186 results about "Luciferase reporter gene" patented technology

The present invention discloses a method for establishing KI-T2A-luciferase cell line based on CRISPR/Cas9 targeted genome modification technology. A T2A-luciferase reporter gene is integrated in the3<rd> end of the mmp 12 gene in a genome by using CRISPR/Cas9 technology. A knock-in cell line of MMP12-T2A-luciferase is established, the site-specific integration of the source gene in the cell lineon the genome is verified. Meanwhile, a reported transcription factor, STAT3, with activation effect on mpp12-T2A is used to transcribe and active the MMP12-T2A-luciferase cell line. The results showthat the expression level of luciferase in MMP12-T2A-luciferase cell line can accurately and sensitively reflect the expression level of MMP12 protein in the cell line. The establishment of the cellline will contribute to the study of the gene function of mmp12 and the screening of small molecule chemical drugs affecting the expression of mmp12, which provides a new experimental thinking and solution for the migration of cancer cells and related researches thereof.

The invention relates to a method for establishing luciferase knock-in cell line based on CRISPR-targeted genome modification technology. The knock-in cell line of SREBP1- T2A-Luciferase was established by in situ integration of the 3'end of SREBP1 gene into the T2A-Luciferase reporter gene using CRISPR/Cas9 technique, the fixed point of the Chinese and foreign source genes of the cell line on thegenome is verified. The transcriptional activation of the SREBP1-T2A-Lucifasse cell line was performed using the reported transcription factor LXR Alpha, which has an active effect on the SREBP1. Theresults show that the expression level of the Luciferase in the SREBP1-T2A-Lucifasse cell line can accurately and sensitively reflect the expression level of the SREBP1 in the cell line. The establishment of the cell line will help to study the function of the SREBP1 gene and to screen the small molecular chemical drugs affecting the expression of SREBP1, and provide a new experimental thought and solution for lipid metabolism and related research.

The invention discloses a method for detecting promoter activity by utilizing double luciferase reporter genes. The method for detecting the promoter activity by utilizing the double luciferase reporter genes comprises step 1, building a pGL3-basic-MSTNpro recombinant which contains 7 sections of MSTN gene 5' control region fragments with different lengths; step 2, performing cultivation and planking on target cells, configuring a mixture of the pGL3-basic-MSTNpro of the step 1 and a lipidosome and enabling a renilla luciferase carrier pRL-TK to be served as an internal reference to perform cell co-transfection; step 3, performing detection on luciferase activity through the double luciferase reporter genes.

A pseudo-virus packaging system of coronaviruses comprises a vesicular stomatitis virus (VSV) vector with a Fluc and EGFP double reporter gene replacing a GP gene and an assembly cell expressing coronavirus spike protein S. The double reporter gene is selected from luciferase and fluorescent protein, and the luciferase reporter gene is preferably a Fluc gene. The fluorescent protein reporter geneis preferably an EGFP gene, and the assembly cell is preferably 293T. The invention further relates to a one-step packaging method of the packaging system; a pseudo-virus with single-cycle infection,low background value and high titer can be quickly packaged through simple, convenient and rapid operation, and the packaging system has the characteristic of being rapid in detection compared with aslow virus mediated pseudo-virus system; the one-step packaging method can be used for researching COVID-19, SARS, MERS and other coronaviruses and other viruses, provides a powerful screening tool for evaluation of antiviral preparations and vaccine, and has wide application value.

The invention provides a method of detection of transcription factor expression activity by a luciferase reporter gene system; the method comprises the steps: based on the luciferase characteristics of being lasting, stable and easy to detect, co-transfecting cells with a luciferase reporter gene vector containing a target gene promoter and a transcription factor expression plasmid; after the cells are cultured, carrying out lysis of a certain number of cells with a cell lysis buffer liquid, collecting a lysate containing luciferase, and centrifuging to take a supernatant; and then real-timely detecting luciferase expression intensity data in a luciferase activity detection buffer liquid, at the same time, detecting the luciferase concentration, correcting, and then calculating to obtain the transcription factor expression quantity, namely the transcription factor expression activity needing to detection. The method is simpler and more feasible in whole operation process, shorter in time consuming, higher in flux, relatively high in repeatability and accuracy, and lower in operating costs, thereby having broad application prospects, and having excellent market potential.

The invention discloses a double-reporter-gene skeleton vector. Double reporter genes of the double-reporter-gene skeleton vector are selected from luciferase and fluorescein, the fluorescein reportergene is preferably an mGFP gene, and the luciferase reporter gene is preferably a Fluc gene. A double-reporter-gene four-plasmid pseudovirus packaging system comprises the double-reporter-gene skeleton vector and three eukaryotic expression packaging plasmids which are respectively integrated with a slow virus gag-pol gene, a slow virus rev gene and an envelope protein gene containing a target virus. A packaged COVID-19 pseudovirus comprises the double-reporter-gene four-plasmid pseudovirus packaging system and a host cell, wherein the host cell is preferably selected from 293T-hACE2 cells. According to the invention, SARS-CoV-2 is specifically implemented, the pseudovirus with single-cycle infection and higher safety can be rapidly packaged, and the vector and the system can be used forresearching coronaviruses such as SARS-Cov2 and the like, provide a powerful screening tool for evaluation of antiviral preparations and vaccines, and have wide application value.

The invention discloses a pregnane X receptor (PXR)-multidrug resistance-associated protection (MRP2) luciferase reporter gene-expressing technical platform high-flux medicament screening method, which screens candidate potential activity pilot medicaments by directly starting from a medicament metabolism dynamic target which is used in a terminal medicament clinic research stage in a new medicament research. In the method, a luciferase reporter gene containing a MRP2 gene promoter sequence and expression plasmids of RXR is constructed and human hepatocellular carcinoma (HepG 2) cells are transfected transiently; and the high-flux screening of in-vitro RXR induction potential activity pilot medicaments is carried out by detecting the activity of the luciferase in a self-luminous detector and reflecting the influences of different potential activity pilot medicaments on the activity of a MRP2 promoter according to the ratio of the fluorescence of a glowworm to the fluorescence of a sea pansy. The method can avoid severe untoward effects aroused by activity medicaments obtained by other medicament screening methods due to medicament interaction generated in a final clinical medication process, save a big amount of preliminary investment in the new medicament research and reduce blindness.

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.

The invention provides a method for determining the receptor affinity of a GLP-1 receptor agonist. The method is based on a tool cell line, namely CHO-GLP-1R-CRE-Luc<+>, when a sample to be determined is combined with GLP-1 receptor on the surface of a cell, the receptor-mediated signal cascade reaction can be activated, cAMP-response element (CRE) is activated specifically, the expression of the luciferase reporter gene is promoted, the quantity of luciferase in cytosol is detected for drawing and fitting to obtain a dose-effect curve of acting of the sample and the GLP-1 receptor; and the half effective concentration (EC50) is calculated. By inspecting and optimizing all influence factors in the determining process, the method for determining the receptor affinity of the GLP-1 receptor agonist is finally established. The method has the advantages of being high in specificity, precision and accuracy, good in durability and convenient to operate, and the like. The method can be used for determining the receptor affinity of the GLP-1 receptor agonist, and thus such type of drug can be fast evaluated and screened.

The invention sets up an anti-inflammatory drug screening primary culture cell model which takes interleukin 1 beta gene of human beings as a target and is high-efficient and reliable, and a screening method thereof. The invention separates and primarily cultures a abdominal cavity macrophagus of the transgenic mouse which is introduced with interleukin 1 beta promoter drive of human beings and expressed by luciferase reporter gene, so that a macrophagus inflammatory model is set up by the inducement of inflammation motivating factor. In the model, the anti-inflammatory activity of the compound can be evaluated by measuring the degree of inhibiting the expression of the reporter gene of the screened compound. The drug screening model set up by the invention is a cell model which is sensitive, high-efficient, reliable and suitable for screening high flux anti-inflammatory drug.

The invention discloses a method for quantitatively determining activity of T4 polynucleotide kinase. The method for quantitatively determining the activity of T4 polynucleotide kinase comprises the following steps: in a reaction system used for phosphorylation, adding ATP by taking oligonucleotide as a substrate for phosphorylating an end 5' of oligonucleotide by utilizing T4 polynucleotide kinase, so as to generate phosphorylated oligonucleotide; then adding a luciferase reporter gene system into the reaction system, wherein luciferase in the luciferase reporter gene system utilizes residual ATP in the reaction system to oxidize luciferin to form oxygenated luciferase and produce chemiluminiscence at the same time; and detecting numerical value of luminescence, and deriving activity degree of T4 polynucleotide kinase by virtue of detection results, wherein the activity degree of T4 polynucleotide kinase is in negative correlation with luminous quantity. Compared with the prior art, the method for quantitatively determining the activity of T4 polynucleotide kinase has the advantages that no radioactive contamination is produced, operation is easy, and quantitative detection analysis can be carried out on the activity of T4 polynucleotide kinase.

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 relates to the field of molecular genetics, in particular to the application of a molecular marker method in pig breeding for disease resistance, wherein according to the molecular marker method, the molecule at a mutation site in a CYP3A88 gene 5' regulatory region of a pig is marked. The inventor of the method discovers that the CYP3A885' regulatory region of a large Chinese streaky-head pig and the CYP3A885' regulatory region of a Duroc long hybrid pig have a plurality of differences, wherein an A-to-T mutation exists at the -78 site, through a luciferase reporter gene system, the fact that promoter activity of the -78 site is lowered remarkably after an A at the -78 site is mutated into a T in a site-directed mutagenesis mode is found, and the promoter activity of the -78 site is the same as the low level of messenger ribonucleic acid (mRNA) expression of a CYP3A88 gene of the porcine reproductive and respiratory syndrome (PRRS)-resistant large Chinese streaky-head pig. Therefore, through genotype detection of the -78 site of the CYP3A88 regulatory region in a pig genome, the genotype of the -78 site of the CYP3A88 regulatory region can be used as a modular marker associated with traits of the PPRS, the molecular marker method not only is simple, convenient and rapid, but also cannot be affected by the environment, and early selection for breeding can be realized.

The invention belongs to the technical field of livestock gene engineering, and relates to construction and an application of a dual-luciferase reporter gene carrier based on a porcine CD163 gene. A key receptor gene, namely the porcine CD163 gene, of a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is amplified and sequenced to obtain a complete fragment of a 3' UTR (Untranslated Region) of the gene, candidate microRNAs (micro Ribonucleic Acids) and an action target of the 3' UTR of the porcine CD163 gene are predicted and regulated, and a sequence of the porcine CD163 gene containing the action target is amplified, and inserted into a dual-luciferase carrier to construct the dual-luciferase reporter gene carrier psi-CHECK2-CD163-3UTR of the 3' UTR of the porcine CD163 gene. The carrier can be used for detecting regulation activity of the candidate microRNAs on porcine CD163 gene expression, and screening the microRNAs inhibiting the porcine CD163 gene expression, and the like.

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 a method for screening and identifying spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) molecular marker MicroRNAs (miRNAs) capable of regulating and controlling the expression of an ATXN3 gene. The method comprises the following steps: obtaining differential expression profile of miRNAs by using miRNA microarray chip technique; further screening and verifying by using a bioinformatics method and a qRT-PCR technique to obtain the miRNAs expressed by regulated ATXN3 gene; determining the target action relation and mechanism of action of the miRNAs and ATXN3 by using qRT-PCR technique and Westernblot; determining target action relation and specific binding site of the miR-25 and ATXN3 gene by using luciferase reporter gene detection technique so as to provide basis for researching and developing diagnostic kit related to SCA3/MJD disease.

The invention discloses an application of STAT3 in porcine ovarian granulosa cells. STAT3 is taken as a research object, and a porcine STAT3 gene promoter double-luciferase reporter gene recombinationplasmid is constructed, and the core promoter region of the STAT3 is found through the expression activity of the STAT3 gene promoter in the porcine ovarian granulosa cell; and then the interaction between the transcription factor C/EBP beta and the core promoter region of the STAT3 is verified; then C/EBP beta superexpression vector is constructed and small interfering RNA (C/EBP beta-siRNA) issynthesized, the effect of C/EBP beta on STAT3 is detected, finally, the expression vector of C/EBP beta and C/EBP beta-siRNA were transfected respectively to the granulosa cells in order to detect the apoptosis and proliferation of the cells. The application of STAT3 in porcine ovarian granulosa cells finds the application of the transcription factor C/EBP beta in the ovarian granulosa cell by finding the transcription factor C/EBP beta in the STAT3 promoter region, and has good application value for researching the ovarian follicular atresia mechanism.

The invention relates to the effect of miR-451a cells in the non-small cell lung cancer. Migration of A549 cells can be significantly inhibited through overexpression of miR-451a in the A549 cells. By means of the bioinformatics analysis means, it is predicted that translation of the target gene mRNA is inhibited or the target gene mRNA is directly degraded by miR-451a through regional complementation with 3'-UTR of the target gene CAB39 mRNA; it is determined that the CAB39 gene is the target gene of miR-451a through double-luciferase reporter gene system analysis and Western Blot experiment verification. Finally, it is verified through the Transwell Assay and Flow Cytometry Assay experimental means that miR-451a performs the function of inhibiting migration of cells by reducing the CAB39 gene in the A549 cells, and a novel accurate treating researching and treating scheme is provided. By means of the effect, important research guidance and application value is provided for using miRNA for diagnosing and treating the non-small cell lung cancer and searching for drug targets in the fields such as tumor immunotherapy clinically.

The invention provides a method for detecting the biological activity of RANKL (receptor activator for nuclear factor-KB ligands) targeted therapy medicines. The method includes steps of firstly, constructing transcription factor NF-kB (nuclear factor-kB) binding sequences, cloning the NF-kB binding sequences into luciferase reporter gene carriers, and guiding luciferase reporter gene transcription by the aid of the NF-kB binding sequences used as components of starters so as to obtain plasmids with the NF-kB binding sequences and luciferase reporter genes; secondly, transfecting cells by theplasmids with the NF-kB binding sequences and the luciferase reporter genes; thirdly, vaccinating the cells, adding RANKL and the RANKL targeted therapy medicines into the cells and then culturing thecells; fourthly, lysing the cells, adding luciferase substrates into the cells and detecting the fluorescence intensity so as to determine the biological activity of the RANKL targeted therapy medicines. The method has the advantage that the biological activity of the denosumab RANKL targeted therapy medicines can be accurately, easily, feasibly and quickly detected by the aid of the method.

The present invention provides a CSF-1R reporter gene cell line and a construction method and an application thereof. The reporter cell line comprises NF-kappa-B reporter gene plasmids; the NF-kappa-Breporter gene plasmids comprise a luciferase reporter gene; and the reporter gene cell line also comprises CSF-1R expression plasmids. The reporter gene cell line expresses human-derived CSF-1R on cell membrane and intracellularly expresses luciferase in response to NF-kappa-B signals, CSF-1/IL-34 can specifically activate the expression of the luciferase of the reporter gene cell line in a dose-dependent manner, at the same time target antibody drugs of the CSF-1R to block the CSF-1/IL-34 and bind the CSF-1R on surfaces of the reporter gene cell line, and have important applications in detection of antibody drug activity.

The invention discloses a Pif promoter capable of being activated by pinctada martensii MSX (muscle segment homeobox) genes and application of the Pif promoter. The nucleotide sequence of the Pif promoter is shown as SEQ ID NO.1. A Pif promoter sequence is obtained through amplification by a genome walking method. The Pif promoter has a binding site 1 and a binding site 2, which can be bound to the MSX genes. Through the establishment of wild type carriers and mutant type carriers of Pif promoters of different fragment lengths, a dual-luciferase reporter gene system is applied to identify the fact that the MSX genes act on the Pif promoter through the binding site 1. The Pif promoter can start the downstream target gene expression, thereby promoting the growth of pearls or pearl oysters.

The invention belongs to the field of biological medicine and relates to applications of ssc-miR-374b-5p in preparation of drugs for reducing fat deposition and/or resisting fat-related diseases. For the first time, according to the invention, ssc-miR-374b-5p is discovered to be able to effectively bind with a transcription product mRNA of a C/EBP-beta encoding gene that is an important transcription factor for adipocyte differentiation, and inhibit activity of a luciferase reporter gene with C/EBP-beta3-UTR. Cellular level experimental results show that the microRNA can bind with C/EBP-beta3-UTR so as to inhibit protein translation of C/EBP-beta, providing experimental basis for reduction of the fat deposition and control of the fat-related diseases. The microRNA provided by the invention is expected to be used as a novel antiobesity-related drug, and has significant values.

The invention belongs to the field of veterinary drug residue analysis, and particularly relates to a detection method of fluoroquinolone drug residues based on a luciferase labelled genetically engineered bacterium, and an application. The invention provides construction and the application of the luciferase labelled genetically engineered bacterium capable of simultaneously detecting 11 fluoroquinolone drug residues. The engineered bacterium is escherichia coli pK12; the preservation number is CCTCC NO (China Center for Type Culture Collection Number):M2013385; and the engineered bacterium is obtained by converting escherichia coli K12 with a plasmid pRecAlux3 comprising an recA promoter and a luciferase reporter gene luxCDABE. Compared with the prior art, the constructed genetically engineered bacterium can simultaneously recognize multiple fluoroquinolone drugs; a detection object of the prior art is widened; and the detection method has the prominent advantages of simplicity, rapidness, sensitiveness, accuracy, semiquantitative function and the like.

The invention discloses a novel epothilone biosynthetic gene P3 promoter as well as a preparation method and application thereof. A nucleotide sequence of the P3 promoter is shown as SEQ ID NO. 1. Thepromoter of a So ce M4 epothilone biosynthetic gene is amplified by adopting a degenerate primer designing method for the first time; the promoter is subjected to cloning and promoter element analysis; and a luciferase reporter gene vector is adopted to verify the functions of the obtained epothilone biosynthetic gene promoter. The obtained novel epothilone biosynthetic gene cluster promoter canlay a molecular biological foundation for transcriptional regulation of sprangium cellulosum epothilone biosynthesis and heterologous expression of an epothilone biosynthetic gene cluster; the improvement of the yield of epothilone is promoted; and wide application of the epothilone in the aspect of biomedicines is promoted.

The invention relates to an application of an miR-34a gene in non-small cell lung cancer. A target gene TGF[beta]R2 of the miR-34a in an H1299 cell line is also provided. The miR-34a is complemented with 3'-UTR of the mRNA of the target gene, such that the translation of the mRNA of the target gene is inhibited, or the mRNA of the target gene is directly degraded. The miR-34a and the TGF[beta]R2 have interaction with each other in the H1299 cell line. The interaction influences life actions such as propagation, apoptosis and the like of the H1299 cells. TGF[beta]R2 is primarily validated as the target gene of the miR-34a by software prediction, vector construction and luciferase reporter gene analysis in HEK293T cells; and then the phenomenon is further validated by a technology of expressing the miR-34a and qRT-PCR in the H1299 cells. The disclosure is the first report that the TGF[beta]R2 in the H1299 cell line is the target gene of the miR-34a. Application values in diagnosis and treatment for the non-small cell lung cancer by using the miRNA clinically and in drug target aspects are provided by the invention.