58 results about "Gene replacement" patented technology

The invention discloses a high-yield lycopene recombinant rhodobacter sphaeroides strain as well as a construction method and application thereof, and belongs to the technical field of biosynthesis and biological energy sources, the construction method comprises the following steps: knocking out a crtC gene of rhodobacter sphaeroides by using a homologous recombination method to obtain a recombinant strain RS01; the method comprises the following steps: on the basis of a strain RS01, replacing a crtI3 gene on a rhodobacter sphaeroides genome with a crtI4 gene (from rhodospirillum rubrum) by using a homologous recombination method to obtain a recombinant strain RS02; the method comprises the following steps: connecting crtB and crtE genes to a pYYDT vector to construct a plasmid pYYDT-crtB-crtE, transferring the plasmid pYYDT-crtB-crtE into escherichia coli S17, and then introducing the plasmid pYYDT-crtB-crtE into a recombinant rhodobacter sphaeroides RS02 in a conjugational transfer manner to obtain a recombinant strain RS03. The recombinant rhodobacter sphaeroides constructed by the invention can be used for efficiently producing lycopene.

The invention discloses an ILTV gD protein antigen epitope expression cassette, a recombinant virus and application, and relates to the field of gene and protein engineering. The ILTV gD protein antigen epitope expression cassette provided by the invention comprises T and B cell epitopes of gD protein derived from an infectious laryngotracheitis virus WG strain. The recombinant virus rH120-gD-T / B is obtained by replacing a 5ab gene of an infectious bronchitis virus H120 strain with a gene for coding an ILTV gD protein antigen epitope expression cassette. The recombinant virus has good genetic stability, can be used for developing a safe and effective IB-ILT bivalent live vector vaccine, and solves the problem of poor stability of the current recombinant IBV.

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 discloses a multivalent expression sheep listeria vaccine for cervical cancer and a preparation method thereof, and the method comprises the following steps: on the basis of replacing sheep listeria LI [delta] ilo:: hly with hemolysin, constructing to obtain auxotrophic sheep listeria LI [delta] ilo:: hly [delta] dal [delta] dat; the fusion antigen is constructed by alternately arranging amino terminal and carboxyl terminal structural domains of four types of HPV6 / 11 / 16 / 18 E6 and E7 proteins and retaining an overlapping region of 16 amino acids at a connecting site; inserting the fusion antigen into a non-resistance plasmid pCW631 which carries a dal gene and replaces an Amp gene with an asd gene, so as to obtain a non-resistance plasmid pCW636; the resistance-free plasmid pCW636 is electrically transferred into the auxotroph sheep listeria monocytogenes LIdeltailo:: hly deltadal deltadat, and the sheep listeria monocytogenes cervical cancer vaccine is obtained. The cervical cancer vaccine provided by the invention can express the E6E7 protein in a multivalent manner, so that multiple protection on cervical cancer is realized.

The invention discloses a Trichoderma reesei engineering bacterium for secreting and expressing plastic degrading enzyme as well as a construction method and application of the Trichoderma reesei engineering bacterium, and belongs to the technical field of gene engineering. An expression plasmid of the plastic degrading enzyme is constructed, trichoderma reesei is converted, and the trichoderma reesei engineering bacterium for secreting and expressing the plastic degrading enzyme is constructed. In the Trichoderma reesei engineering bacteria, an endogenous protein coding gene cbh1 with the highest secretory expression quantity in Trichoderma reesei is replaced with a gene for coding the plastic degrading enzyme. The Trichoderma reesei engineering strain for secreting and expressing the plastic degrading enzyme is successfully constructed for the first time, and the Trichoderma reesei engineering strain constructed by the invention can significantly increase the productivity of the plastic degrading enzyme, and has potential to support future large-scale application.

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.

The present invention relates to the technical fields of fermentation engineering and genetic engineering, and particularly relates to a method for synthesizing lactic acid monomer using sucrose as a raw material. Through the genetic recombination technology of site-directed gene replacement, the promoter sequence of the sucrose metabolic operon in the production strain is corrected, enabling it to maintain a high level of expression throughout the production process of lactic acid monomer, demonstrating an obvious ability to highly catabolize sucrose to synthesize lactic acid monomers with high optical purity and high chemical purity (D-lactic acid or L-lactic acid). For example, strains DSAPW5 and LSAPW5 are fermented for 28-30 h under the condition of 25-50 °C, and the fermentation levels of D-lactic acid and L-lactic acid can reach 158.58 g / L and 159.65 g / L respectively, and the optical purity can reach 99.94% and 99.95%. The present invention can be applied to the industrial production of lactic acid monomer using sucrose or sucrose-containing raw materials.

The invention discloses a recombinant rhodobacter sphaeroides strain for synthesizing lycopene by photocatalytic immobilization of CO2 and a construction method and application thereof, and the construction method comprises the following steps: knocking out an endogenous crtC gene of rhodobacter sphaeroides by using a homologous recombination method, and replacing a crtI3 gene of rhodobacter sphaeroides by using a crtI4 gene from rhodobacter rubrum to obtain a recombinant strain RS-1; a crtB gene and a crtE gene are connected to a pYYDT vector, transferred into escherichia coli S17 and then introduced into a recombinant rhodobacter sphaeroides RS-1 through conjugational transfer, and a recombinant strain RS-2 is obtained. The recombinant rhodobacter sphaeroides constructed by the invention can be used for de novo synthesis of lycopene by using CO2 as a carbon source through photocatalysis. The method is simple to operate, and meanwhile, high-yield lycopene can be obtained.