10 results about "Firefly Luciferases" patented technology

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.

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 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.

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 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.