9 results about "FLAG-tag" patented technology

The invention discloses a G protein coupled receptor family membrane receptor GPR40 related to eukaryotic expression and glycometabolism effect, and a method for extracting and detecting the concentration of a compound combined with the GPR40 receptor in a cell based on the affinity of receptor protein. The detection method comprises the following steps: eukaryotically expressing a membrane receptor GPR40 protein with a Flag tag in a human embryonic kidney cell HEK293, and after perfluorooctane sulfonic acid (PFOS) is exposed, extracting and purifying a GPR40-PFOS compound in a cell lysis solution by utilizing Protein G magnetic beads and a Flag antibody; after uncombined PFOS is washed away by a cracking buffer solution containing a protease inhibitor, acetonitrile is added, and PFOS in the compound is extracted; and centrifuging and treating with a 0.22 mu m filter membrane, and detecting the concentration of the PFOS by using a high performance liquid chromatography-mass spectrometer (HPLC-MS / MS). The method can directly detect the concentration of PFOS combined with the GPR40 receptor, can be used for screening other perfluorinated compounds and environmental pollutants which potentially act on the GPR40 receptor to play a glycometabolism effect, can simplify the steps of effect-oriented analysis, and plays an important role in compound recognition and screening.

The invention discloses an eurytopic chlamydomonas reinhardtii expression vector based on a PSAD promoter and a 3 * Flag tag as well as a construction method and application of the eurytopic chlamydomonas reinhardtii expression vector. The nucleotide sequence of the eurytopic chlamydomonas reinhardtii expression vector is shown as SEQ ID No.1; the vector comprises a PSAD promoter, a target gene insertion site EcoR V, a 3 * Flag tag at the 3'terminal, an rbcS2 terminator, a TUB2 promoter, a hygromycin B screening gene and an rbcS2 terminator. The construction method comprises the following steps: constructing a skeleton of a pHyg-PSAD-3Flag vector; constructing a sequence fragment fused with a 3 * Flag tag; and connecting the sequence fragment fused with the 3 * Flag tag with a plasmid skeleton fragment through T4 DNA (deoxyribonucleic acid) enzyme so as to construct the pHyg-PSAD-3Flag vector. According to the invention, the expression plasmid of the chlamydomonas pHyg-PSAD-3Flag can be used for efficiently screening algae strains capable of stably expressing target protein.

A method for endogenously extracting a Mycobacterium smegmatis protein nanocage is provided. The method includes the following steps: introducing a recombinant plasmid containing a CFP29 gene and a 1×Flag affinity tag into a Mycobacterium smegmatis strain to obtain a recombinant Mycobacterium smegmatis strain; extracting a total protein solution of the recombinant Mycobacterium smegmatis strain, and subjecting the total protein solution of the recombinant Mycobacterium smegmatis strain to Flag tag affinity column chromatography purification to obtain a crude extract of the Mycobacterium smegmatis protein nanocage; and subjecting the crude extract of the Mycobacterium smegmatis protein nanocage to gel exclusion chromatography purification to obtain a pure product of the Mycobacterium smegmatis protein nanocage. The method is simple to operate and convenient to implement during extraction and purification, and an obtained Mycobacterium smegmatis background protein nanocage has a high yield, excellent purity, and stable properties.

The invention provides a construction method and application of an animal model for specifically driving LSL-RCAN1-P2A-EGFP-Flag tag to express by using a CAG (chorionic gonadotropin) promoter. The method is realized by inserting a CAG promoter sequence, an LSL sequence, an RCAN1 gene coding sequence, an EGFP sequence, a Flag tag sequence and a PolyA sequence into a Rosa26 site of a mouse genome. When the RCAN1-2A-EGFP gene is hybridized with a mouse with tissue or cell specific expression CRE enzyme, a Stop sequence between loxP sites can be cut off, so that overexpression of the RCAN1-2A-EGFP protein driven by a tissue or cell specific CAG promoter is realized, the translated protein can be separated into two independent proteins for performing functions, and an animal model is provided for researching the function of the RCAN1 in specific tissue or cell.

The invention discloses a construction method and application of an IER2-Flag gene knock-in mouse model, and the construction method comprises the following steps: determining a target site of an IER2 gene to be knocked in a mouse, and designing sgRNA with a sequence as shown in SEQ ID No: 2; the method comprises the following steps: carrying out recombination on double-stranded sgRNA formed by annealing and CRISPR-Cas9 plasmid PX459, and constructing a PX459-sgRNA recombinant plasmid of which the sequence is as shown in SEQ ID No: 3-4; the method comprises the following steps: by taking a recombinant plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification on a double-stranded DNA (Deoxyribose Nucleic Acid) fragment of a T7 promoter sequence + sgRNA + tracr RNA, and carrying out in-vitro transcription to obtain single-stranded sgRNA + tracr RNA, a single-chain sgRNA + tracr RNA, a Donor sequence containing a 3 * Flag tag and Cas9 Protein are co-injected into a male pronucleus of a mouse fertilized egg, an F0-generation IER2-Flag gene knock-in mouse can be obtained until an F3-generation IER2-Flag gene knock-in mouse is obtained, and the IER2-Flag gene knock-in mouse model is obtained. According to the invention, the expression of the Flag tag is detected for the first time to further identify that the IER2 protein is continuously and strongly expressed in the enamel cell, and an important animal model is provided for the research on revealing a mechanism for regulating and controlling enamel mineralization by the IER2 gene.

This invention discloses a method for constructing a TrpA1-3×FLAG-P2A-iCre gene knock-in mouse. A fragment containing a 3×FLAG tag, a P2A sequence, and an iCre recombinase sequence is inserted into the 27th exon region of the mouse TrpA1 gene using a CRISPR / Cas9 system, achieving genetic labeling and Cre functionalization in TrpA1-expressing cells. The donor vector provided by this invention contains a 5' homologous arm and a 3' homologous arm that are homologous to the TrpA1 genome sequence. Stably inherited knock-in mice are obtained by microinjecting the CRISPR / Cas9 system and the donor vector into fertilized eggs.

The invention belongs to biological medicines, and discloses an enhanced exosome for expressing an antigen-targeted antibody as well as a preparation method and application of the enhanced exosome. An antigen targeting antibody is expressed on the surface of the exosome, and the antigen targeting antibody comprises: 1) a signal peptide; (2) a histidine tag (His-tag) and a Flag tag; 3) a single-chain antibody region of a targeted antigen; 4) a hinge area; 5) transmembrane area; and 6) a fluorescent protein. The enhanced exosome is prepared by a gene engineering method, and surface modification of the exosome is realized by utilizing a lentiviral vector, so that the enhanced exosome has definite antigen targeting property and preparation sustainability.

An assay for predicting severe acute kidney injury is disclosed. The assay detects cleavage of a peptide or protein with an internal sequence that mimics all or some of the central linker region of insulin-like growth factor binding protein 1. The amount of cleavage of the peptide or protein predicts the likelihood that a patient will develop severe acute kidney injury. The peptide or protein used in the assay may contain a terminal His tag and a terminal Flag tag to immobilize the amino-terminal and carboxy-terminal ends of the peptide on a test strip of the assay after cleavage.

The invention relates to a CRISPR / dCas9 (clustered regularly interspaced short palindromic repeats / dCas9) vector for improving BnNAC022 gene expression as well as a construction method and application of the CRISPR / dCas9 vector. The CRISPR / dCas9 vector is a CRISPR-SL-U6-26-TG1-TG2 vector, the nucleotide sequence of the CRISPR / dCas9 vector is shown as SEQ ID NO.7, and the CRISPR / dCas9 vector contains an expression cassette with two cores; the expression box 1 comprises an arabidopsis thaliana U6-26 promoter, a target spot 1, an sgRNA2.0 support sequence, an At-tRNAGly coding sequence, a target spot 2 and an sgRNA2.0 support sequence in sequence from the 5 '-3' end; and the expression box 2 sequentially comprises a constitutive promoter, a 3 * Flag tag coding sequence, a first SV40 NLS, a dCas9 coding gene, an NLS nuclear localization signal, a VP64 coding gene, a coding gene of self-splitting polypeptide T2A, an MS2 coding gene, a second SV40 NLS, a P65 coding gene, an HSF1 coding gene and an NOS transcription terminator from the 5 '-3' end. The recombinant vector CRISPR-SL-U6-26-TG1-TG2 constructed by the invention is an activation vector of a targeted BnNAC022 gene promoter, so that the expression level of the BnNAC022 gene in the brassica napus can be remarkably improved, and the aphid resistance of the brassica napus can be enhanced.