172 results about "Plasmid Vector" patented technology

The invention discloses application of scylla serrata antibacterial peptide Scycin in preparation of an anti-inflammatory composition and a preparation method of the scylla serrata antibacterial peptide Scycin. The nucleotide sequence of the scylla serrata antibacterial peptide Scycin is shown as SEQ ID NO.01. The invention further discloses a preparation method of the scylla serrata antibacterial peptide Scycin. In an LPS (lipopolysaccharide)-induced RAW 264.7 macrophage inflammation model, the recombinant expression plasmid vector containing the scylla antibacterial peptide Scin remarkably inhibits an inflammation cascade reaction by down-regulating expression of proinflammatory factors such as TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6), the anti-inflammatory effect of the recombinant expression plasmid vector is equivalent to that of a mesalazine positive control group, and obvious cytotoxicity is not observed.

The invention discloses a novel plasmid vector capable of increasing CHO foreign protein expression quantity and construction and application thereof, and belongs to the technical field of gene engineering. The plasmid vector pCHO6GS belongs to a brand-new plasmid system, and the expression quantity of a common monoclonal antibody and recombinant protein (such as H5HA trimer) difficult to express in CHO can be increased by at least more than one time, so that the expression quantity of foreign protein is increased beyond expectation through the novel plasmid vector; and the method also plays an extremely important role in reducing cost and improving efficiency of various protein products taking CHO as a cell factory.

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 discloses an engineered exosome for promoting hair regeneration and a preparation method and application thereof, Wnt10b gene is inserted into a plasmid vector to construct a target plasmid, the target plasmid and a lentivirus packaging plasmid are co-transfected to packaging cells to generate lentivirus particles, and then target stem cells are infected. Screening to obtain a cell strain capable of stably expressing a target gene, culturing the stably transfected strain, and collecting the engineered exosome generated by the stably transfected strain. The exosomes are rich in Wnt10b protein and can promote proliferation and differentiation of hair follicle stem cells on a molecular level, so that hair follicle tissues in a resting period are activated and enter a growing period again, and hair regeneration is realized. Meanwhile, the exosome is high in safety, and adverse reactions caused by traditional chemical synthesis drugs are avoided. The exosome can be applied to the preparation of hair regeneration products, and the accurate symptomatic treatment capability of the exosome provides a hair growth method aiming at pathogenesis for alopecia patients, and provides a new hope for patients who seek safe and effective hair growth solutions.

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

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 present invention relates to a plasmid vector for synthesizing a self-circularization RNA structure in an in vitro transcription (IVT) process and to a DNA fragment for preparing same. The present invention can be used in a method for efficiently and economically producing circular RNA in large quantities under an STS system.

The invention relates to the technical field of recombinant proteins, and relates to a recombinant XII type collagen as well as a preparation method and application thereof. The recombinant XII type collagen is formed by connecting 5-30 repetitive units in series, and the amino acid sequence of each repetitive unit is as shown in SEQ ID NO: 1. The nucleic acid molecule is used for coding the recombinant XII type collagen; a vector comprises the nucleic acid molecule. According to the invention, a nucleotide sequence for coding the amino acid sequence is inserted into a vector to obtain a recombinant plasmid vector, then the recombinant plasmid vector is introduced into a host cell for culture, and then separation and purification are carried out to obtain the recombinant XII type collagen. The recombinant XII type collagen provided by the invention is easy to obtain, can realize efficient secretory expression, and meets the requirements of industrial production; the biological activity is good, and the proliferation and migration of human immortalized cartilage cells can be promoted.

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 discloses a pig whole sperm membrane protein antigen specific nano antibody library construction method which comprises the following steps: S1, immunizing alpaca with pig whole sperms, and separating peripheral blood cells after immunization; s2, RNA in peripheral blood cells is extracted, cDNA is obtained through reverse transcription, and a VHH coding sequence of an antibody is amplified; s3, cloning the amplified VHH coding sequence to a bacteriophage plasmid vector to prepare a recombinant bacteriophage vector; s4, transforming the recombinant bacteriophage vector into escherichia coli and carrying out enlarged culture to obtain a bacteriophage display library; and S5, performing panning on the phage display library for a plurality of times by using the pig X / Y sperms, and constructing the pig single sex sperm specific nano antibody library. According to the method, the pig whole sperm is adopted as an immunogen for immunizing alpaca for the first time, the capacity of the constructed antibody library reaches 3.4 * 10 < 8 > pfu, the abundance of the antibody library is higher, the coverage degree of whole sperm antigens is higher, and the variety is more complete, so that the capture success rate of the pig sex control specific antibody is increased, and a foundation is laid for industrial application of the pig sperm specific antibody.

The invention is applicable to the technical field of biotechnology and cell biology, and provides a method for quickly constructing an IgG antibody high-expression stably-transfected CHO cell strain, which comprises the following steps: S1, constructing antibody plasmids; s2, carrying out cell resuscitation, passage and plasmid transfection; s3, screening positive cells; s4, preparing, namely screening high-expression stably transfected single cell clones; s5, cell feeding batch culture; s6, verifying the stability of the cell strain expressed by the antibody, according to the method provided by the invention, after the plasmid vector enters the cell through electrotransfection and standing culture is carried out for 48 hours, shaking culture is carried out, the cell recovery time can be shortened, the recovery speed is higher than that of traditional standing culture, and the cell state is better than that of a standing state; according to the method, high-density cells are inoculated during cell fed-batch culture, and under the high-density inoculation, the logarithmic phase of the cells is shortened, and the cells enter a protein production phase earlier, so that higher expression quantity can be achieved, the overall production period is greatly shortened, and a large amount of cost is saved.

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.

Generally, the present disclosure is directed to methods of producing Resolvin-loaded extracellular vesicles, compositions comprising Resolvin-loaded extracellular vesicles, and methods of treating diseases using such compositions. In some embodiments, methods described herein comprise providing a population of cells, transfecting the population of cells with at least one plasmid vector encoding human cyclooxygenase-2 and 5-lipoxygenase, applying DHA, EP A, and ASA to the transfected population of cells, and isolating the Resolvin-loaded extracellular vesicles from culture media of the transfected population of cells. In some cases, a composition described herein comprises a plurality of extracellular vesicles comprising at least one Resolvin packaged within an interior of the EVs. In some embodiments, upon disposing a plurality of Resolvin-loaded extracellular vesicles within a biological compartment of the patient, inflammation from an inflammatory condition or disease, such as acute lung respiratory distress syndrome, is mitigated or resolved.

The invention relates to a linear covalent closed DNA vaccine, which comprises a plasmid vector containing an IHNV virus antigen expression unit, the sequence of the antigen expression unit is shown as SEQ ID NO.1, and the DNA structural form is a linear terminal covalent closed form. The invention also relates to a preparation method and application of the linear covalent closed DNA vaccine. Compared with a traditional DNA vaccine, the linear covalent closed DNA vaccine has no redundant bacterium source nucleic acid sequence, and has good safety and effectiveness for preventing and treating infectious hematopoietic necrosis of rainbow trout.

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 discloses a positive quality control product for detecting breast cancer and ovarian cancer gene large fragment deletion and a preparation method thereof, the gene comprises breast cancer and ovarian cancer related genetic genes BRCA1 / BRCA2 / ATM / PALB2, the positive quality control product is a recombinant plasmid containing four gene segments including a first exon of a to-be-detected gene, a plant sequence, a last exon of the gene and RNase P; the method comprises the following steps: inserting a human genome RNase P sequence into a plasmid vector according to a fragment ratio of 2: 1, and verifying the copy number of a to-be-detected gene by adopting a fluorescent quantitative qPCR (quantitative polymerase chain reaction) method so as to judge whether the obtained plasmid can be used as a positive quality control product for detection or not, thereby completing the quality inspection of the positive quality control product; the preparation method provided by the invention is simple and easy to operate and good in stability, the positive quality control product obtained by adopting the preparation method can be used as a positive internal standard quality control material for detecting breast cancer and ovarian cancer gene large fragment deletion, the whole experiment process is monitored, and the accuracy of experiment and detection results is improved.

The present disclosure provides methods and tools that allow for the production of large quantities of double-stranded RNA (dsRNA) in bacterial cells, including plasmid vectors that can be used to transform Gram-positive or Gram-negative bacterial cells, such that the cells produce high yields of target dsRNA.

The invention discloses a plukenetia volubilis FAD2 gene promoter. The nucleotide sequence of the plukenetia volubilis FAD2 gene promoter proPvFAD2.4 is shown in the specification. The proPvFAD2.4 comprises TATA-box and CAAT-box, and the TATA-box and the CAAT-box are located at the upstream of a transcription start site (TSS), so that basic transcription support is provided for positioning of RNA polymerase II, and the basic expression level of the gene is ensured. Comprising seed specific elements of AE-box and O2-site, and specific expression of PvFAD2 in the seed development stage is combined with DOF (OBP2) and MADS-box transcription factors to drive grease to be quickly accumulated. And the recombinant plasmid vector has a strong starting effect, so that the promoter has a function of starting downstream gene expression in a tobacco leaf cell environment, and an experimental foundation is laid for subsequent analysis of a regulation mechanism of the promoter.

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 present invention discloses a plasmid vector and cell line that can induce targeted genomic DNA double-strand breaks. The present invention constructs a nuclease AscI expression plasmid based on a lentiviral vector backbone by integrating the Tet-on chemically inducible transcription control system and the ERT2 ligand-dependent nuclear localization control system. The vector regulates the transcription of AscI through the Tet-on system and uses the 4-hydroxytamoxifen-induced ERT2 system to achieve spatiotemporal specific nuclear translocation of the endonuclease AscI. The present invention also constructs and screens a cell line that stably expresses AscI. With the help of small chemical molecules, the spatiotemporal expression of the AscI endonuclease in cells can be precisely regulated, thereby inducing DNA double-strand breaks at specific sites in the genome. The present invention provides an efficient and accurate research tool for the field of DNA damage repair, which can be widely used in studying DNA damage repair mechanisms and high-throughput screening of related drugs.

The invention belongs to the technical field of biology, and relates to a zero-background carrier as well as a preparation method and application thereof. The zero-background vector provided by the invention comprises an optimized escherichia coli CcdB gene and a corresponding escherichia coli promoter, and the nucleotide sequence of the zero-background vector is shown as SEQ ID NO.1. The zero background vector is applicable to any plasmid vector; both a cloning vector plasmid and an expression vector plasmid have relatively high connection efficiency and positive rate, and a connection product can be directly converted into a common escherichia coli strain, so that a target gene cloning vector can be effectively prepared; the target plasmid inserted with the zero background carrier has high short fragment insertion efficiency, and can also be used for long fragment insertion at the same time. In the genome assembly process, no matter whether a small fragment or a large gene fragment is inserted, the method can ensure an efficient assembly effect, especially in small fragment gene cloning, the efficiency and the positive rate can be remarkably improved, and meanwhile generation of false positive cloning is reduced.

The invention discloses a plasmid vector containing cucumber mosaic virus Fny isolate mutant RNA1 and application of the plasmid vector, and belongs to the technical field of plant virology and molecular biology. The plasmid vector contains a CMVFny isolate mutant RNA1 (ribonucleic acid 1), and the nucleotide sequence of the CMVFny isolate mutant RNA1 is as shown in SEQ ID NO. 1. According to the present invention, the structure of the RNA1 of the cucumber mosaic virus Fny isolate is modified so as to provide the cucumber mosaic virus Fny isolate with weak toxicity; and when being used as a vaccine vector, the vaccine vector at least can accommodate 200bp exogenous fragments and maintain genetic stability, thereby providing a basis for developing multivalent attenuated vaccines.

The invention belongs to the technical field of biology. The invention provides a recombinant protein, an amino acid sequence of the recombinant protein is formed by repeatedly connecting dominant epitopes of canine parvovirus VP2 protein in series, the amino acid sequence of the canine parvovirus recombinant protein is converted into a corresponding nucleotide sequence by adopting escherichia coli preferred codons, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed; therefore, the expression quantity of the recombinant protein in escherichia coli is improved. The invention also relates to a method for establishing a bacteriophage library by immunizing a mouse with the recombinant protein, obtaining a corresponding canine parvovirus VP2 protein single-chain antibody scfv sequence through panning screening, constructing a complete mouse IgG1 antibody sequence expression vector from the obtained scfv sequence, expressing a monoclonal antibody through transient HEK293F cells, and purifying the monoclonal antibody, thereby obtaining the canine parvovirus VP2 protein single-chain antibody. An optimal monoclonal antibody pairing combination is determined through an immunofluorescence orthogonal experiment.

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;

The invention relates to the technical field of antibody detection, in particular to a recombinant antigen and application of a transfected living cell immunofluorescence method of the recombinant antigen in anti-myelin oligodendroglia glycoprotein IgG antibody detection. The method comprises the following steps: constructing a myelin sheath oligodendroglia glycoprotein expression plasmid, with an expression plasmid vector being pcdna3.1-target gene-linker-* 3Flag-P2A-EGFP, an amino acid sequence of the expression plasmid gene being as shown in SEQ ID NO: 1, a base sequence of the expression plasmid gene being as shown in SEQ ID NO: 2, carrying out cell transfection to make the expression plasmid enter a target cell, culturing for 16-28 h to obtain a cell immobilized with the expression plasmid, and purifying the cell to obtain the myelin sheath oligodendroglia glycoprotein. The kit can be used for detecting the anti-myelin oligodendroglia glycoprotein IgG antibody by an immunofluorescence staining method. The problems of antigen spatial conformation change, epitope shielding or cell shrinkage of cells are solved, and the detection sensitivity is 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 provides an engineering strain for synthesizing isoleucine 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-isoleucine 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-isoleucine is improved; meanwhile, by overexpressing or strengthening a plurality of L-isoleucine biosynthetic pathway related genes from different sources and knocking out or weakening L-isoleucine degradation pathway related genes, efficient fermentation production of the L-isoleucine is realized.

The invention discloses a sgRNA sequence for targeted knockout of an O antigen gene cluster OL101 and application of the sgRNA sequence. The invention relates to an sgRNA sequence for targeted knockout of an O antigen gene cluster OL101. The sequence comprises oagen-sgRNA-F as shown in SEQ ID NO. 1 and oagen-sgRNA-R as shown in SEQ ID NO. 2. The invention relates to a CRISPR / Cas9 and lambda Red plasmid vector system for targeted knockout of an O antigen gene cluster OL101, the plasmid vector system is a sgRNA sequence for targeted knockout of the O antigen gene cluster in claim 1 and a recombinant expression plasmid vector of lambda Red and CRISPR / cas9 vectors, and the sequence of the plasmid vector system is as shown in SEQ ID NO.3. According to the invention, the Klebsiella pneumoniae OL101 can be specifically knocked out in a targeted manner, so that an OL101 knocked-out strain is obtained, and the specific functions of the Klebsiella pneumoniae O antigen OL101 can be clarified. According to the system for targeted knockout of the Klebsiella pneumoniae O antigen OL101, O antigen gene clusters of strains can be effectively knocked out, the gene knockout period is short, and the method is simple.