35 results about "Plasmidome" patented technology

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 system and application thereof to construction of swine-derived recombinant cells with insulin receptor substrate gene defects. The invention provides an sgRNA (ribonucleic acid) combination which is composed of sgRNAIRS1-1, sgRNAIRS1-3, sgRNAIRS2-2 and sgRNAIRS2-3. The invention provides a plasmid combination. The plasmid combination is composed of four plasmids, and the four plasmids are respectively subjected to transcription to obtain sgRNAIRS1-1, sgRNAIRS1-3, sgRNAIRS2-2 and sgRNAIRS2-3. Theapplication of the sgRNA combination or the plasmid combination is as follows: preparation of recombinant cells; preparation of a diabetic cell model; preparation of a diabetic animal model. Accordingto the invention, sgRNAIRS1-1 is as shown in SEQ ID NO:8; sgRNAIRS1-3 is as shown in SEQ ID NO:10; sgRNAIRS2-2 is shown as SEQ ID NO:14; and sgRNAIRS2-3 is as shown in SEQ ID NO:15. A solid foundation is laid for the preparation of diabetic pig models, and important application value for the research and development of diabetic medicaments is achieved.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.

The invention provides an escherichia coli engineering bacterium for producing 2'-fucosyllactose. Through a CRISPR / Cas9 gene editing system, related genes of an original strain 2'-fucosyllactose in ananabolic pathway can be knocked out; and a modular metabolic pathway is constructed, the expression level of phosphomannomutase (ManB), mannose-1-phosphateguanylyltransferase (ManC), GDP-mannose-6-dehydrogenase (Gmd), GDP-fucose-synthase (WcaG), L-fucose 1-kinase / GDP-fucose pyrophosphorylase (Fkp) and 2'-fucosyllactose synthetase (FucT2) in the metabolic pathway can be regulated and controlled through different plasmid combinations, so that 2'-fucosyllactose with higher concentration can be accumulated in cells. A method for efficiently producing the 2'-fucosyllactose is also provided.

The invention discloses a gene vector system of a redox sensitive shielding system having targeting function, its preparation and application in the field of gene therapy. The gene vector system disclosed herein comprises a redox sensitive shielding system having targeting function, a cationic high-molecular material and plasmid DNA; wherein the cationic high-molecular material and plasmid DNA form compound particles, the redox sensitive shielding system having targeting function shields the compound surface by electrostatic action, thus the toxicity of the vector can be reduced, the loaded genetic material is transferred into the cell successfully, the expression of the genetic material is realized, the transfection process is completed, the targeting of gene transfection can be raised, and simultaneously the gene transfection efficiency is raised.

The invention discloses a genetic vector system of a nanoparticle with multiple oxidation-reduction stimulus response as well as a preparation method and application of the genetic vector system. The genetic vector system is composed of a shielding system, a cation material and foreign plasmid DNA (deoxyribonucleic acid); and the shielding system and the cation material both have oxidation-reduction stimulus response, so that a ternary complex nanoparticle with multiple oxidation-reduction stimulus response is formed. The genetic vector of the ternary complex nanoparticle is low in cytotoxicity, and capable of well compressing and compounding the plasmid DNA under physiological conditions, successfully transferring a supported genetic substance into a cell and entering the cell; and the reduction environment in the cell can be more rapidly broken to release the supported gene, so that the expression of gene substances is realized, the transfection process is finished, and the gene transfection efficiency can be remarkably increased.

The invention relates to a genetically engineered bacterium for producing 2'- fucosyllactose and application of the genetically engineered bacterium, and belongs to metabolic engineering and food fermentation technologies. The expression levels of phosphomannomutase, mannose-1-phosphoguanine transferase, GDP-mannose-6-dehydrogenase, GDP-fucosyl synthase, alpha-1,2-fucosyl transferase and lactose permease in a metabolic pathway are regulated and controlled by changing the expression of a transcription regulation factor in escherichia coli, so that an optimal plasmid combination is obtained; beta-galactosidase and UDP-glucose lipid carrier transferase in an escherichia coli synthetic route are knocked out through a CRISPR-Cas9 gene editing system, and therefore, the accumulated amount of 2'-fucosyllactose is increased; and the recombinant escherichia coli obtained by the invention can be used for synthesizing 2'-fucosyllactose by utilizing glycerol or glucose, is stable in heredity and high in expression level, and has obvious industrial production potential.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.

The invention discloses an [alpha]-bisabolol synthetic plasmid as well as a construction method and an escherichia coli engineered strain thereof. An IPP and DMAPP synthetic plasmid pS-MVA and the [alpha]-bisabolol synthetic plasmid are constructed, and the two plasmids are introduced into an escherichia coli strain E.coli DH5[alpha]. The [alpha]-bisabolol synthetic plasmid takes a pTrc99A vectoras a skeleton plasmid and is assembled with an escherichia coli gene ispA and an artificially synthetic [alpha]-bisabolol synthetase gene or a wild-type [alpha]-bisabolol synthetase gene from different sources; the artificially synthetic [alpha]-bisabolol synthetase gene is selected from one of AaBBS, LdBBS, MrBBS, ScBBS and SsBBS; and the wild-type [alpha]-bisabolol synthetase gene is selected from one of LdBBS, ScBBS and MrBBS. According to the invention, the [alpha]-bisabolol is synthesized through a biological method; and the [alpha]-bisabolol is high in yield, high in activity and is green-going and environment-friendly.

The present invention belongs to the technical field of molecular biology, relates to a method for constructing a FSCN1 gene stable knockout cell line, a plasmid or a plasmid combination and an application thereof, aims at instability and incompleteness of a method for siRNA transient knockout of FSCN1 gene and provides a method for stable knockout of the FSCN1 gene from genome. The method is usedfor studying functions of the FSCN1 and can also be used for constructing the FSCN1 gene knockout cell line. The provided method for constructing the FSCN1 gene knockout cell line is based on a CRISPR-Cas9 system. The method uses "N18 or N20" of a fragment meeting 5'-G-N18-NGG-3' or 5'-G-N20-NGG-3' or 5'-CCN-N18-C-3' or 5'-CCN-N20-C-3' sequence regular array in a protein coding sequence in a first exon and a protein coding sequence in a fifth exon of the human FSCN1 gene as a target sequence; and N represents any one of A, T, C and G, wherein "N18" and "N20" are 18 and 20 deoxynucleotides,respectively.

The present invention relates to gene therapy medicine. The present invention provides one kind of tumor-targeting gene therapy medicine composition qmIL2-SON2 / NfBS-MiniCMV-PEIIImut comprising non-viral protein carrier qmIL2-SON2 and specific tumor tissue killing gene eukaryon expressing plasmid NfBS-MiniCMV-PEIIImut. The present invention also provides one kind of specific tumor tissue regulating sequence NfkB-MiniCMV promoter containing the coding sequence of Nf-kB protein combining site and shortened miniCMB promoter. The present invention provides all the relevant research results, including the use of composition qmIL2-SON2 / NfBS-MiniCMV-PEIIImut assembled with the non-viral protein carrier and the killing gene recombinant in killing IL2R enriching tumor cell specifically without killing normal cells.

The invention relates to a lentivirus stable packaging cell line and a preparation method thereof, the lentivirus stable packaging cell line comprises a packaging plasmid group, and the packaging plasmid group is composed of a pPuro.coTetR plasmid, a pVSVG plasmid and a pGagPol-RRE-NES-cINT plasmid, wherein the pPuro.coTetR plasmid comprises a CoTetR gene, the pVSVG plasmid comprises a VSVG gene,and the pGagPol-RRE-NES-cINT plasmid comprises a GagPol gene and a Rev gene. The invention further discloses the preparation method of the recombinant plasmid. The lentivirus stable packaging cell line is relatively stable in the aspects of passage, virus production and genetic gene copy number, and the lentivirus produced by using the lentivirus stable packaging cell line is high in titer and lowin impurity content.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced with reverse DNA gyrase or reaction (d) is not performed.

The invention discloses a method for randomly inserting a DNA fragment into a bacillus subtilis chromosome and application of the method. The invention relates to a composition, which consists of a transposon element or a plasmid containing the transposon element and a plasmid pUSIGH. The composition is applied to randomly inserting the target DNA fragment into the bacillus subtilis chromosome. After the composition is co-transformed into bacillus subtilis, the transposon element or a plasmid containing the transposon element can integrate the transposon element into a host chromosome through homologous recombination, and pUSIGH can be normally and independently copied in a host. Transposase gene expression on pUSIGH is induced by using xylose, and the transposon is randomly inserted into the chromosome under the action of transposase. Then, clones with relatively high target protein expression level can be screened out.

The invention discloses a plasmid carrying nanoparticle for preventing and treating influenza virus and a preparation method thereof. The plasmid carrying nanoparticle comprises a transport carrier and a plasmid, wherein the plasmid comprises a plasmid combination for inhibiting H1N1 virus and applied to a CRISPRa system and an SP-dCas9-VPR plasmid; and the transport carrier is a polymer plasmid transport carrier or a lipid plasmid transport carrier. According to the invention, the plasmid carrying nanoparticle for up-regulating IFNIII gene expression through a prepared CRISPRa plasmid systemcan be used for preventing influenza virus infection in advance and treating influenza virus infection in the early stage, has universality for influenza virus subtypes, has great application value and is worthy of vigorous promotion.

The invention relates to a plasmid DNA entrapped cationic liposome compound. The plasmid DNA entrapped cationic liposome compound consists of a cationic liposome, and chitosan and plasmid DNA wrapped by the cationic liposome, wherein the cationic liposome consists of cationic phospholipid DOTAP, neutral phospholipid HSPC or EPC, auxiliary phospholipid DOPE, cholesterol Chol, and functional phospholipid DSPE-PEG2000-X or DOPE-PEG2000-X; the plasmid DNA is a recombinant photosensitive transcription factor expression gene and diphtheria toxin A fragment (DT-A) transcription unit; the plasmid DNA for light-operated expression of diphtheria toxin is firstly compressed by chitosan, and then entrapped into the cationic liposome; and various groups can be modified on the surface of the cationic liposome, so that a drug delivery system has a certain targeting effect, and the treatment effect on malignant tumors is good.

The invention discloses a CRISPR / Cas9 system and application of the CRISPR / Cas9 system in construction of swine-derived recombinant cells for resisting amyotrophy protein gene defects. The invention provides an sgRNA combination, the sgRNA combination is composed of sgRNADMD-Ug3 (the target sequence binding region is shown as the 1-20th nucleotide in SEQ ID NO: 8) and sgRNADMD-Dg3 (the target sequence binding region is shown as the 1-20th nucleotide in SEQ ID NO: 11). The invention provides a kit, the kit is composed of a plasmid pKG-U6gRNA (DMD-Ug3) of sgRNADMD-Ug3 obtained through transcription, a plasmid pKGU6gRNA (DMD-Dg3) of sgRNADMD-Ug3 obtained through transcription, and a plasmid pKG-GE3. The sgRNA combination or the kit can be used for preparing recombinant cells or preparing a Duchenne muscular dystrophy animal model. The recombinant cells are anti-amyotrophy protein gene defect cells and can be used for preparing an animal disease model through somatic cell cloning. A solidfoundation is laid for the preparation of a Duchenne muscular dystrophy swine model, and has important application value for the research and development of Duchenne muscular dystrophy medicines.

The invention discloses a genetic vector system of a nanoparticle with multiple oxidation-reduction stimulus response as well as a preparation method and application of the genetic vector system. The genetic vector system is composed of a shielding system, a cation material and foreign plasmid DNA (deoxyribonucleic acid); and the shielding system and the cation material both have oxidation-reduction stimulus response, so that a ternary complex nanoparticle with multiple oxidation-reduction stimulus response is formed. The genetic vector of the ternary complex nanoparticle is low in cytotoxicity, and capable of well compressing and compounding the plasmid DNA under physiological conditions, successfully transferring a supported genetic substance into a cell and entering the cell; and the reduction environment in the cell can be more rapidly broken to release the supported gene, so that the expression of gene substances is realized, the transfection process is finished, and the gene transfection efficiency can be remarkably increased.

The invention provides an engineering strain of Escherichia coli that produces 2'-fucosyllactose, through the CRISPR / Cas9 gene editing system, the relevant genes in the synthetic and metabolic pathway of the original strain 2'-fucosyllactose are knocked out; Modular metabolic pathway, phosphomannose mutase (ManB), mannose‑1‑phosphate guanyltransferase (ManC), GDP‑mannose‑6‑dehydrogenase ( Gmd), GDP-fucose synthase (WcaG), L-fucose 1-kinase / GDP-fucose pyrophosphorylase (Fkp) and 2'-fucosyllactose synthase (FucT2) expression level, so that a higher concentration of 2'-fucosyllactose can be accumulated in the cell. The present invention also provides a method for efficiently producing 2'-fucosyllactose.

The invention belongs to the technical field of genetic engineering and molecular precision improvement and breeding of poultry breeds, and discloses a gene editing method for introducing a plasmid vector group, a DNA group and an exogenous gene, including: P1 plasmid, P2 plasmid and P3 plasmid; DNA components include: The RNA-mediated Cas9 endogenous endonuclease gene, the specific site-mediated sgRNA gene and the site of introduction of the gene fragment and the insertion site-specific homologous DNA fragment. The method of the present invention can integrate the target DNA fragment into the appropriate position of the genome of the poultry organism, and it is helpful to insert the target DNA fragment into the appropriate position of the animal cell genome, thereby obtaining transgenic or chimeric animal individuals. The carrier group of the present invention has the characteristics of easy construction, easy mass production, safety and high efficiency.

The invention discloses a plasmid composition, a DNAPK gene knockout rat model building method and application. A pair of specific transcription activator like effectors for specifically recognizing two sections of adjacent nucleotide sequences on rat DNAPK genes are built; the pair of transcription activator like effectors are used for obtaining a pair of transcription activator like effector nucleases; the rat DNAPK genes are subjected to accurate and efficient targeting; the DNAPK gene knockout rat model is further obtained.

The invention relates to the field of eukaryotic saccharomycetes gene modification and fermentation, and provides a recombinant plasmid combination, genetically modified saccharomycetes and a method for producing odd-chain fatty acid, the recombinant plasmid combination comprises at least one expression plasmid; the expression plasmid comprises at least one polynucleotide encoding a polypeptide having threonine deaminase activity, fatty acid synthase activity, propionyl-CoA synthase activity, aldehyde dehydrogenase activity or keto acid decarboxylase activity; the recombinant plasmid combination can be used for carrying out genetic engineering modification on microorganisms, so that expression regulation of key enzymes can be carried out in the modified microorganisms to establish a synthetic route of odd-chain fatty acid, the odd-chain fatty acid can be directly synthesized by utilizing L-threonine and (or) glucose of propionic acid or non-propionic acid, the fermentation cost is low, and the yield and proportion of odd-chain fatty acid can be further improved by a gene modification method.

The present invention relates to a lentiviral stable packaging cell line and a preparation method thereof. The lentiviral stable packaging cell line comprises a packaging plasmid group consisting of pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid Composition; the pPuro.coTetR plasmid comprises the CoTetR gene, the pVSVG plasmid comprises the VSVG gene, and the pGagPol-RRE-NES-cINT plasmid comprises the GagPol and Rev genes. The lentivirus stable packaging cell line is relatively stable in terms of passage, toxin production and genetic gene copy number, and the lentivirus produced by using the lentivirus stable packaging cell line has high titer and less impurity content.

The invention discloses a cationic liposome complex for encapsulating plasmid DNA, which is composed of cationic liposome and the encapsulating plasmid DNA, wherein the cationic liposome is composed of DOTAP and auxiliary lipid, and the plasmid DNA It is a plasmid carrier for at least one cytokine that promotes immune response; the principle of immunotherapy is applied in the present invention, and through the protection of the liposome, the recombinant gene of the pro-immune cytokine and anti-angiogenic protein can be transported in the blood, and the The liposome complex has the function of causing ischemic necrosis on tumor cell tissue and helping the body to recognize tumor antigens. The liposome GM csf complex of the present invention has a cure rate of 100% for early EMT6 breast cancer cell lung metastases, and a cure rate of 33% for late EMT6 cell lung metastases; the combination of GM csf gene and recombinant VEGFR gene The combined application of liposome complex can increase the cure rate to 50% for advanced EMT6 cell lung metastases.

Provided in the present disclosure is a method for using a four-plasmid system to prepare modified immune effector cells. The method comprises: forming a lentivirus by using four plasmids within 293T cells, extracting and obtaining the lentivirus, then transfecting immune effector cells by using the lentivirus, and expressing a chimeric antigen receptor. Also provided in the present disclosure is a use of the immune effector cell obtained by using the described method and of a composition containing the immune effector cell.

Provided in the present disclosure is a method for using a four-plasmid system to prepare modified immune effector cells. The method comprises: forming a lentivirus by using four plasmids within 293T cells, extracting and obtaining the lentivirus, then transfecting immune effector cells by using the lentivirus, and expressing a chimeric antigen receptor. Also provided in the present disclosure is a use of the immune effector cell obtained by using the described method and of a composition containing the immune effector cell.

The invention discloses a plasmid carrying nanoparticle for preventing and treating influenza virus and a preparation method thereof. The plasmid-carrying nanoparticles include transport vectors and plasmids, and the plasmids include a combination of plasmids and SP-dCas9-VPR plasmids that are used to suppress H1N1 viruses in the CRISPRa system, and the transport vectors are polymeric plasmid transport vectors or lipids Plasmid transfer vector. The prepared CRISPRa plasmid system of the present invention up-regulates the expression of the IFNⅢ gene by the plasmid-carrying nanoparticles, which can be used to prevent influenza virus infection in advance and treat the initial stage of influenza virus infection. This kind of prevention or treatment for influenza virus is suitable for influenza virus subtypes. It is universal, has great application value, and is worthy of vigorous promotion.

The invention discloses a gene expression regulation system and application thereof. The gene expression regulation and control system comprises a plasmid composition for gene expression regulation and control, the plasmid composition comprises a plasmid 1 and a plasmid 2, and the plasmid 1 is a plasmid with an expression nucleotide sequence as shown in SEQ ID NO.1; the plasmid 2 is a plasmid for expressing mini-CRISPR, and the mini-CRISPR contains a target sequence and a nucleotide sequence as shown in SEQ ID NO.5 at the 5'end and the 3 'end of the target sequence. The I-F type CRISPR-Cas double-plasmid system provided by the invention can accurately identify and combine a promoter region of a target gene, and effective inhibition on transcription expression of the target gene can be realized through conventional plasmid transformation. The system has obvious simplicity and convenience in bacterial gene function research, and meanwhile has wide application prospects in the aspects of bacterial physiology research, metabolic pathway transformation, natural compound synthesis, drug target discovery, novel drug development, high-throughput screening and the like.

The invention discloses an [alpha]-bisabolol synthetic plasmid as well as a construction method and an escherichia coli engineered strain thereof. An IPP and DMAPP synthetic plasmid pS-MVA and the [alpha]-bisabolol synthetic plasmid are constructed, and the two plasmids are introduced into an escherichia coli strain E.coli DH5[alpha]. The [alpha]-bisabolol synthetic plasmid takes a pTrc99A vectoras a skeleton plasmid and is assembled with an escherichia coli gene ispA and an artificially synthetic [alpha]-bisabolol synthetase gene or a wild-type [alpha]-bisabolol synthetase gene from different sources; the artificially synthetic [alpha]-bisabolol synthetase gene is selected from one of AaBBS, LdBBS, MrBBS, ScBBS and SsBBS; and the wild-type [alpha]-bisabolol synthetase gene is selected from one of LdBBS, ScBBS and MrBBS. According to the invention, the [alpha]-bisabolol is synthesized through a biological method; and the [alpha]-bisabolol is high in yield, high in activity and is green-going and environment-friendly.

The invention discloses a TALE library expression vector as well as a preparation method and application thereof, a PFUS-A + 10 * NH plasmid mutant, a PFUS-B7 + 7 * NH plasmid mutant, a pLR-NH plasmid mutant and a TALE-VP64 skeleton plasmid are assembled into the TALE library expression vector, the assembly is rapid and modularized, the generated RVD content and the whole DNA binding domain composition can be accurately controlled, and the TALE library expression vector has the advantages of high specificity, high specificity, high specificity, high specificity and high efficiency. The TALE library expression vector can be combined with any 18bp target DNA, and the TALE library expression vector disclosed by the invention has a wide application value in the technical fields of gene editing and biology; and researchers can directly use the material as an experimental material, so that a large amount of time is saved for the majority of TALE researchers.