34 results about "Renilla luciferase" 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 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 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.

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 liposome medicinal composition with tumor targeting, in-vivo tracing and treating functions and a preparation method thereof. The medicinal composition is a liposome targeting medicament which is resistant to CD44 antibody coupling, has a molecular imaging function simultaneously, and can be used for monitoring the in-vivo distribution of a medicament in real time in a living body state. In particular, plasmids containing three fusion genes, including renilla luciferase, red fluorescent proteins and suicide gene thymidine kinase, are coupled to CD44 antibody mediation-resistant immunoliposome, the specificity of liposome nanoparticles in a liver cancer in-situ model of an in-vivo targeting NOD / SCID (Non-Obese Diabetic / Severe Combined Immune-Deficiency) mouse is monitored by detecting a renilla luciferase signal with a living body imaging system, and apoptosis of liver cancer cells is induced by applying target thymidine kinase of ganciclovir; and moreover, a targeted liposome can be coated with adriamycin for inducing apoptosis of the liver cancer cells. The liposome medicinal composition provided by the invention does not have any toxic or side effect, has small damage and a good effect, and is suitable to be applied for a long time.

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 a dual-luciferase reporter gene plasmid applicable to screening of an orphan nuclear receptor activity regulating agent. The plasmid is a chimeric plasmid (p-TK(DR1)<3>-Fluc-hRluc) of DR1-containing firefly luciferase and the internal reference renilla luciferase. A construction method for the plasmid is to insert a fragment as shown in SEQ ID No. 1 into a hRluc-containingframework plasmid obtained after NheI / BglII double-digestion of the commercial plasmid pmirGLO. High-efficiency screening of the orphan nuclear receptor activity regulating agent can be realized through cotransfection of cells with the plasmid and an orphan nuclear receptor-overexpressed plasmid and activity detection via dual luciferases. The method is simple to operate, and the dual-luciferase reporter gene plasmid has high sensitivity and good specificity.

The invention relates to an establishment method of a renilla luciferase-carrying duck Tembusu reported virus and a product and application thereof. The establishment method comprises the following steps: restructuring an infectious cloned plasmid containing full-length cDNA of the duck Tembusu virus, inserting a gene of renilla luciferase into a site between 5'UTR and a structural gene of the TMUV virus to establish an infectious cloned plasmid, into which the RLuc gene is inserted, of the full-length TMUV reported virus, then obtaining a transcript of the reported virus by using a high-quality automatically-capped T7 in-vitro transcription kit, and transfecting BHK21 cells to rescue the reported virus. The renilla luciferase is excellent in sensitivity and convenient to use and can be quantitatively detected, so that the replication condition of the virus can be reflected by detecting the activity of the RLuc; and the renilla luciferase-carrying duck Tembusu reported virus can be applied to research in multiple aspects such as the life cycle and the replication mechanism of the TMUV, an anti-TMUV test, interaction between a viral protein and a host and other multi-aspect mechanisms.

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 the technical field of biology, in particular to a verification method for detecting ath-miRNA170-3p targeted MSH2 by using a tobacco dual luciferase report system. The tobacco dual luciferase reporting system comprises fluorescein luciferase (F-Luc) and renilla luciferase (R-LuC). The R-Luc is used as an internal reference, and restriction enzyme cutting sites AvrII and AgeI are embedded into a 3 'UTR region of the FLuc. The method disclosed by the invention is beneficial to clarification of an arabidopsis thaliana MSH2 gene expression mechanism regulated and controlled by the athmiRNA170-3p and screening of the biomarker, and has important significance on research on response to adversity stress of the arabidopsis thaliana MSH2 gene regulated and controlled by the ath-miRNA170-3p.

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 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.

A kit is used for diagnosing NPM1 antibodies, ranilla luciferase genes and NPM1 antigen genes are recombined into fusion vectors, and through a cell transformation technology, the fusion vectors are guided into a mammalian cell system to conduct luciferase-NPM1 antigen fusion protein expression; gene recombination cells are disrupted, luciferase-NPM1 antigen fusion proteins are obtained, NPM1 antigens are coated with a 96 elisa plate, and to-be-detected serum is added; the antibodies in the serum are combined with the antigens on the plate to be adsorbed to the plate; and the luciferase-NPM1 antigen fusion proteins are added, through an immunoprecipitation reaction, the luciferase-NPM1 antigen fusion proteins are combined with the NPM1 antibodies in the serum of a patient and are adsorbedto the plate, and finally a luciferase substrate is added to detect the fluorescence intensity. According to the fluorescence intensity, the content of the NPM1 antibodies can be reflected to providereference for diagnosis of prostatic cancer; and a method is quick, simple and convenient and has high sensitivity.

The invention discloses application of Arabidopsis mutant protoplasts in the analysis of signal transduction pathway. The steps of analyzing the signal transduction pathway are as follows: (1) constructing a protein fusion reporter gene expression vector, an effect vector and an internal control vector, wherein the protein fusion reporter gene expression vector expresses a fusion protein containing a protein tag A, the fusion protein consists of a target protein A and firefly luciferase, the effect vector expresses a target protein B containing a protein tag B, and the internal control vectorexpresses Renilla luciferase; and (2) transiently expressing the protein fusion reporter gene expression vector, the effect vector and the internal control vector in Arabidopsis mutant protoplasts andthen analyzing the signal transduction pathway. The invention provides a new strategy for reconstruction and research of plant hormone signal pathways in Arabidopsis protoplasts and has important application value.

The invention discloses a transgenic cell with in-vivo tracking and oncotherapy functions and a preparation method thereof. The transgenic cell is a mesenchymal stem cell from a human umbilical cord, is capable of performing oncotherapy, has a molecular imaging function, and is capable of monitoring the body distribution and survival condition in real time in a living body state after being subjected to intratumor injection. Particularly, a renilla luciferase-red fluorescence protein-suicide gene thymidine kinase fusion gene is transfected in the mesenchymal stem cell from the human umbilical cord, and a fluorescein signal is collected by means of a living body imaging system to monitor targeted therapy of a Nude mouse breast cancer model; a mouse injected with the mesenchymal stem cell transfected with the fusion gene is administrated with a substrate ganciclovir of thymidine kinase, and the death of tumor cells around the mesenchymal stem cell is induced through the bystander effect.

The invention discloses a construction method for stably expressing a Zika virus replicon containing renilla luciferase Rluc and an application thereof. According to the invention, a replication subsystem of the Zika virus containing the renilla luciferase Rluc is constructed, so that the replication subsystem can be stably expressed in cells, and the replication condition of the virus can be detected by detecting a renilla luciferase Rluc signal. Meanwhile, the method can be used for antiviral drug screening.

The invention discloses a high-throughput drug screening model using a human pregnane X receptor (hPXR)-mediated dual-luciferase reporter gene technique. The invention discloses a method for screening a human pregnane X receptor stimulant and an antagonist; by virtue of the method which is a new drug screening method, active drug ligands having regulating effect on a target gene, namely cytochrome CYP450 gene, of the PXR. A human embryonic renal HEK293T cell is subjected to the instantaneous co-transfection of an hPXR expression plasmid, a firefly luciferase reporter gene plasmid and an internal reference renilla luciferase plasmid by virtue of the method. The activities of the firefly luciferase and the renilla luciferase are simultaneously detected to show the regulating effect of the drug on the PXR activity, so as to establish a method for screening the hPXR stimulant and the hPXR antagonist. On one hand, the method can screen out compounds having an hPXR stimulating activity at high throughput from a massive compound library so as to avoid a risk that adverse drugs interact caused by the combined use of new drugs; and on the other hand, the method can screen out compounds having PXR antagonistic activity to inhibit the inducing effect of ligands on drug metabolic enzyme and transporter, so as to reduce the occurrence of adverse DDI and to reverse the drug resistance of anticancer drugs.

The kit of the invention is used for diagnosing NPM1 antibody, the Renilla luciferase gene and the NPM1 antigen gene are recombined into a fusion vector, and the fusion vector is introduced into the mammalian cell system by using the cell transformation technology, and the luciferase-NPM1 antigen fusion protein is carried out. Expression; lyse the gene recombinant cells to obtain a luciferase-NPM1 antigen fusion protein, coat the NPM1 antigen in a 96 microtiter plate, and add the serum to be tested. The antibody in the serum will bind to the antigen on the plate and attach to the plate; the luciferase-NPM1 antigen fusion protein is added, and the luciferase-NPM1 antigen fusion protein is combined with the NPM1 antibody in the patient's serum through immunoprecipitation. And adsorbed on the plate, and finally added the luciferase substrate to detect the fluorescence intensity. According to the fluorescence intensity, the content of NPM1 antibody can be reflected, which can provide reference for the diagnosis of prostate cancer. The method is fast, simple, and highly sensitive.

The invention discloses a PPARγ receptor agonist and its preparation method and application. The first receptor agonist is ursolic acid extracted from Cornus officinalis. Specifically, it is implemented according to the following steps: preparing the cornus officinalis extract; step 2: separating and purifying the cornus officinalis extract obtained in step 1 to obtain a receptor agonist. The present invention discloses a novel natural PPARγ receptor agonist. At a concentration of 3 μM, the relative average intensity of firefly luciferase activity and Renilla luciferase activity of the compound is the same as that of the full agonist of PPARγ receptor Rosiglitazone, the results of this study provide a lead compound for the development of new natural antidiabetic drugs.

The invention discloses a PPAR gamma receptor stimulant as well as a preparation method and application thereof. One receptor stimulant is ursolic acid extracted from asiatic cornelian cherry fruit. The preparation method specifically comprises the following steps: step 1, preparing an asiatic cornelian cherry fruit extract; and step 2, separating and purifying the asiatic cornelian cherry fruit extract obtained in the step 1 to obtain the receptor stimulant. The invention provides a novel natural PPAR gamma receptor stimulant, and the relative average intensity of the firefly luciferase activity and the internal control renilla luciferase activity of the compound is equivalent to that of the full stimulant rosiglitazone of the PPAR gamma receptor under the concentration of 3 microns; theresearch result provides a lead compound for research and development of novel natural antidiabetic drugs.

The invention relates to an establishment method of a renilla luciferase-carrying duck Tembusu reported virus and a product and application thereof. The establishment method comprises the following steps: restructuring an infectious cloned plasmid containing full-length cDNA of the duck Tembusu virus, inserting a gene of renilla luciferase into a site between 5'UTR and a structural gene of the TMUV virus to establish an infectious cloned plasmid, into which the RLuc gene is inserted, of the full-length TMUV reported virus, then obtaining a transcript of the reported virus by using a high-quality automatically-capped T7 in-vitro transcription kit, and transfecting BHK21 cells to rescue the reported virus. The renilla luciferase is excellent in sensitivity and convenient to use and can be quantitatively detected, so that the replication condition of the virus can be reflected by detecting the activity of the RLuc; and the renilla luciferase-carrying duck Tembusu reported virus can be applied to research in multiple aspects such as the life cycle and the replication mechanism of the TMUV, an anti-TMUV test, interaction between a viral protein and a host and other multi-aspect mechanisms.

A cell with in vivo tracing and tumor treatment functions and a preparation method. The cell is a human umbilical cord-derived mesenchymal stem cell, which can be used for tumor treatment and has molecular imaging function, and can monitor its distribution and survival in vivo in real time after intratumoral injection. Specifically, the Renilla luciferase-red fluorescent protein-suicide gene thymidine kinase triple fusion gene was transfected into human umbilical cord-derived mesenchymal stem cells, and the luciferin signal was collected by an in vivo imaging system to monitor its effect on the mammary glands of Nude mice. Targeted therapy in cancer models; administration of the thymidine kinase substrate ganciclovir to mice injected with mesenchymal stem cells transfected with the triple fusion gene induces the death of tumor cells surrounding mesenchymal stem cells through the bystander effect.

The invention discloses a recombinant plasmid containing an HDAC1 (histone deacetylase 1) gene promoter and a reporter gene as well as a construction method and the application of the recombinant plasmid. A promoter specific sequence of a histone deacetylase 1 gene is cloned and recombined with the promoter specific sequence to a firefly luciferase reporter gene vector so as to construct a recombinant plasmid vector of the HDAC1 reporter gene; the constructed vector and a reporter gene vector of renilla luciferase are co-transfected into human prostate cancer cells PC3, and the transcriptional activity of an active reaction HDAC1 gene promoter of firefly and renilla luciferase is detected at the same time, so that a method for targeted screening of tumor inhibiting drugs is established. The method has excellent application prospects in the aspects of screening tumor inhibiting drugs, evaluating the possible influence of to-be-detected drugs on tumors, researching the action mechanism of the drugs and the like.

The invention discloses a recombinant plasmid containing MRP (Multidrug Resistance-associated Protein)-2 genes 3' UTR (Untranslated Region) and reporter genes and application thereof. Constructed MRP-2 gene 3' UTR and reporter gene carriers and pRL-SV40 carriers and microRNA (Ribonucleic Acid) to be screened are co-transfected into a drug-resistance colon cancer cell line; inhibitory activity of microRNA to MRP-2 gene 3' UTR region is responded by simultaneously detecting activities of firefly and renilla luciferase; and thus, a method for targeted screening of reversal tumor multidrug resistance microRNA is established. The method has excellent application prospect in aspects of screening the reversal tumor multidrug resistance microRNA, researching acting mechanism between the reversal tumor multidrug resistance and microRNA and researching the mechanism of the tumor cell multidrug resistance microRNA level.

The invention belongs to the technical field of molecular biology, and particularly relates to a dual-luciferase-labeled bovine ephemeral fever virus protein compound and application thereof in detection of bovine ephemeral fever virus infection antibodies and vaccine antibodies. The protein compound disclosed by the invention comprises a firefly luciferase labeled bovine ephemeral fever virus P protein and a renilla luciferase labeled GNS-alpha2-alpha3-beta fusion protein. The protein complex can respectively capture structural protein antibodies generated by bovine ephemeral fever virus infected cattle or vaccine immunized cattle and virus infection specific GNS, alpha2, alpha3 and beta protein antibodies, can realize detection of the two types of antibodies in a single hole by utilizing the difference of two luciferase substrates, and can be used for differential diagnosis of bovine ephemeral fever virus infection and immunization.

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 provides a recombinant plasmid containing WSB1 gene promoter and reporter gene, its construction method and application. Specifically, the WSB1 gene promoter-specific sequence was cloned, recombined into the vector sequence containing the firefly luciferase reporter gene, and a recombinant plasmid containing the WSB1 gene promoter and reporter gene was constructed, and then the constructed recombinant plasmid was combined with Renilla luciferase The reporter gene plasmid pRL-TK was co-transfected into tumor cells to make it stably expressed. Finally, the activity of WSB1 gene promoter to initiate transcription was reflected by detecting the activity of dual luciferase, so as to be applied to targeted screening of anti-tumor drugs. The recombinant plasmid constructed by the present invention adopts the WSB1 gene promoter, and selects a specific promoter sequence. The drug screening model constructed has high specificity, high sensitivity and high accuracy, and realizes the antibacterial drug targeting WSB1. High-throughput screening of cancer drugs.

The invention discloses a reference internal type dual-luciferase reporter vector and an application thereof. Firefly luciferase gene is fused between the renilla luciferase gene of the pRL-TK vector and the ampicillin resistance gene, the two luciferases are on the same vector, and the firefly luciferase gene is used as reference. The one-step binary bridging coupling long-distance polymerase chain reaction (PCR) and the Escherichia coli vivo homologous recombination method are utilized to place the firefly luciferase gene and the renilla luciferase gene on the same vector; and monoclonal sites are introduced in the 3' untranslated region of the renilla luciferase gene for conveniently cloning the target segment, thus the reference internal type dual-luciferase reporter vector can be constructed. The vector of the invention has the advantages of high repeatability, little multiple-pore variation, convenient operation and precise quantification. The vector of the invention is suitable for the screening, identification and confirmation of the miRNA target molecule and also suitable for the quantitative analysis of the activity change of miRNA in cellular level.

The invention belongs to the technical field of biology and specifically relates to an NF-kappa B dual-luciferase reporter cell system capable of stably expressing pig-origin RIG-I receptor genes anda construction method thereof. The cell system is an HEK293-NF-kappa B cell which the pig-origin RIG-I (pRIG-I) receptor genes are transferred into, and the HEK293-NF-kappa B cell is an HEK293 cell which NR-kappa B firefly luciferase and internal reference renilla luciferase are guided into. The stable cell system disclosed by the invention can be applied to researching detection on downstream NF-kappa B promoter reporter genes activated by pRIG-I. The invention further provides a biological material for an RNA-Seq technology to screen stable cell internal differential expression genes under silent and active states of the pRIG-I in high throughput; furthermore, a foundation and a platform are established for further researching species specificity of the pRIG-I.