135 results about "Firefly Luciferases" patented technology

The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2 we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). It is shown herein that Neh2 domain is sufficient for recognition, ubiquitination and proteasomal degradation of Neh2-luciferase fusion protein. The novel Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time-course of reporter activation. The novel reporter was used to screen a library of compounds to identify activators of Nrf2. The most robust and yet non toxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin-induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

The invention relates to a method for preparing a chitosan-silk fibroin composite nano-fiber multifunctional patch for promoting myocardial tissue regeneration and monitoring stem cells. The method comprises the following steps: preparing a cellulose nano-fiber basal plate by an electrostatic spinning technology; alternately assembling positively charged chitosan (CS) and negatively charged silk fibroin (SF) to the surface of nano-fibers layer by layer by adopting a layer-by-layer assembly technique, and assembling 5.5-10.5 layers to form a CS-SF composite nano-fiber membrane; and planting seed cells of adipose tissue-derived stromal cells or cardiac progenitor cells labeled by green fluorescent protein and firefly luciferase on the surface of the CS-SF composite nano-fiber membrane, preparing the patch by three-dimensional co-culture. The patch has excellent biocompatibility, can be used as a cell vector, has an effect of resisting oxidative stress to improve the survival rate and treatment efficiency of stem cells, and is capable of evaluating the number, distribution and function states of transplanted stem cells, effectively preventing occurrence of post-myocardial infarction heart failure and reducing the death rate of ischemic cardiomyopathy.

The invention relates to a method and apparatus for fast testing the surface cleaning degree and microorganism pollution. The ATP sulfurylase can promote the PPi on the condition of existing adenosine-5'-phosphosulfate APS to generate ATP so as to reduction the hydrolysis pyrophosphoric acid ATP. It adds firefly luciferase stabilizer to simplify the preserving condition and adds adenosine triphosphatase restrainer to improve the test accuracy and sensibility.

The invention relates to a gene encoding of firefly luciferase, its preparation method and application, which comprises the following steps: 1. introducing mutation sites of mutant enzymes; 2. identifying recombinant plasmids; 3. sequencing to obtain polymerase chain reaction (PCR) fragment with 1060 mutation sites; 4. obtaining escherichia by using restriction endonucleasecoli purifying after recovering and purifying through gel; 5. identifying recombinant plasmids to obtain a separating protein gene; 6. culturing escherichia coli, 7. converting recombinant plasmids into BL21 competent cell to obtain bacterial strain capable of expressing mutant enzymes, 8. culturing the obtained BL21 expression bacterial strain; 9. centrifuging bacteria liquid of protein, centrifuging to obtain supernatant through ultrasonic fragmentation; 10. determining to obtain firefly luciferase with high heating stability and high enzyme activity. The invention relates to application of firefly luciferase gene on foodstuff, medicine, thalline detection. The luminous intensity of the mutant enzyme is about 15 to 20 times than that of wild type, and the mutant enzyme has good heat stability than that of the wild type.

The invention belongs to the biological technical field and relates to a method of screening a tumor multidrug resistance reversal agent. In the method, a promoter specific sequence of a multidrug resistance (MDR1) gene is cloned and regrouped into a firefly luciferase report gene vector promotor sequence, therefore, an MDR1 report gene vector is constructed. The constructed vector and the sea pansy luciferase report gene vector are cotransfected into a sensitive and drug resistant colon cancer cell line. The activities of fireflies and the luciferase are detected simultaneously to reflect the promotor transcription activity of a MDRI gene promoter. Therefore, the method of screening the tumor multidrug resistance reversal agent in a targeted way is constructed. The method has perfect application prospect in screening tumor multidrug resistance reversal agents, researching the mechanism of the tumor multidrug resistance reversal agents and mechanisms of chemotherapeutic drugs inducing humor cells to generate drug resistance, etc.

The invention discloses a novel multifunctional dual-luciferase reporter gene plasmid. The plasmid contains a luc firefly luciferase gene, a Rluc renilla luciferase gene, a SV40 poly(A) termination signal part and two independent multiple cloning sites, wherein the two independent multiple cloning sites are located on the upstream and the downstream of a luc firefly luciferase gene coder frame respectively. The dual-luciferase reporter gene plasmid containing detection gene and reference gene is successfully constructed, not only can the detection gene be transferred into a subject cell conveniently, but also the reference gene can be taken in correspondingly, the complexity of experiment operation and experiment errors are greatly reduced, the plasmid has the multiple cloning sites at thetwo ends of the firefly luciferase gene, the multiple cloning site located on the upstream of firefly luciferase can introduce a promoter sequence which can be used for genetic transcription adjustment, control and detection, and the multiple cloning site located on the downstream of the firefly luciferase can introduce a 3'UTR sequence which can be used for miRNA target identification, so that switching of different application of the same plasmid is achieved.

This invention relates to a method for constructing 9LLUC cell strain capable of stably expressing firefly luciferase PGL3. This invention provides a cell strain 9LLUC, which is obtained by transfecting glioma cell 9L with lentivirus vector containing firefly luciferase PGL3 gene, and screening. Cell strain 9LLUC can be transplanted into different animals to obtain relative glioma animal models. Though stable expression of PGL3, the generation, development, infiltration, metastasis of glioma cell, and the curative effects of relative therapies can be real time observed. The glioma animal models can be used for studying the development of glioma, and screening relative drugs and therapies.

The invention relates to an A549 nude mouse model of stably expressed luciferase and building and application thereof. The nude mouse model leads firefly luciferase genes to human lung adenocarcinoma A549 cell strains through the gene recombination technology and lentivirus infection, tumor clone cell strains of the stably expressed luciferase are obtained through subcloning screening, and recombinational A549 cells are inoculated in a mouse to obtain the A549 nude mouse model. Compared with a traditional tumor model drug effect detection method, the A549 nude mouse model enables observation to be visual and convenient, can be used for somatoscopy without damaging animals, is reliable in result, and has good application prospect.

The invention discloses a firebug luciferase gene and applications thereof, the gene of the luciferase gene is a DNA molecule shown in sequence 1 of the sequence list. A recombination expression vector can be established by using the firebug luciferase gene, and then is transduced into host cells, thus obtaining the firebug luciferase. Compared with the present production methods of firebug luciferase, the method of the invention is characterized by being easy purified, high yield and purity, and good preserved activity; the invention is a high effective production method of firebug luciferase and suitable for being applied in industrial production.

The invention discloses a dual fluorescent reporter gene vector for identifying miRNA targets, a preparation method thereof, and an application thereof in identifying the miRNA targets in coding regions and detecting functions of the miRNA on the coding regions. The carrier has a renilla luciferase gene and a firefly luciferase gene, and multiple cloning sites (MCS) are located between a terminal amino acid codon and a stop codon of a firefly luciferase reporter gene, thereby facilitating insertion of the miRNA targets into the coding regions of the firefly luciferase reporter gene, so as to identify the miRNA targets in coding regions and detect the functions of the miRNA on the coding regions. The preparation method of the carrier provided by the invention does not require a special treatment for terminals of the carrier or target fragments, is free of connection reaction and PCR amplification, and is simple to operate, short in experiment period and high in efficiency.

A process for reversible chemical modification of the luciferase, a process for the covalent conjugation of a reversibly modified luciferase to a chemical moiety (a protein or a binding partner such as biotin or an antibody), a process for reactivation of the reversibly-modified and inactivated luciferase, a process for making the said luciferase conjugates and a bioluminescent assay method that uses covalently conjugated firefly luciferase are taught. The present invention also relates to a composition comprising a reversibly modified luciferase, as well as a composition comprising a reversibly modified luciferase covalently conjugated to a chemical moiety.

The invention discloses a cell sifting moudle for fast sifting of antineoplastic- histone precursor histone deacetylase inhibitor compound from compound storage during new drug development. The moudle makes use of speciality that the precursor histone deacetylase inhibitor can activate some starting subsequence, constructs te oexpression vector containing special starting factor and firefly luciferase reporting gene, and builds cos- 7 clone that stably expresses said vector. The cell contains starting subsequence with different degree of activity for luciferase, the starting factor can be acitvated by histone precursor histone deacetylase inhibitor and then the expression of luciferase reporting gene is intensified, and so the detection for histone precursor histone deacetylase inhibitor activity in sample through detection of luciferase activity change can be finished in a short time. The cell sifting moudle can be used for fast and high- flux sifting of new antineoplastic and histone precursor histone deacetylase inhibitor compound from compound storage during new drug development.

The invention provides a method for detecting duck IFN-beta promoter activity by using double luciferase reporter genes. The method comprises the following steps: A, constructing firefly luciferase recombinant plasmids containing a duck IFN-beta promoter target sequence; B, premixing the firefly luciferase recombinant plasmids containing the duck IFN-beta promoter target sequence with an irritant,internal reference plasmids and lipidosomes, and then carrying out transfection on target cells, wherein the internal reference plasmids are sea cucumber luciferase plasmids; and C, carrying out cracking after cultivation on the target cells after transfection, and detecting luciferase activity so as to obtain the duck IFN-beta promoter activity. The method is simple and practicable in operationand high in flux, and has relatively high repeatability and accuracy, and an effective detection tool is provided for researching duck IFN-beta passages.

The invention discloses a method for detecting plasmids of polycyclic aromatic hydrocarbons in an environment and an application thereof, and relates to the fields of molecular biology and environmental biotechnology. According to the method provided by the invention, by extracting the promoter of a humanized AHR (Aromatic Hydrocarbon Receptor) gene, using a CV060 plasmid including a firefly luciferase and renilla luciferase reporter gene as a carrier and inserting the AHR gene promoter with a specific sequence, a recombined lentivirus plasmid of a reference internal type dual-luciferase reporter gene including the AHR gene promoter is built. The detection method provided by the invention is good in repeatability, high in sensitivity, reliable in detection result, and simple and convenient to operate, and can be widely applied to the detection of the polycyclic aromatic hydrocarbons in the environment.

The invention discloses fusion protein for screening and evaluating an anti-enterovirus 71 (EV 71) medicine and application of the fusion protein. According to the fusion protein, firefly luciferase (Fluc) is used as a reporter gene, two micromolecule polypeptides which can be bonded tightly pepA and pepB are bonded with the N end and the C end of the firefly luciferase respectively, the middles of the firefly luciferase are connected by an EV713C protease effect substrate, and a fusion gene is inserted into an eukaryotic expression vector and is expressed to generate the fusion protein. Simultaneously, enhanced green fluorescent protein (EGFP) which is inserted reversely can indicate the transfection efficiency of the EGFP in cells which are transfected in vitro by observing the condition of green fluorescence. By utilizing an indication vector provided by the fusion protein, the reproduction condition of EV 71 can be indicated simply, quickly, flexibly and quantitatively, and the indication vector also can be applied to the screening and evaluation of the anti-EV 71 medicine, has high actual application value and has a broad application prospect in the field of medical science.

The invention provides a polyethyleneimine derivative and a preparation method thereof. The polyethyleneimine derivative is a cationic polymer and can be used as a gene delivery carrier. The polyethyleneimine derivative provided by the invention can be effectively combined with DNA (Deoxyribonucleic Acid), and nanoparticles with particle diameter potential can be obtained. With the polymer, the plasmid DNA of the green fluorescent protein and firefly luciferase protein can be effectively transfected into HEK293 cells; extremely high transfection efficiency is shown; and even the transfection efficiency is higher than that of the commercial polyethyleneimine. The derivative is easy and practical in synthesis method and can be subjected to mass production.

The present invention provides a lentiviral vector, and the lentiviral vector is obtained by inserting firefly luciferase coding gene sequence in the 2180bp of pLVX-IRES-ZsGreen1 lentiviral vector. The invention also relates to a host cell containing the lentiviral vector, a construction method of the lentiviral vector, and application of the lentiviral vector in analysis of function or regulation effect of mammalian cell promoters and / or enhancers. The lentiviral vector provides a fast, efficient, intuitive, simple and easy method for finding, analysis and application of target protein expression or expression level regulation promoters and / or enhancers.

The invention relates to a tetrahymena cell line containing luciferase gene, a construction method and applications thereof. According to the present invention, a PCR technology is adopted to optimize a N-terminal sequence and a C-terminal sequence of firefly luciferase gene into tetrahymena preference codons, the optimized firefly luciferase gene LUC is linked to a carrier pBX to obtain firefly luciferase gene-containing recombinant plasmid pBX-LUC, a gene gun particle bombardment method is adopted to transform the firefly luciferase gene-containing recombinant plasmid pBX-LUC into tetrahymena thermophila B2086 cells, intracellular homologous recombination and paromomycin resistance screening are performed, and MTT1 gene in the cells are replaced by the LUC gene to obtain the engineered cell line B2086-LUC, wherein the engineered cell line B2086-LUC expresses luciferase under specific heavy metal induction, coloration is performed after a fluorescent substrate D-Luciferin is added, and the enlarged fluorescence intensity is detected on so as to reflect conditions of heavy metal pollutants in a reaction environment through quantitative data, such that the detection method is rapid and sensitive, and easy to operate. The tetrahymena cell line containing luciferase gene has a wide application prospect in biological monitoring of water body environment heavy metal pollution.

The invention relates to firefly luciferase and an encoding gene and an obtaining method thereof. The defect that the original firefly luciferase in North America is poor in heat stability and cannot be used at a high temperature is overcome; and the original firefly luciferase in North America is mutated, so that mutative enzyme of which the heat stability is obviously improved is obtained; and the application value is improved. An amino acid sequence is SEQ ID NO.1; and a protein deoxyribonucleic acid (DNA) sequence in encoding is SEQ ID NO.2. An encoding gene and a preparation method of firefly luciferase mutant in North America are provided. By the method, a recombinant expression vector containing a wild firefly luciferase gene is built; and the protein is purified by using an affinity chromatography method. The method is simple and feasible, simple to operate, and low in cost.

The invention discloses an up-conversion carbon quantum dot-protoporphyrin IX compound photosensitizer capable of being triggered by bioluminescence and a preparation method thereof. A carbon quantumdot with up-conversion fluorescence emission performance is used as a carrier, and forms the novel compound photosensitizer together with protoporphyrin IX through a typical EDC / NHS reaction. The photosensitizer is excellent in water solubility; and when the photosensitizer is used for testing on human hepatocellular carcinoma cells (SMMC-7721), after firefly luciferase and a substrate thereof areadded to form bioluminescence, the yield of active oxygen in the SMMC-7721 cells is obviously increased, and the photosensitizer has a certain killing effect on cancer cells.

The invention provides a novel infection model based on EBOV (Ebola Virus) virus-like particles (VLP) and taking firefly luciferase (Fluc) as a reporter protein. The model enters cells with the help of an EBOV envelope protein (GP); the Fluc can be introduced into the cells after the model enters the cells, so that cell infection can be quantified according to signal intensity of the Fluc in the cells; mouse infection can be quantified through biological imaging according to signal intensity of bioluminescence in a mouse body. Therefore, the inventor can utilize the model to carry out in-vitro and in-vivo pre-screening experiments on a neutralizing antibody and an entered inhibitor of the GP outside a BSL-4 (Biosafety Level-4) laboratory.

The invention discloses a gene knockout test kit and method for rapidly screening sgRNA. The test kit includes a kit body, a reagent A and a reagent B. The test kit utilizes pCAG-mCherry-Luc plasmids obtained through fusion of full-length sequences-containing firefly luciferase and green fluorescent protein C terminals, targeted genomic sequences are inserted in the fronts of green fluorescent protein initiation codons, finally the plasmids and expression plasmids of sgRNA and CRISPR-Cas9 transfect HEK293FT cells together, through red fluorescence protein brightness observation and luciferase viability measurement, qualitative or quantitative measurement of sgRNA mediated genome modification activity is quickly conducted, and through a conventional method in the field, the effectiveness of an sgRNA screening system in a slow virus system is verified. The test kit and the method have the advantages that an analog genome of any target gene can be constructed simply and rapidly, and the sgRNA activity is evaluated more quickly and simply and accurately quantified.

The invention discloses application of MYB6 protein and a coding gene thereof in regulating verticillium wilt resistance of plants. Through a co-immunoprecipitation and firefly luciferase complementary test, it is proved that MYB6 and full-length PUB25 and PUB26 react in vivo, through a phenotypic experiment, it is proved that an myb6 mutant after MYB6 mutation is more sensitive to verticiliumdahliae, and the verticillium wilt resistance of the transgenic plants overexpressing the MYB6 is improved, which proves that the MYB6 participates in the regulation of the immune response of the plants to the verticiliumdahliae, and has great value in improving disease resistance of the plants, especially verticillium wilt resistance, and plant breeding.

The invention relates to the fields of tumor models and establishment and application of tumor models and particularly relates to an establishment method and application a radiotherapy model for mousein-situ bearing cancer. The radiotherapy model can be used for evaluating radiotherapy effects. The establishment method comprises the steps of introducing firefly luciferase genes into a mouse Louislung cancer cell strain (LLC) by virtue of a gene recombination technique by taking a wild mouse as a carrier, carrying out subcloning screening so as to obtain a tumor cloning cell strain capable ofstably expressing luciferase, and inoculating recombinant LLC cells to a lung of the mouse. Compared with a traditional tumor model method, the establishment method has the advantages that the pathological and physiological environments of the lung cancer can be stimulated; the method has very important significances to the diagnosis and treatment of the lung cancer; and the model contains luciferase genes and can be used for imaging in a small animal living body imaging instrument, the development process of the lung cancer can be dynamically monitored in real time, the observation is relatively visible and convenient, living bodies can be observed without injuring the animals, the result is relatively reliable, and the model has very good application prospects.

This invention relates to: the development of a mutant firefly luciferase in order to use dATP as a DNA polymerase substrate upon pyrosequencing, such luciferase being subjected to substrate specificity modification in a manner such that the dATP-induced activity alone is decreased while the ATP-induced activity is maintained; and a mutant firefly luciferase for which the proportion of activity induced by dATP to activity induced by ATP (dATP / ATP) is lower than that for the wild-type firefly luciferase, in which an amino acid identified based on homology analysis as corresponding with the 421st amino acid (glycine) of the amino acid sequence of the wild-type North American firefly (Photinus pyralis) luciferase has been substituted with a polar amino acid.

The invention relates to a genetically encoded formaldehyde reactive unnatural amino acid. Specifically, the invention discloses a genetically encoded formaldehyde reactive unnatural amino acid PrAK,a preparation method and application thereof in formaldehyde detection and imaging. According to the formaldehyde reactive unnatural amino acid PrAK provided by the invention, biomacromolecules such as EGFP (Enhanced Green Fluorescent Protein) and firefly luciferase (fLuc) can be specifically introduced into sites in a protein translation process; a reaction type biomacromolecule formaldehyde fluorescent probe and a bioluminescence probe are constructed, and formaldehyde in a biological sample can be effectively detected or imaged.

The invention discloses a method for making firefly luciferase, comprising the following steps of: inserting the firefly luciferase gene into multiple cloning sites of a prokaryotic expression vector; obtaining a recombinant expression vector containing the firefly luciferase gene; introducing the recombinant expression vector into a colon bacillus cell to obtain a recombinant cell containing the recombinant expression vector; culturing the recombinant cell to obtain the firefly luciferase from the expression. The method for making the firefly luciferase has the advantages of simple process, short growth period, low cost, easy purification, high enzyme activity and stable performance and so on. The method can relieve a situation that the firefly luciferase mainly depends on import, and can promote the development of a fluorescent microorganism rapid detection system and the application of the firefly luciferase in medicine and environment science and so on, thereby providing excellent enzyme preparation for the rapid detection and applications in other aspects of ATP bioluminescence microorganism.

The invention discloses a construction method of a human APP promoter-containing Luci cell line, and the specific method is as follows: construction of an expression vector containing a human APP promoter region-luciferase chimeric gene; cotransfection of constructed pGL3-APPP Luc-Puro plasmids and pRL-SV40 plasmids into a human embryonic kidney cell line HEK-293T cell line; laying and planting of transfected HEK-293T cells after trypsinization into a 10cm culture dish, puromycin pressurization screening to obtain five cell lines with genomes capable of stably integrating the human APP promoter region-luciferase chimeric gene, and detection of activity of firefly luciferase and renilla luciferase in the five cell lines to obtain the cell line containing the human APP promoter region-luciferase chimeric gene. The cell line prepared by the cell line construction method can be used for screening of new anti senile dementia compounds and therapeutic drugs for inhibiting the expression of human APP.

The invention discloses an ATP (adenosine triphosphate) fluorescence detection reagent with remarkable heat stability. A specific proportion of heat stability enhancing reagent is added to an enzyme reaction basic solution system and a buffer solution; the ATP fluorescence detection reagent comprises the enzyme reaction basic solution system, a buffer system and the heat stability enhancing reagent, wherein the enzyme reaction basic solution system contains luciferase, fluorescein and metal ions, and preferably, luciferase is firefly luciferase, fluorescein is D-luciferin potassium salt, concentrations of firefly luciferase and D-luciferin potassium salt are 10 mg / L and 40 mg / L respectively; metal ions are Mg<2+> and concentration of the metal ions Mg<2+> is 12.5 mM preferably. According to the ATP fluorescence detection reagent with remarkable heat stability, the heat stability of the detection reagent is greatly enhanced, reagent activity can still be maintained in a certain period of time under the condition of extreme high temperature being 75 DEG C, and therefore, the storage time of the detection reagent is remarkably prolonged.