44 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 invention relates to the technical field of biomedicine, and particularly discloses a dynamic monitoring method for ATP (adenosine triphosphate) level in cardiac muscle cells, recombinant cardiac muscle cells and application. The preparation method comprises the following steps: cloning an iATPSnFRs gene as shown in SEQ ID NO.3 into multiple cloning sites of a pLVX-AcGFP1-N1 plasmid, so as to construct an iATPSnFR1 recombinant plasmid; then transfecting myocardial cells with the iATPSnFR1 recombinant plasmid to obtain recombinant myocardial cells; and monitoring the ATP level change in the iATPSnFR1 recombinant myocardial cells in real time by using a high content imaging system. The recombinant myocardial cell containing the iATPSnFR1 is obtained through construction, so that the iATPSnFR1 can be specifically combined with ATP in the cell and generate a fluorescence signal, real-time and dynamic monitoring of the ATP level can be realized, and the recombinant myocardial cell has the advantages of non-invasiveness, high spatial resolution and the like.

The invention discloses a CAR (chimeric antigen receptor) recombinant lentiviral expression vector suitable for CIK (cytokine-induced killer) cells and NK (natural killer) cells to express EpCAM antibodies, IL-21 and CCL19, and belongs to the technical field of genetic engineering and biology. According to the expression vector, a CAR (chimeric antigen receptor) gene structure, an IL-21 (interleukin-21) gene expression structure and a CCL19 (chimeric antigen receptor 19) gene expression structure are connected between multiple cloning sites Nhe1 and Not1 of a vector pCHD-CMV-MCS-EF1-copGFP-T2A-puro, so that a recombinant plasmid pCDH-21 * 19 EpCAM-CAR is obtained. And transforming the recombinant plasmid into escherichia coli DH5alpha to obtain the recombinant microbial cell. The pCDH-21 * 19 EpCAM-CAR recombinant lentiviral expression vector is packaged, lentiviral particles are concentrated, CIK cells and NK cells are infected, and eukaryote transgenic cells are obtained. The CAR vector aiming at the solid tumor colorectal cancer is constructed, the design of the vector aims at improving the activating and killing capacity of CIK cells and NK cells and solving the problems of tumor microenvironment immunosuppression and the like, and a foundation is further laid for the research of treating solid tumors by applying a CAR technology.

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 relates to the technical field of biology, in particular to construction and application of an lnc30 overexpression strain. Comprising the following steps: (1) carrying out enzyme digestion on an original plasmid pLN-ENR-GFP by utilizing Hind III restriction enzyme, recovering a larger fragment, and then carrying out fragment self-connection to obtain a pBSD plasmid; (2) construction of a pBSD-30 plasmid: carrying out PCR (Polymerase Chain Reaction) to obtain and purify a U6 promoter gene, and connecting the gene between a 5'cloning site ApaI and a 3 'cloning site Avr II of the PLN-ENR-GFP plasmid; the method comprises the following steps: synthesizing an lnc30 full-length gene, and connecting the lnc30 gene between a 5'cloning site Avr II and a 3 'cloning site Afl II of a PLN-ENR-GFP plasmid; the lnc30 is connected with a U6 promoter, so that high expression of the lnc30 in cells is ensured. Experiments prove that lnc30 overexpression can significantly enhance the growth advantage of plasmodium falciparum and the ability of merozoite invading erythrocytes and participate in regulation of the erythroid stage development process of plasmodium, but knock-down of lnc30 may cause death of plasmodium, which indicates that the lnc30 overexpression strain is successfully constructed and can be used for research of a plasmodium falciparum gene expression regulation mechanism, and the lnc30 overexpression strain can be used for preparing the plasmodium falciparum gene expression regulation mechanism. The method is used for screening and researching drugs for inhibiting growth and development of plasmodium falciparum, and has remarkable scientific value and application prospect.

Provided is a precursor plasmid used for preparing a screening-tag-free plasmid, the precursor plasmid comprising: (1) a conditioned replication initiation site having a plasmid replication initiation capacity that is dependent on regulatory proteins, wherein the conditioned replication initiation site has a first replication initiation state in the presence of a first regulatory protein and a second replication initiation site in the presence of a second regulatory protein, and has a stronger ability to initiate plasmid replication in the second replication initiation state than in the first replication initiation state; 2) a first regulatory protein expression cassette expressing the first regulatory protein; 3) a sequence encoding a repressor protein; 4) a screened tag gene; 5) a target gene, or a cloning site for inserting the target gene; and 6) paired recombination sites. Also provided are a host cell suitable for the recombination of the precursor plasmid and the production of a screening-tag-free plasmid, and a method for large-scale production of the screening-tag-free plasmid.

The invention discloses an EpCAM gene high expression vector based on a lentiviral vector and a cell strain thereof, and belongs to the technical field of gene engineering and biology. According to the vector, an EpCAM gene expression structure is connected between multiple cloning sites Xba1 and Nhe1 of a vector pCHD-CMV-MCS-EF1-RFP-T2A-puro, high-level expression is carried out under the driving of a CMV (cytomegalovirus) promoter, and a recombinant plasmid pCDH-OE-EpCAM is obtained. And transforming the recombinant plasmid into escherichia coli DH5alpha to obtain the recombinant microbial cell. The recombinant plasmid is respectively transfected with colorectal cancer cell strains HT-29 and HT-115 through a Lipo2000 transfection reagent, and then monoclonal cells are screened, so that transgenic cells are obtained. The vector for overexpression of the human EpCAM gene and the colorectal cancer cell strain for overexpression of the EpCAM gene are constructed, and a very important foundation is laid for molecular mechanism research of generation and development of the EpCAM gene and colorectal cancer.

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 application discloses a gene expression vector pJXNU for actinomycetes, a construction method and application thereof. The sequence of the vector is shown as SEQ ID NO. 2. The vector is based on a pSET152 plasmid, and loaded with kanamycin and thiostrepton antibiotic resistance genes, and a 533bp DNA fragment containing three gene expression element sequences and two sets of multiple cloning sites (MCS) shown as SEQ ID NO. 1. The vector has two autonomously expressed strong gene expression elements and one thiostrepton-induced expression element, can realize autonomous high-efficiency expression of two or two groups of genes and / or induced expression of one or one group of genes on one vector, and can realize multi-type functional gene research and application in an actinomycete host.

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 present invention relates, in certain embodiments, to a fusion protein comprising a DEEP fusion tag and a target protein (e.g., protein of interest). In various other embodiments, the invention provides methods of producing a fusion protein comprising a DEEP fusion tag and a target protein, constructs comprising a nucleotide sequence encoding a DEEP fusion tag and a cloning site for introducing a nucleotide sequence encoding a target protein, and kits comprising such DNA constructs.

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;

The present application belongs to the technical field of genetic engineering, and particularly relates to a method for preparing recombinant deoxyribonuclease by prokaryotic expression. The method comprises the following steps: S1, connecting a target gene into a multiple cloning site of an expression vector to construct a recombinant expression vector, wherein the target gene comprises an X X A tag coding gene and a deoxyribonuclease coding gene; S2, introducing the recombinant expression vector into E. coli; S3, culturing the recombinant E. coli and inducing expression; and S4, collecting bacterial bodies after centrifugation of the recombinant E. coli culture solution, ultrasonic crushing, centrifugal collection of cell crushing supernatant, and then affinity purification to obtain a deoxyribonuclease fusion protein. The present application fuses the X X A tag with the deoxyribonuclease, adopts a prokaryotic expression system, has high host bacterium activity in the process of inducing expression, realizes high-efficiency expression of DNase, and significantly improves the soluble expression level, and has the advantages of good DNase biological activity and high stability.

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 application discloses an expression plasmid system of antigen-loaded PHA nanoparticles and preparation and application thereof. The expression plasmid system comprises pACYC-PhaA-PhaB plasmid and pET28a-PhaC-Target plasmid, nucleotide sequences of the plasmid and the pET28a-PhaC-Target plasmid are respectively shown as SEQ ID NO: 4 and SEQ ID NO: 5; the pACYC-PhaA-PhaB plasmid is a PhaA and PhaB protein expression frame inserted into a multiple cloning site of a vector pACYCDuet-1 and driven by two T7 promoters respectively; the pET28a-Phac-Target plasmid is a PhaC-vaccine antigen fusion protein expression frame inserted into a multiple cloning site of a vector pET-28a. The application lays a good foundation for preparation of PHA nanoparticle vaccines and related research.

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 biology, and particularly relates to a phage vector, a construction method and application of the phage vector in construction of a high-titer and high-insertion-rate phage antibody library. The phagocytosis vector comprises a functional region and a non-functional region, the functional region comprises multiple cloning sites, the multiple cloning sites comprise single-type restriction enzyme cutting sites, and the restriction enzyme cutting sites are used for connecting a target gene sequence. The construction of the single-enzyme digestion phage plasmid vector and the phage library constructed by the single-enzyme digestion phage plasmid vector have the advantages that the insertion rate of gene fragments is higher, the storage capacity is higher, the titer reaches 1010 cfu / mL, and the optimized single-enzyme digestion system is more suitable for construction of the phage library than a double-enzyme digestion system.

The present invention provides a VIGS silencing system of AmPDS gene of Allium mongolicum and its application, which belongs to the technical field of plant genetic engineering. The AmPDS gene cloned fragment is connected to the multiple cloning site of tobacco rattle virus vector to obtain a recombinant plasmid. TRV2‑AmPDS , transformed into Agrobacterium to obtain recombinant plasmid TRV2‑AmPDS Agrobacterium was used to infect leaves of Allium mongolicum; the AmPDS gene is represented by SEQ ID NO. 3. The AmPDS gene-silenced plants obtained by constructing the VIGS silencing system for the AmPDS gene of Allium mongolicum provided by the present invention exhibited an albino phenotype within 20 days, with significantly reduced AmPDS gene expression and a silencing rate reaching a maximum of 56.7%. The establishment of this system provides important support for in-depth research on stress-resistant genes in Allium mongolicum.

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 provides a multi-copy plasmid of Rhodotorula mucilaginosa, Rhodotorula mucilaginosa transformation, a Rhodotorula mucilaginosa strain with efficient overexpression of a target gene and a construction method of the Rhodotorula mucilaginosa strain, and relates to the technical field of genetic engineering. The construction method of the plasmid comprises the following steps: screening geneticin as a resistance marker of wild Rhodotorula mucilaginosa; taking plasmid YEp352 as a template and P1-F / R as primers to amplify a linearized plasmid fragment; taking a DNA sequence of a geneticin resistance expression box with a homologous arm as a template and G1-F / R as primers to obtain a target gene fragment with a homologous sequence; assembling the linearized plasmid fragment and the target gene fragment with the homologous sequence, and then transforming into an E. coli competent cell; and finally, positive colony identification is performed. The multi-copy plasmid provided by the application has multiple cloning sites and a selection marker, so that the genome modification in the Rhodotorula mucilaginosa is more flexible and convenient, and the target gene can be stably and efficiently expressed.