39 results about "FLAG-tag" patented technology

An objective of the present invention is to provide antibodies that recognize HLA class I and have significant cell death-inducing activity. To achieve the above objective, first, the present inventors immunized mice with soluble HLA-A to which a FLAG tag is attached. Four days after the final immunization, spleen cells from the mice were fused with mouse myeloma cells. Then, hybridoma screening was carried out using the cell aggregation-inducing activity and cell death-inducing activity as an index. Thus, the monoclonal antibody F17B1 that has strong cell death-inducing activity was selected. The sequences of the H chain and L chain variable regions of the mouse monoclonal antibody F17B1 were determined to humanize this antibody. Then, the humanized anti-HLA class I antibody H2-G1d_L1-k0 was assessed for cell death induction. The result showed that H2-G1d_L1-k0 suppressed the growth of the IM9 cell line in a concentration dependent manner.

The invention relates to a novel high-efficiency method for screening an angiotensin invertase inhibitor. According to the method, a FLAG label is utilized to further purify angiotensin invertase overexpressed in mammal cells and immobilize the angiotensin invertase on a magnetic bead, and then the magnetic bead is applied to screening of active components of crude drugs; and high performance liquid chromatography-mass spectrography is employed for tracking assessment of invertase inhibition activity, so the screening method is more sensitive. The method provided by the invention overcomes problems in rapid preparation of pure enzyme; the functions of enzyme are maximally guaranteed through overexpression of target enzyme via eukaryotic cells; and compared with extraction of enzyme from animal tissue, the method is simpler to operate, so inactivation of enzyme caused by complex operation is mitigated and purifying cost for enzyme preparation is substantially reduced.

The invention relates to the biological field, and especially relates to a substrate, a method and a kit for in-vitro quantitative determination of the activity of vWF-CP enzyme. The substrate comprises a vWF functional area amino acid sequence. The end N and the end C of the vWF functional area amino acid sequence are respectively combined with a HIS label amino acid sequence and a FLAG label amino acid sequence. The method comprises the following steps: the holes of an ELISA plate are coated with an antibody in advance; and then, the substrate, a sample to be tested, an ELISA antibody, and chromogenic agent are added, and the absorbance of liquid in the holes of the ELISA plate is measured using an ELISA reader. The kit comprises an ELISA plate, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution, or comprises an ELISA plate, an antibody, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution. The substrate, the method and the kit for in-vitro quantitative determination of the activity of vWF-CP enzyme have the advantages of high sensitivity, high accuracy, simple operation and low price, and are suitable for broad patient screening.

The invention provides a eukaryotic cell line capable of stably co-expressing recombinant human sweet taste receptor proteins His-T1R2 and FLAG-T1R3. (1) The eukaryotic cell line stably carries and codes a nucleotide sequence of the human sweet taste receptor protein T1R2 with a His tag and codes a nucleotide sequence of the human sweet taste receptor protein T1R3 with an FLAG tag; (2) the eukaryotic cell line is prepared from human embryonic kidney cells HEK293. The invention further discloses a preparation method of the eukaryotic cell line and a preparation method of a recombinant human chimera protein G15-gustducin44 with an HA tag expressed in the eukaryotic cell line. The 'sweetness' of exogenous sweet molecules can be detected on a cellular level, and a method capable of rapidly, effectively and sensitively detecting sweet substances can be established.

The invention discloses a construction method for a zebra fish model using hepcidin to treat high-iron inhibition of bone formation. The method comprises the following steps: with zebra fish oosperms as a model, carrying out intervention by using ferric ammonium citrate (FAC) of different concentrations, carrying out alizarin red staining and calculating bone mineralization areas at the 4th day and selecting the optimal intervention concentration of FAC; choosing zebra fish oosperms which are in cell stage 1 to 2 under a microscope, injecting hepcidin mRNA with Flag labels and of different concentrations, placing injected oosperms in an FAC culture solution with a concentration of 100 mu g / mL, carrying out intervention and then carrying out alizarin red staining at the 4th day so as to obtain the zebra fish model using hepcidin to treat high-iron inhibition of bone formation. In recent years, considerable research shows that iron metabolism is related to bone metabolism; a zebra fish is advantageous as a model since the zebra fish develops osteoclasts in 20 d after fertilization, and the zebra fish model can be used for effective research on influence of iron metabolism on bone formation. Meanwhile, commercially available hepcidin has the disadvantages of a high price, few manufacturers and uncertain biological activity at present; hepcidin used in the model is originated from a zebra fish sequence and has certain biological activity.

The invention discloses an expression vector of a nuclease protein Cas9 and a construction method and expression purification of the expression vector of the nuclease protein Cas9. According to the expression vector of the nuclease protein Cas9, an original thioredoxin sequence in pET32a is replaced with cDNA of an amplified thioredoxin-histidine-tag-tobacco-etch-virus-protease recognition site, and codons are optimized to a Cas9 gene sequence which is easy to express in a host and has nuclear localization signal peptide and 3xFLAG tag sequence fused in a C-terminal to be integrated into the thioredox in sequence. According to the expression vector of the nuclease protein Cas9 and the construction method and expression purification of the expression vector of the nuclease protein Cas9, codon optimization and thioredoxin fusion expression are used for obtaining a large amount of the nuclease Cas9 protein, under the condition that only an LB medium is used, 10 mg of a highly purified active Cas9 protein can be finally obtained per gram of escherichia coli Tuner (DE3) wet bacteria, and high expression of the high-fidelity nuclease protein Cas9 at a low cost is realized; and the requirements on the host are not high, and the obtained protein can be used for large-scale biological in vitro assembly and in vivo gene editing experiments.

The invention discloses recombinant adeno-associated virus increasing targeted transduction efficiency of adeno-associated virus, and application of the recombinant adeno-associated virus. The recombinant adeno-associated virus contains modified VP2 protein of the adeno-associated virus and primary VP1 protein, primary VP2 protein and primary VP3 protein of the adeno-associated virus, wherein the modified VP2 protein of the adeno-associated virus is formed in the way that a section of FLAG tag sequence is inserted into N terminus of the VP2 capsid protein. The invention further discloses a construction method of the recombinant adeno-associated virus and the application of the recombinant adeno-associated virus in gene therapy and in preparation of tissue engineering scaffold materials. The recombinant adeno-associated virus disclosed by the invention contains FLAG epitope, thus being capable of effectively infecting target cells and enhancing transduction efficiency. Compared with the conventional virus vector, the recombinant adeno-associated virus has higher transduction efficiency and better targeting performance, thus being applicable to multiple aspects of the gene therapy, construction of virus delivery systems, the preparation of the tissue engineering scaffold materials, and the like.

The invention discloses a 2019-nCoV S-antigens for generating 2019-nCoV neutralizing antibodies and a method for preparing the 2019-nCoV S-antigens. According to the 2019-nCoV S-antigens and the method, the 2019-nCoV S-antigens comprise 2019-nCoV S-antigen trimers combined with a cell membrane based on a Flag tag. The 2019-nCoV S-antigens can generate high-titer neutralizing antibodies so as to more effectively immunize animals.

The invention relates to the technical field of biomedicine, in particular to an EGFP-Wnt2 fusion protein antigen, a Wnt2 monoclonal antibody and an application of a Wnt2 monoclonal antibody. The EGFP-Wnt2 fusion protein antigen is mainly a fusion protein of EGFP and a full-length amino acid sequence of a humanized Wnt2 signal-erasing peptide. The EGFP-Wnt2 fusion protein antigen comprises an EGFPamino acid sequence, a TEV restriction enzyme cutting site and a FLAG tag sequence, and the Wnt2 signal-erasing peptide full-length amino acid sequence, a 6* His tag sequence and an anti-Wnt2 monoclonal antibody can be used for inhibiting immune escape and growth of tumor cells. When the Wnt2 monoclonal antibody disclosed by the invention is combined with the human Wnt2 antigen secreted by cells,the promotion of the cell on the formation of an inhibitory immune microenvironment in a tumor is antagonized, and the immune escape and growth of the tumor cell are inhibited. The Wnt2 monoclonal antibody provided by the invention can be used in immune treatment of solid tumors such as esophageal squamous carcinoma, gastric cancer, pancreatic cancer, colorectal colon cancer, lung cancer, breastcancer, glioma, liver cancer and the like.

The invention discloses a method for constructing a recombinant insect baculovirus expression vector of PVC2 (porcine circovirus type 2) Cap-labeled protein. The method comprises the following steps:(1) cloning whole genes of a PCV2GZ-RH1 strain into a pMD19-T vector, designing a primer to introduce a V5Tag or Flag tag into the ORF2 tail end of PCV2 with a PCR technology on the basis of the recombinant T vector as a template; (2) subcloning the ORF2 carrying the tag into pFastBacHTA and pFastBacDual donor plasmid, and transforming the positive plasmid obtained by subcloning into DH10Bac competent cells for Bacmind bacmid preparation; (3) transfecting insect cells with Bacmind bacmid to obtain recombinant insect baculovirus and performing amplification on the recombinant insect baculovirusto P3-generation virus.

The present invention relates to a novel endogenous gene expression substituting lentivirus vector. According to the present invention, a U6 promoter is inserted between the 5' LTR and the 3' LTR of lentivirus so as to perform transcription of a segment of a shRNA sequence, and the shRNA insertion region contains double restriction enzyme cutting sites such as BamHI and XhoI so as to insert the shRNA sequence after annealing; an EFla promoter is used for transcriptional expression of a Flag tag fused exogenous gene, the Flag tag is fused on the N terminal of the exogenous gene, and the multiple cloning site contains three restriction enzyme cutting sites such as HpaI, EcoRI and NheI3 so as to insert the exogenous gene, wherein the HpaI is the blunt-ended restriction enzyme cutting site so as to ensure insertion of any genes; IRES is used for starting the EGFP reporter gene; and the production of two proteins through the one RNA transcripted from the EFla can be achieved. In addition, the disadvantages of the conventional vectors are overcome, and the fast and efficient intracellular genetically modified way is provided.

A method for separating an RAVE compound from Saccharomyces cerevisiae cells by using an FLAG label relates to modification and purification of an RAVE compound protein in Saccharomyces cerevisiae, and belongs to the fields of the molecular biology and proteomics. The invention provides a method for separating the RAVE compound from a Saccharomyces cerevisiae recombinant strain by using the affinity chromatography principle of the FLAG label, and lays a solid foundation for the subsequent researches of the structure of the compound. The affinity chromatography principle of the FLAG label is used to separate easily biodegradable multimeric regulatory protein RAVE compound from a large amount of a cell homogenates supernatant, so the concentration and the purity of the RAVE compound are greatly improved; the FLAG label only contains 8 amino acids, and has a very small influence on the structures and the functions of proteins, so the activity of the separated RAVE compound is maximally guaranteed; and a series of proteins interacting with the RAVE compound by separating through the FLAG label affinity chromatography, and the identification by combining with mass spectrum can enrich the research data of RAVE compound related proteomics.

A preparation method for polypeptide nano-bubbles, comprising the following steps: constructing a recombinant plasmid, which includes a Flag tag, an adipose tissue-targeting polypeptide and the coding gene of nano-bubble marker membrane protein CD63; and transfecting the recombinant plasmid into cells which secrete nano-bubbles through lipidosome for culturing, collecting a cell culture solution, and extracting the polypeptide nano-bubbles by ultracentrifugation. The present invention further discloses the polypeptide nano-bubbles and application thereof in the preparation of drugs for treating obesity. The polypeptide nano-bubbles bring great convenience for targeted therapy of anti-obesity drugs. The polypeptide nano-bubbles have good biocompatibility and are capable of carrying different kinds of bioactive substances.

The invention discloses a nuclear localization signal F4NLS and application thereof in improvement of basic group editing efficiency and expansion of an editable basic group range. The nuclear localization signal F4NLS is composed of a nuclear localization signal A and a nuclear localization signal B: the nuclear localization signal A comprises 3*Flag tag protein and NLS protein; and the nuclear localization signal B comprises NLS protein. The amino acid sequence of the 3*Flag tag protein is the 1st to 22nd sites of a sequence 10; the amino acid sequence of the NLS protein is the 1st to 7th sites of a sequence 11. Experiments prove that the nuclear localization signal F4NLS can improve the basic group editing efficiency and expand the editable basic group range, and has a good applicationprospect in the field of biological genome editing.

The invention provides a preparation method of a 3 * FLAG tag fusion expression vector, which comprises the following steps: using a KpnI / XhoI double-enzyme digestion pGL3 Basic vector to obtain a recovered product of the KpnI / XhoI double-enzyme digestion pGL3 Basic vector, using a pRGEB32Bar-Cas9 plasmid as a template to obtain a 2 * 35S promoter PCR recovered product, carrying out a ligation reaction to obtain an intermediate vector pGL-35S, carrying out double-enzyme digestion, connecting an MCS annealing primer to obtain an intermediate vector pGL-35S-MCS, carrying out double-enzyme digestion, connecting a Nos Ter sequence to obtain the 3 * FLAG tag fusion expression vector. The preparation method comprises the following steps: firstly, performing double enzyme digestion on an intermediate vector pGL-35S-MCS-Nos to obtain the intermediate vector pGL-35S-MCS-Nos, connecting a connecting peptide Linker primer subjected to denaturation annealing after double enzyme digestion to obtain the intermediate vector pGL-35S-MCS-Linker-Nos, and connecting a 3 * FLAG tag annealing primer after double enzyme digestion to obtain the 3 * FLAG tag fusion expression vector. The 3 * FLAG tag fusion expression vector prepared in the invention has a small framework, has a full length of 4088bp, and is suitable for plant protoplast transformation.

The invention relates to a novel eukaryotic recombinant protein with a tumor inhibition effect and a preparation method thereof and belongs to the field of medical bioengineering. The novel eukaryotic recombinant protein with a tumor inhibition effect comprises 132 amino acids, 1th-25th amino acids form an NLS sequence, 26th-39th amino acids form a His-FLAG label sequence, 42th-121th amino acids form an N240 sequence, 122th-132th amino acids form a TAT sequence and the His-FLAG label sequence is bonded with the N240 sequence through two RS amino acids. The novel eukaryotic recombinant protein solves the problem that the existing small molecular drug cannot obstruct bonding of TCF4 and other cancer genes but beta-catenin so that the existing small molecular drug cannot effectively inhibit tumor stem cells, realizes comprehensive blocking of bonding of TCF4 and a protooncogene, effectively inhibits tumor stem cells and improves chemotherapeutic drug sensitivity.

The invention belongs to the field of biological medicines, and particularly relates to a method for screening CTL (cytotoxic T lymphocyte) epitopes by an independently constructed SLA-2-HB01-pCDH / sT2 cell line. According to the invention, an exogenous SLA-2-HB01 gene is transfected to an sT2 cell, and a stable SLA-2 gene expression model is established. The method comprises the following steps: loading EB155 and other positive epitope peptides on the surfaces of cells, detecting an SLA-2-peptide-beta2m complex expressed on the surfaces of the cells through FACs, and judging that the SLA-2-HB01-pCDH / sT2 has the function of presenting exogenous polypeptide epitopes. In order to promote the formation of cell surface complexes, the addition of the beta2m is beneficial to the improvement of the presentation efficiency of the antigen polypeptide. In the result of the invention, As63, EB155 and Hu62 peptides can be presented by an SLA-2-HB01-pCDH / sT2 cell line, As63 is presented in the SLA-2-HB01-pCDH / sT2, SLA-2-HB01-Flag-pCDH / sT2 and SLA-2-HB01-3 * Flag-pCDH / in the sT2 cell line, and the presentation efficiency is basically consistent, which indicates that a series of sT2 for expressing the SLA-2-HB01 gene constructed by the invention has the function of presenting the antigen, and the SLA-2-HB01-3 * Flag-pCDH / sT2 can be used for expressing the SLA-2-HB01 gene. Therefore, the SLA-2 heavy chain molecule can be used for screening polypeptide epitopes of the swine viruses on the cellular level, and it is further proved that the antigen presentation function of a cell line is not affected when a Flag tag is added to the C-terminal of the SLA-2 heavy chain molecule.

The invention discloses an inducible-expression shuttle expression tool vector for borrelia and application thereof. The sequence of the shuttle expression tool vector is shown in SEQ ID NO.1. The obtained shuttle expression tool vector contains the following components: (1) a replicon capable of replicating in Escherichia coli; (2) a replicon capable of replicating in borrelia; (3) a streptomycin resistance gene (aadA) controlled by a strong promoter flaB of borrelia and used for vector screening; (3) a coding region of repressor protein (LacI) and a promoter of repressor protein (LacI); (4) a binding site LacO of repressor protein; (5) polyclone sites inserted by using exogenous genes; and (6) a N-terminal fused FLAG tag and (or) a C-terminal fused HA tag. The vector can controllably express target genes in borrelia, so that the development of scientific research on borrelia is facilitated. The vector can be directly applied to gene function related researches on spirochetes by scientific researchers of the field, so that research process is accelerated.

The present invention belongs to the field of protein separating and purifying technology, and provides one kind of tandem affinity purified vector for mammal cell and its preparation process and usage in purifying protein composition. Tandem affinity purification (TAP) is used in separating protein composition and researching protein-protein interaction in physiological condition. The present invention has two Flag sequences to replace the protein A flag sequence in initial TAP vector and the decreased flag molecular weight is favorable to the effective fusion expression between the foreign target gene and the TAP vector and the high affinity combination to the Flag resisting monoclonal antibody. Therefore, the TAP vector is used in separating protein composition of higher mammal cell effectively and simply.

The invention belongs to the technical field of biology, and discloses a method for quickly positioning a T-DNA insertion site by a flag tag of a sam file. The method comprises the following steps: preparing a sample; carrying out whole genome re-sequencing; according to a sequencing result, obtaining a joint-removed clean_data; comparing a sequencing result with a transgenic vector sequence; according to the flag label of the comparison result file, removing sequences which are completely compared and cannot be compared to a carrier, and simultaneously removing labels which are compared for multiple times; extracting reads IDs and sequences of the screened flag tag values; and carrying out sequence alignment on the obtained sequence and an arabidopsis genome. According to a comparison result, a sequence which can be completely compared to an arabidopsis genome is removed; the remaining reads sequences are screened, and insertion sites are obtained; the false positive value in the insertion site is rejected and a final insertion site is obtained.

The invention belongs to the technical field of molecular biology, and in particular relates to a method for extracting polysome from rice on the basis of immune-precipitation. The method comprises the steps: by mainly adopting an immunization method, enriching a Flag tag-labeled ribosome-mRAN (messenger ribonucleic acid) compound by using affinity chromatography, chelating other metal ions in tissue homogenate by using EGTA (ethylene glycol tetraacetic acid), and inhibiting the dissociation of ribosome and mRNA by adding chloramphenicol and cycloheximide, wherein the formulas of an extraction solution and an elution solution as well as the material homogenate and concentration modes are improved; the ribosome-mRAN compound is eluted by using EDTA with the final concentration of 0.1M. The method disclosed by the invention is expected to separate 2-4 important new functional genes involving in low-temperature stress, ensures the normal growth and proliferation of plants under the low-temperature stress, and finally plays an important role in yield development.

The invention relates to the technical field of electronic tags, and particularly relates to a miniature high-frequency tag and a preparation method thereof. An etching copper antenna of the PI or PET base material and the high-precision size is adopted so that the tag is enabled to has the characteristics of being small in size structure, long in the service life, wide in range of application, stable in performance and high-temperature-resistant and corrosion-resistant. Besides, the miniature high-frequency tag can be used in the severe environment and the condition which cannot be realized by a large tag to realize an RFID read function.