26 results about "Cloning Site" patented technology

The invention provides an hpRNA interference vector for laver and application of the hpRNA interference vector, and belongs to the technical field of genetic engineering and algae biology. According to the hpRNA interference vector, a binary vector pBI121 is used as a skeleton vector, a laver endogenous Actin promoter and a multiple cloning site segment are inserted, the multiple cloning sites allow target gene segments to be cloned in a forward and reverse repetition mode, the target gene segments are spaced by introns, and therefore an efficient hpRNA structure is formed after transcription, and the efficient hpRNA interference vector is obtained. The gene can be directly used for silencing important genes related to growth and development, metabolic regulation, stress response and the like in the laver. The invention further provides a construction method of the hpRNA interference vector and application of the hpRNA interference vector in silencing laver genes and provides a method for silencing laver gene expression, the gene silencing effect is remarkable, experimental results can be repeated, and a powerful tool is provided for molecular breeding and genetic improvement of laver.

The present application relates to the field of genetic engineering, and in particular to a vector for analyzing plant promoter expression specificity, a preparation method and application thereof. The T-DNA region of the vector comprises, from 5' end to 3' end, a callus-specific promoter-driven selection marker gene expression cassette and a reporter gene expression cassette with a multiple cloning site; the callus-specific promoter-driven selection marker gene expression cassette comprises a callus-specific promoter, a selection marker gene coding sequence and a terminator. The vector of the present application utilizes a rice callus-specific promoter to drive the expression of a selection marker gene, can greatly reduce the non-specific interaction with the promoter of a target gene, improve the specificity of the expression of the target gene, and effectively reduce the biological safety risk caused by the selection marker gene in a transgenic plant, and has important application value in basic theoretical research and molecular breeding.

The application provides a construction of a fluorescent protein labeled microtubulin and a microtubule binding protein universal bivalent carrier, and the process is as follows: a first stage is to construct a GFP-alpha tubulin carrier, and a second stage is to construct a GFP-alpha tubulin-mCherry universal bivalent co-expression carrier; in the second stage, 35S, mCherry and NOS sequences are inserted into a KpnI enzyme cutting site of a multiple cloning site of the GFP-alpha tubulin carrier constructed in the first stage, and a single nucleic acid enzyme cutting site is inserted into the 5' end and the 3' end of the mCherry sequence, respectively, and the single nucleic acid enzyme cutting site is XbaI, KpnI / Acc65I and AscI sequences, respectively, so as to obtain the universal bivalent carrier. The universal bivalent carrier provided by the application can express two target genes simultaneously, has the fluorescent signals of GFP and mCherry, can quickly and accurately identify a transgenic plant, is convenient for positive seedling screening, can be used for a tobacco transient expression experiment, can shorten an experimental period, and saves time and effort.

This invention relates to genetic engineering technology and aims to provide a method for preparing and applying bacteriophage capsid protein MS2-armored PRRSV antigen mRNA particles. The method includes: inserting the MS2 bacteriophage capsid protein gene and an mRNA expression cassette into a double multiple cloning site expression vector; transforming the recombinant expression plasmid into host bacteria; inducing expression to allow the MS2 capsid protein to express and self-assemble into virus-like particles; utilizing the specific recognition of the MS2 capsid protein on the packaging stem-loop structure, packaging the transcribed mRNA inside the virus-like particles to form MS2-armored PRRSV antigen mRNA particles; lysing the host bacteria, separating the supernatant, and enriching and purifying the particles. This invention significantly enhances the resistance of mRNA to nuclease degradation and improves formulation stability; it can increase the efficiency of mRNA entering antigen-presenting cells and enhance immunogenicity; it can be mass-produced with controllable processes and relatively low cost.

The invention provides a method for prokaryotically expressing cat allergen Fel d 1 recombinant protein, which comprises the following steps of: cloning a cat allergen Fel d 1 gene to multiple cloning sites of an expression vector pGEX-4T-1 to obtain a recombinant expression vector pGEX-4T-1-Fel d 1, transforming the recombinant expression vector into escherichia coli Rosetta (DE3) competent cells to construct recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), and carrying out prokaryotic expression on the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) to obtain the cat allergen Fel d 1 recombinant protein. IPTG (isopropyl-beta-d-thiogalactoside) is used for inducing protein expression, and a GST affinity chromatography medium is used for capturing high-purity soluble GST-Fel d 1 fusion protein. According to the invention, a pGEX-4T-1-GST label system is combined with Escherichia coli Rosetta (DE3), so that the problem of soluble expression of the cat allergen Fel d 1 protein is fundamentally solved.

The application discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is constructed by introducing an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, a MCS (multiple cloning site), a 3'-UTR sequence and a T7 terminator sequence on the basis of part of a sequence of an E. coli plasmid pUC19. The pSY1A plasmid vector has a MCS into which an exogenous gene can be inserted, and the MCS and a transcription assembly constitute a transcription module, so that the transcription module can be preserved by preserving the plasmid, and in-vitro transcription of the wheat germ cell-free expression system becomes more convenient and faster.

The invention discloses a pSY1A plasmid vector for a wheat germ cell-free expression system, which is characterized in that on the basis of partial sequence of escherichia coli plasmid pUC19, an ampicillin resistance gene, an Ori replicon, a T7 promoter, a translation enhancer, MCS multiple cloning sites, a 3 '-UTR sequence and a T7 terminator sequence are introduced to construct the plasmid vector pSY1A. According to the pSY1A plasmid vector provided by the invention, the vector has multiple cloning sites into which exogenous genes can be inserted, and the multiple cloning sites and a transcription component form a transcription module, so that the transcription module can be stored by storing plasmids, and the in-vitro transcription of a wheat germ cell-free expression system becomes more convenient and quicker.

The invention discloses a dsRNA fragment capable of improving wheat amylopectin content and a synthesis method and application thereof, and relates to the field of wheat starch content regulation, pET28a is used as an initial skeleton, a sequence containing a reverse T7 promoter, multiple cloning sites and ISA1 or ISA2 gene fragment is inserted, a recombinant vector is constructed through restriction enzyme digestion connection of restriction enzymes SalI and EcoRI, and the recombinant vector is used as a recombinant vector for improving wheat amylopectin content. And transforming the recombinant expression vector into an escherichia coli HT115 competent cell, and synthesizing the dsRNA fragment after induced expression. According to the invention, a non-transgenic transient silencing technology is adopted, and target gene expression is inhibited in a targeted manner, so that the problems of long period, low efficiency, dependence on complex gene cloning and vector construction processes and potential off-target effect and ecological risk existing in the traditional gene technology are avoided.

A method for preparing interaction plasmid library and a method for testing interactions between cDNA libraries are provided. The cDNA libraries could be inserted into the first multiple cloning site and the second multiple cloning site of the dual expression vector for testing the interactions between the different cDNA libraries.

The invention discloses an overexpression and interference lentiviral vector constructed based on miR-486-5p as well as a construction method and application of the overexpression and interference lentiviral vector. According to the lentiviral vector, an oligonucleotide sequence is constructed on multiple cloning sites of a plasmid skeleton, and lentivirus is used for packaging; the nucleotide sequence of the oligonucleotide is one selected from the group consisting of miR-486-5p-shRNA30, miR-486-5p-shRNA451, Anti-miR-486-5p TuD and Anti-miR-486-5p, and the nucleotide sequence of the oligonucleotide is one selected from the group consisting of miR-486-5p and Anti-miR-486-5p; and the plasmid skeleton contains a promoter and a plasmid skeleton of MCS multiple cloning sites. According to the present invention, the hairpin structure of pri-miR-451 is innovatively adopted as the skeleton, the AGO2 non-classical processing pathway and the miRNA cavernous body adsorption principle are combined to construct the regulation and control vector adapted to the miR-486 characteristics, and the core regulation and control effect is provided in the muscle cell development through the gene knockout or overexpression;

A dual expression vector and a method are provided. The dual expression vector has a first multiple cloning site and a second multiple cloning site. The genes of the different proteins could be inserted into the first multiple cloning site and the second multiple cloning site of the dual expression vector for testing the interaction between the different proteins.

The invention belongs to the technical field of gene engineering, and particularly relates to a proline hydroxylase controllable expression vector, a construction method of proline hydroxylase expression controllable engineering bacteria and application of the proline hydroxylase expression controllable engineering bacteria. The invention constructs a recombinant vector. The recombinant vector comprises a Trp promoter, a ribosome binding site, a multiple cloning site and a marker gene, wherein the nucleotide sequence of a part of gene segments of the Trp promoter is as shown in SEQ ID NO. 2. By adopting the recombinant vector, a controllable expression vector of the target protein can be further constructed, the expression level of the target protein can be accurately controlled by changing the tryptophan concentration in a culture medium, high tryptophan inhibits expression, and low tryptophan promotes expression. The regulation mechanism allows reduction of ineffective expression of the target protein in a thallus growth stage, reduces metabolic load, can realize real-time regulation in different stages, and provides a solution for efficient production of high-quality target protein.

The application discloses a method for expressing BirA enzyme by using prokaryotic Escherichia coli and purifying the BirA enzyme, and relates to the technical fields of genetic engineering and protein engineering. A recombinant expression vector for expressing the BirA enzyme is a pET-28A-BirA plasmid which is constructed by inserting a BirA enzyme coding gene into a multiple cloning site of a pET-28A vector. An 8× histidine tag coding nucleotide sequence is fused to a C terminal of the BirA enzyme coding gene. The nucleotide sequence of the BirA enzyme coding gene is shown as SEQ ID NO:1. The prokaryotic expression system is simple in culture condition, short in cycle, and low in cost, and is suitable for large-scale preparation. The 8× histidine tag is introduced into the C terminal of the BirA enzyme gene, so that the expression product can be purified by one-step nickel column affinity chromatography, and the purification process is simple and high in efficiency.

The disclosure provides a thermostable enzyme-linked biosensor expression cassette comprising a nucleic acid comprising a nucleotide sequence encoding a β-glucosidase reporter. The enzyme-linked biosensor expression cassette of the disclosure comprises a nucleic acid comprising a nucleotide sequence encoding (i) a transcription factor, (ii) a promoter, (iii) a terminator, and (iv) a cloning site for a gene of interest. The disclosure further provides novel variants of β-glucosidase that function as the reporter enzyme and exhibit superior properties (e.g., without limitation, pH stability and thermal stability) compared to existing β-glucosidase, providing improved biosensor expression cassettes.

The invention relates to the technical field of plant genetic transformation, and provides a plant genetic transformation system based on direct delivery, a genetic transformation method and application. An original vector pMKV057 is directionally modified to obtain a modified vector, and the modification method comprises the following steps: replacing an LUC expression cassette of the vector with a modular expression cassette integrated with an epitope tag; the method comprises the following steps: designing multiple cloning sites on a vector, introducing restriction enzyme cutting sites HindIII and EcoRI or HindIII and XbaI, and inserting a Kozak sequence into a 5'end upstream adjacent region of a nucleotide sequence of the multiple cloning sites to obtain a modified vector. And transforming the modified vector into agrobacterium tumefaciens to carry out plant genetic transformation. Through the technical scheme, the problems of low plant genetic transformation efficiency and long transformation time in related technologies are solved.

The invention relates to a universal plasmid vector for in vitro transcription (IVT) DNA template preparation. The universal plasmid vector comprises a) a gene expression cassette; b) telomerase recognition sequences: setting TelN telomerase recognition sequences at two ends of the expression cassette; and c) a skeleton region which contains a plurality of specific endonuclease recognition sequence sites which are connected in series. A specific telomerase recognition sequence, an optimized multi-cloning site structure and a removable skeleton region are introduced into plasmid design, so that a high-purity and low-residue DNA template can be quickly obtained under the condition that large-scale bacterial fermentation is not needed, and a more efficient template source is provided for the fields of mRNA vaccines, gene therapy, cell therapy and the like.

The invention belongs to the technical field of biological medicines, and particularly relates to an AAV vector for regulating smooth muscle contraction as well as a construction method and application of the AAV vector. The AAV vector for regulating and controlling the contraction of the smooth muscle, disclosed by the invention, is obtained by inserting a gene for regulating and controlling the contraction of the smooth muscle between restriction enzyme cutting sites of multiple cloning sites of a pssAAV-CBA-LUC-P2A-GFP-bGH-141 vector. The obtained AAV vector expresses an exogenous gene, such as a gene ACTA2 for regulating smooth muscle contraction, and has the advantages of long time and good stability. Animal experiments show that the AAV vector can directly increase the contractility of smooth muscle by regulating expression of smooth muscle contraction related genes such as ACTA2, so that the aim of treating related diseases such as gastroesophageal reflux disease, fecal incontinence and the like is fulfilled.

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 application discloses a nucleic acid delivery carrier, a delivery system and application thereof. The nucleic acid delivery carrier is constructed by inserting a CpG sequence into a multi-cloning site of a eukaryotic expression carrier, and the nucleic acid delivery system is constructed by the delivery carrier and probiotics. Based on the delivery carrier, the nucleic acid to be delivered is inserted into the delivery carrier and then transformed into probiotics to construct a recombinant bacteria preparation. The nucleic acid to be delivered can be delivered into the body by orally taking or injecting the recombinant bacteria preparation, which overcomes the deficiency that the nucleic acid is difficult to be efficiently delivered into the body by only using the recombinant expression carrier to transform the recombinant bacteria. The nucleic acid delivery carrier or the delivery system can be used to deliver the nucleic acid with therapeutic or immune effect into the body, and the nucleic acid delivery carrier or the delivery system can be used to prepare a medicine or an immune preparation for treating diseases by delivering the nucleic acid, and has wide application.

The invention relates to the technical field of vaccines, in particular to a canine parvovirus-rabies bivalent dog oral vaccine based on a replication-defective canine parvovirus packaging vector and preparation. The preparation method comprises the following steps: inserting a full-length sequence of a codon-optimized rabies virus glycoprotein gene into multiple cloning sites of a replication-defective canine parvovirus packaging vector genome, and carrying out cell transfection and virus packaging to obtain the canine parvovirus-rabies bigeminal canine oral vaccine based on the replication-defective canine parvovirus packaging vector. The vaccine can prevent and control two canine pathogens at the same time, and compared with an injection vaccine, the vaccine has better compliance to animals.

The invention discloses a recombinant vector for expressing bacteriophage lyase and a construction method and application of the recombinant vector. The recombinant vector for expressing the bacteriophage lyase is based on a pichia pastoris expression system. The recombinant vector for expressing the bacteriophage lyase comprises a bacteriophage lyase gene, a multiple cloning site, a replication start site, a promoter, a terminator, an alpha-factor signal peptide and a resistance selection marker. According to the recombinant vector of the bacteriophage lyase provided by the invention, the lyase gene is recombined onto the vector, so that the expressed lyase can be used for developing a novel feed additive for preventing necrotic enteritis of poultry directly or after being prepared into a preparation, and a food preservative for controlling clostridium perfringens pollution in meat; or as a therapeutic drug for local or systemic infections.

The invention relates to a linear mRNA (messenger ribonucleic acid) vaccine expression vector and application thereof, and the linear mRNA vaccine expression vector is constructed by the following method: a framework of the linear mRNA vaccine expression vector is a pcDNA (complementary deoxyribonucleic acid) vector framework; comprising the following elements from the 5'end to the 3 'end: a promoter, a 5' UTR sequence, a Kozak sequence, a signal peptide sequence, a cloning site sequence, a tag sequence, a 3 'UTR sequence, a Poly (A) tail sequence and a linearization site. The vector sequence is designed and optimized, the target gene is easy to clone and detect, the mRNA molecule is synthesized through in-vitro transcription, the stability of the mRNA molecule and the translation efficiency of the coded polypeptide are improved, the vector can be used for expression of linear mRNA molecules, and more and better choices are provided for development of mRNA vaccines.

The invention discloses an engineering bacterium for producing L-piperidine acid and application of the engineering bacterium in preparation of antidepressant drugs, and belongs to the technical field of biological medicines. According to the engineering bacteria, a promoter J23105, a lat gene optimized through prokaryotic expression and a proC gene are sequentially inserted into multiple cloning sites of a kanamycin-resistant pRSF-Duet-1 plasmid, and synthetic operons arranged in the same direction are formed; the operon is spliced with a linearized pRSF-Duet-1 plasmid vector in a homologous recombination or Gibson assembly mode, the spliced operon and the linearized pRSF-Duet-1 plasmid vector are transformed into escherichia coli for amplification, after it is confirmed through sequencing that the sequence and the direction of an insertion fragment are correct, the insertion fragment is transferred into a chassis bacterium, and the operon is obtained. Researches prove that colonization of the engineering bacteria can improve anxiety and depression behaviors of mice and improve social scores of the mice, so that the engineering bacteria can be used for preventing and treating depression. Therefore, the engineering bacterium disclosed by the invention can be used for preparing anti-depression drugs, such as production of viable bacteria preparations.

The present invention provides an improved lentiviral backbone for use in gene therapy. The disclosed lentiviral backbones may have a sequence with at least 90% similarity to SEQ ID NO: 1. The disclosed lentiviral backbones may comprise a multiple cloning site that allows for the insertion of a transgene of interest, e.g., for use in the treatment of genetic disorders.

Provided are recombinant nucleic acids (e.g., vectors), and related methods, for expression of a target gene in a host cell. The recombinant nucleic acids comprise a promoter comprising a nucleic acid sequence having at least 70% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; a cloning site for insertion of a nucleic acid encoding the target gene; and at least one non-promoter regulatory element required for the expression of the target gene in the host cell.

The application provides a protein purification method and application thereof. The method comprises the following steps: (1) constructing a nucleotide fragment encoding AeBlueM-protease recognition site-target protein, and cloning the nucleotide fragment into a basic vector at a multiple cloning site by using a homologous recombination method to obtain a carrier; (2) constructing a nucleotide fragment encoding AeBlueM-Im7, and cloning the nucleotide fragment into the basic vector at the multiple cloning site by using the homologous recombination method to obtain the carrier; (3) constructing a nucleotide fragment encoding CL7-protease recognition site-target protein, and cloning the nucleotide fragment into the basic vector at the multiple cloning site by using the homologous recombination method to obtain the carrier. The purification method is not dependent on traditional resins and chromatography columns, is simple to operate, is easy to scale up, and is low in cost.