383 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 belongs to the technical field of enzyme engineering and biology, and particularly relates to a fungal luciferase mutant and application of the fungal luciferase mutant in improving the bioluminescence intensity of fungi or plants, and the amino acid sequence of the fungal luciferase mutant is shown as SEQ ID NO.1. The fungal luciferase mutant NnLuzT138P is obtained by mutating fungal luciferase NnLuz, the NnLuzT138P and other FBP luminescence pathway genes are co-expressed in fungi or plants, the bioluminescence intensity of the fungi and the plants can be remarkably improved, and compared with the prior art, the light quantum intensity is improved by more than two times. The invention provides a feasible scheme for construction of enhanced sustainable luminous fungi and creation of luminous plants.

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.

Provided herein are bioluminescent polypeptides and compositions and methods for the assembly of a tripartite or multipartite bioluminescent complex. In particular embodiments, a bioluminescent complex is formed upon the interaction of three or more peptide and / or polypeptide components.

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 discloses a cell and a method for detecting the activity of a GLP-1R / GIPR double-target agonist drug. The invention relates to a cell for co-expressing GLP-1R, GIPR and CRE-miniP-luciferase reporter genes, and provides a method for detecting the biological activity of a GLP-1R / GIPR double-target agonist drug based on the cell, and the method is simple to operate, short in test period, sensitive in reaction, small in variability, strong in specificity and high in accuracy, can be used for release inspection of products, and can be used for detecting the biological activity of the GLP-1R / GIPR double-target agonist drug. The method has important significance on quality control and clinical application of GLP-1R / GIPR double-target agonist drugs.

Cas-cleavable reporter systems, CRISPR-Cas systems thereof, and methods of use thereof in CRISPR-Cas based diagnostics. Cas-cleavable reporter systems may generate a luminescent, fluorescent, or other detectable signal upon Cas-collateral cleavage of one or more reporter system components. CRISPR-Cas systems may comprise a Cas protein having collateral cleavage activity, guide molecules, and the Cas-cleavable reporter system. Cas-cleavable reporter systems may be a split luciferase reporter system, at least one element of which is bound to beads. For multiplexing, CRISPR-Cas systems may comprise a Cas-cleavable quenched reporter system, optionally a Cas-cleavable quenched fluorescent reporter system, a Cas protein having collateral cleavage activity, and target-specific guide molecules attached to color-coded beads. CRISPR-Cas systems may further comprise amplification reagents. Methods may apply said CRISPR-Cas systems to flow cell, well-plate, or other devices, and may measure detectable signals by microscopic, plate reader, or other methods.

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 method for detecting insect dsRNA degrading enzyme activity based on a dual luciferase report system, and relates to the technical field of biological medicine. The method comprises the following steps: constructing an insect dsRNA degrading enzyme into a pAc5.1 / V5-His B plasmid to form a recombinant plasmid, co-transfecting the recombinant plasmid and a psiCHECKTM-2 plasmid into an insect cell for culturing, then respectively adding dsLuc and dsGFP into the cell, continuing culturing, finally collecting the cell, and detecting the enzyme activity of the cell. The system is constructed based on a dual-luciferase report system, and qualitative and quantitative enzyme activity is realized through three steps of cell culture, transfection and detection. Compared with in-vitro heterologous protein expression, agarose detection after in-vitro degradation reaction or RNA extraction and then fluorescent quantitative PCR (RT-qPCR) detection, the method provided by the invention is faster and more accurate, and is not limited to the first step in which the protein is difficult to obtain.

The present invention discloses an application of a nano-luciferase fusion antigen in the detection of African swine fever virus antibodies. The fusion antigen is composed of LgBiT-p30 and SmBiT-p30 proteins, which are fused and expressed by two subunits of nano-luciferase, LgBiT and SmBiT, respectively, with the African swine fever virus p30 protein. The fusion antigen can bind to a specific antibody against the African swine fever virus p30 protein to form a functional luciferase, which reacts with a substrate to produce a bright luminescent signal. The fusion antigen has high specificity and sensitivity in the detection of African swine fever virus antibodies, making the antibody detection process more efficient and rapid. The present invention realizes simple, rapid, sensitive and specific detection of African swine fever virus antibodies, and provides an innovative and reliable solution for the serological diagnosis of African swine fever.

The invention discloses a dual-luciferase reporter gene detection system and application thereof. The detection system comprises Gaussian luciferase, Pleuroma xiphias luciferase and a substrate, and the substrate is coelenteran or a derivative of coelenteran. Compared with other dual-luciferase reporter gene detection systems, the dual-luciferase reporter gene detection system has stronger signals and simple reaction conditions.

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.

An isolated polynucleotide encoding a modified luciferase polypeptide and substrates. The OgLuc variant polypeptide has at least 60% amino acid sequence identity to SEQ ID NO: 1 and at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID NO: 1. The OgLuc variant polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to the corresponding polypeptide of the wild-type Oplophorus luciferase.

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 discloses a group of chicken PGC-1alpha gene promoters as well as a construction method and application thereof, and belongs to the field of gene engineering. The chicken PGC-1 alpha gene promoter is any one of A1-A5 sequences of which the nucleotide sequences are shown as SEQ ID NO: 1 to SEQ ID NO: 5. The promoter is applied to the promotion of expression of firefly luciferase in chicken preadipocytes, and is applied to the regulation and control of the chicken PGC-1 alpha gene promoter by a specific transcription factor PPAR gamma. The five PGC-1alpha gene promoters have promoter activity in chicken preadipocytes, and can directly regulate and control transcription expression of the chicken PGC-1alpha gene. The chicken PGC-1alpha promoter disclosed by the invention can predict the transcriptional activity of the PGC-1alpha at the in-vitro cellular level, so that the chicken PGC-1alpha promoter has potential application values for revealing a transcriptional regulation mechanism of the chicken PGC-1alpha, breeding low-abdominal-fat broilers, revealing molecular mechanisms after various human diseases occur and developing drugs for targeting PGC-1alpha expression.

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 construction method and application of a Uusu figure virus report replicon. According to the invention, a low-copy plasmid pACYC-177B is used as a carrier, all non-structural proteins and non-coding region genes of USUV are retained, 38 amino acids before the N terminal of C protein are retained in the structural protein part, 45 amino acids behind the C terminal of E protein are retained in the structural protein part, and a NanoLuc luciferase tag is inserted between the retained C and E protein genes to obtain a pACYC-TMN-Nlucc-Rep report replicon. The Uu figure virus report replicon pACYC-TMN-Nluc-Rep constructed in the invention can normally work on cells such as BHK21, Vero, DF-1, Hela and the like, and can be effectively translated and replicated. Therefore, the USUV report replicon pACYC-TMN-Nlucc-Rep constructed in the invention can be used as an important research tool to be applied to the research of the USUV.

The invention discloses application of phenazine-1-carboxylic acid or medicinal salt thereof in preparation of drugs for inhibiting African swine fever virus. According to the invention, a double-report virus rASFV-Glucc / EGFP for co-expressing green fluorescent protein and Gaussian luciferase is used, and screening from 246 natural small molecule compounds finds that phenazine-1-carboxylic acid has a remarkable inhibition effect on the replication of ASFV; the inhibition effect of the PCA is further evaluated, and it is found that the half maximum cytotoxicity concentration of the PCA to primary porcine alveolar macrophages of target cells is 470.5 mu M, and the half maximum inhibition concentration of the PCA to ASFV in PAMs is 1.59 mu M. According to a dose-dependent inhibition experiment result, when the PCA concentration is 25 [mu] M, the ASFV inhibition effect can reach 100 times or more. The invention has application prospects in preparation of anti-ASFV drugs or preparations and the like.

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 discloses a double-report plasmid system and a construction method and application thereof. The double-report plasmid system comprises a translation report plasmid and an error folding response plasmid; the translation reporter plasmid comprises a target protein gene sequence, an mRNA secondary structure sequence and a luciferase gene sequence; the misfolding response plasmid comprises a heat shock pressure promoter and a fluorescent protein gene. The double-report plasmid system can quantitatively and visually detect translation characteristics and wrong folding conditions of the recombinant protein, the whole operation process is carried out based on a 96-well plate, the double-report plasmid system has the characteristics of low cost, high timeliness, high flux and the like, and the recombinant protein can be rapidly and efficiently soluble (positive: complete soluble; negative: completely insoluble; and in an intermediate state, the solubility is relatively poor), so that the efficiency of recombinant protein expression research is greatly improved.