72 results about "Luciferase reporter" patented technology

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.

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, and specifically relates to a molecular marking method for a pig MX1 gene 5' control region mutable site, and an application thereof in pig disease resistance breeding. According to the invention, a plurality of differences are found in MX1 5' control regions of different breeds of pigs. Among Chinese local pig breeds, a 275bp segment inserted gene is a dominant genotype. Among western pig breeds, the frequency of the genotype is low. With a luciferase reporter assay system, it is found that, the activity of a promoter with an inserted275bp segment is substantially higher than that of another. The existence of the 275bp segment in a pig genome MX1 control region can serve as a molecular mark related to pig disease resistant characters. The method is simple and fast, and is not influenced by the environment. With the method, early-stage breed selection can be realized.

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 invention relates to a miR-506 targeting PENK gene of triple negative breast cancer cells and application of the miR-506. By screening miRWalk2.0, miRBase, TargetScanHuman 7.0 databases and detecting tissue samples of triple negative breast cancer patients by using a qRT-PCR technology, it is confirmed that the expression of miR-506 in the triple negative breast cancer is decreased significantly, and is averagely down-regulated by 88.95%, a dual-luciferase reporter experiment confirms that miR-506 can directly interact with Penk, and it is indicated that miR-506 can target PENK. Accordingto the miR-506 targeting the PENK gene of the triple negative breast cancer cells and the application of the miR-506, it is proved that miR-506 affects the proliferation, metastasis and invasion of triple negative breast cancer cells by increasing the content of penk in a targeting mode; the effectiveness of miR-506 targeting penk in inhibiting the progression of the triple negative breast canceris detected through a nude mouse breast cancer model; and the results show that the miR-506 can inhibit the development of the triple negative breast cancer through targeting penk.

The invention discloses a DNA fragment and an application thereof in preparation of an H5N1-subtype flu Guassia luciferase reporter virus. The nucleotide sequence of the DNA fragment is composed of following sequences in a successive series manner: an H5N1-subtype flu virus NA segment 3'-terminal noncoded region coding gene sequence, an H5N1-subtype flu virus NA gene encoded region coding sequence, a PTV-1 virus 2A peptide encoded gene sequence, a Gaussia luciferase encoded gene sequence, a H5N1-subtype flu virus NA segment 5'-terminal packaged signal encoded gene sequence, and a H5N1-subtype flu virus NA segment 5'-terminal noncoded region coding sequence. By means of the recombined H5N1-subtype flu reporter virus prepared with the DNA fragments, quantitative detection on viruses in a sample can be objectively carried out quickly and accurately through activity of luciferase in cellular supernatant or animal serum. The H5N1-subtype flu Guassia luciferase reporter virus can not only save time but also improve accuracy and reliability of a detection result, thereby providing a new convenient and effective tool for drug screening and vaccine assessment of anti-H5N1-subtype flu virus drugs.

The present invention relates generally to methods to monitor the transport of proteins through the secretory pathway, and methods to monitor ER stress. In particular, the present invention relates to methods to monitor, in real-time, the processing of protein through the secretory pathway, which can be monitored both at a subcellular level by florescence visualization and quantitatively by detecting the secreted luciferase reporter protein. The present invention also relates to methods to assess biological processes in cells, in particular the secretory pathway and ER stress, as well as methods to identify agents which augment or inhibit the secretory pathway and / or ER stress. The present invention also relates to compositions and nucleic constructs encoding a secreted luciferase-fluorescent protein conjugate for methods to monitor protein trafficking in the cell by simultaneous detection of fluorescence and luciferase secretion.

The invention provides a method for identifying the active region of an ATP5B (adenosine triphosphate 5B) gene promoter. The method comprises the following steps: (1) cloning an ATP5B gene promoter sequence; (2) constructing an ATP5B gene 5' end promoter series deletion fragment luciferase reporter vector; and (3) performing cell transfection and luciferase activity analysis on the ATP5B gene 5' end promoter series deletion fragment luciferase reporter vector in the step (2) respectively, and indicating the active area of the ATP5B gene promoter is an area between the two adjacent deletion fragments if the activity of the promoter between the adjacent two deletion fragments is obviously changed. The active area of the ATP5B gene promoter is identified by a luciferase activity analysis method, a regulating and control component for regulating and controlling the transcriptional activity of the ATP5B gene promoter is further identified, and a MyoD and GATA transcription factor binding site is determined to be the regulating and control component for regulating and controlling the transcriptional activity of the ATP5B gene promoter.

The invention discloses an influenza luciferase reporter virus-based animal model building method and application. In the method, a mouse is mainly infected with Gaussia luciferase gene-carrying recombinant influenza virus, and the infection degree of the mouse is evaluated by detecting the expression level of luciferase in a lung tissue of the mouse. The invention further discloses an influenza luciferase reporter virus technology-based method for screening and evaluating an anti-influenza drug. According to the method, anti-influenza drugs with prevention / treatment effects are screened by mainly detecting the influence of antiviral treatment on the expression level of the luciferase in the lung tissue of the mouse. Detection of positive drugs ribavirin and oseltamivir phosphate shows that the method disclosed by the invention has a wide application value in the aspect of screening, researching, developing and evaluating the novel anti-influenza drug.

Inhibitors of luciferase enzymes are disclosed and find use in multiplexed assays using multiple luciferases and multiple inhibitors, in both in vitro and in vivo embodiments.

The present invention relates to a systems and methods of using expression of one or two luminescent proteins in a plant root cell to visualize plant root structure as well as to determine how stressors affect gene expression in plant roots while maintaining the natural soil habitat

The invention discloses a dual-luciferase reporter gene vector of human TLR4 gene 3' untranslated region and a building method and application thereof and belongs to the field of molecular genetics. The dual-luciferase reporter gene vector is pGLTlr4 / PSV40 / 3UTR, and a DNA sequence of the vector is shown as SEQ ID NO.5; the vector comprises a human TLR4 gene 3' untranslated region sequence which isshown as SEQ ID NO: 3 and cloned to luc gene downstream in a dual-luciferase reporter vector pGL3-Promoter. The vector can be used for detecting impact of SNP sites on the TLR4 gene 3' untranslated region on TLR4 gene expression and detecting targeting relation between the SNP sites on the TLR4 gene 3' untranslated region and miRNA.

The invention discloses a verification method of key transcription factors in the core region of the Nanos2 promoter, comprising the following steps: firstly, carrying out PCR amplification of the sequence of the 5' flanking region of Nanos2 to construct an eukaryotic expression vector pNanos2-EGFP, and respectively transfecting pNanos2-EGFP, pEGFP-N1 and pLinker-EGFP into DF-1 cells to qualitatively analyze the promoter region of Nanos2; respectively constructing vectors pGL3-1187, pGL3-959, pGL3-788, pGL3-493 and pGL3-155 with different lengths deleted at 5' end of the promoter and co-transfecting the vectors and pRL-SV40 into DF-1 cells, carrying out luciferase reporter gene assay for identification of the core region of the Nanos2 promoter, constructing a vector with the core region deleted, and performing luciferase activity analysis to observe whether the promoter activity is lost or remarkably reduced after the core region is deleted; carrying out transcription factor binding site prediction on the core region of the Nanos2 promoter, and performing Dual luciferase reporter assay and CHIP test to determine the key transcription factors. Male germ cell differentiation is detected by methods of cell morphology observation, qRT-PCR, cellular immunochemistry and flow cytometry.

The invention provides an identification method of a regulatory element for regulating the transcriptional activity of an FATP1 gene promoter. The method includes the steps of: cloning an FATP1 gene promoter sequence; constructing a series of deletion FATP1 gene 5' end promoter fragment luciferase reporter vectors; transfecting cells with the luciferase reporter vectors respectively for luciferaseactivity analysis, and determining the activity area of the FATP1 gene core promoter; and analyzing a transcription factor binding site in the activity area, conducting site-directed mutagenesis on the transcription factor binding site, and according to the activity change condition of the FATP1 gene promoter after mutagenesis, determining the regulatory element for regulating the transcriptionalactivity of the FATP1 gene promoter. The method provided by the invention identifies the regulatory element for regulating the transcriptional activity of the FATP1 gene promoter, and finally determines the KLF15 transcription factor binding site as the regulatory element for regulating the transcriptional activity of the FATP1 gene promoter.

The invention provides a preparing method of a brain blood vessel endothelial cell line for stably expressing TOP-Flash / Renilla dual-luciferase reporter genes. The preparing method comprises the following steps of 1, selecting cerebral microvascular endothelial cells, infecting the cerebral microvascular endothelial cells with a slow virus carrying a Renilla gene, and conducting screening to obtain a cell line for stably expressing Renilla; 2, infecting the cell line obtained in step 1 with a slow virus carrying a TOP-Flash gene, and conducting screening to obtain the brain blood vessel endothelial cell line for simultaneously stably expressing the Renilla and TOP-Flash dual-luciferase reporter genes. The brain blood vessel endothelial cell line can be used for screening active substancesfor activating the activity of a Wnt signal channel in the brain blood vessel endothelial cells, the screening efficiency and accuracy are improved, and the preparing method is simple.

The invention discloses a multifunctional dual luciferase reporter gene vector based on human TLR4 gene, a construction method and an application thereof, which belong to the field of molecular genetics. The multifunctional dual luciferase reporter gene vector of the present invention is pGLTlr4 / -3494+235 / 3UTR, and a DNA sequence of the vector is shown as SEQ ID NO.5; the multifunctional dual luciferase reporter gene vector comprises the human TLR4 gene-3494 to +235 sequence and the complete 3'-UTR sequence, the human TLR4 gene-3494 to +235 sequence is shown as SEQ ID NO: 1, and the complete 3'-UTR sequence is shown as SEQ ID NO: 2; and the human TLR4 gene-3494 to +235 sequence and the 3'-UTR complete sequence are cloned into upstream and downstream of the firefly luciferase luc gene in the pGL3-Basic plasmid respectively. The method seamlessly clones the upstream and downstream non-coding sequences of the TLR4 gene into the upstream and downstream of the firefly luciferase luc gene, respectively, the structural composition of the vector is closer to the real situation than the traditional luciferase reporter plasmid, and the negative effect of an exogenous promoter on the experimental results is effectively avoided.

The invention discloses a dual-luciferase reporter gene carrier based on a human TLR4 gene promoter region and a construction method and application of the dual-luciferase reporter gene carrier, and belongs to the field of molecular genetics. By designing a primer TPprimer 1 and a primer TPprimer 2 with the 5' tail ends including homologous arms in the upstream and downstream sequences of an interface, the -3494 sequence to the +235 sequence of a TLR4 gene are obtained through amplification, the -3494 sequence to the +235 sequence of the TLR4 gene are seamlessly cloned to NcoI loci of a dual-luciferase reporter carrier pGL3-basic through a Gibson assembling method so as to construct the dual-luciferase reporter gene carrier pGLTlr4 / -3494+235 in the promoter region at the 5' end of the humanized TLR4 gene. The dual-luciferase reporter gene carrier can be digested through restriction enzymes such as BglII, KpnI and EcoRI and then reconnected to construct the derivative reporter gene carrier including promoters with different lengths, thus the derivative reporter gene carrier is used for analyzing the promoter sequence of the TLR4 gene in cells of different tissue sources, and specificity regulating and controlling mechanisms for promoter cells of the TLR4 gene and the like are investigated.

The invention belongs to the technical field of animal gene engineering, and particularly relates to a goat miR-27a (micro Ribonucleic Acid-27a) site-specific modification system and application thereof. The goat miR-27a site-specific modification system constructed by the invention can be used for effectively identifying and modifying a binding site of a transcription factor CP2 in a goat miR-27a promoter region. The binding site of the miR-27a promoter of the goat miR-27a site-specific modification system has a nucleotide sequence as shown in SEQ ID NO: 1 in a sequence table. The goat miR-27a site-specific modification system is used for analyzing the binding site of the transcription factor CP2 in the sequence of the goat miR-27a promoter region; a wild dual-luciferase reporter vector pGL3-W of the binding site is constructed; afterwards, the base mutation modification is carried out on the binding site; a mutant dual-luciferase reporter vector pGL3-M is constructed and obtained. The two dual-luciferase reporter vectors constructed by the goat miR-27a site-specific modification system can be used for researching the regulation and control mechanism of the follicular development of a goat, and have important significance for breeding a goat variety with a high-production lamb quantity.

The method for detecting that LRP6 is the target gene of miR-29a by using a dual-luciferase reporter gene, the purpose of the present invention is to confirm that miR-29a can be negatively expressed by targeting the 3'UTR of the LRP6 gene through a dual-luciferase reporter gene system Regulates the expression of LRP6. A method for detecting LRP6 as a target gene of miR-29a using a dual-luciferase reporter gene, the method comprising the following steps: (1) constructing a plasmid vector and performing point mutations: (2) dual fluorescence detection. The present invention The beneficial effects are as follows: provide a new method for verifying that miR-29a can negatively regulate the expression of LRP6 by targeting the 3'UTR of the LRP6 gene, and contribute to miR-29a, LRP6 and Wnt / -catenin signaling pathways Elucidation of the pathogenesis of osteogenesis-related diseases involved; contribute to the development of potential drugs for osteogenesis-related diseases involved in miR‑29a, LRP6 and Wnt / ‑catenin signaling pathways and the screening of biomarkers.

The invention belongs to the technical field of diabetic nephropathy treatment, and particularly relates to a research method of a protection mechanism of BMSC-Exo on high glucose induced HK2 cell damage. The experimental steps of the protection mechanism of BMSC-Exo on high glucose induced HK2 cell damage include HK2 cell grouping and processing, construction of a miR-886-5p knocked-out HK2 cellline by a Crispr / Cas9 technology, an RNA pull down technology, an FISH experiment, DN establishment and PAS glycogen staining. Bioinformatics prediction is combined with a luciferase reporter vector experiment to prove the interaction between miR-886-5p and a target gene, the RNA pull down experiment and the FISH experiment further prove the binding relationship between miR-886-5 p and the targetgene, and a cure experiment proves that the target gene can reverse the function of miR-886-5p and confirm the mechanism of miR-886-5p in HK2 cells.

The invention belongs to the field of biological technology, and in particular relates to an NF-kappaB dual-luciferase report cell line capable of stably expressing a pig derived TLR8 receptor gene, and a construction method of the NF-kappaB dual-luciferase report cell line. The cell line is an HEK293-NF-kappaB cell, in which a pTLR8 gene is transferred; and the HEK293-NF-kappaB cell is an HEK293cell, in which NF-kappaB luciferase and endodontic luciferase are introduced. The invention provides a stable cell line, which can be used for researching pTLR8 activated downstream NF-kappaB promoterreport gene detection. The invention also provides a biological material for an RNA-Seq technology to perform high-throughput screening on differential expression genes in the stable cell under the bTLR8 silent and activated states, and lays the foundation and a platform for further researching the species specificity of TLR8.

The invention relates to an anguilla japonica cytokine IL-6 gene promoter and application thereof. The anguilla japonica genome is compared and analyzed through a first exon sequence of an IL-6 gene open reading frame, an IL-6 gene promoter sequence is cloned by analyzing and predicting a 5' flanking region sequence of the IL-6 gene through a touchdown PCR method, and the IL-6 gene promoter pGL3-IL-6-pro luciferase reporter plasmid is successfully constructed. Experiments prove that the IL-6 gene promoter can be induced and activated by poly I: C, LPS and aeromonas hydrophila, and it is found that the important signal channel inflammation regulation factor Caspase-1 can significantly up-regulate the luciferase activity of the IL-6 gene promoter. The invention provides a good experimental system for researching the expression regulation mechanism of the anguilla japonica cytokine IL-6 gene and the regulation mechanism of important fish inflammation-related NF-kappa B, MAPK and I-type interferon signal path network, and also can efficiently express exogenous genes after constructing an expression vector by using the promoter; or the promoter is applied to construction of transgenic fish and has important theoretical and practical significance.

The present invention encompasses modified luciferases, methods for making modified luciferases, and assays utilizing modified luciferases. Modified luciferases of the invention show increased activity over wildtype luciferases and also show increased stability of signal. The present invention also encompasses multiplex assays utilizing multiple luciferases reporters with different emission spectra and different substrates for simultaneous luciferase measurements.

The invention belongs to the technical field of biology and in particular relates to an NF-kB dual-luciferase reporter cell for stably expressing swine TLR5 receptor genes and a construction method thereof. A cell line refers to HEK293-NF-kB cells in which the swine TLR5 receptor genes are transferred, and the HEK293-NF-kB cells refer to HEK293 cells in which NF-kB firefly luciferase and renilla luciferase are transferred. The invention provides the stable cell line used in detection for researching pTLR5 activated downstream NF-kB promoter report genes. The invention also provides a biological material for performing high-throughput screening on differentially expressed genes in stable cells in quiesced and activated states in an RNA-Seq technology, and lays a foundation and platform forfurther researching species specificity of TLR5.

The invention relates to a preparation method and application of a dual luciferase reporter expression vector pFireRluc. According to the invention, the problem that a testing system is affected by cell activity and transfection efficiency of two different vectors in the transfection process can be effectively solved; a Rluc sequence fragment is obtained through amplification by taking a pmirGLO vector as a template and taking R1 / R2 as a primer; SalI restriction enzyme cutting sites are added at two ends of the sequence, a pGL4.10 vector is subjected to SalI single restriction digest, and theRluc sequence fragment is inserted into the pGL4.10 vector, and thus a recombinant pFireRluc dual luciferase reporter vector is constructed; the recombinant pFireRluc dual luciferase reporter vector is effectively used for expression and identification of eukaryotic cells and activity testing of the dual luciferase reporter expression vector pFireRluc. The method disclosed by the invention has theadvantages that novelty and uniqueness are realized, the credibility of experimental data is improved, the influence of cell activity and transfection efficiency on experiment result is reduced or can be eliminated, and further the stability and the reliability of a detection result are improved.

The invention belongs to the technical field of biology and particularly relates to an NF-kB dual-luciferase reporting cell line for stably expressing human TLR8 receptor gene and a construction method thereof. The cell line is HEK293-NF-kB cells with transgenosis of the human TLR8 gene, and the HEK293-NF-kB cells are HEK293 cells with introduction of NF-kB luciferase and internal reference ranilla luciferase. The NF-kB dual-luciferase reporting cell line and the construction method also provide a biological material for studying the action of TLR8 ligands in tumor treatment and utilizing an RNA-Seq technology to screen genes for stabilizing differential expression in the cells under the silent and activated states of hTLR8 by high flux, and lays foundation and platform for further studying the species specificity of the TLR8.

The invention discloses a tracking targeting plasmid of a vaccinia virus Tiantan strain. The plasmid includes upstream homologous recombinant arms VTT-TKL and downstream homologous recombinant arms VTT-TKR of vaccinia virus Tiantan strain TK genes. Luciferase reporter genes and EGFP green fluorescent protein marker genes, which are expressed through promoters P 7.5, are inserted between the upstream homologous recombinant arms VTT-TKL and the downstream homologous recombinant arms VTT-TKR. The invention further discloses a preparation method of the tracking targeting plasmid of the vaccinia virus Tiantan strain. According to the tracking targeting plasmid of the vaccinia virus Tiantan strain, the targeted removal of the TK genes in the vaccinia virus Tiantan strain can be achieved, and a recombinant Tiantan strain virus with the TK genes removed is prepared.

The invention provides a duck circovirus tandem repeated sequence and application thereof and belongs to the technical fields of bioengineering and molecular biology. The invention firstly discovers aQTR sequence in which DuCV specifically exists, and the tandem repeated sequence does not appear in other circoviruses. QTR can serve as a gene I type and II type DuCV genetic typing new molecular marker; meanwhile, the invention proves the effect of regulating and controlling mRNA stability by the QTR serving as a DSE component by a dual-luciferase reporter system and a semi-anchored reverse mutation PCR technology, and defines the important effect of regulating and controlling virus gene expression by the QTR. The invention provides important technical and theoretical basis for researchingDuCV genotyping and virus replication regulation and control mechanism.

The invention pertains to a novel cell line, an HIV tat-rev dependent GFP-Gaussia luciferase Reporter cell line, known henceforth as the GGR cell line, that detects pseudotype and replication competent HIV (cloned or uncloned isolates, in cell media or human serum) rapidly and with high sensitivity. This GGR cell line provides an improved method of characterizing the entry phenotype of HIV envelope genes, and detecting and examining primary HIV samples in the context of laboratory research, clinical trial monitoring, and medical diagnostics. Examples include, but are not limited to, determining the functional HIV viral load, responsiveness to treatment, characterization of viral co-receptor usage (testing for viral co-receptor usage, i.e., CCR5 vs CXCR4, as required prior to prescribing FDA-approved CCR5 inhibitors), and characterization of other viral or drug resistance phenotypic properties to guide treatment.

The invention an analysis and identification method on the miRNA key target gene of blood fluke in the specific growth period, and belongs to the field of life medicine science. Bioinformatics is adopted to analyze the blood fluke genome so as to find certain miRNA target gene groups in the blood fluke genome. Then Solexa is utilized to analyze the blood fluke expression difference gene groups in the high expression period of miRNA and the low expression period of miRNA. The overlapped part between the blood fluke expression difference gene groups and the miRNA target gene groups is the candidate key gene groups. Then KEGG analysis is used to find the important genes in the candidate key gene groups so as to determine the key target gene group primarily. Then the miRNA mimic is utilized to carry out in-vitro experiments to further screen out the key target genes, and finally the target relationship between the miRNA and the key target genes is further confirmed through a luciferase reporter system. The establishment of the provided method can help the identification of the key target genes of miRNA in the specific period, help the disclosure of the modulation effect of miRNA, and provide an ideal route for miRNA function researches. At the same time valuable references are provided for the similar research methods on other species.