24 results about "Shuttle vector" patented technology

The present disclosure relates to the seminal discovery of a generation and use of genetically engineered Vibrio sp. Provided is the use of the genetically engineered bacteria for the construction, maintenance, manipulation, and / or propagation of DNA constructs; protein expression; protein secretion; vectors and other metabolic tools; metabolic engineering; expression of cellular extracts for cell-free biology; shuttle vectors; cloning vectors; and for synthetic biology applications. The disclosure also relates to the use of the replication machinery of Vibrio sp. as a cloning or expression vector for replication of recombinant DNA constructs. The disclosure also relates to methods of use of the above.

The invention relates to the technical field of biological genetic engineering, in particular to construction of recombinant lactobacillus plantarum of an African swine fever virus antigen, and the method comprises the following steps: constructing a lactobacillus plantarum shuttle vector containing different ASFV antigen genes; and referring to a genome sequence of a Chinese isolate CADCHN09 of ASFV disclosed in GenBank. The method has the advantages that an inducer is not needed, antigen protein is efficiently expressed, the dendritic cell (DC) binding peptide for enhancing immune cell uptake and processing is subjected to fusion expression, the oral immunization mode is simple, mucosal immunity is stimulated, and ASFV infection is effectively prevented and controlled; probiotics, namely lactobacillus plantarum, which naturally exist in animal intestinal tracts and can be colonized and copied in digestive tracts for a long time are used as a carrier; key epitopes of proteins such as nucleocapsid protein p72, outer envelope protein CD2v, inner envelope protein p54, p22, pE199L and pE248R, which play an important role in adsorption, internalization, endocytosis and membrane fusion of ASFV, are preferably fused with dendritic cell binding peptide and then are displayed on the surface of lactobacillus plantarum.

The invention relates to the field of PQQ production, in particular to an engineering bacterium for producing pyrroloquinoline quinone and a preparation method of the engineering bacterium. According to the invention, different broad-host shuttle vectors are used, PQQ is expressed in denitrifying hyphomicrobe to synthesize related gene clusters, denitrifying hyphomicrobe engineering bacteria with high yield of PQQ are successfully constructed, and the engineering bacteria have the characteristics of rapid growth, high fermentation yield and the like, and lay a foundation for realizing green production of PQQ.

This invention discloses a recombinant adenovirus vaccine targeting the Tp0326 antigen and its preparation method, comprising a recombinant adenovirus vector and the Tp0326 antigen expressed therein. The recombinant adenovirus vector uses a replication-defective human adenovirus type 5 (Ad5) as a backbone, inserting the full-length Tp0326 antigen gene of *Treponema pallidum* into the adenovirus shuttle vector pshuttle-IRES-rGFP-1, and obtaining it through homologous recombination with the adenovirus backbone plasmid pAdEasy-1. This invention, using a replication-defective Ad5 as a vector, provides a recombinant adenovirus vaccine with high safety and efficient expression of the full-length Tp0326 antigen. The expressed antigen has a natural conformation, retains the ECL4 immunodominant epitope, and is more likely to induce a specific immune response, resulting in stronger immunogenicity. The preparation method involves constructing a recombinant adenovirus vector through homologous recombination, packaging it in HEK293T cells, and purifying it by CsCl density gradient centrifugation to obtain a high-purity, high-titer recombinant adenovirus vaccine suitable for large-scale production. The recombinant adenovirus vaccine can be administered via intramuscular or intranasal injection.

The invention discloses a green fluorescence report system for screening a Listeria monocytogenes quorum sensing inhibitor as well as a construction method and application of the green fluorescence report system. The report system is constructed through the following steps: synthesizing a DNA fragment according to a listeria monocytogenes AGR system pII promoter sequence, connecting the DNA fragment with a green fluorescent protein pEGFP plasmid, amplifying an EGFP-pII target fragment through PCR, and connecting the target fragment to a pKSV7 shuttle vector plasmid through homologous recombination to obtain a pKSV7-EGFP-pII plasmid, and then transferring the gene into a competent cell of listeria monocytogenes ATCC BAA-679, and screening to obtain a report system. The report system disclosed by the invention can be used for intuitively evaluating the expression condition of the quorum sensing system of the listeria monocytogenes and screening out the quorum sensing inhibitor.

The present disclosure provides a recombinant adenovirus for expressing African swine fever virus (ASFV) EP153R-EP402R protein and a construction method thereof, and belongs to the technical field of genetic engineering. In the present disclosure, a recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R is obtained through a series of intermediate processes using a recombinant adenovirus shuttle vector pENTRE-EGFP-TOPO; the recombinant adenovirus vector is linearized to transfect AD293 cells, a recombinant virus is screened according to cytopathy formed by adenovirus infection, an adenovirus packaging process is achieved, and the recombinant adenovirus for expressing ASFV EP153R-EP402R protein is obtained, laying a foundation for the construction of a recombinant adenovirus vaccine for expressing the ASFV EP153R-EP402R protein.

The present invention addresses the problem of providing a novel vector using a replication initiation point derived from Thermus thermophilus. The problem is solved by providing a shuttle vector that includes a specific first replication initiation point derived from Thermus thermophilus and a second replication initiation point derived from a biological species other than the Thermus thermophilus.

The present application provides a shuttle vector, which can be applied to various host cells for operation, thereby providing a novel tool in the field of genetic engineering. The present application further provides a prokaryotic host cell containing the shuttle vector, so as to use the shuttle vector to construct an expression vector containing a gene to be expressed, thereby allowing mass production of proteins in different hosts by using a single vector.

The invention belongs to the technical field of information, and discloses a copper inducible plasmid which comprises a yeast shuttle vector, a Candida glabrata-derived CEN / ARS element loaded on the yeast shuttle vector, a copper ion inducible promoter loaded on the yeast shuttle vector and a target gene, the target gene is connected to the downstream of the copper ion induced promoter. After being transfected into candida glabrata, the plasmid can show the following advantages: the plasmid is stable, the stability of host bacteria cannot be influenced on the basis of controllable expression of the dosage of an inducer, and simultaneous expression of various target proteins can be realized on the basis of loads of different target genes of the plasmid. Meanwhile, the invention also discloses an inducible expression method of the target gene.

The invention discloses a method for improving hypoxia damage resistance of islet cells, and belongs to the technical field of biology. According to the invention, islet cells in a packaging bracket and a culture medium containing a mesenchymal stem cell exosome are incubated together, and the mesenchymal stem cell exosome is loaded with a miRNA mimics composition and a miRNA inhibitor; the miRNA mimic composition is prepared from one or more of miR-539-3p mimic, miR-21-5p mimic, miR-27a-3p mimic and miR-155-5p mimic, and the miRNA mimic composition is prepared from one or more of miR-539-3p mimic, miR-21-5p mimic, miR-27a-3p mimic and miR-155-5p mimic; the miRNA inhibitor is a miR-485-3p inhibitor, and the miRNA inhibitor is a miR-485-3p inhibitor. According to the present invention, the exosome is adopted as the natural shuttle vector, the miRNA mimics and / or the inhibitor are / is loaded into the islet cells, the hypoxia damage resistance gene expression of the islet cells is subjected to targeted regulation, the survival of the islet cells under the hypoxia condition is promoted, and the activity of the islet cells under the hypoxia condition can be effectively improved by using the method provided by the present invention.

The invention discloses a novel efficient clostridium pasteurianum genetic transformation method which comprises the following steps: culturing and collecting clostridium pasteurianum thalli, centrifuging, washing, adding SMP buffer for resuspension, adding carrier plasmids or methylated plasmids, uniformly mixing, carrying out ultrasonic treatment on the obtained suspension under an ice bath condition, and carrying out freeze drying to obtain the clostridium pasteurianum genetic transformation. And transferring the suspension subjected to ultrasonic treatment into an electric revolving cup for electric shock treatment, quickly adding the suspension into a culture medium after electric shock, resuscitating, coating resuscitated bacterial liquid onto an RCM culture medium solid plate containing chloramphenicol resistance, culturing, and counting the growth condition of bacterial colonies. A novel transformation system is adopted for the clostridium pasteurianum, ultrasonic treatment is performed before genetic vector electric shock transformation, shuttle vector plasmids are subjected to methylation treatment, the obtained transformation efficiency is far higher than that of single ultrasonic treatment or single electric shock treatment, and a new way for transferring exogenous genes into the clostridium pasteurianum is developed; the method is simple, high in operability and good in stability.

The present invention belongs to the field of molecular biology and provides a shuttle vector for expression in Escherichia coli (E. coli) and Bacilli, said shuttle vector comprising a high-copy replication origin functional in E. coli, a low to medium-copy ORI functional in Bacilli, and a synthetic constitutive regulatory nucleic acid that confers reduced constitutive expression compared to the corresponding initiation regulatory nucleic acid molecule in bacterial cells.

The present invention relates to an L-tryptophan-producing engineered strain of Clostridium butyricum and use thereof in the preparation of a drug for treating tumors, and pertains to the technical field of biomedical engineering. The tryptophan operon gene trpEDCBA is overexpressed in Clostridium butyricum by means of a thl promoter and a pMTL82151 shuttle vector to construct an engineered strain of Clostridium butyricum with a high L-tryptophan yield. The strain can specifically target and colonize tumors in mice without additional treatment. The strain secretes butanoic acid and tryptophan metabolites to inhibit IDO enzyme expression, regulate CD8+ T cell metabolism, and change the immune microenvironment of tumors, thereby achieving the purpose of inhibiting tumor growth. In addition, the strain will not affect the physiological structures of the major organs in the body, showing a favorable safety profile.

The application discloses application of a broad-spectrum type III esterase derived from Rhodococcus in phthalate ester pollution bioremediation, and overcomes the limitation of low degradation efficiency of a natural strain; through genetic engineering technology, the esterase 5359 gene is cloned into a Rhodococcus-E.coli shuttle vector pNV18 to construct a recombinant Rhodococcus engineering bacterium WT-pNV18-5359 overexpressing the esterase. Experimental results show that the engineering bacterium significantly improves the degradation performance: in single PAEs degradation test, the degradation rates of long-chain PAEs such as DEHP, DnOP and DiNP are respectively increased by 15.71%, 13.99% and 12.82% compared with the wild type; in a mixed PAEs system, the degradation of long-chain PAEs is obviously superior, and the engineering bacterium has good genetic stability and does not affect growth. Through genetic engineering to strengthen the application of known enzymes, efficient and stable PAEs pollution bioremediation is realized.

The invention discloses a shuttle vector system based on blue-green algae endogenous plasmids, and belongs to the technical field of genetic engineering. The shuttle vector system comprises a ternary replication element, and the ternary replication element is a replication part of a synechococcus plasmid pANS or a replication part of a synechococcus plasmid pANL, a replication part of an escherichia coli plasmid pUC19 and a replication part of a saccharomyces cerevisiae plasmid pRS416 or a replication part of a saccharomyces cerevisiae plasmid pRS415. The shuttle vector system can shuttle and copy among three different hosts, namely synechococcus, escherichia coli and saccharomyces cerevisiae, and simultaneous expression of heterologous genes is realized by utilizing expression advantages of different hosts. The shuttle vector system has the advantages of being easy to operate, short in preparation period and convenient for large-scale preparation, and more possibilities are provided for available genetic tools of blue-green algae genetic engineering.

The invention discloses an artificial synthesis method and application of a vesicle virus shuttle vector. The shuttle vector of the vesicular virus takes a whole genome of the vesicular virus of the budworm as a basis, and 13 gene segments from AC1 to AC13 are obtained through segmented PCR (Polymerase Chain Reaction) amplification. The method comprises the following steps: dividing gene segments into four groups, transforming the four groups into yeast protoplast spheres, and obtaining four large gene segments from AC-1-pGF-TAR to AC-4-pGF-TAR by virtue of a transformation-associated recombination cloning (TAR) technology, wherein the four large gene segments are obtained from the AC-1-pGF-TAR to the AC-4-pGF-TAR; according to the invention, the vesicular virus shuttle vector is artificially synthesized on the basis of the whole genome of the insect vesicular virus, can be used as a molecular operation platform of the insect vesicular virus, obtains a rapid and efficient insect vesicular virus editing system, and plays an important role in theoretical research and production application.

The invention discloses an engineered nitrogen-fixing vibrio natrieae and a preparation method and application thereof, and relates to the field of synthetic biology, a nitrogen-fixing gene nif gene cluster is cloned and assembled on an escherichia coli-yeast shuttle vector to form recombinant plasmids, the plasmids are transformed and extracted by escherichia coli and then transformed into wild vibrio natrieae, and the engineered nitrogen-fixing vibrio natrieae is obtained. And obtaining the nitrogen-fixing vibrio natriivibrio. The invention further discloses the obtained engineered nitrogen-fixing vibrio natriticus and application thereof. The invention develops novel vibrio natriticus capable of efficiently fixing nitrogen in a broad spectrum as well as a preparation method and application of the novel vibrio natriticus.

To provide plasmid DNA with a novel replication origin that can stably introduce foreign genes into actinomycetes. [Solution] The plasmid vector according to the present disclosure comprises (a) a nucleotide sequence having a specific sequence, or (b) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted in the nucleotide sequence having the specific sequence.

This invention provides a chimeric adenovirus vector Ad5F35 and a recombinant CCR5-32 mutant gene, its construction method, and its application, belonging to the field of gene therapy technology. The construction method provided by this invention includes the following steps: (1) inserting the CCR5Δ32 mutant gene into the multiple cloning site of a shuttle vector to obtain a shuttle plasmid; (2) co-transfecting cells with the shuttle plasmid and the Ad5F35 adenovirus backbone plasmid to generate an Ad5F35 chimeric recombinant adenovirus capable of expressing the CCR5Δ32 gene. By inducing the inability of the CCR5 protein on the host cell to be normally expressed on the cell membrane surface, the binding of HIV-1 gp 120 to the CCR5Δ32 mutant gene is effectively prevented, thus preventing the HIV-1 virus from entering the host cell for replication, thereby achieving the goal of treating AIDS.

The invention relates to the technical field of biology, in particular to a novel recombinant vaccinia virus shuttle vector and application thereof.The shuttle vector comprises homologous recombination arms composed of an upstream gene sequence and a downstream gene sequence of vaccinia virus thymidine kinase TK, and the homologous recombination arms are VACV-093 and VACV-095 respectively; the kit further comprises three exogenous gene expression cassettes, and the three exogenous gene expression cassettes respectively contain a pTK11 heterozygous promoter, a pSE / L promoter and a p7.5 K promoter. The invention innovatively provides a new strategy for completely removing a TK open reading frame, retaining a pTK promoter and constructing a hybrid strong promoter, and solves the problem of promoter limitation during multi-gene expression and the problem of low screening efficiency of recombinant vaccinia virus.

The invention discloses a method for constructing a recombinant baculovirus for expressing L-amino acid oxidase of yellow-spotted bluefish. The method comprises the following steps: cloning a gene with a DNA sequence as shown in SEQ ID NO: 1 into a vector pMD18-T to construct a recombinant plasmid; using restriction endonucleases EcoRI and XhoI to digest the recombinant plasmid and a donor plasmid, respectively, and ligating the digestion products to obtain a recombinant donor plasmid; transforming the recombinant donor plasmid into Escherichia coli, allowing the target gene to specifically transpose with a baculovirus shuttle vector contained in the Escherichia coli, and screening and extracting the recombinant bacmid; and transfecting the recombinant bacmid into insect cells using a liposome transfection method. After the insect cells become pathological, the recombinant baculovirus is harvested.

The present application provides a shuttle vector that can be operated in a variety of host cells, thereby providing a new tool in the field of genetic engineering. On the other hand, the invention further provides a prokaryotic host cell containing the shuttle vector, so that the shuttle vector is utilized to construct an expression vector containing a gene to be expressed, and a single vector can be utilized to massively produce proteins in different hosts.

The invention provides a resistance marker-free auxotrophic bacillus subtilis and a construction method and application thereof, the newly constructed strain takes bacillus subtilis 168 as a starting strain, indole-3-glycerophosphate synthase gene trpC of the bacillus subtilis 168 is knocked out, synthesis of tryptophan in the growth and metabolism process of the strain 168 is eliminated, and the resistance marker-free auxotrophic bacillus subtilis is obtained. The shuttle vector YH46 is constructed by taking pMD-19T as a framework, so that the shuttle vector YH46 can normally grow only in the presence of exogenous tryptophan, and meanwhile, a universal auxotroph shuttle vector YH46 is constructed by taking pMD-19T as the framework, so that convenience is provided for high-efficiency expression of genes in bacillus subtilis; the tryptophan auxotrophic strain BS-TR is constructed through double exchange by using a homologous recombination principle, the construction process is simple and easy to implement, and the strain BS-TR does not contain a resistance gene selection marker, can be used as a food-grade engineering bacterium, and can be used for intracellular expression or extracellular secretion of target protein for production.

The invention relates to a construction method of a recombinant baculovirus shuttle vector Bacmid '. The method comprises the following steps: cloning a lacZ alpha-attTn7 sequence into a baculovirus transfer vector subjected to reverse amplification, so as to obtain a recombinant plasmid pBacPAK8-lacZ alpha-attTn7; the recombinant virus AcNPV-lacZalpha-attTn7 is obtained by co-transfecting Sf9 cells with a defective virus Bacmid, and a recombinant virus AcNPV-lacZalpha-attTn7 is generated; carrying out amplification and ultra-high-speed centrifugation on the obtained recombinant virus, and extracting through phenol chloroform to obtain virus DNA (Deoxyribonucleic Acid); the recombinant baculovirus shuttle vector Bacmid'is obtained by co-transforming competent cells of recombinant virus DNA (deoxyribonucleic acid) and pBvBAC (obtained by cloning Vcat, miniF and Chitinase to pUC19), and carrying out recombinant cloning by utilizing blue-white spot screening. According to the present invention, the miniF replicon co-localized with lacZalpha-attTn7 is separated, such that the recombinant baculovirus shuttle vector Bacmid'with the high gene cloning stability is successfully constructed; according to the technology, the expression stability of the exogenous gene is remarkably improved, and a powerful research tool is provided for vaccine development and virology application based on the baculovirus vector.