44 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.

Mice that comprise a replacement of endogenous mouse IL-6 and / or IL-6 receptor genes are described, and methods for making and using the mice. Mice comprising a replacement at an endogenous IL-6Rα locus of mouse ectodomain-encoding sequence with human ectodomain-encoding sequence is provided. Mice comprising a human IL-6 gene under control of mouse IL-6 regulatory elements is also provided, including mice that have a replacement of mouse IL-6-encoding sequence with human IL-6-encoding sequence at an endogenous mouse IL-6 locus.

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 discloses a neurotoxicity-free VSV vector recombinant oncolytic virus carrying Marburg virus deficient G protein, which is characterized in that a G protein coding gene of wild VSV is replaced by a coding gene of Marburg virus deficient G protein through a reverse genetic manipulation technology, so as to save and obtain a recombinant oncolytic virus VSV-MARG-delta MLD capable of being autonomously replicated. The invention has the advantages of no neurotoxicity and efficient oncolytic activity.

This invention discloses a *Lactobacillus paracasei* S-NB gene deletion mutant strain, its construction method, and its application. The S-NB gene deletion mutant strain uses *Lactobacillus paracasei* S-NB as the starting strain, and the mutant strain contains *Lactobacillus paracasei* S-NB gene deletion mutants. cps The upstream segment of the gene was knocked out by the plasmid. cps Upstream homologous arm gene substitution in *Lactobacillus paracasei* S-NB cps The downstream gene fragment carried by the knockout plasmid cps Gene substitution in the downstream homologous arm of the gene; among which, cps The gene base sequence of the upstream homologous arm is shown in SEQ ID NO:1. cps The downstream homologous arm gene base sequence is shown in SEQ ID NO:2. Compared with traditional methods, the success rate of knockout vector construction in this invention reaches over 95%, and the operation is simple, time-saving, and the operation cycle is only 6-7 days; the knockout vector constructed using this invention can knock out Lactobacillus paracasei S-NB. cps Genes, the result cps Gene deletion mutants are unaffected in growth, but their capsule layer becomes thinner and their ability to produce biofilm decreases.

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 chimeric Kunjin virus of tamabi virus prM-E and a preparation method and application thereof. On the basis of a reverse genetic manipulation system of the Kunjin virus KUNV, the KUNV prM / E gene is replaced by the DTMUV prM / E gene to construct a chimeric DTMUV virus infectious clone plasmid with the KUNV as a skeleton, which is named as pUC19-KUNV-DTMUV prM / E. Then, the chimeric tamabi virus prM-E Kunjin virus is obtained through virus rescue, and is named as rKUNV-CHv-prM / E (CHv). Analysis on the virus characteristics shows that the CHv can effectively proliferate in BHK-21 and DEF cells, and the duck embryo virulence is weaker than that of the KUNV and the DTMUV. The animal experiment results show that the CHv only detects obvious virus levels in the brain, and does not produce viremia; the CHv after immunization can produce similar IgG antibody levels to those after immunization of the KUNV and the DTMUV. Therefore, the recombinant virus of the chimeric DTMUV prM / E with the KUNV as a skeleton prepared by the application can induce the duckling to produce sufficient immune protective neutralizing antibody levels, and can be applied to preparation of a duck tamabi virus vaccine.

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 application discloses a protein mutant and application of a biomaterial thereof in production of L-isoleucine, and belongs to the technical field of biotechnology.The technical problem solved by the application is how to improve the yield of L-isoleucine.The sequence of the disclosed protein mutant is SEQ ID No.4.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 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 multivalent expression listeria monocytogenes cervical cancer vaccine and a preparation method thereof. The method comprises the following steps: constructing attenuated listeria monocytogenes of which actA and plcB virulence genes are knocked out; on the basis of the attenuated listeria monocytogenes, constructing to obtain auxotroph listeria monocytogenes LM [delta] dal [delta] dat; the fusion antigen is constructed by alternately arranging amino terminal and carboxyl terminal structural domains of multiple types of E6 and E7 proteins of HPV (human papillomavirus); inserting the fusion antigen into a non-resistance plasmid pCW631 which carries a dal gene and replaces an Amp gene with an asd gene through PCR (Polymerase Chain Reaction) amplification, gel recovery and seamless cloning technologies to obtain a non-resistance plasmid pCW636; the non-resistance plasmid pCW636 is electrically transferred into the auxotroph listeria monocytogenes LM [delta] dal [delta] dat, and the listeria monocytogenes cervical cancer vaccine is obtained after PCR (polymerase chain reaction) screening, product verification and sequencing. The cervical cancer vaccine for multivalent expression of the E6E7 protein is constructed on the basis of the attenuated listeria monocytogenes, and multiple protection on cervical cancer can be achieved.

The application discloses a recombinant orf virus for expressing PCV3 Cap protein, and the recombinant orf virus is obtained by the following steps: firstly, replacing the 132 gene of the orf virus with a nucleotide sequence, such as the gene sequence 1 of SEQ ID NO: 5, to obtain a recombinant virus 1; and secondly, replacing the nucleotide sequence, such as the gene sequence 1 of SEQ ID NO: 5, in the recombinant virus 1 with a nucleotide sequence, such as the gene sequence 2 of SEQ ID NO: 6, to obtain the recombinant orf virus. The application replaces the 132 gene of the ORFV virus with the optimized Cap gene of the PCV3 to prepare the recombinant orf virus. The recombinant orf virus can stably express the PCV3 Cap protein, and provides a vaccine candidate strain for preventing the PCV3 related diseases and a feasible technical platform for developing the recombinant orf virus of other viruses.

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 provides RNA, oligonucleotide and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising them, methods of synthesizing them and methods for gene replacement, gene therapy, gene transcription suppression and delivery of therapeutic proteins to tissue in vivo comprising the molecules. The present invention also provides methods of reducing the immunogenicity of RNA, oligonucleotide and polyribonucleotide molecules.

The invention provides an MET2 gene knockout saccharomyces cerevisiae strain, a construction method and application, and belongs to the technical field of gene engineering. The MET2 gene knockout saccharomyces cerevisiae strain is obtained by replacing the MET2 gene in a saccharomyces cerevisiae strain BSPC040 with a hygromycin B resistance gene and transferring the hygromycin B resistance gene into a pJFE3-XI plasmid. The MET2 gene knockout saccharomyces cerevisiae strain can obviously improve the sugar alcohol conversion rate of a saccharomyces cerevisiae BSPC040 strain carrying a xylose metabolic pathway. The MET2 gene knockout saccharomyces cerevisiae strain has the advantages that the MET2 gene knockout saccharomyces cerevisiae strain has the advantages that the MET2 gene knockout saccharomyces cerevisiae strain has the advantages that the MET2 gene knockout saccharomyces cerevisiae strain is 23.1% higher in ethanol yield under the condition of consumption of equivalent xylose as compared with MET2 gene knockout saccharomyces cerevisiae strains, and novel theoretical basis and gene editing strategies are provided for strain optimization in industrial production of lignocellulosic ethanol.