80 results about "Plasmid Vector" patented technology

The invention relates to a high-throughput cis-acting element screening carrier and a screening method. Specifically, the invention provides a plasmid vector system containing bar codes, each bar code in the system is in one-to-one correspondence with a candidate cis-acting element, and the activation multiple of the candidate cis-acting element can be obtained by measuring the abundance of the bar codes; in order to eliminate the influence of a bar code on the vector on a detection result, an exogenous intron which can be cut off during transcription is inserted into a coding region in the vector and is used for distinguishing vector DNA and RNA obtained by transcription. The screening method of the biological cis-acting element has high efficiency, wide applicability and high throughput, and has outstanding application value in biological research and breeding.

The invention relates to recombinant escherichia coli for de novo synthesis of piceatannol as well as a construction method and application thereof, and belongs to the field of synthetic biology and metabolic engineering. According to the invention, an L-tyrosine production strain is used as an original strain, five key genes required for synthesizing piceatannol are combined and connected to a plasmid vector, and a recombinant plasmid is introduced into the original strain, so that an engineering strain for heterologous synthesis of piceatannol is successfully constructed. According to the invention, the yield of piceatannol is further obviously improved through copy number optimization.

The invention relates to a strong promoter P7 suitable for streptomyces and application thereof, and relates to the field of genetic engineering and microbial metabolism engineering. The nucleotide sequence of the strong promoter P7 is as shown in SEQ ID No.1, and the strong promoter comprises a plasmid vector of the strong promoter; a host cell comprising the plasmid vector; the invention also discloses application of the strong promoter, the plasmid vector and the host cell in starting expression of a target gene. Compared with the prior art, the characterization of the strong promoter P7 provides an effective tool element for streptomyces strong promoter engineering and high-efficiency gene expression, and has important significance on streptomyces silent gene characterization, high-efficiency gene expression, metabolite synthesis, metabolic pathway reconstruction and the like. The strong promoter can be applied to common streptomyces type strains, and has important significance on high yield of important proteins including enzymes and important metabolites from actinomycetes.

The invention discloses a limonium bicolor gene LbZFP8 and application thereof, and relates to the technical field of biological genetic engineering. The invention relates to a limonium bicolor gene LbZFP8 and application thereof. The limonium bicolor gene LbZFP8 is a gene for coding a protein composed of an amino acid sequence as shown in SEQ ID NO: 1; the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a virus vector or engineering bacteria. The gene provided by the invention can significantly improve the salt tolerance and oxidation resistance of a target plant by transforming plant cells. Experiments show that under the salt stress condition, the survival rate of the LbZFP8 overexpressed arabidopsis thaliana plant is increased by about 45% compared with that of a control group. Meanwhile, the compound plays a key role in regulating and controlling an active oxygen scavenging system, and the activity of SOD and POD enzymes can be effectively enhanced. The technology provides an important gene resource for cultivating new varieties of crops with strong stress resistance, and has a wide application prospect.

The invention provides a method for increasing the yield of L-isoleucine. Comprising the steps that an engineering strain containing an expression plasmid vector or an expression plasmid vector combination is cultured, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, and the first group of polynucleotides comprise at least one polynucleotide encoding L-isoleucine synthetic pathway enzyme; the second group of polynucleotides comprising at least one polynucleotide encoding an enzyme that modulates the balance of NAD + / NADH and NADP + / NADPH; and a backbone plasmid capable of autonomously replicating in a host cell. According to the invention, the yield of L-isoleucine and the conversion rate of glucose are improved.

The invention discloses a method for synthesizing alpha-arbutin through biotransformation, and belongs to the technical field of biochemistry. A gene cloning technology is utilized, sucrose phosphorylase Spase in leuconostoc mesenteroides and sucrose phosphorylase gtfa in bifidobacterium adolescentis are screened and found through a Deep Molecules and DLKcat algorithm on a Uniprot database, novopro is utilized for codon optimization, then the sucrose phosphorylase Spase and the sucrose phosphorylase gtfa are expressed on a plasmid vector PET-28a and are converted into an escherichia coli BL21 (DE3) strain, and the sucrose phosphorylase Spase and the sucrose phosphorylase gtfa in the bifidobacterium adolescentis are obtained. An engineering strain for heterologous expression of the gene is constructed by taking the gene as a chassis cell. According to the process, sucrose and hydroquinone are taken as co-substrates, and bioconversion is carried out under the action of sucrose phosphorylase from different sources to obtain alpha-arbutin.

The invention provides a method for improving the genetic stability of PolyA tail of mRNA (messenger ribonucleic acid). The method for improving the genetic stability of the PolyA tail in the preparation process of the mRNA containing the PolyA tail comprises the steps that a plasmid vector containing three-segment PolyA polynucleotides A1-G-A2-G-A3 is transferred into a host cell to be cultured, A1, A2 and A3 are each independently composed of 30-45 continuous A, and the host cell is selected from DH5a, Stbl3 and JM109; and separating and recovering a plasmid vector containing PolyA polynucleotide, carrying out enzyme digestion linearization to obtain an mRNA transcription template, and carrying out transcription to prepare mRNA. According to the method, the high genetic stability of the plasmid PolyA tail sequence can be achieved, the stability of mRNA is not affected, the mRNA protein translation efficiency is not affected, and the Oligo-dT purification process is not affected.

The invention belongs to the technical fields of immunotherapy, bioengineering, gene therapy and cell therapy, and provides a preparation method of an enhanced CAR-T cell for overexpressing LYAR, and the enhanced CAR-T cell and application thereof in order to solve the problems that the killing function of the CAR-T cell in solid tumor treatment is limited, the durability is poor, and function depletion is likely to occur. CDNA of the LYAR gene is cloned to an overexpression plasmid vector and packaged into lentivirus, T cells are jointly infected by the lentivirus and the CAR expression virus, the LYAR gene is forced to express under the driving of an EF-1alpha promoter, and stable overexpression of the LYAR in the CAR-T cells is achieved; and the CAR is a CAR targeting EGFR (epidermal growth factor receptor) and CD19. According to the present invention, the gene coding LYAR and the CAR gene are simultaneously transferred into the T cell through the lentivirus infection technology, and are compulsively expressed under the action of the EF1 alpha promoter, such that the anti-tumor function of the CAR-T cell can be significantly enhanced, and the activation and killing functions of the immune cell can be improved;

A process for producing three recombinant enzymes capable of hydrolyzing each class of organophosphate, carbamate, and pyrethroid insecticides, as well as a formulation containing these enzymes provides the recombinant plasmid DNA vector, in which the protein secretory system has been modified to enhance the secretion of recombinant protein into the periplasmic space or culture medium. A nucleotide sequence encoding PelB (pectate lyase B) signal sequence for periplasmic localization is deleted and replaced with a nucleotide sequence encoding LamB (maltoporin or phage lambda receptor) signal sequence to enable a more efficient secretory recombinant protein. Three recombinant plasmid DNA vectors for the expression of recombinant proteins that are constructed using the modified plasmid DNA vector described above. The recombinant proteins include a recombinant organophosphate-hydrolyzing enzyme; a recombinant carbamate-hydrolyzing enzyme; and a recombinant pyrethroid-hydrolyzing enzyme.

A method for improving the replication of covalent closed circular plasmids is provided. [Solution] The method comprises the step of preparing a covalent closed circular plasmid having a Pol I-dependent origin of replication and an insert containing a structured DNA sequence selected from the group consisting of a reverse repeat sequence, a directional repeat sequence, a homopolymer repeat sequence, a eukaryotic origin of replication, or a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also comprises the step of modifying the covalent closed circular recombinant molecule so that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, thereby improving the replication of the covalent closed circular plasmid with the resulting Pol III-dependent origin of replication. A covalent closed circular recombinant DNA molecule without antibiotic markers is also provided.

The invention relates to a plasmid vector capable of easily detecting the copy number of plasmids and a detection kit thereof. According to the plasmid vector, a plasmid replicon ORI gene and a single copy conserved gene of an escherichia coli genome are constructed on the same plasmid. The detection kit contains the plasmid vector or a single-copy quality control product prepared from the plasmid vector. The method has the advantages of high detection result accuracy, high reproducibility and simple detection process, and effectively solves the related problems existing in the traditional escherichia coli plasmid copy number detection method.

This invention provides a method for constructing a visualized mouse model characterizing hepatocyte necrosis and apoptosis, belonging to the field of biomedical technology. The invention involves disassembling firefly luciferase (Fluc) into inactive N-terminal (Nluc) and C-terminal (Cluc) fragments, which are then fused and expressed at specific locations on the MLKL molecule, forming two independent plasmid vectors, MLKL-Nluc and MLKL-Cluc. These two plasmid vectors are then injected into mouse hepatocytes via tail vein high-pressure hydrodynamic injection to construct the visualized mouse model characterizing hepatocyte necrosis and apoptosis. This invention allows for non-invasive and dynamic monitoring of the execution process of necrosis and apoptosis—MLKL oligomerization—in animal models, providing a powerful and specific tool that fills the technological gap in real-time visual monitoring of necrosis and apoptosis in pan-apoptotic research.

The application discloses a novel plasmid vector with improved CHO exogenous protein expression amount and construction and application thereof, and belongs to the technical field of genetic engineering. The plasmid vector pCHO6GS belongs to a novel plasmid system, and can at least improve the expression amount of general monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO by more than 1 times, that is, the unexpected improvement of the exogenous protein expression amount is realized by the novel plasmid vector, and the novel plasmid vector has an extremely important role in cost reduction and efficiency increase of various protein products with CHO as a cell factory.

The invention provides a construction method and application of bacillus amyloliquefaciens capable of enhancing yhcA expression, and the construction method comprises the following steps: taking bacillus amyloliquefaciens XH7 genome DNA as a template, carrying out PCR amplification on a transport protein coding gene yhcA, and connecting a promoter and a terminator to obtain a target gene segment; respectively carrying out double enzyme digestion on a free plasmid vector and a target gene segment by adopting the restriction enzyme to obtain a linear plasmid segment and an enzyme digestion gene segment; connecting the enzyme-digested gene fragment and the linear plasmid fragment through DNA ligase to obtain a free expression vector; and finally, transferring the free expression vector into a competent cell of bacillus amyloliquefaciens XH7, and adding a resistance selection marker to obtain a positive transformant. The research proves that the enhanced expression of the transporter gene yhcA is also a very effective method for improving the inosinic acid yield of bacillus amyloliquefaciens.

The invention belongs to the technical field of biology, and discloses a multivalent mRNA antigen construction method for an anti-periodontitis red complex, which comprises the following steps: screening a core antigen group of key pathogenic bacteria of the periodontitis red complex from a database, carrying out codon optimization and UTR and Poly (A) tail sequence modification, constructing a modular plasmid vector, and carrying out screening to obtain the multivalent mRNA antigen of the anti-periodontitis red complex. The high-purity multivalent mRNA antigen is obtained through fermentation purification, co-transcription capping and tailing and affinity chromatography. According to the invention, a multi-target collaborative immune design is adopted, so that the immune protection range is wide; the non-replicating mRNA does not contain viable bacterium components, uses modified nucleotide, and is high in safety; through a sequence optimization and transcription process, the mRNA translation efficiency and stability are excellent; the method has the advantages of simple operation, modular construction and standardized production process, strong controllability and easy large-scale production, provides a safe, lasting and specific technical scheme for preparation and prevention of periodontitis-related drugs, and has important clinical application value.

This disclosure provides compositions, systems, and methods for targeting, editing, modifying, or manipulating the genome of a host cell at one or more locations in a DNA sequence within a cell, tissue, or object. A genetically modified system for treating phenylketonuria (PKU) is described. In one embodiment, this disclosure relates to a pharmaceutical composition comprising the above system and a pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of plasmid vectors, viral vectors, vesicles, and lipid nanoparticles.

The application discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is constructed by introducing an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, a MCS (multiple cloning site), a 3'-UTR sequence and a T7 terminator sequence on the basis of part of a sequence of an E. coli plasmid pUC19. The pSY1A plasmid vector has a MCS into which an exogenous gene can be inserted, and the MCS and a transcription assembly constitute a transcription module, so that the transcription module can be preserved by preserving the plasmid, and in-vitro transcription of the wheat germ cell-free expression system becomes more convenient and faster.

The invention provides an engineering strain for synthesizing homoserine by using ethylene glycol and application of the engineering strain. The engineering strain comprises an expression plasmid vector or an expression plasmid vector combination, the vector or combination comprises a first group of polynucleotides and a second group of polynucleotides, and the first group of polynucleotides comprises at least one polynucleotide encoding an enzyme promoting ethylene glycol utilization; the second set of polynucleotides comprising at least one polynucleotide encoding an L-homoserine synthesis pathway enzyme; and a backbone plasmid capable of autonomously replicating in a host cell. By introducing the first group of polynucleotides, not only is the glucose utilization rate of the engineering strain improved, but also the yield of the L-homoserine is improved; meanwhile, by overexpressing or strengthening a plurality of L-homoserine biosynthetic pathway related genes from different sources and knocking out or weakening L-homoserine degradation pathway related genes, efficient fermentation production of the L-homoserine is realized.

The present invention relates to an isolated polypeptide having amino acylase activity and comprising an amino acid sequence having at least 87% sequence identity over its full length to the amino acid sequence set forth in SEQ ID NO: 1. The invention further relates to isolated nucleic acid molecules comprising a nucleotide sequence encoding such an aminoacylase, plasmid vectors comprising said nucleic acid molecules, recombinant host cells comprising isolated nucleic acid molecules or vectors, and methods of producing said aminoacylase. The invention also encompasses the use of an aminoacylase according to the invention for the N-acylation of an amino acid or a salt thereof, as well as a method for producing an N-acylamino acid or a salt thereof using an aminoacylase. Furthermore, the present invention relates to the N-acylamino acids obtained, to compositions comprising them, and to the use of the N-acylamino acids produced in cosmetics, home care products or industrial and / or institutional products.

This invention provides novel compositions and methods for effectively stimulating antiviral immunity. [Solution] A composition comprising (a) a vector comprising a plasmid encoding at least one viral antigen; and (b) a vector comprising a CD1d recognition antigen; and (c) at least one pharmaceutically acceptable carrier, wherein at least one of vectors (a) and (b) is an intact bacterial minicell or a dead bacterial cell.

The application discloses a DNA polymerase III mutant and application thereof. The mutant contains an amino acid sequence shown in SEQ ID No. 1, and the mutant can be applied to improving the mutation frequency of a host strain genome. In the application, the Escherichia coli is used as a starting strain, a high-efficiency evolution auxiliary plasmid is introduced, and the mutation frequency of the host is improved; combined with competitive pressure directional screening of a tryptophan structural analog 5MT, finally, the Escherichia coli engineering strain with high yield of tryptophan is obtained. The auxiliary plasmid comprises a pBad24 plasmid vector skeleton and an engineered Escherichia coli DNA polymerase III danQ nucleotide fragment. The high-efficiency evolution auxiliary plasmid provided by the application can significantly improve the mutation rate of the host genome, combined with the directional screening of 5MT, the traditional mutagenesis cycle can be greatly shortened, the engineering strain with high yield of tryptophan can be efficiently screened, and the application has important application value.

The invention relates to a plasmid vector for detecting the translation condition of amino acid sequence protein and application of the plasmid vector, and belongs to the technical field of biology. The plasmid vector for detecting the protein translation condition of the amino acid sequence, provided by the invention, is loaded with an exogenous amino acid sequence, and the amino acid sequence contains an amino acid repetitive sequence; a fluorescent protein coding gene of one color is inserted in the upstream of an amino acid repetitive sequence coding region, and a fluorescent protein coding gene of another color is inserted in the downstream of the amino acid repetitive sequence coding region. According to the invention, the translation process of the protein containing the amino acid repetitive sequence in the cell can be monitored in real time according to the expression condition of the fluorescent protein, and the formation process of the truncated object after translation blocking is captured in real time, so that a favorable tool is provided for researching and evaluating the translation condition of the protein containing the amino acid repetitive sequence in the cell; the method can also be further applied to pathogenesis research of related diseases such as Huntington's disease and / or neuronal intracellular inclusion body disease.

The invention discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is characterized in that on the basis of partial sequence of escherichia coli plasmid pUC19, an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, MCS multiple cloning sites, a 3 '-UTR sequence and a T7 terminator sequence are introduced to construct the plasmid vector pSY1A. According to the pSY1A plasmid vector provided by the invention, the vector has multiple cloning sites into which exogenous genes can be inserted, and the multiple cloning sites and a transcription component form a transcription module, so that the transcription module can be stored by storing plasmids, and the in-vitro transcription of a wheat germ cell-free expression system becomes more convenient and quicker.

The present invention relates to the field of in-vitro transcription, in particular to a plasmid backbone capable of improving the stability of the polyadenylic acid tail. Provided is an expression cassette, sequentially comprising: a rop gene, a replicon ori, and a resistance gene, wherein the rop gene, the replicon ori, and the resistance gene are connected by means of linker fragments. Low-copy pmRVacSL and medium-copy pmRVacM plasmid vector systems are established by means of artificial modification. Since pmRVacSL is a low-copy vector, the plasmid DNA yield is relatively low. The pmRVacM plasmid backbone addresses the problem of low plasmid DNA yield of pmRVacSL. The vector system not only enables stable replication of polyA tail sequences of 150 bp or more, but also allows for rapid addition of genes of interest and polyadenylic acid (polyA) sequences, efficiently and rapidly obtaining the target DNA template for mRNA in vitro transcription.

The application discloses a single-chain circular DNA virus GyH1 recombinant polyepitope adenovirus vector vaccine and application thereof, and belongs to the technical field of vaccines.The antigen epitopes of GyH1 VP1, VP2 and VP3 proteins are screened through an online bioinformation analysis server, the antigen epitopes are connected by using a linker to construct a polyepitope vaccine GyMEV, and the nucleotide sequence of the GyMEV is obtained through reverse translation and codon optimization.The antigen polypeptide GyMEV nucleotide sequence is cloned into a plasmid vector pMD19-T through artificial synthesis, the GyMEV is directionally cloned into a linearized adenovirus vector by using seamless cloning technology to obtain a recombinant adenovirus plasmid rAd5-GyMEV, and the recombinant plasmid is transfected into HEK 293 cells to obtain the recombinant polyepitope adenovirus vector vaccine GyMEV.The results show that the GyMEV can more effectively stimulate the immune response of the body and has stronger protection.The vaccine also has the safety of a subunit vaccine and can adopt various immunization modes, can stimulate the immune response of the body in all directions, and thus has a wide development and application prospect.

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a D-psicose-3-epimerase mutant, which is obtained by performing point mutation on one site in the 79th, 115th, 141st, 193rd or 202nd amino acid of a D-psicose-3-epimerase parent; specifically, a mutation primer for point mutation is designed, a vector carrying a wild-type D-psicose-3-epimerase gene is used as a template to perform point mutation and construct a plasmid vector containing the mutant, and then the plasmid vector is transformed into a host cell to obtain the D-psicose-3-epimerase mutant; and the D-psicose-3-epimerase mutant is applied to the production of psicose. The thermal stability and catalytic activity of the D-psicose-3-epimerase mutant are significantly improved, and the D-psicose-3-epimerase mutant has good industrial application value.

The invention discloses an engineering strain for increasing supply of cofactors and application of the engineering strain in improving production of epsilon-polylysine, and belongs to the technical field of microbial genetic engineering. The method comprises the following steps: by taking streptomyces albus for producing epsilon-polylysine as an original strain, simultaneously connecting a nicotinic acid ribose phosphate transferase gene pncB, a phosphoenolpyruvate carboxylase gene pck and a pyruvate dehydrogenase gene pdh in the strain with a pIB139 vector by utilizing a homologous recombination method to obtain a recombinant plasmid vector pIB139-pncB-pck-pdh, and introducing the recombinant plasmid vector into a competent cell to obtain the recombinant plasmid vector pIB139-pncB-pck-pdh. The engineering bacterium BP3 is obtained. The shake flask epsilon-PL yield of the engineering bacterium BP3 reaches 1.61 g / L and is improved by 30.89% compared with that of an original strain; the yield of fed batch fermentation reaches 37.21 g / L, the yield reaches 4.85 g / (L.d), and the maximum thallus concentration DCW reaches 43.47 g / L.

The invention discloses a Grin2b stably transfected cell strain, a construction method and application thereof and a method for screening pollutant high-throughput neurotoxicity or learning and memory disorders, and belongs to the technical field of biologication.The construction method comprises the following steps that 1, cells are cultured; (2) constructing a plasmid vector, wherein the plasmid vector comprises a Grin2b coding sequence; (3) virus coating and virus collection; (4) testing the titer of the lentivirus; (5) stable plant screening; and (6) carrying out stable plant transformation function verification. The Grin2b stably transfected cell strain based on the Grin2b related ion channel constructed by the invention provides a brand new technical platform for high-throughput neurotoxicity recognition, learning memory screening and mechanism research of organic pollutants and degradation products thereof.

The invention aims to provide a technical scheme of yeast high-efficiency expression of recombinant camel source serum albumin, X33, GS115, S288C and SMD1168H yeast strains are selected as an expression chassis, codon optimization is combined with a double-promoter regulation strategy of an AOX1 inducible promoter and a GAP constitutive promoter, a recombinant expression system is constructed based on a PICZ alpha A plasmid vector, and the recombinant camel source serum albumin is obtained. According to the method, the problems of incomplete folding, insufficient solubility and the like of the recombinant camel serum albumin in a prokaryotic expression system are solved, efficient and stable expression of target protein is realized, the recombinant camel serum albumin with high purity, high biological activity and high safety is finally obtained, and technical support is provided for industrial production of human serum albumin substitute products of the recombinant camel serum albumin.

The invention discloses a method for preparing a collagen heterotrimer by an intracellular one-step method, and belongs to the technical field of genetic engineering. According to the method, three collagen heterotrimer single chains are constructed in a plasmid vector for co-expression, the sequence of the heterotrimer single chains is changed by inserting a tag, and the collagen heterotrimer is efficiently synthesized in cells by a one-step method by using recombinant bacteria for expression and purification. According to the method disclosed by the invention, one-step preparation of the heterotrimer collagen with different A, B and C chains, which is high in thermal stability and relatively high in yield, is realized; on the basis of the method disclosed by the invention, the heterotrimer collagen composed of any collagen fragment can be theoretically prepared, and the heterotrimer collagen comprises type I collagen; on the basis, according to the collagen prepared by an intracellular one-step method, the Tm value of a heterotrimer prepared by intracellular folding is 42 DEG C, and the heat stability is relatively high; and the intracellular folded collagen heterotrimer structure is obviously superior to in-vitro folded collagen heterotrimer structure.