beta is inserted into a VSV vector genome, so that the anti-tumor enhanced neurotoxicity-free VSV vector recombinant oncolytic virus is obtained, and the anti-tumor enhanced neurotoxicity-free VSV vector recombinant oncolytic virus is obtained. And saving to obtain the anti-tumor enhanced neurotoxicity-free VSV vector recombinant oncolytic virus VSV-LUJG-IFN-beta which can be autonomously replicated. The compound provided by the invention has the advantages of no neurotoxicity, good safety, and strong tumor inhibition effect.

The application discloses application of NCgl0762 protein and mutants thereof in production of L-isoleucine and belongs to the technical field of biotechnology.The application solves the technical problem of how to improve the yield of L-isoleucine.The sequence of the disclosed protein is SEQ ID No.4 or SEQ ID No.2.The yield of L-isoleucine can be improved by replacing the coding gene of the protein shown in SEQ ID No.2 with the coding gene of the protein shown in SEQ ID No.4 or improving the content or activity of the protein shown in SEQ ID No.2 or SEQ ID No.4 in the original biological cell.

The application provides a MET2 gene knockout Saccharomyces cerevisiae strain, a construction method and application, and belongs to the technical field of genetic engineering. The MET2 gene knockout Saccharomyces cerevisiae strain is obtained by replacing the MET2 gene in the Saccharomyces cerevisiae strain BSPC040 with a hygromycin B resistance gene and then introducing the pJFE3-XI plasmid. The MET2 gene knockout Saccharomyces cerevisiae strain can significantly improve the glycol alcohol conversion rate of the Saccharomyces cerevisiae BSPC040 strain carrying a xylose metabolic pathway. In the case of consuming the same amount of xylose, compared with the Saccharomyces cerevisiae strain without the MET2 gene knockout, the ethanol yield of the MET2 gene knockout Saccharomyces cerevisiae strain is increased by 23.1%, which provides a new theoretical basis and gene editing strategy for strain optimization in the industrial production of lignocellulosic ethanol.

In one embodiment, the invention provides a dual-cassette gene vehicle comprising cassettes for expression of both a mini-dystrophin gene and NF-κB / p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue, which is an adeno-associated viral (AAV) vector, wherein the mini-dystrophin gene is operably linked to a construct comprising a muscle-specific first promoter and a modified Mcken (MCK) enhancer and wherein the NF-κB / p65-shRNA gene is under the control of a second promoter. Also are provided pharmaceutical compositions comprising such gene vehicles and a method for ameliorating Duchenne muscular dystrophy (DMD) employing such gene delivery vehicles and pharmaceutical compositions.

The invention discloses a glucose metabolism reconstruction-based engineering strain for efficiently synthesizing rhamnolipid and a construction method thereof, the construction method comprises the following steps: taking pseudomonas putida KT2440 or a flag gene cluster knockout strain thereof as an original strain, optimizing the genome of the original strain through gene modification, and introducing recombinant plasmids to obtain the engineering strain for efficiently synthesizing rhamnolipid; the modification comprises at least one of (1) to (6): (1) heterologous expression of rhamnolipid synthesis related genes; (2) replacing the rmlA gene with the gene rmlA *; (3) knocking out related genes for coding Gad; (4) heterologous expression of a galP gene encoding glucose transporter protein; (5) heterologous expression of the glf gene encoding the glucose transporter; and (6) overexpressing and coding the glk gene of glucokinase. The method disclosed by the invention has the beneficial effects that efficient directional distribution of glucose to a rhamnolipid synthesis route is realized, and the synthesis efficiency of the rhamnolipid is remarkably improved.

This invention relates to the field of plant genetic engineering, specifically to the MMR1 protein, its encoding gene, and its applications in regulating ROS homeostasis in the root tip meristem of plants. This invention involves knocking out the MMR1 gene to obtain stable transgenic lines of Arabidopsis thaliana. mmr1-1, mmr1-2 and build MMR1 Gene replacement lines and MMRI Gene overexpression lines; mutants compared to wild type mmr1-1, mmr1-2 The results showed shortened root length, shortened root tip meristem, and reduced root tip division ability; reintroduced lines indicated that the MMR1 protein was located in the mitochondria; the roots of the overexpressing lines were longer than those of the wild type, indicating... MMR1 The gene plays an important role in root elongation, root apical meristem development, and root apical meristem cell activity in Arabidopsis thaliana. This invention fills a gap in the field of mitochondrial-localized protein regulation of root development and ROS homeostasis, providing new genetic resources for root improvement and transgenic breeding.

The application discloses a construction method of saccharomyces cerevisiae for producing squalene. The recombinant saccharomyces cerevisiae disclosed by the application co-locates farnesyl pyrophosphate synthetase (FPPS) and squalene synthetase (SQS) on the surface of a lipid droplet, replaces an HMG1 gene with a truncated tHMG1 gene, knocks out OPI3 and CHO2 genes, and expresses an exogenous PLIN3 gene. The application co-locates the key enzymes FPPS and SQS for synthesizing squalene on the surface of a lipid droplet of saccharomyces cerevisiae by using a molecular localization technology, shortens the spatial distance of the enzymes involved in continuous catalysis, stores squalene by using a lipid droplet, reduces the toxicity of squalene to a host cell, and further adjusts the size and quantity of the lipid droplet, so that the yield of squalene is increased by 6 times.

The application discloses a method for improving the yield of gamma-polyglutamic acid. The method is to replace the rapA gene in a gamma-polyglutamic acid producing strain by a mutant gene rapA, so that the yield of gamma-polyglutamic acid of the producing strain is significantly improved. The nucleotide sequence of the mutant gene rapA is shown in SEQ ID NO. 1, and the base at the 575th position is mutated from A to G. The bacillus velezensis PGA-224 contains the mutant gene rapA, and the preservation number is GDMCC No. 62295. The mutant rapA gene is used to replace the rapA gene in the gamma-polyglutamic acid producing strain, so that the yield of gamma-polyglutamic acid is significantly improved.

Embodiments of the present application provide a kind of based on the digital asset synthesis method and device of blockchain, the method includes any blockchain node in detecting digital asset synthesis transaction, the gene sequence that k digital assets to be synthesized has is XOR operation, generates initial offspring gene sequence, when determining that initial offspring gene sequence exists on blockchain, based on the random gene sequence that is not used, the i th gene replacement is carried out to initial offspring gene sequence, generates the ordered offspring candidate set belonging to the i th gene replacement, if it is determined that j candidate gene sequences all exist in blockchain, the i+1 th gene replacement is carried out to initial offspring gene sequence, until after the n-1 th gene replacement is carried out to initial offspring gene sequence, so as to determine the gene sequence that new digital asset has.It is thus, the execution result that this scheme can effectively ensure that on different blockchain nodes for the gene sequence that new digital asset has is consistent.

The application discloses a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Marburg virus defective G protein, and is characterized in that: a coding gene of a wild-type VSV G protein is replaced by a coding gene of a Marburg virus defective G protein through reverse genetic manipulation technology, so that a recombinant oncolytic virus VSV-MARG-ΔMLD capable of autonomous replication is obtained. The application has both non-neurotoxicity and high oncolytic activity.

The application discloses a Cryptosporidium parvum CpDHR1 gene replacement strain and a construction method and application thereof. The application researches and finds that the CpDHR1 gene of Cryptosporidium parvum is related to virulence, the CpDHR1 gene of a high-virulence strain is replaced by the CpDHR1 gene of a low-virulence strain through a gene editing technology, and a Cryptosporidium parvum CpDHR1 gene replacement strain is successfully constructed. Research shows that after the CpDHR1 gene of a high-virulence wild-type Cryptosporidium parvum IIdA20G1 subtype strain is replaced, the strain can still be cultured in vitro, but the in-vitro growth of the strain is significantly reduced, the virulence of the strain is weakened, the oocyst excretion intensity of the strain is weakened, the oocyst load in mice is reduced, the clinical symptoms of the mice are alleviated, the survival time of the mice is prolonged, the virulence of the strain is significantly weakened, the strain can be used for preparing a Cryptosporidium parvum low-virulence vaccine product, and Cryptosporidium parvum infection can be better prevented.

The invention discloses a recombinant serum type 3 duck adenovirus and a construction method and application thereof, the recombinant serum type 3 duck adenovirus replaces a non-structural protein gene ORF52 in the serum type 3 duck adenovirus with an exogenous target gene or inserts the exogenous target gene into the non-structural protein gene ORF52, and the nucleotide sequence of the non-structural protein gene ORF52 is shown as SEQ ID NO. 1. The recombinant serum type 3 duck adenovirus for expressing green fluorescent protein is constructed by targeting an exogenous gene EGFP to a non-structural protein gene ORF52 of the duck adenovirus type 3 by utilizing a gene editing technology, and the non-structural protein gene ORF52 is proved to be capable of serving as an insertion site of the exogenous gene, so that the recombinant serum type 3 duck adenovirus can be used as an insertion site of the exogenous gene. And a foundation is laid for developing a genetic engineering recombinant DAdV-3 multi-combined multivalent vaccine based on taking DAdV-3 as a virus vector.

This invention discloses an engineered Trichoderma reesei strain that secretes and expresses lysostaphin, its construction method, and its applications, belonging to the field of genetic engineering technology. This invention obtains the lysostaphin proenzyme and its encoding gene by modifying lysostaphin with a secretory peptide. The encoding gene, promoter, and terminator are then ligated into a shuttle plasmid to obtain a lysostaphin expression plasmid. This plasmid is transformed into Trichoderma reesei, and the endogenous protein encoding gene cbh1, which has the highest secretion expression level in Trichoderma reesei, is replaced with the aforementioned encoding gene, thus constructing an engineered Trichoderma reesei strain that secretes and expresses lysostaphin. This invention is the first successful construction of an engineered Trichoderma reesei strain capable of secreting and expressing lysostaphin, which simplifies the production process of lysostaphin, significantly reduces its production cost, and lays the foundation for large-scale industrial production of lysostaphin.