512 results about "Cloning Site" patented technology

The invention provides an mVSV virus vector, i.e., attenuated mVSV obtained after multiple modification mutations occur to an M protein amino acid site of a wild Indiana strain VSV, and an optimized heterologous antigen gene is preferentially integrated to a double cloning site area of an mVSV packaging core plasmid pmVSV-Core at the same time. The mVSV virus vector vaccine comprises a heterologous antigen gene which fuses or embeds a target virus between G and L genes of an mVSV vector envelope, wherein the antigen gene comprises an enveloped and embedded antigen gene encoding the target virus, an embedded combination antigen gene or a fused antigen gene; the mVSV virus vector is embedded or fused with a dominant antigen of spike protein S of an SARS-CoV-2 pathogen; the dominant antigen is preferably selected from a receptor binding domain of spike protein S, namely RBD; and a COVID-19 vaccine based on mVSV mediation is formed. The vaccine has good prevention or treatment effect on COVID-19 infected people.

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.

An object of the present invention is to provide a transformation system for Labyrinthulomycota that allows the elucidation of biosynthetic mechanisms of lipids such as PUFA and carotenoids as well as for the construction of a high production system and the design and development of novel functional lipid molecules by the control of the mechanisms. The present invention provides a vector for the transformation of Labyrinthulomycota with a transgene, which comprises at least (1) a nucleotide sequence which is homologous to a part of chromosomal DNA of Labyrinthulomycota and is capable of homologous recombination with the chromosomal DNA, (2) a selection marker gene having a promoter sequence located upstream and a terminator sequence located downstream, and (3) a cloning site for transgene insertion having a promoter sequence located upstream and a terminator sequence located downstream.

A protein expression vector containing (a) a nucleotide sequence encoding an IgG(κ) or a trypsin secretory signal peptide, (b) a nucleotide sequence encoding a polyhistidine amino acid sequence, (c) a nucleotide sequence encoding an amino acid sequence comprising amino acid residues 36–40 of SEQ ID NO:19 (Asp-Asp-Asp-Asp-Lys), which is cleavable by an enterokinase, and (d) a cloning site into which a nucleotide sequence encoding a target protein can be inserted, wherein (a), (b), (c) and (d) are assembled within the vector in the order recited.

A process for the production of humanized chimera antibody, wherein the chimera antibody is produced easily without changing any of the amino acids of its mouse antibody variable region, which comprises the steps of: (1) constructing a cassette vector by inserting a cDNA coding for a heavy chain constant region of human antibody into an expression vector for animal cell use and establishing a cloning site in the upstream region of the heavy chain constant region of said cassette vector for inserting a cDNA which encodes a heavy chain variable region of nonhuman animal antibody; (2) digesting a cDNA coding for the heavy chain variable region of nonhuman animal antibody with restriction enzymes; (3) inserting said cDNA coding for the heavy chain variable region of nonhuman animal antibody into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5'-end side of said heavy chain constant region of human antibody and a base sequence corresponding to the 3'-end side of said heavy chain variable region of nonhuman animal antibody and is possessed of the restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody heavy chain expression vector in which said cDNA coding for the heavy chain constant region of human antibody and said cDNA coding for the heavy chain variable region of nonhuman animal antibody are linked together through said synthetic DNA; (4) constructing a cassette vector by inserting a cDNA coding for a light chain constant region of human antibody into an expression vector for animal cell use and establishing a cloning site in the upstream region of the light chain constant region of said cassette vector for inserting a cDNA which encodes a light chain variable region of nonhuman animal antibody; (5) digesting a cDNA coding for the light chain variable region of nonhuman animal antibody with restriction enzymes; (6) inserting said cDNA coding for a light chain variable region of nonhuman animal antibody into the cassette vector using a synthetic DNA which comprises a base sequence corresponding to the 5'-end side of said light chain constant region of human antibody and a base sequence corresponding to the 3'-end side of said light chain variable region of nonhuman animal antibody and is possessed of the restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody light chain expression vector in which said cDNA coding for the light chain constant region of human antibody and said cDNA coding for the light chain variable region of nonhuman animal antibody are linked together through said synthetic DNA; (7) introducing these expression vectors into host cells to obtain a transformant; and (8) culturing said transformant in an appropriate culture medium, thereby allowing the transformant to produce and accumulate a humanized chimera antibody, and collecting said humanized chimera antibody from the resulting culture broth.

A colibacillus-corynebacterium inducible expression carrier pDXW-8 and a building method thereof belong to the technical field of genetic engineering. The inducible expression carrier pDXW-8 can be copied and stably exists in colibacillus and corynebacterium and comprises a tac promoter, a terminator rrnBT1T2, a resistance marker card kanamycin mark resistance gene, a negative regulation gene laclPE104 and a multi-cloning site, wherein the tac promoter plays a transcript promoting function in the colibacillus and the corynebacterium, the terminator rrnBT1T2 has a transcript stopping function, the resistance marker card kanamycin mark resistance gene is used for selecting transformants of the corynebacterium, the negative regulation gene is used for severely controlling expression of the tac promoter, and the multi-cloning site includes 11 signal limited incision enzyme sites. In the corynebacterium, the carrier is moderate in foreign protein expression, and is particularly adaptable to study on corynebacterium metabolic engineering.

Disclosed herein are cloning vectors which include: (a) a cloning site which permits the cloning of a nucleic acid in defined orientation; (b) at least one cleavage site adjacent to the cloning site, the cleavage site being rarely-occurring in nucleic acids; and (c) a long region which is located on the side of the cloning site opposite to the cleavage site (b), wherein the long region and the region between the cloning site and the cleavage site (b) contain neither the cloning site nor at least two frequently-occurring cleavage sites.

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 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 vector used in a Kluyveromyces marxianus auxotrophic strain, and a preparation method and application thereof. The recombinant vector sequentially comprises a PKD1 vector, an inulase promoter, an inulase signal peptide, a polyclone site, an inulase terminator, a nutrient gene promoter and a nutrient gene open reading frame. The constructed recombinant vector and preparation method thereof can be used for constructing a transformant, thereby implementing expression of the exogenous gene.

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.

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 discloses a method for constructing a secretory expression carrier and secretory expression system of Chlammydomonas reinhardtii. In the invention, the method comprises the steps of predicating a signal peptide gene sequence by using an amino acid sequence from a secretory protein of the Chlammydomonas reinhardtii through signal peptide predicating software; artificially synthesizing a signal peptide gene obtained by predicating; and inserting the signal peptide gene between a promoter and a cloning site of the Chlammydomonas reinhardtii expression carrier to construct the secretory expression carrier of the Chlammydomonas reinhardtii; meanwhile, detecting the expression efficiency of the constructed secretory expression carrier of the Chlammydomonas reinhardtii by using an exogenous gene; converting the secretory expression carrier of the Chlammydomonas reinhardtii, containing the exogenous gene, to the Chlammydomonas reinhardtii by using a Chlammydomonas reinhardtii genetic conversion method; and screening the Chlammydomonas reinhardtii to obtain a transgene engineering strain capable of expressing the exogenous gene in a secretory way. By using the invention, the basis is laid for using the Chlammydomonas reinhardtii as the secretory expression exogenous gene of a bioreactor.

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 present invention relates to methods for producing a recombinant polypeptide of interest, the method comprising the steps of: a) providing a polynucleotide library encoding one or more polypeptides of interest, wherein the library was prepared in an expression cloning vector comprising at least the following elements: i) a polynucleotide encoding a selectable marker; ii) a fungal replication initiation sequence, preferably an autonomously replicating sequence (ARS); and iii) a polynucleotide comprising in sequential order: a promoter derived from a fungal cell, a cloning-site into which the library is cloned, and a transcription terminator; b) transforming a mutant of a parent filamentous fungal host cell with the library, wherein the frequency of non-homologous recombination in the mutant has been decreased compared to the parent; c) culturing the transformed host cell obtained in (b) under conditions suitable for expression of the polynucleotide library; and d) selecting a transformed host cell which produces the polypeptide of interest.

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.

An Actinobacillus succinogenes plasmid vector which provides a means to overexpress proteins in A. succinogenes. The plasmid can be transformed efficiently by electroporation, and replicates in a stable manner in A. succinogenes. The plasmid comprises at least one marker gene, operably linked to a first promoter functional in Actinobacillus succinogenes, an origin of replication functional in Actinobacillus succinogenes, a second promoter isolated from Actinobacillus succinogenes, and a cloning site downstream from the second promoter. Plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are disclosed. The pckA gene polypeptide sequence and nucleic acid sequence of Actinobacillus succinogenes, including the promoter and ribosome binding site, is disclosed. Furthermore, a method for producing a recombinant Actinobacillus succinogenes is described, including a method of transformation. Additionally, a recombinant Actinobacillus succinogenes is disclosed and a method for producing succinate utilizing this recombinant Actinobacillus succinogenes is described.

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.

An object of the present invention is to provide a transformation system for Labyrinthulomycota that allows the elucidation of biosynthetic mechanisms of lipids such as PUFA and carotenoids as well as for the construction of a high production system and the design and development of novel functional lipid molecules by the control of the mechanisms. The present invention provides a vector for the transformation of Labyrinthulomycota with a transgene, which comprises at least (1) a nucleotide sequence which is homologous to a part of chromosomal DNA of Labyrinthulomycota and is capable of homologous recombination with the chromosomal DNA, (2) a selection marker gene having a promoter sequence located upstream and a terminator sequence located downstream, and (3) a cloning site for transgene insertion having a promoter sequence located upstream and a terminator sequence located downstream.

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 relates to a CAR-NK cell as well as a preparation method and an application thereof. The method comprises the following steps: S1, linking a specific antibody coding gene into a cloning site region of a modified CAR skeleton so as to obtain a CAR gene, wherein the modified CAR skeleton comprises a CD244 episporium signal region, the cloning site region, a CD244 extracellular hinge region, a CD244 transmembrane part region and a CD244 intracellular part region which are sequentially connected; and S2, infecting the CAR gene with NK cell through lentivirus, so that the NK cell expressing the CAR protein is obtained, namely the CAR-NK cell. The prepared tumor immnuotherapy cell, namely the CAR-NK cell, of the invention, besides a patient with cancer, is also applicable to allosome infusion; therefore, the prepared tumor immnuotherapy cell, in comparison with existing CAR-T, is wider in application scope and is lower in preparation cost.

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 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.