598 results about "Multiple cloning site" patented technology

The invention relates to a mammal cell high-efficiency expression vector pSNEO. The vector is constructed on the basis of neomycin resistance screening gene NEO by combined strategy of weakening screening gene expression and reinforcing target gene expression. The screening gene weakening comprises the following two sides: introducing deletion mutant into the promoter to implement transcription weakening of the screening gene, and introducing a hairpin structure sequence before the translation initial site of the screening gene to implement the translation weakening of the screening gene. The expression reinforcement of the target gene is implemented by adding a transcription control element WPRE sequence between polyclone site and polyA tail of the target gene expression frame. The pSNEO vector can conveniently and quickly complete the construction of the stable high-expression cell strain of the target gene, and provides a new tool for preparing the high-polymer recombinant protein.

A group of modular cloning vector plasmids for the synthesis of a transgene or other complicated DNA construct, by providing a backbone having docking points therein, for the purpose of gene expression or analysis of gene expression. The invention is useful for assembling a variety of DNA fragments into a de novo DNA construct or transgene by using cloning vectors optimized to reduce the amount of manipulation frequently needed. The module vector contains at least one multiple cloning site (MCS) and multiple sets of rare restriction and / or unique homing endonuclease (“HE”) sites, arranged in a linear pattern. This arrangement defines a modular architecture that allows the user to place domain modules or inserts into a PE3 transgene vector construct without disturbing the integrity of DNA elements already incorporated into the PE3 vector in previous cloning steps. The PE3 transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

The present invention concerns methods and compositions for the construction of a series of stable vectors for genomic library construction useful in Gram negative species. In certain embodiments, the vectors contain the pBBR1 replicon, capable of to stable replication in a broad range of Gram negative species. In various embodiments, the plasmid vectors may also contain bidirectional, rho-independent transcriptional terminators flanking the multiple cloning site, which allows for greater insert stability, and thus, greater genomic representation. Each vector may vary in its selection marker region, mobilization function, and promoter used to express insert sequences. These vectors are of use in the screening of highly representational genomic libraries in a broad variety of Gram negative species.

The invention provides a simple and convenient Agrobacterium Tumefacies dual carrier containing double T-DNA structural regions, and a method of using the carrier system to cultivate transgenic rice without resistance selection label. With the help of the principle of co-conversion mediated by root nodule Agrobacterium Tumefacies, the system contains two separate T-DNA structural sections, where the first T-DNA region contains antibiotic resistance selection label gene and the second one contains a universal polyclonal site able to be arbitrarily inserted with destination gene. The double-T-DNA carrier has small molecular weight, easy to clone and after the destination gene and necessary regulation and control series are cloned in the T-DNA region containing the polyclonal site, it realizes rice co-conversion; by selfing, it selects transgenic individual with destination gene but without selectin label gene from the self-bred progeny, thus eliminating the negative effect on transgenic plant commercialized production, etc, possibly caused by selection label gene.

The present invention provides new lentiviral vectors, including lentiviral transfer plasmids and infectious lentiviral particles. Lentiviral vectors of the invention were designed to offer a number of desirable features including reduced size, convenient cloning sites (including multiple cloning sites and sites for particularly useful restriction enzymes), loxP sites, self-inactivating LTRs, etc. Certain of the vectors are optimized for expression of reporter genes and / or for expression of siRNAs or shRNAs within eukaryotic cells. The invention also provides three and four plasmid lentiviral expression systems. In addition, the invention provides a variety of methods for using the vectors including gene silencing in cells and transgenic animals, and methods of treating disease.

The invention discloses an RNA (Ribonucleic Acid) interference vector and application thereof. The RNA interference vector provided by the invention comprises FAD2 (fatty acid desaturase 2) gene intron and an upstream polyclone site and a downstream polyclone site on two sides of the FAD2 gene intron, wherein the FAD2 gene intron is the 60th-1190th site nucleotide from the 5' terminal of the sequence 1 in a sequence table. Proven by experiments of the invention, the invention provides an RNA interference vector suitable for plants; shRNA (short hairpin RNA) formed by expression of the RNA interference vector in a receptor is diced by Dicer into siRNA (small interfering RNA); and the siRNA can specifically inhibit the expression of a target gene, thereby achieving the aims of controlling the plant height of paddy and studying the CYP90D2 / D2 gene function.

A group of modular cloning vector plasmids for the synthesis of a transgene or other complicated DNA construct, by providing a backbone having docking points therein, for the purpose of gene expression or analysis of gene expression. The invention is useful for assembling a variety of DNA fragments into a de novo DNA construct or transgene by using cloning vectors optimized to reduce the amount of manipulation frequently needed. The module vector contains at least one multiple cloning site (MCS) and multiple sets of rare restriction and / or unique homing endonuclease (“HE”) sites, arranged in a linear pattern. This arrangement defines a modular architecture that allows the user to place domain modules or inserts into a PE3 transgene vector construct without disturbing the integrity of DNA elements already incorporated into the PE3 vector in previous cloning steps. The PE3 transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

The invention provides a shRNA lentiviral expression vector for specifically inhibiting hepatic cell CYP2E1 gene expression, comprising basic sequences, resistance gene sequences, multiple cloning site sequences, promoter sequences of PLKO.1-puro expression vector, and shRNA oligonucleotides sequences of target CYP2E1 genes; the shRNA oligonucleotides sequences are inserted into multiple clone sites forwards. shRNA lentiviral expression vector provided by the invention has the advantages of high transfection efficiency, little dosage, sustainability, high efficiency, stability and specificityfor inhibiting hepatic cell CYP2E1 gene expression, and can be used for preparing medicines for treating diseases related to abnormal CYP2E1 gene expression as powerful tools.

The invention provides a recombinant adenovirus vector for expressing an African swine fever virus (ASFV) EP402R gene, a construction method of the recombinant adenovirus vector and a preparation method of a recombinant adenovirus, belonging to the technical field of genetic engineering. The method provides recombinant adenovirus plasmids pAD-EP402R for expressing the EP402R gene, a pAD-EF1alpha-GFP adenovirus expression vector is used as basis, and the EP402R gene is introduced at a multiple cloning site. The invention also provides the preparation method of the recombinant adenovirus for expressing the EP402R gene; the contracted plasmids pAD-EP402R are mixed with an adenovirus packaging system PEI so as to realize the packaging process of the adenovirus, so that the recombinant adenovirus capable of directly infecting eukaryotic cells is obtained; therefore, the aim of the normal expression of the EP402R gene in the eukaryotic cells is realized, and a foundation is laid for the study of a recombinant adenoviral vector candidate vaccine based on expression of the EP402R.

The invention discloses a carrier and engineering bacteria to express the human serum albumin. The carrier is the plasmid which inserts the encoding human serum albumin sequence into the multi clone locus of the Pichia pastoris expression carrier. The 5'end of the encoding sequence of the serum albumin is connected to the encoding sequence of the secretory signal peptide and the leading peptide which the 5' end is connected to the sequence GAAACG of the AOX1 gene for the Pichia pastoris. The bacteria including the expression carrier especially the Pichia pastoris HSA75-10 CGMCCNo.1360 can be cultured to get the high yield of the human serum albumin (10g / L of the supernate) which is 80% of the secretory protein.

A chimeric virus-like granular DNA vaccine able to generate stronger B cell activity, exciting high antibody level, improving cell's immunizing level by cross presentation, and activating T cell, toxic T cell and complete immunoreaction is a recombinant eucaryotic expression carrier of coding gene with chimeric protein. Its preparing process is also disclosed.

The invention relates to a gene deletion system for the security control of transgene monocotyledon, comprising two homodromous loxP and FRT fused specificity recognition sites loxP-FRT, wherein a section of multiple clone site sequence for inserting a target gene to be led in plant is contained between the two specificity recognition sites. The gene deletion system can completely delete foreign genes in a specific plant organ by a plant expression vector which is formed by leading a monocotyledon tissue-specific promoter sequence, and can achieve the deletion efficiency of 99.8 percent, thereby being used for preparing the safe transgene monocotyledon.

The invention provides fowlpox virus vector shuttle plasmid pTGP3 which comprises recombinant arms TKL and TKR, a bidirectional promoter PE / L, a fluorescent protein expression cassette, and a resistant marker gene and replication origin ori; the upstream and the downstream of the bidirectional promoter PE / L are respectively provided with cloning sites MCSL and MCSR; and both ends of the fluorescent protein expression cassette are provided with loxp sequences. The plasmid of the invention has two different screening markers, and the recombinant fowlpox virus prepared with the plasmid can express 1 to 3 types of gene with different meshes in the whole processes of the early and the later periods; the strong composite promoter with expression activity in the early and the later periods is applied so as to realize the all-process high-efficiency expression of a target gene; and the loxp sequences are introduced into both ends of the fluorescent protein expression cassette, so as to knock out the exogenous recombinant fowlpox virus screening markers. The invention lays foundation for the series and the scale application of the recombinant fowlpox virus in vaccine and biological drug research and development fields.

The invention relates to a cloning vector and preparation and application thereof and discloses a cloning vector pUC57-ccdB which is a modified vector with ccdB genes inserted at multiple cloning sites of a pUC57 vector, wherein the ccdB genes have blunt-end restriction enzyme digested recognition sites. By means of the lethal effect of CcdB protein on escherichia coli not containing F plasmids, the ccdB genes having the restriction enzyme Sma I digested sites are inserted on the pUC57 plasmids through the molecular biological technology to obtain the vector pUC57-ccdB. Blunt ends are generated through Sma I digestion and are connected with the genes needing to be cloned so that insertion of the genes needing to be cloned can be achieved. Meanwhile, bacterial colonies with empty vectors are avoided. The cloning vector plays an important role in the fields of molecular biology and genetic engineering.

The invention provides a signal peptide-free recombinant vector for exogenous gene expression in a Kluyveromyces marxianus nutritional deficient strain as well as a preparation method and application thereof. The recombinant vector sequentially comprises an ampicillin resistance gene, a PKD1 vector, an inulase promoter, multiple cloning sites, an inulase terminator, a nutritional gene promoter and a nutritional gene open reading frame. The signal peptide-free recombinant vector and the preparation method thereof, constructed by the invention, can be used for constructing transformants to realize exogenous gene expression.

The invention provides a recombinant vector used for carrying out foreign gene secretory expression in an auxotrophic kluyveromyces marxianus strain as well as a preparation method and application of the recombinant vector. The recombinant vector comprises ampicillin resistance genes, a PKD1 vector, inulase promoters, signal peptides, multiple cloning sites, inulase terminators, nutritional gene promoters and a nutritional gene open reading flame according to the sequence. The constructed recombinant vector used for carrying out foreign gene secretory expression and the preparation method of the recombinant vector can be used for constructing a transformant to achieve secretory expression of foreign genes.

The invention discloses a single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof. The bacillus subtilis shuttle expression vector provided by the invention comprises the following elements: a bacillus subtilis formed strong promoter P43, a sequence which encodes signal peptide, multiple cloning sites, an antibiotics resistance gene (kalamycin or chloramphenicol), a bifunctional promoter sequence which can start resistance gene expression of kalamycin or chloramphenicol in escherichia coli and bacillus subtilis, and copy homing sequences of bacillus subtilis and escherichia coli. The expression vector disclosed by the invention not only can be stably copied in escherichia coli, but also can be stably copied in bacillus subtilis, and expresses homologous and heterologous proteins. All vectors just contain one resistance gene, the plasmid of which is less than 400bp, so that gene operation and plasmid conversion are easy to carry out, and the conversion ratio is high. Besides the expression vector of an exogenous gene, the vector can be further used for screening promoters and constructing a gene knock-out vector.

The invention provides a promoter optimized lentiviral expression system. The promoter optimized lentiviral expression system is characterized in that an MSCV promoter is adopted; the genetically modified T cells of the promoter optimized lentiviral expression system are enabled to express an anti-tumor T cell receptor (TCR), and are characterized in that TCRalpha, furin, an interval sequence, a 2A peptide and a TCRbeta sequence are orderly inserted in multiple cloning sites; and a chimeric antigen receptor (CAR) expression vector constructed by use of a promoter optimized lentiviral vector is characterized in that a CAR sequence is inserted in the multiple cloning sites. The recombinant lentiviral vector can be applied to, for example, expressing the recombinant TCR in the T cells and expressing the CAR in T or NK cells to achieve the purpose of specifically killing tumors, and also can be applied to clinical oncotherapy.

The invention provides a method for establishing a recombinant adenovirus vector with Africa swine fever EP153R and P54 gene coexpression and packaging adenovirus and belongs to the technical field ofgene engineering. According to the adenovirus vector of coexpression of the EP153R gene and the P54 gene, with a pShuttle-CMV eukaryotic expression vector as a basis, the CTLA4, EP153R and P54 genesare introduced at multiple cloning sites. The invention further provides recombinant adenovirus with coexpression of the EP153R and P54 genes. The established pAD-Shuttle-CMV-CTLA4-EP153R-P54 vector is used for achieving the adenovirus packaging process, the adenovirus which can directly infect animals or eukaryocyte is obtained, the aim of coexpression of the EP153R and P54 genes in eukaryocyte is achieved, and the basis is laid for research on adenovirus vaccines of coexpression of the EP153R and P54 genes.

The invention discloses an optimization gene for coding chicken interferon alpha and application thereof in preparing the chicken interferon alpha. The optimization gene is DNA (deoxyribonucleic acid) shown as the sequence 2 in a sequence table. The expression ability of the gene is far higher than that of the traditional gene; the gene is inserted into the polyclone site of pET28b(+) to obtain a recombinant plasmid, and the recombinant plasmid is imported into escherichia coli BL21 to obtain a recombination strain. A large amount of ChIFN alpha protein can be prepared through carrying out simple induction culture on the recombination strain. The gene, the recombinant plasmid and the recombination strain has economic value for the production of the chicken interferon alpha and also has great value for broiler culture industry.

The invention relates to a replication defective recombinant adenovirus Ad41 vector system and an application thereof in the field of gene therapy and recombinant vaccines. Specifically, the vector system comprises a backbone plasmid, a shuttle plasmid and a packaging cell line, wherein, the backbone plasmid contains an adenovirus Ad41 genome which deletes an encoding region required for packaging adenovirus, the shuttle plasmid contains multiple cloning sites used for inserting a target gene and an adenovirus Ad41 genomic fragment used for carrying out homologous recombination with the backbone plasmid, and the packaging cell line integrates genes required for packaging adenovirus Ad41 in the cell genome and can stably express the packaging genes. The generated recombinant Ad41 virus has broad application prospects in intestinal gene therapy and research of oral recombinant vaccines.

The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo / GFP and Hyg / RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.

The invention relates to a bacillus thuringiensis cry1Ah / cry1Ie bivalent gene expression vector and an application thereof, belonging to the technical field of biology. pCIABIA3301 is taken as a vector framework, and m2-cry1Ah and m2-cry1Ie genes are inserted on multiple cloning sites of the pCIABIA3301 to get the expression vector pMAhIeb. An agrobacterium-mediated transformation method is utilized for transforming the constructed expression vector to a species of a maize inbred line to get a high-resistance transgenic bivalent gene maize transformation event. The research of expression and genetic stability of a Cry1Ah protein of the two transformation events shows that the Cry1Ah protein can realize normal and stable expression in T1-T2 generations of maize plants; and furthermore, the expression level is very high, and the resistance of the plants to pyrausta nubilalis in a field is also genetic stably. The obtained insect-resistant transgenic maize material has great application value and can be used as a candidate material for next-step Bt insect-resistant maize breeding work.

The present invention relates to gene engineering bacteria, and is especially one expression vector for high efficiency secreting expression on mannase gene and its multicopy body. Primer is designed according to the cloning site of Pichia yeast pAO815, the polyclonal site region of the target expression vector is replaced into Not I, XhoL I and EcoR I to form polyclonal sites for easy cloning, the alpha-factor signal peptide and the target gene are fused and connected to the expression vector for the expression vector containing the target gene to secrete and express mannase, and the restriction enzymes Bgl II and BamH I with complementary sticky ends are cleaved, linked and converted to obtain multicopy expression unit plasmid containing mannase gene. The multicopy body after transforming Pichia yeast can express mannase in high efficiency and is used to prepare mannase from konjaku meal.

The invention discloses a Lac shuttle vector, comprising at least (a) a region which regulates a plasmid copy number; (b) an eukaryotic gene expression cassette, which comprises at least an eukaryotic gene transcriptional promoter sequence, a multiple cloning site and a transcriptional terminator sequence; (c) a lactic acid bacteria plasmid sequence, which comprises a plus origin of replication, and a nucleic acid sequence encoding for a protein which relates to the lactic acid bacteria plasmid replication; and (d) a non-antibiotic resistance selection gene and the promoter sequence thereof. The Lac shuttle vector features a non-antibiotic resistance gene as a selection marker, which is useful in pharmaceuticals and foods.

The invention discloses a late promoter targeting adjustment and control oncolytic adenovirus pCN305 vector, a construction method and application thereof. The vector is established on a pCN103 vector with targeting property; all exogenous genes for killing and inhibiting various cancers, such as IL-24, TRAIL, SOCS3, cpp-SOCS3, SOCS1, cpp-SOCS1and so on, are adjusted, controlled and expressed by late promoters of viruses through induction of internal ribosome into IRES sites and polyclonal sites; and the pCN305 vector has good anticancer effect through experiment in vivo and vitro. All double-target replicating oncolytic adenovirus established by application of the pCN305 vector, such as AdCN305-IL-24, AdCN305-TRAIL, AdCN305-SOCS3, AdCN305-cpp-SOCS3, AdCN305-SOCS1, AdCN305-cpp-SOCS1 and so on, can be used for treating various tumors. Moreover, the invention can develop a novel virus-gene anticancer medicine for effectively treating the tumors.

The invention discloses a recombinant porcine pseudorabies virus strain (belonging to Herpesviridae) and a preparation method thereof. The recombinant porcine pseudorabies virus strain has the accession number of CCTCC NO: V201345. Based on porcine pseudorabies virus vectors, green fluorescent protein labels are used so that green fluorescent protein expression gG-less universal transfer vector PG is constructed, and the recombinant porcine pseudorabies virus strain contains a pseudorabies virus self-late gene gG promoter, a CMV promoter and SV40Poly(A), has upstream flanks of 0.8kb and downstream flanks of 1.7kb and completely satisfies homologous recombination demands. Enhanced green fluorescent protein (EGFP) can be used as a gene expression indicator and comprises porcine IL-18 gene inserted into multiple cloning sites of the EGFP so that connection construction of different expression cassettes by a common vector is realized and two expression cassettes are individually expressed. The exogenous gene expression quantity is closely related to the connection direction of the two expression cassettes.

The invention relates to lactic acid galactococcus food-grade secretion expression vector, and the preparation method and the application thereof, in particular to the lactic acid galactococcus food-grade secretion expression vector which comprises the following components: a thyA gene selection marker, replicons of plasmid pWV01, multiple cloning sites, promoter used for secretion expression, a ribosome bind site of Usp45, a signal peptide sequence of Usp45 and a partial Usp45 mature peptides coded sequence.

The invention belongs to the field of molecular biology, and relates to an efficient and safe transposon integration system and use thereof. The invention also relates to a nucleic acid construct and use thereof. Preferably, the nucleic acid construct comprises the following elements in order: a 5′-terminal repeat sequence of a transposon, a multiple cloning site, a polyA tailing signal sequence, a 3′-terminal repeat sequence of a transposon, a sequence encoding a transposase and a promoter controlling expression of the transposase; wherein the multiple cloning site is used for operably inserting an exogenous gene and optionally a promoter controlling expression of the exogenous gene; the polyA tailing signal sequence has a polyA tailing signal function in both forward and reverse directions; and the direction of the expression cassette of the transposase is opposite to the direction of the exogenous gene expression cassette. The nucleic acid construct is useful for mediating efficient and safe expression of an exogenous gene in a host cell.

The invention discloses a universal gene-knockout suicide vector for vibrios and a construction method theroef and provides an application thereof in gene knockout of the vibrios. The universal gene-knockout suicide vector pLP12 is a ring-shaped vector and comprises a PBAD promoter, a repressor protein gene araC, an RP4 transferring initiation site, a chlorampenicol resistant gene, an R6K duplicating initiation site, a multiple-cloning-site area and a lethal gene vmi480; the multiple-cloning-site area at least contains two AhdI restriction enzyme digestion sites; the suicide vector pLP12 is subject to AhdI restriction enzyme digestion to form linearized suicide vector pLP12T. The universal gene-knockout suicide vector adopts entirely-new reverse selection genes vmi480 and is used for replacing the common sacB gene. Foreign fragments carried by the pLP12T are transferred to vibrio cells to be mutated by a jointing mode, under the pressure of antibiotics and reverse selection of products of lethal gene vmi480, first-time homologous recombination and second-time homologous recombination are carried out on the vibrios successively, and finally the mutant strain with deletion of target genes is generated.