311 results about "Luciferases" patented technology

The invention provides an UTR (Untranslated Region) element H2202 P1-G as well as a construction method and application thereof, and relates to the technical field of mRNA (messenger ribonucleic acid). According to the present invention, the ribosome load prediction and the secondary structure optimization are performed on the natural 5 'UTR of the HIV TAT 202 gene through the BaidleHelix platform, and the obtained HTAT 202 P1 sequence avoids the inhibitory hairpin structure so as to significantly improve the luciferase expression quantity compared to the natural UTR; an ncRNA sequence without a secondary structure is introduced on the basis of the HTAT 202 P1, translation inhibition of a 5 'cap region is further relieved, and the protein expression quantity of the constructed H2202 P1-G mutant (the DNA sequence of the H2202 P1-G is as shown in SEQ NO 1, and the RNA sequence is as shown in SEQ NO 2) is further improved.

The invention discloses a protein optimization design and screening method and device based on an artificial intelligence algorithm, and the method takes fungal luciferase as an example, and integrates multi-dimensional bioinformatics analysis and deep learning technology to realize efficient protein engineering transformation and optimization. The method comprises the following steps: firstly, identifying a binding domain of luciferase and fluorescein by utilizing an AI-driven molecular docking simulation method, and determining a key conservative site by combining literature, evolutionary analysis and structural prediction; then, generating a protein functional domain skeleton under the constraint of a fixed site by adopting a diffusion model, and performing protein sequence prediction by utilizing a graph neural network model; and finally, scoring and screening the generated sequences to obtain high-stability candidate variants. According to the method, a conservative site dynamic fusion strategy is innovatively constructed, a fixed region is optimized through logic of structure prediction, evolution site intersection priority and literature site union set expansion, the diversity and adaptability of a protein design sequence are met, and the efficiency bottleneck of a traditional scheme is broken through.

The invention provides an UTR element NHP1 as well as a construction method and application thereof, and relates to the technical field of mRNA. A 5 'UTR with a good expression effect is designed by integrating dominant sequences of a human high-expression gene and a pathogen natural UTR, a chimeric structure NHP1 with high ribosome load is predicted through a calculation model, a DNA sequence of the NHP1 is as shown in SEQ NO 1, and an RNA sequence of the NHP1 is as shown in SEQ NO 2; an EGFP report system is adopted on the DNA level to rapidly screen UTR; the translation efficiency is quantitatively evaluated on the RNA level through luciferase mRNA (N1-methyl pseudouridine modification); and the particle size is controlled by a microfluidic technology, so that the optimized UTR-mRNA is efficiently expressed after being delivered.

The invention discloses a recombinant fusion protein of a human lambda light-chain single-chain antibody, a DNA (deoxyribonucleic acid) molecule, a library building method and application, and the recombinant fusion protein of the human lambda light-chain single-chain antibody is the recombinant fusion protein formed by connecting the human lambda light-chain single-chain antibody, phage Piii protein and luciferase, the amino acid sequence of the recombinant fusion protein of the human lambda light-chain single-chain antibody is shown as SEQ ID NO: 5, the recombinant fusion protein library of the human lambda light-chain single-chain antibody has large capacity, the established single-chain antibody TG1 library is more than 1 * 10 < 9 > cfu / mL, positive clones capable of being combined with ligands can be screened and enriched for multiple rounds, and the recombinant fusion protein library has the characteristics of high sensitivity, high signal-to-noise ratio and the like.

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of circ0005704 and related biological elements thereof in diagnosis and treatment of recurrent spontaneous abortion with unknown reasons. Researches find that differential expression and autophagy level of ULK1 in trophoblast cells of URSA patients are increased. High-throughput transcriptome sequencing is combined with bioinformatics analysis, and it is found that miR-26a-5p is down-regulated in URSA patients, is responsible for up-regulation of ULK1 and promotes autophagy of trophoblast cells, so that occurrence of URSA is increased. Through a high-throughput transcriptional set screening strategy and dual luciferase reporter gene analysis, it is found that circ0005704 enhances the expression of ULK1 through miR-26a-5p. In a word, the research of the invention shows that the circ0005704 / miR-26a-5p / ULK1 signal axis participates in the pathogenesis of URSA by adjusting the migration of the trophoblast cells, and a new target and scientific evidence are provided for the clinical treatment of URSA.

The invention belongs to the technical field of plant biology, and particularly relates to a gene for regulating and controlling powdery mildew resistance of cucumbers and application of the gene, two cswrky31 mutants are obtained based on a cucumber Tnt1 reverse transcription transposon mutant library, and the two cswrky31 mutants both show high susceptibility to powdery mildew bacteria. A target gene Csa5G551250 which is directly regulated and controlled by CsWRKY31 is screened by virtue of a high-throughput transcriptome sequencing technology and a DNA affinity purification sequencing technology in combination with a yeast one-hybrid experiment. A dual luciferase report experiment shows that the CsWRKY31 positively regulates and controls the expression of the Csa5G551250, and the overexpression of the Csa5G551250 gene promotes the resistance of the cucumber to powdery mildew bacteria. A new reference gene resource is provided for cultivation of cucumber disease-resistant varieties, and a new thought is provided for molecular mechanism research of transcription factor mediated plant immunity.

The invention discloses a monoluciferase-based ADCP biological activity detection method, which comprises the following steps: separating a mononuclear cell from a human peripheral blood mononuclear cell as an effector cell, phagocytizing a Raji cell transfected by Luciferase as a target cell by the mononuclear cell, acidifying and degrading the Raji cell in a phagocytic body, and degrading the Luciferase so as to attenuate a light-emitting signal, so that the ADCP biological activity is detected. The ADCP biological activity of the antibody therapeutic drug is accurately quantified by detecting the weakening of the light-emitting signal. According to the present invention, the method only needs to separate the mononuclear cells and perform Luciferase infection on the target cells, and only needs to add the luciferase substrate after the phagocytosis process, such that the luminescence value can be read by using the microplate reader, the operation process is simple, the steps are less, the time consumption is short, the high-throughput screening is achieved, and the new direction is provided for the ADCP detection.

The invention discloses osmanthus transcription factors OfbHLH10 and OfWRKY78 for regulating synthesis of a flower fragrance component beta-ionone as well as a coding gene and application of the osmanthus transcription factors OfbHLH10 and OfWRKY78, and belongs to the field of plant molecular biology. The amino acid sequence of the osmanthus fragrans transcription factor OfbHLH10 provided by the invention is as shown in SEQ ID NO. 2, and the amino acid sequence of the transcription factor OfWRKY78 interacting with the osmanthus fragrans transcription factor OfbHLH10 is as shown in SEQ ID NO. 4. According to the invention, carriers constructed by CDs of OfWRKY78 and a promoter of OfCCD4 are jointly injected into Ben's tobacco leaves, a dual-luciferase experiment is carried out, and a result shows that OfbHLH10 has a promoting effect on expression of a synthetase gene OfCCD4 of beta-ionone, OfWRKY78 has an inhibiting effect on expression of OfCCD4, and OfbHLH10 and OfWRKY78 interact with each other and has an antagonistic effect on expression of OfCCD4. The gene provided by the invention can effectively regulate and control the content of beta-ionone in osmanthus fragrans fragrance, and has a good application prospect.

The invention relates to the technical field of biology and biology, in particular to a screening method of thermostable mutation sites of a G protein coupled receptor and application of the thermostable mutation sites of the G protein coupled receptor, and the screening method comprises the following steps: S1, replacing a third intracellular ring segment in a wild type G protein coupled receptor with a third intracellular ring sequence of a kappa opioid receptor; marking a SmBiT peptide fragment label on the wild type G protein coupled receptor; s2, mutating a wild type G protein coupled receptor according to a mutation site in the G protein coupled receptor mutant to be detected; s3, incubating the G protein coupled receptor modified in the step S2 at different temperatures to obtain modified G protein coupled receptor samples treated at different temperatures; s4, carrying out mixed incubation on the modified G protein coupled receptor samples treated at different temperatures and a nano antibody-LgBiT compound, then adding luciferase to act as a substrate, and detecting; the method is high in screening efficiency, low in cost and short in time.

The invention provides a honeysuckle MYB gene family transcription factor 13476. A honeysuckle HQT2 gene, an HQT2 gene promoter and a transcription factor 13476 are cloned respectively, and a yeast one-hybrid experiment proves that 13476 can directly interact with the HQT2 promoter. Furthermore, the regulation of the 13476 protein on the transcriptional activity of the HQT2 gene is analyzed by using a dual luciferase system in tobacco, and the 13476 is proved to have an inhibiting effect on the promoter of the HQT2. Tobacco transient transformation results show that the content of chlorogenic acid in plant cells can be increased by 30-40% through heterologous transient expression of the LjHQT2 gene, and the content of chlorogenic acid reaches the level of 1.59 mg / g. The RNAi method is adopted, molecules aiming at Lj13476 are transiently expressed in honeysuckle, the content of chlorogenic acid can be increased by 10%-20%, the content of isochlorogenic acid A can be increased by 5%-30%, and the content of isochlorogenic acid C can be increased by 7%-20%. The interaction mechanism of the honeysuckle HQT2 gene and the transcription factor 13476 is studied, a certain basis is provided for the regulation mechanism of chlorogenic acid, and the HQT2 gene and the transcription factor 13476 are of great significance for breeding high-quality honeysuckle in the future.

The invention discloses a key gene AaMYB114 closely linked with the color character of an actinidia arguta fruit and application of the key gene AaMYB114. The AaMYB114 gene disclosed by the invention is positioned at 5237694-5239966bp of a chromosome 9 of an actinidia arguta genome, and the full length of the AaMYB114 gene is 2273bp. According to the application, all-red type and all-green type actinidia arguta are used as materials for transcriptome sequencing, and the gene AaMYB114 related to the formation of the peel color of the actinidia arguta is explored. Instantaneous conversion of the actinidia arguta fruit proves that the overexpression of the AaMYB114 increases the anthocyanin content in the actinidia arguta peel. Yeast one-hybridization and dual-luciferase report tests prove that the AaMYB114 not only interacts with a promoter of AaGST, activates the expression of the promoter and regulates anthocyanin transport of peel tissues of actinidia arguta, but also is combined with promoters of anthocyanin synthesis structural genes AaPAL, AaCHI and AaF3H and activates the expression of the promoters to promote anthocyanin synthesis. Therefore, the AaMYB114 gene plays a dual regulation and control role in the formation of peel color and luster, and can be used for cultivating red-peel actinidia arguta.

The invention relates to the technical field of bacterial detection, in particular to a bacterial drug sensitivity detection assembly based on an ATP luciferase method and a using method.The bacterial drug sensitivity detection assembly comprises a supporting assembly, a sampling assembly, an ATP fluorescence detector and a 96-hole drug sensitivity plate, and a sealing block is arranged at the top of the inner wall of the ATP fluorescence detector; the supporting assembly comprises a base, a controller is arranged on one side of the base, and an electric push rod is arranged in the middle of the upper surface of the base. The device effectively realizes detection of bacterial drug sensitivity of a sample, is high in detection precision and good in detection effect, can also mix and stir the sample in the 96-hole drug sensitive plate, and avoids precipitation of bacteria in the 96-hole drug sensitive plate due to long-time standing; the sampling quantity of samples can be accurately detected, and the accuracy and the stability of subsequent detection are ensured; when the sample in the sampling tube is mixed with the reaction liquid, the flowing efficiency of the sample can be ensured, and the problems of high liquid feeding difficulty, poor mixing effect and the like caused by negative pressure are avoided.

The invention belongs to the technical field of microorganisms, and particularly relates to a method for rapidly screening antibacterial peptides in chlamydomonas based on luciferase labeling and application of the method. Specifically, a plasmid with a luciferase tag and ubiquitin protein is utilized, antibacterial peptide is introduced into the plasmid, and a chlamydomonas reinhardtii strain capable of stably secreting the antibacterial peptide to the outside of a cell in an exosome form is developed. The ubiquitin secretion efficiency of the exosome is further optimized and improved, so that the protein secretion efficiency of the antibacterial peptide is improved, and the method has good practical application prospect and value.

The invention provides a chicken PGC reporter gene cell strain for detecting the biological activity of FGF2 as well as a construction method and application of the chicken PGC reporter gene cell strain, and belongs to the technical field of biological activity detection. The cell strain is a recombinant cell which is stably integrated with a reporter gene expression cassette responding to an FGF signal channel in a chicken PGC genome; the expression cassette comprises a serum response element (SRE), a minimum promoter and a reporter gene (such as a luciferase gene). The construction method comprises the following steps: introducing a donor vector containing the elements into chicken PGC by utilizing a PiggyBac transposon system, and screening to obtain a stably expressed cell strain. The cell strain provided by the invention retains endogenous signal channel characteristics of chicken PGC, and can specifically and highly sensitively respond to FGF2 stimulation. The method can be used for biological activity quantitative determination and thermal stability evaluation of the FGF2 protein and the mutant thereof, quality control of culture medium components and screening of an FGF signal channel regulator.

A plasmid capable of automatically inserting a membrane-localized luciferase reporter gene into a chromosome of a bacterium / recipient bacterium. After the plasmid inserts a luciferase reporter gene into a chromosome of a bacterium / recipient bacterium, the plasmid can be lost. Since the plasmid can be lost, the bacterium / recipient bacterium into whose chromosome the luciferase reporter gene is inserted does not require the addition of antibiotics during subsequent use, providing convenience and reducing interference.

The invention provides a mechanism for regulating and controlling AsA content of kiwi fruits by AcMYB73 and AcMYB108 transcription factors and application of the mechanism. According to the application disclosed by the invention, after GABA is used for treating kiwi fruits, transcriptome sequencing is carried out, and AcMYB73 and AcMYB108 transcription factors which are most likely to regulate and control ascorbic acid-related metabolic genes are screened from numerous transcription factors with most obvious abundance change; experiments such as LUC / REN dual luciferase, yeast single impurity, gel migration and the like prove that both the AcMYB73 and the AcMYB108 can positively regulate and control the ascorbic acid synthesis gene and the circulating gene of the kiwi fruit and reversely regulate and control the ascorbic acid degradation gene of the kiwi fruit at the same time. The invention also successfully constructs overexpression vectors and interference vectors of AcMYB73 and AcMYB108, and successfully realizes regulation and control of the content of ascorbic acid in kiwi fruits.

Coelenterazine analogs, methods for making the analogs, kits comprising the analogs, and methods of using the compounds to detect luminescence in luciferase-based assays are described. For example, the compounds can be used for in vivo imaging.

The invention provides a single-chain and double-protein-expressed circular RNA construct and a preparation method and application thereof, and the single-chain and double-protein-expressed circular RNA construct comprises 5'and 3 'introns used for realizing RNA self-splicing and cyclization as a first kind of introns; the 5'and 3 'homologous arms are used as pairing sites in RNA molecules; the exon 1 and the exon 2, which are connected after being spliced, can be spliced and connected together under the action of the first type of introns to form a complete and continuous open reading frame; an interval sequence for providing spatial and adjustment sequence structures; the IRES is used for starting translation; a glycine-serine linker as a flexible linker; a green fluorescent protein for gene expression monitoring; a luciferase for producing light by a catalytic chemical reaction. All the advantages of the double-IRES system for generating natural complete protein are reserved, and the core defect of low efficiency can be thoroughly overcome.

The invention relates to a novel copepod luciferase mutant and application thereof. The luciferase mutant takes an amino acid sequence of wild type Gaussian luciferase or an amino acid sequence of luciferase having at least 70% homology with the Gaussian luciferase SEQ ID NO: 3 as a reference, and compared with the amino acid sequence shown as SEQ ID NO: 3, the amino acid sequence of the luciferase mutant has the advantages that the amino acid sequence of the wild type Gaussian luciferase mutant is more than the amino acid sequence shown as SEQ ID NO: 3; at least one of the following positions is mutated: a tenth position, a 12th position, a 19th position, a 20th position, a 21st position, a 22nd position, a 23rd position, a 35th position, a 44th position, a 45th position, a 59th position, a 62nd position, a 67th position, a 78th position, a 85th position, a 86th position, a 87th position, a 93th position, a 107th position, a 144th position and a 121th position.

The present invention relates to novel cell lines for the expression of the human LHCG receptor and for the expression of the human LHCG receptor and a luciferase reporter gene. Methods for producing such cell lines are also described. The cell lines of the present invention, which express the LHCG receptor and a luciferase reporter gene, can be used in methods for determining the presence and biological activity of r-hLH and r-hCG samples.

The application discloses a use of phenazine-1-carboxylic acid or a pharmaceutically acceptable salt thereof in preparation of a medicine for inhibiting African swine fever virus. The application uses a double reporter virus rASFV-Gluc / EGFP co-expressing green fluorescent protein and Gaussia luciferase to screen 246 natural small molecule compounds, and finds that phenazine-1-carboxylic acid has a significant inhibiting effect on replication of the ASFV; further evaluation of the inhibiting effect finds that the half maximal cytotoxicity concentration of PCA on target cells of primary porcine alveolar macrophages is 470.5 muM, and the half maximal inhibitory concentration of PCA on the ASFV in PAMs is 1.59 muM. According to a dose-dependent inhibiting experiment result, when the concentration of PCA is 25 muM, the inhibiting effect on the ASFV can reach more than 100 times. The application has application prospects in preparation of medicines or preparations against the ASFV.

Provided are a dual-luciferase reporter gene detection system and use thereof. The detection system includes Gaussia luciferase, Pleuromamma xiphias luciferase, and a substrate. The substrate is coelenterazine or a derivative thereof. Compared with other dual-luciferase reporter gene detection systems, the present dual-luciferase reporter gene detection system produces stronger signals and requires simple reaction conditions.

The present invention relates to a method for screening compounds for bactericidal activity using thermostable luciferase and based on a real-time bioluminescence measurement. The present invention further relates to a method for determining the sensitivity of a bacterial sample originating from a subject suffering from a bacterial infection to a group of known antibiotics and to a method for assessing the minimum inhibitory concentration (MIC) of a bactericidal compound.

The present disclosure provides polypeptides that can be used in a complementation assay as a biosensor. In certain aspects, the polypeptide is a larger component of a split luciferase, wherein the luciferase is derived from LuxSit. The polypeptide may comprise an amino acid sequence having at least 85% identity to the amino acid sequence: of SEQ ID NO:1 and comprises D at position 30, N at position 46, R at position 58, R at position 61, F at position 64, T at position 68, E at position 101, and A at position 103; G at position 9, D at position 30, D at position 54, F at position 64, K at position 68, and E at position 101; D at position 30, D at position 46, K at position 68, and E at position 101; S at position 9, D at position 30, L at position 43, D at position 46, G at position 54, F at position 64, K at position 68, E at position 101, and T at position 103; D at position 30, D at position 46, F at position 64, K at position 68, E at position 101, and T at position 103; N at position 30, D at position 46, E at position 101, and A at position 103; or D at position 30, N at position 46, F at position 64, T at position 68, E at position 101, and A at position 103, wherein the numbering of the amino acid positions is based on the numbering of the amino acids in SEQ ID NO:1.

The application discloses a molecular marker, a primer, a detection method and application related to pig muscle fiber, rib number and back fat thickness, and relates to the technical field of molecular markers. The molecular marker is located in a MYLPF gene, and a sequence of the molecular marker is shown as SEQ ID NO: 4. The sequence is located at a first exon of a 202 transcript of the MYLPF gene, and is used for detecting SNP site mutation. The application determines a cause mutation affecting the circumference, area of pig muscle fiber, rib number and average back fat thickness by using a MYLPF gene promoter constructed to replace a double luciferase reporter vector. A specific primer is designed on the mutation site of the promoter and amplification is performed. The site is detected for polymorphism by using a restriction endonuclease NlaIII. According to the result of the polymorphism detection, differences in the circumference, area of muscle fiber, rib number and average back fat thickness among pig individuals are distinguished, and a large population scale sampling and determination is not needed.

The application discloses an anti-influenza virus high-sensitivity antibody and a preparation method and application thereof. The application provides an antibody for binding to an influenza virus, named VHH108-Fc-Nluc antibody, which comprises three segments, namely, a VHH108 antibody, Fc and NLuc luciferase; the VHH108 antibody has a segment shown in positions 3-125 in SEQ ID NO:1. The application further provides a kit for detecting the influenza virus, which comprises the VHH108-Fc-Nluc antibody and a VHH57 antibody. The VHH57 antibody has a segment shown in positions 230-350 in SEQ ID NO:3. The application is suitable for detecting various subtypes of influenza A viruses, has the advantages of high sensitivity and strong specificity, and has market value and application value.

The invention discloses a method for detecting an AAV neutralizing antibody. The method comprises the following steps: preparing a recombinant AAV vector AAV-Gluc carrying a Gaussian luciferase reporter gene; the method comprises the following steps: diluting AAV-Gluc according to a certain dilution step to prepare an AAV-Gluc working solution; diluting the sample to be detected; carrying out cell planking, culturing the target cells to form a uniform cell suspension, and adding the cell suspension into a cell culture plate; mixing the sample diluent with the AAV-Gluc diluent, and carrying out incubation; adding the incubated sample mixed solution into the cell culture plate, and carrying out virus infection and culture; obtaining a cultured cell supernatant, adding a detection solution, and detecting signal intensity; and analyzing and sorting the data to obtain the neutralization titer. The secreting type Gaussian luciferase reporter vector is used, target protein is secreted outside cells after being expressed, and detection is facilitated; the infected supernatant is directly sucked for detection, and the operation is convenient and fast; cell lysis and extraction are not needed, protein loss is reduced, and detection accuracy and precision are improved.

The present disclosure is directed to compositions comprising mitochondrial optogenetics-based gene therapies and their methods of use. In some embodiments, a composition described herein comprises an expression vector comprising a first nucleic acid sequence encoding a channelrhodopsin fusion protein and a second nucleic acid sequence encoding a luciferase protein. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence. In some instances, the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS). In some implementations, when the expression vector is expressed, the luciferase protein is localized to the cytosol. In some cases, the IMM-MLS comprises a leading sequence from a mitochondrial inner membrane protein selected from ABCB10, ABCB140, Cytochrome C, and renal outer medullary potassium channel (ROMK).

The invention provides a human brain organoid drug screening platform related to Huntington's disease, and the platform comprises: (1) a first brain organoid, which is formed by differentiation culture of a visual cell line containing a plurality of CAG repetitive sequences of HTT, and (2) a second brain organoid, which is formed by differentiation culture of the visual cell line containing a plurality of CAG repetitive sequences of HTT; and (2) a second brain organoid which is formed by carrying out differentiation culture on a quantifiable cell line, wherein the quantifiable cell line contains a plurality of CAG repetitive sequences of HTT. The human brain organoid drug screening platform related to the Huntington's disease is an organoid model with a fluorescence reporter gene d2GFP and luciferase, and an enhanced synaptic active response element (E-SARE) promoter is used for marking neuron activity; in addition, secretory luciferase can detect subtle changes of neuron activity.