24 results about "Firefly Luciferases" patented technology

The invention discloses a construction method and application of a rabies virus RNA polymerase dual-luciferase report system, and belongs to the technical field of biology. In order to solve the technical problem that a report system for detecting the RABV RNA polymerase activity needs to be developed urgently, a Firefly luciferase fragment and a vector pcDNA3.1-SRV9-M-G obtained by amplifying M protein and G protein-deleted rabies virus SRV9 strain full-length plasmid are seamlessly connected, a recombinant luciferase report plasmid is constructed, the recombinant luciferase report plasmid and an auxiliary plasmid are combined, and the recombinant luciferase RNA polymerase activity detection system is constructed. And the RABV RNA polymerase dual-luciferase report system is formed by the Renilla luciferase report plasmid and the T7 promoter plasmid, and the RABV RNA polymerase dual-luciferase report system comprises the Renilla luciferase report plasmid and the T7 promoter plasmid. The system can accurately and efficiently detect the activity of RABV polymerase, and by virtue of the function, effective antiviral targets and therapeutic drugs can be screened out in an assisted manner.

The invention provides application of microbial metabolites in tumor immune response and an intestinal microbial metabolite screening method. A screening system is constructed by combining firefly luciferase with PD-1 / SHP-2, various intestinal microbial metabolites are independently co-cultured with the screening system, the screened microbial metabolites are glycodeoxycholic acid and taurocholic acid, and the influence of various metabolites on PD-1 pathway signal transduction is reflected by measuring the activity of luciferase. The invention constructs a reliable, rapid and effective screening method for discovering intestinal microbial metabolites influencing a PD-1 signal channel and evaluating the influence efficacy of the metabolites, and screening out metabolites having effects on intestinal cancer immune response, thereby providing an experimental basis for enhancing intestinal cancer treatment.

This invention provides a method for constructing a visualized mouse model characterizing hepatocyte necrosis and apoptosis, belonging to the field of biomedical technology. The invention involves disassembling firefly luciferase (Fluc) into inactive N-terminal (Nluc) and C-terminal (Cluc) fragments, which are then fused and expressed at specific locations on the MLKL molecule, forming two independent plasmid vectors, MLKL-Nluc and MLKL-Cluc. These two plasmid vectors are then injected into mouse hepatocytes via tail vein high-pressure hydrodynamic injection to construct the visualized mouse model characterizing hepatocyte necrosis and apoptosis. This invention allows for non-invasive and dynamic monitoring of the execution process of necrosis and apoptosis—MLKL oligomerization—in animal models, providing a powerful and specific tool that fills the technological gap in real-time visual monitoring of necrosis and apoptosis in pan-apoptotic research.

The application belongs to the field of plant synthetic biology and plant gene expression regulation, and particularly relates to a vector for plant promoter double system quantitative characterization, a standardization method, a product and application. The method combines a protoplast transient expression system and an agrobacterium-mediated leaf transient expression system, uses a compact promoter characterization plasmid (pLIP) containing a firefly luciferase module driven by a to-be-detected promoter and a renilla luciferase internal reference module driven by a 35S promoter, and the two modules are located in a T-DNA interval. The ratio of the two luciferases is calculated, the RPU (relative promoter unit) method is introduced, a standard promoter (35S) is used as a reference, and normalization and direct comparison of promoter activities in different systems are realized. The scheme significantly improves the repeatability and cross-system compatibility of promoter activity data, and is suitable for efficient screening and standardized characterization of regulatory elements in plant synthetic biology.

The application discloses a firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit, which comprises a luciferase lysis solution, a firefly luciferase detection reagent, a NanoLuc luciferase detection buffer, a NanoLuc luciferase substrate concentrate, an AkaLuc luciferase detection buffer and an AkaLuc luciferase substrate concentrate. The firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit prepared by the application has high detection sensitivity, a wider linear detection range, higher signals, a smaller signal attenuation degree, a slower light signal decline trend, and is more suitable for application in high-throughput detection.

A cell-based screening system and method for identifying compounds that selectively modulate the expression of CAG repeat-containing RNA associated with spinocerebellar ataxias and related disorders. The system comprises a human HEK293T cell line engineered to co-express two reporter constructs: a CAG repeat-expanded polyglutamine-nanoluciferase fusion protein with at least 60 CAG repeats, and a control firefly luciferase with no CAG repeats. Each construct contains a unique probe-binding sequence downstream of the repeat region, enabling independent quantification via multiplex RT-qPCR with fluorescent probes, as well as dual luciferase assays. The cell line is optimized for high-throughput screening to identify therapeutic compounds that reduce pathogenic CAG repeat RNA levels while sparing control transcripts. The invention further encompasses methods for screening, validating, and identifying candidate therapeutics for CAG expansion disorders, including spinocerebellar ataxias and Huntington's disease.

This invention relates to a quantitative method for ultrasonic disruption of *E. coli* cells, specifically a method based on firefly luciferase in *E. coli* cells. The method involves mixing *E. coli* expressing firefly luciferase as an internal standard with a suspension of target protein-expressing bacteria, followed by ultrasonic disruption of the cells. The degree of disruption of the target protein-expressing bacterial suspension is quantitatively calculated by measuring the activity of the firefly luciferase. The target protein-expressing bacterial suspension uses *E. coli* expressing the target protein. Compared with existing technologies, this invention overcomes the shortcomings of complex experiments, low efficiency, and unstable accuracy in existing technologies. It achieves efficient and highly accurate quantification of the degree of ultrasonic disruption of *E. coli* cells, reduces experimental complexity, and improves research and development efficiency and accuracy. This provides a scientific basis and new approach for the development of new feed protein resources and livestock breeding research, and has broad application potential.

The invention discloses a subtropical estuary benthic fish mullet chubil AHR1b gene recombinant expression plasmid and application thereof, and belongs to the technical field of environmental toxicology. The AHR1b gene recombinant expression plasmid is prepared by introducing an AHR1b gene into an expression vector. The invention also discloses a luciferase reporter gene system, which comprises an AHR1b gene recombinant expression plasmid, an ARNT1 gene recombinant expression plasmid and a firefly luciferase reporter plasmid containing a mouse CYP1A1 gene promoter. According to the invention, a luciferase reporter gene system is utilized to carry out comprehensive characterization on TCDD toxicity and evaluate the sensitivity and the specificity of a mugilogobius chuchuyi AHR1b and ARNT1 subtype dependency reporter gene system, so that a theoretical basis is provided for supporting high-throughput and high-sensitivity environment monitoring, and meanwhile, a species-specific early warning technology is provided for offshore and estuary DLCs pollution.

The invention provides a chikungunya virus invasion model, an establishment method and application, according to the invasion model, chikungunya structural protein expression plasmids and firefly luciferase reporter gene plasmids containing specific packaging signals are co-transfected to mammalian cells, and the chikungunya structural protein expression plasmids and the firefly luciferase reporter gene plasmids containing the specific packaging signals are co-transfected by utilizing a packaging mechanism of the packaging signals. And self-assembling to form the high-simulation virus-like particle capable of efficiently bearing the reporter gene. According to the method, evaluation of chikungunya virus invading cells without depending on a BSL-3 experimental environment is achieved, and drugs / vaccines for resisting the chikungunya virus can be quantitatively screened through firefly luciferase. The method has the advantages that the whole process that viruses invade host cells can be efficiently and accurately simulated, meanwhile, high sensitivity and operation convenience are achieved, and a stable, reliable and high-drug-effect-resolution experimental platform is provided for deeply researching the invasion mechanism of CHIKV and developing targeted inhibitor high-throughput screening.

The application discloses a luciferase substrate, a preparation method and use thereof. The luciferase substrate has the advantages of long emission wavelength, high light intensity and high detection sensitivity after acting on firefly luciferase, and has good performance in live cell and live animal imaging, and can be used as an important analysis tool in the fields of live bio-luminescence imaging and protein quantitative detection.

The invention discloses a method for identifying the translation activity of a 5 '-end cap analogue of mRNA (messenger Ribonucleic Acid), which is characterized in that a luciferase expression plasmid containing a 5' UTR (Untranslated Region), a firefly luciferase open reading frame, a 3 'UTR and a PolyA tail is used as a transcription system, and the translation activity of the 5'-end cap analogue is evaluated according to the activity of firefly luciferase expressed by a reporter gene. According to the method, differentiated translation activities can be identified for the 5 '-end cap analogues with different structures, a methodological basis is provided for accurate configuration of mRNA translation, and the method can also be used for high-throughput screening of the 5'-end cap analogues with intracellular translation activities.

The application discloses a uORF element for regulating chicken insulin receptor expression and application thereof, belongs to the field of molecular biology and genetic engineering, and the nucleotide sequence of the uORF element is shown in any one of SEQ ID NO. 1-2; the application comprises any one of the following: (1) regulating the expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts and chicken preadipocytes; (2) being applied to genetic breeding of chickens, and the genetic breeding is breeding of meat chickens with reduced insulin resistance; (3) application in preparation of a reagent for regulating the expression of chicken insulin receptors. The application can regulate the expression level of INSR, improve insulin sensitivity, and provide material support for research on the mechanism of insulin in regulating blood glucose.

The invention belongs to the field of virus detection, and discloses a novel coronavirus detection kit and application thereof in drug screening. The kit comprises a sample buffer solution, a positive control reagent, a detection reagent, a sampling label and a pipette. A modified firefly luciferase is used, the modified firefly luciferase carries the most effective site of action of 3CL at a non-critical site, and when coronavirus or host cells carrying viral components are present in a test sample, the 3CL enzyme in the sample destroys the modified firefly luciferase, breaks the enzyme into two parts, resulting in inactivation thereof. The coronavirus infection is diagnosed by measuring the specific enzyme activity of the coronavirus in a clinical sample, and the method is suitable for large-scale rapid preliminary screening and suitable for medical units such as community clinics with simple condition facilities; the positive sample provided by the invention needs to be further typed and diagnosed, such as typed and diagnosed by using a PCR (Polymerase Chain Reaction) method.

Aging is a process that the structure, functions and various physiological systems of an organism gradually decline after the organism experiences long-term physiological and metabolic activities, and is closely related to various chronic diseases and degenerative diseases. The aging process usually accompanies the phenomena of cell cycle stagnation, DNA damage, telomere shortening and the like. Existing senescence detection methods such as SA-beta-gal staining method, gene expression analysis and the like can provide certain senescence marker detection, but have limitations in the aspects of monitoring the senescence process in real time and capturing dynamic cell changes. Therefore, the development of an efficient, sensitive and real-time senescence detection technology is particularly important. The invention relates to an aging drug screening platform established on the basis of dual fluorescein plasmids, and real-time and accurate monitoring of cell molecular events in an aging process is realized by utilizing plasmids marked by two different fluoresceins and simultaneously using two fluorescently-marked genes, namely firefly luciferase and Renilla luciferase. By installing the promoter of the senescence marker gene hP21 or hGLB1 into pGL4.17 and pRL plasmids, senescence can be detected with high precision and high sensitivity, and the defects of a traditional detection method are overcome. The technology not only can reflect the cell change in the aging process in real time, but also can be widely applied to large-scale aging-related effective drug screening, and serves for obtaining drugs which can be applied to clinical treatment.

An ultrasensitive and long-lasting luminescence cascade can be used for point-of-care detection. A reaction chamber can receive and incubate a volume of a sample and a volume of magnetic tag solution before the combination is washed. Then, a working solution including d-luciferin-6-O-β-d-galactopyranoside (LUGAL), firefly luciferase (Fluc), and a luminescence buffer comprising ATP can be added to the reaction chamber. When the reaction chamber also includes tagged target components including beta galactosidase (GAL) then the GAL supplies luciferin intermediates that catalyze the cleavage of a protecting group from the LUGAL, generating luciferin, and the Fluc catalyzes the oxidation of D-luciferin in a presence of the ATP and O2, yielding oxyluciferin and luminescence. Repeated catalytic cycles increase a measurable signal of the luminescence. An optical sensor can capture an image of the luminescence and the presence of tagged target components can be determined based on an amount of luminescence in the image.

The invention belongs to the field of plant synthetic biology and plant gene expression regulation and control, and particularly relates to a vector for plant promoter dual-system quantitative characterization, a standardization method, a product and application. According to the method, a protoplast transient expression system and an agrobacterium-mediated leaf transient expression system are combined, a promoter characterization plasmid (pLIP) with a compact structure is used, a firefly luciferase module driven by a promoter to be detected and a renilla luciferase internal reference module driven by a 35S promoter are contained, and the firefly luciferase module and the renilla luciferase internal reference module are located in a T-DNA interval. Normalization and direct comparison of the activities of promoters in different systems are realized by calculating the ratio of dual luciferase and introducing an RPU (Relative Promoter Unit) method and taking a standard promoter (35S) as a reference. According to the scheme, the repeatability and cross-system compatibility of promoter activity data are remarkably improved, and the method is suitable for efficient screening and standardized characterization of regulatory elements in plant synthetic biology.

The invention discloses a firefly luciferase Fluc mutant and application of the firefly luciferase Fluc mutant, wild firefly luciferase Fluc from North America fireflies is taken as a research object, single mutation or combined mutation is carried out on a protein sequence of the firefly luciferase Fluc to obtain a mutant protein, and the mutant protein is used for preparing the firefly luciferase Fluc mutant. Compared with wild type Fluc, the heat stability and activity of the Fluc mutant protein are improved to a certain degree, compared with the wild type Fluc, the heat stability of the preferable mutant protein is improved by 13.2-14.5 DEG C, the activity is improved by 1.4-2.5 times, the yield is improved by 2.4-3.9 times, the purity of the obtained preferable mutant protein is higher than 99%, and it can be seen that the preferable mutant protein can be applied to the field of industrial production. The luciferase Fluc mutant protein provided by the invention has higher enzyme activity, better thermal stability and higher yield, widens the application conditions of luciferase Fluc, has higher practical application value, and is more suitable for large-scale production and industrial use.

The application discloses a subtropical estuary benthic fish Oxyeleotris marmoratus AHR1b gene recombination expression plasmid and application thereof, and belongs to the technical field of environmental toxicology. The AHR1b gene recombination expression plasmid is prepared by introducing the AHR1b gene into an expression vector. A luciferase reporter gene system is also disclosed, which comprises the AHR1b gene recombination expression plasmid, an ARNT1 gene recombination expression plasmid and a firefly luciferase reporter plasmid containing a mouse CYP1A1 gene promoter. The luciferase reporter gene system is used to comprehensively characterize TCDD toxicity, to evaluate the sensitivity and specificity of the Oxyeleotris marmoratus AHR1b and ARNT1 subtype-dependent reporter gene system, to provide a theoretical basis for supporting high-throughput and high-sensitivity environmental monitoring, and to provide a species-specific early warning technology for offshore and estuary DLCs pollution.

The invention discloses an AHR2a and ARNT1 gene recombinant expression plasmid of subtropical estuary benthic fish mullet chubil and application of the recombinant expression plasmid, and belongs to the field of environmental toxicology. According to the recombinant expression plasmids, AHR2a and ARNT1 genes are respectively connected with an expression vector to construct a recombinant expression plasmid I and a recombinant expression plasmid II. The invention also constructs a luciferase reporter gene system containing the two recombinant expression plasmids and a firefly luciferase reporter plasmid containing a mouse CYP1A1 gene promoter. By utilizing the luciferase reporter gene system, the toxicity of dioxin and dioxin-like chemical substances and complex environmental wastewater can be comprehensively represented, the sensitivity and the specificity of the mugilogobius chuenensis AHR2a and ARNT1 subtype dependency reporter gene system are evaluated, and a theoretical basis is provided for supporting high-throughput and high-sensitivity environmental monitoring; and a species-specific early warning technology is provided for offshore and estuary DLCs pollution.

The invention provides a dual-luciferase reporter gene detection kit suitable for a plant sample, and belongs to the technical field of biological detection, the kit comprises a lysate containing PIPES-KOH, PVPP, NP-40, Triton-X100, ascorbic acid, DTT, EDTA, sucrose and glycerin, a firefly luciferase detection working solution and a renilla luciferase detection working solution. The lysate provided by the invention can promote the lysis of a plant sample and reduce the inhibition on firefly luciferase and renilla luciferase, and is matched with a firefly luciferase detection working solution and a renilla luciferase detection working solution; the extraction efficiency, the activity stability and the detection efficiency of firefly luciferase and renilla luciferase in a plant sample to be detected are remarkably improved, so that the detection sensitivity of the plant sample is improved.

The present invention belongs to the field of drug detection and specifically discloses a method for detecting the antibody-dependent cell-mediated cytotoxic biological activity of human immunoglobulin, comprising the following steps: 1) taking a target cell suspension and adding it to a well plate, then adding a series of concentrations of human immunoglobulin solutions for incubation, and finally adding an effector cell suspension for incubation; 2) taking the well plate incubated in step 1) to detect fluorescence, draw a dose-effect curve and calculate the half-maximal effective concentration; the target cells are CHO-K1 cells expressing the SARS-CoV-2 Spike protein, and the effector cells are Jurkat cells stably expressing CD16a on the cell membrane and stably transfected with the firefly luciferase gene driven by the NFAT response element. The detection method of the present invention can accurately and reliably detect the antibody-dependent cell-mediated cytotoxic biological activity of two different human immunoglobulin preparations, IVIG and SCIG.

The invention discloses a mouse ovarian cancer carboplatin drug-resistant cell strain as well as a construction method and application thereof, and belongs to the field of biomedicine. According to the invention, a firefly luciferase gene is transfected and introduced into a mouse epithelial ovarian cancer ID8 cell strain through a lentiviral vector, and a cell strain (ID8-Luc) capable of stably expressing the luciferase gene is obtained through puromycin screening. In-vitro induction is carried out by gradually increasing the dosage of carboplatin, the carboplatin-resistant ID8-Luc cell strain is successfully constructed, the drug-resistant strength is high, and the drug-resistant stabilization time is long. The invention provides an efficient tool cell strain for exploring a tumor drug resistance mechanism and screening anti-tumor drugs and novel targeted drug-resistant drugs, and has a good application prospect.

The invention provides a MAFG transcriptional activation luciferase reporting system based on an MYC promoter, relates to the technical field of biomedicine, and is technically characterized in that the MAFG transcriptional activation luciferase reporting system based on the MYC promoter is provided and comprises a reporter gene plasmid constructed by an MYC promoter truncation body and firefly luciferase, the MYC promoter truncation body comprises a sequence as shown in SEQ ID NO: 01. The invention provides a dual-luciferase report system (Dual-Luciferase report) based on an MYC promoter, and the dual-Luciferase report system (Dual-Luciferase report) is used for detecting the activation transcription capability of a transcription factor MAFG. The luciferase does not need post-translation processing, so that report activity is generated immediately once translation is carried out; and the light product quantum efficiency is high, so that the detection is sensitive and rapid. The method is mainly applied to miRNA target gene verification, transcription factor regulation verification and the like at present. The system can accurately reflect the transcriptional activity of MAFG based on luciferase of an MYC promoter region.

The invention discloses a uORF element for regulating and controlling expression of a chicken insulin receptor and application of the uORF element, and belongs to the field of molecular biology and genetic engineering, and the nucleotide sequence of the uORF element is shown as any one of SEQ ID NO.1-2; the application comprises any one of the following applications: (1) regulating and controlling expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts and chicken preadipocytes; (2) the method is applied to genetic breeding of chickens, and the genetic breeding refers to breeding of broiler chickens capable of reducing insulin resistance; and (3) application in preparation of a reagent for regulating expression of the chicken insulin receptor. The expression level of INSR can be regulated and controlled, insulin sensitivity is improved, and material support is provided for research on analysis of a mechanism for regulating and controlling blood sugar by insulin.