122 results about "T7 RNA polymerase" patented technology

The invention mainly belongs to the technical field of higher organism genome editing, and particularly relates to an establishment method of a T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 gene editing system. The method is based on the highly specific recognition principle for the T7 RNA polymerase and T7 promoter, and comprises the following steps: establishing T7 promoter-sgRNA, transforming the T7 promoter-sgRNA, a Cas9 expression plasmid and a T7 RNA polymerase expression plasmid into cells, and catalyzing the T7 promoter to promote the transcription of sgRNA by using the T7 RNA polymerase so as to guide the Cas9 to perform cutting at the target, thereby completing the gene editing process. By using the T7 promoter to express the sgRNA, the method simplifies the design steps of the CRISPR / Cas9 system, so that more people can quickly and easily research the interested gene locus by using the CRISPR / Cas9 gene editing tool.

The invention discloses a CRISPR nucleic acid detection kit for detecting a novel coronavirus (2019-nCoV). The invention provides a kit for detecting the novel coronavirus. The kit comprises a CRISPR-Cas13a system for detecting the novel coronavirus and lateral flow test paper matched with the CRISPR-Cas13a system for use. The CRISPR-Cas13a system comprises a1) a crRNA protein and an LwCas13a protein which are independently packaged; a2) a reporter RNA consisting of 20 U; and a3) an RT-RAA amplification primer used for amplifying a target sequence of a sample to be detected, wherein a 5'tail end of one primer in the RT-RAA amplification primer is provided with an area recognized by T7 RNA polymerase. According to the CRISPR nucleic acid detection test paper, high-sensitivity, high-specificity and convenient detection on a novel coronavirus nucleic acid can be realized through the CRISPR-Cas13a system, and the sensitivity reaches 10copies / test.

The invention discloses an optimized in vitro cell-free protein synthesis system. The system comprises a cell extract, a carbohydrate material, a phosphate compound, a buffering agent and a DNA molecular template for encoding an exogenous protein, wherein the cell extract is a yeast cell extract inserted into a T7 RNA polymerase gene; the carbohydrate material is a mixture of glucose and maltodextrin; the buffering agent is a trihydroxymethyl aminomethane buffering agent; and the DNA molecule template is prepared by a nucleic acid isothermal amplification method, and a sequence as shown in SEQID NO.1 is inserted into the upstream of the coding sequence of the exogenous protein in the DNA molecular template. By optimizing, the cost of in vitro protein synthesis is reduced and the yield ofthe target protein is increased.

The present invention relates to method for obtaining optimum expressed proteins in a transformed gram positive bacteria by specific integration of T7 RNA polymerase gene into the chromosome of a gram positive bacteria exhibiting resistance to aminoglycosides, said method comprising:- digesting an E. coli plasmid with a restriction enzyme- digesting the genomic DNA of said gram positive bacteria- ligating the said digested plasmid to the digested genomic DNA of said gram positive bacteria- transforming the said gram positive bacteria protoplasts with the said ligation mixture of step 2 to yield transformed gram positive bacteria (transformants),- screening the said transformants for kanamycin resistance and aminoglycoside sensitivity to ensure that the targetting of the said plasmid vector into the chromosome of the said gram positive bacteria is successful- cloning of the desired gene in the said vector—culturing the transformant in a suitable culture medium- isolating the expressed proteins from the culture medium

The invention relates to a method for sensitively detecting alkaline phosphatase by double-signal amplification mediated by a transcription reaction initiated by dephosphorylation. The method comprises the steps that a 5'-phosphorylated T7 promoter single chain is designed, alkaline phosphatase can catalyze 5'-phosphorylated T7 promoter to be dephosphorylated to protect the T7 promoter chain frombeing digested by Lambada exonuclease, the remaining T7 promoter can activate transcription reaction mediated by T7 RNA polymerase, and thus a large number of RNA transcripts are produced; and subsequently, the RNA transcripts complement and pair with Taqman probes to form a RNA-DNA duplex, duplex-specific nucleases is introduced to initiate circular cutting of the Taqman probes, and a significantly enhanced fluorescence signal is produced. The method is simple and convenient to operate, has high sensitivity and high specificity, and can be applied to the screening of target inhibitors in complex biological samples and the quantitative detection of targets in cervical cancer cells.

The present invention provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.

A system for ligase-free cloning and / or expressing a target gene is described herein. A preferred version of the invention includes an E. coli host. The host preferably includes a T7 RNA polymerase gene comprising a T7gpl coding sequence, a lacUV5 promoter, and a lac operator. The host preferably further includes a lacI gene comprising a lacI coding sequence with an ATG start codon, a promoter derived from the lacql allele, and a translational enhancer derived from a 5′ RNA leader sequence of T7 gene 10. The invention further includes a low-copy plasmid vector comprising a T7 promoter a lac operator operationally linked to the T7 promoter. The system is configured to inhibit target gene expression when uninduced and to permit gene expression upon induction by auto-induction.

The invention discloses a cell line ST / T7 capable of stably expressing T7RNA polymerase, the microorganism preservation number of which is: CGMCC No.24444. In the invention, a T7RNA polymerase gene is inserted into a eukaryotic expression vector pIRES2-EGFP to get the pIRES2-EGFP-T7 to transfect ST cells to obtain the cell line ST / T7 capable of stably expressing T7RNA polymerase. Through RT-PCR detection, expression plasmid containing a red fluorescent protein gene controlled by T7 promotors can be used to transfect the cell line so as to observe the expression of red fluorescent protein in a transcription product amplified form a ST / T7 cell to the T7RNA polymerase. The cell line ST / T7 can provide reverse T7RNA polymerase for use in reverse genetic manipulation of RNA-virus such as hog cholera virus, etc., and can be used as transcription and expression systems in vitro for research on genic structures and functions.

Disclosed is a T7 RNA polymerase mutant having improved thermal stability and / or specific activity in comparison with wild-type T7-like bacteriophage RNA polymerase, wherein at least one amino acid residue corresponding to at least one of the amino acid residues selected from the group at least consisting of glutamine at position 768, lysine at position 179 and valine at position 685 of the amino acid sequence that composes wild-type T7 RNA polymerase shown in SEQ ID NO: 6, is substituted with another amino acid.

The present invention relates to T7 RNA polymerase variants with improved affinity for 2′-modified nucleotides compared to the wildtype as well as methods for their production and methods of using them. The present invention also relates to the 2′-modified RNA molecules produced according to the methods of the invention.

The invention discloses an iterative gene circuit based on a vibrio fischeri quorum sensing system and a T7 expression system. The iterative gene circuit comprises a signal molecular protein gene luxIof vibrio fischeri, a receptor protein gene luxR, a promoter sequence PluxI(1) for sensing a signal molecular and receptor protein complex, a T7 RNA polymerase gene T7 RNApoly and a green fluorescentprotein egfp for representing the circuit intensity. The gene circuit does not rely on an inducer and can utilize the T7 expression system to spontaneously and forcefully initiate target gene expression in any host bacterium; and gene elements are constructed on different expression vectors, the iterative gene circuit is constructed in engineering bacteria, the T7 expression system is utilized toamplify signals of the vibrio fischeri quorum sensing system, and the iterative gene circuit has the function of utilizing the T7 expression system to spontaneously and forcefully initiate target gene expression, and has a lower leakage ratio and higher initiating strength.

Disclosed is a T7 RNA polymerase mutant having improved thermal stability and / or specific activity in comparison with wild-type T7-like bacteriophage RNA polymerase, wherein at least one amino acid residue corresponding to at least one of the amino acid residues selected from the group at least consisting of glutamine at position 768, lysine at position 179 and valine at position 685 of the amino acid sequence that composes wild-type T7 RNA polymerase shown in SEQ ID NO: 6, is substituted with another amino acid.

The invention provides a T7 RNA polymerase mutant by introducing a novel mutation. The T7 RNA polymerase mutant is selected from the mutant (R632C) with arginine at the position 632 in the amino acid sequence as shown in SEQ ID NO: 1 constituting a wild type T7 RNA polymerase substituted by cysteine. The T7 RNA polymerase mutant has DNA-dependent RNA polymerase activity, and can use various 2'-modified nucleoside triphosphates as a synthetic substrate compared with the wild type T7 RNA polymerase. The invention further provides methods and kits for synthesizing the mutant and a nucleic acid containing one or more modified nucleotides.

The invention discloses a controlling method for constructing a recombination bacterial BL21(BAD) for evoked preparation of T7 RNA polymerase by levorotation arabinose, the chromosome of the bacterial includes a T7 gene 1 controlled by an araBAD promoter, the T7 RNA polymerase produced by the recombination bacterial can evoke and activate the T7 promotor, thus manufacturing the destination gene product cloned at the downstream of the promotor.

The invention discloses a simultaneous amplification and testing reagent kit for VC (vibrio cholerae). The simultaneous amplification and testing reagent kit for the VC comprises a capture probe, a VC testing primer T7, a VC testing primer nT7, a VC testing probe, an M-MLV reverse transcriptase, a T7 RNA (ribonucleic acid) polymerase and the like. The simultaneous amplification and testing reagent kit for the VC can test VC RNA in food, has the advantages of high specificity, high sensitivity (capable of reaching 100 CFU / ml), low contamination (an amplified product RNA is easy to degrade in the natural environment) and rapidness in testing (testing can be completed within 50 minutes conventionally), plays an important role in rapid testing of the VC and is wide in application prospect.

The present invention provides engineered RNA polymerase variants and compositions comprising these variants. The present invention further provides engineered T7 RNA polymerase variants and compositions comprising these variants. These variants have been evolved for selective incorporation of the m7G(5′)ppp(5′)m7G cap analog over GTP at the initiation of in vitro transcription. The present invention also provides methods for selective capping of RNA transcripts.

The invention discloses an RNA isothermal amplification nucleic acid detection kit aiming at Escherichia coli 0157. The kit comprises a capture probe, a pair of 0157 detection primer T7 and n T7 primer, a 0157 detection probe, M-MLV reverse transcriptase enzymes, T7 RNA polymerases and other reagents. The kit provided by the invention can detect 0157 RNA in foods, has the characteristics of high specificity, high sensitivity (which can reach 103 CFU / mL), low pollution (as the amplification product RNA is susceptible to degradation in the natural environment), accurate result (as false positives can be avoided) and quick detection (as the detection can be completed within 40 minutes conventionally ), plays an important role in the quick detection of the Escherichia coli 0157, and has a wide application prospect.

The invention discloses an enterovirus type 71 (EV71) real-time fluorescent nucleic acid isothermal amplification detection kit comprising reagents such as a capturing probe, EV71 amplification primers T7 primer and nT7 primer, a EV71 detection probe, M-MLV reverse transcriptase, T7 RNA polymerase, and the like. The kit can be used for detecting EV71 RNA in throat swab or stool, and has the characteristics of high specificity, high sensitivity (reaching 10copies / reaction), low pollution (amplification product RNA can be easily degraded under natural environment), and fast detection (conventionally detection can be finished within 60min). The kit can perform important effect in clinical diagnosis of EV71 early-stage infection, and can be widely applied.

The invention discloses a single-subunit RNA polymerase and its purification method and application in RNA synthesis. The single-subunit RNA polymerase is a single-subunit RNA polymerase derived froma phi KMV phage or an other phage single-subunit RNA polymerase with a protein sequence which is 25% or more homologous to the single-subunit RNA polymerase derived from the phi KMV phage, contains acharacteristic amino acid sequence shown in the formula of SEQ ID NO. 1 in the sequence table and has the total amino acid sequence number of 800 and 830. The research result shows that the RNA polymerase has a long-distance relationship with the existing RNA tool enzyme and high transcription efficiency. Compared with the existing T7 RNA polymerase and Syn5 RNA polymerase, the single-subunit RNApolymerase solves the problem that the synthesis of RNA rich in a stable and high-level structure from the T7 RNA polymerase and Syn5 RNA polymerase produces complex products. The single-subunit RNA polymerase produces same transcription products.

The invention provides a specific and high-performance interferometric technique for treating RNA infected by CSFV, which designs specific siRNA sequence for dissimilar genes of hog cholera virus and obtains high-performance and specific interfering RNA molecule for reducing hog cholera virus by T7 RNA polyase external rerecording system and plasmid expression system, so is suitable for commercial manufacture. The biological agent produced by this method has 92.9-99.0% high depression efficiency and can appreciably prevent CSFV affection inside sensitized animals to make the animals relieve from morbidity and death.

The invention provides novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers. A respiratory virus conservative area is selected, and a promoter sequence capable of being recognized by T7 RNA (ribose nucleic acid) polymerase is formed at the 5' terminal of one of the synthesized primers. The invention further provides a novel high-sensitivity respiratory virus nucleic acid NASBA detection method. T7 RNA polymerase promoter sequences are added to the 5' terminals of the two primers complementary to two ends of a respiratory virus RNA sequence, so that when the primers enter a circulation phase, RNA sequences [RNA(+)] consistent with a template sequence can be synthesized, RNA sequences [RNA(-)] complementary to a template RNA sequence can be synthesized, and the detection sensitivity of viral nucleic acid is greatly improved; a throat swab sample of a patient can be directly used for amplification, a reverse transcription process is not required, and the operation time is saved.

The invention relates to an induced T7 RNA (ribonucleic acid) polymerase, particularly a light or small-molecule-compound induced-regulation T7 RNA polymerase. Specific amino acid sequence positions in the T7 RNA polymerase are used as splitting sites to perform splitting, and the split segments are fused with light-sensitive or small-molecule-compound-sensitive structure fields, proteins or peptides to implement the following induced regulations: a. regulating T7 RNA polymerase activity under the light or small-molecule-compound mediated protein-protein interactions; b. restoring the T7 RNA polymerase activity by split segment self-assembly through light-induced structure regulation; and c. embedding the light-sensitive structure field into the T7 RNA polymerase, and regulating the polymerase activity through the light-induced structure. The induction system provided by the invention implements accurate regulation on the gene expression in time and space dimensions, and the diversification of the T7 RNA polymerase splitting system provides powerful reference values for modular regulatory gene expression.

The invention discloses a Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit comprising reagents such as a capturing probe, CA16 amplification primers T7 primer and nT7 primer, a CA16 detection probe, M-MLV reverse transcriptase, T7 RNA polymerase, and the like. The kit can be used for detecting CA16 RNA in throat swab or stool, and has the characteristics of high specificity, high sensitivity (reaching 10copies / reaction), low pollution (amplification product RNA can be easily degraded under natural environment), and fast detection (conventionally detection can be finished within 60min). The kit can perform important effect in clinical diagnosis of CA16 early-stage infection, and can be widely applied.

The invention discloses a controlling method for constructing a recombination bacterial BL21(G2) for evoked preparation of T7 RNA polymerase by heat, the chromosome of the bacterial includes a T7 gene controlled by lambda P[L] and P[R] double promoters and a cI857 inhibitory gene, the T7 RNA polymerase produced by the recombination bacterial can evoke and activate the T7 promotor, thus manufacturing the destination gene product cloned at the downstream of the promotor.

The invention belongs to the biological technical field and in particular relates to a nonreactive expression system expressed by utilizing bacillus subtilis and a construction method. Light background T7 RNA polymerase expressed by induction of a xylose operon is utilized, the T7 RNA polymerase specifically transcribes a T7 promoter, then a cascade amplification expression system of a target gene is transferred into a bacillus subtilis genome, and a selective marker gene used in an intermediate process is deleted. The construction method overcomes the defects that an antibiotic selective marker gene can drift easily, a plasmid expression system is unstable, expression can not be induced and expression quantity is not high, and a nonreactive expression system with light background, controllable induction and efficient expression is constructed. By combining the advantages that a bacillus subtilis expression system is low in fermentation cost, large scale production can be realized, fermentation products can be secreted into fermentation broth and endotoxin is not contained, a brand new expression system is provided for expression of target protein.

The invention relates to an expression system comprising a prokaryotic cell which contains a polynucleotide construct that encodes a T7 RNA polymerase under the control of a T7 promoter and under the control of an inducible promoter. The invention also relates to an expression vector which contains a gene that encodes a protein to be expressed under the control of a T7 promoter, characterized in that said expression vector comprises a plasmid stabilization system.

The invention belongs to the technical field of veterinary biological products, and particularly relates to swine-borne BVDV-2 strain infectious cDNA (complementary deoxyribonucleic acid) clone and application thereof. The swine-borne BVDV-2 strain infectious cDNA clone contains swine-borne BVDV-2 seed virus full-length cDNA, T7 RNA (ribonucleic acid) polymerase promoters are inserted in 5' terminals of the swine-borne BVDV-2 seed virus full-length cDNA, and SbfI restriction enzymes are inserted in 3' terminals of the swine-borne BVDV-2 seed virus full-length cDNA. The invention further discloses a plasmid pASH28 with the swine-borne BVDV-2 strain infectious cDNA. The plasmid is linearized and then is subjected to in-vitro transcription to obtain RNA, MDBK (Madin-Darby bovine kidney) cells are transfected by the plasmid, and bovine viral diarrhea viruses can be successfully rescued. The swine-borne BVDV-2 strain infectious cDNA clone, the application and the plasmid have the advantage that the swine-born BVDV-2 strain infectious cDNA clone can be applied to research on functional difference between different animal-borne BVDV-2 proteins and also can be used for genetically modifying and preparing high-titer attenuation BVDV vaccine.

The invention relates to a recombinant expression plasmid, especially to a recombinant expression plasmid based on a T7 promoter. The invention also relates to a transformant containing the recombinant expression plasmid, and application of the transformant, particularly application of the transformant to fermentation production of lysine decarboxylase and the like and production of 1,5-pentamethylene diamine. The recombination expression plasmid comprises a plasmid skeleton; a replicon; a target gene and the T7 promoter controlling the expression of the target gene; and a T7 RNA polymerase gene and a sugar-induced stringent promoter controlling the expression of the T7 RNA polymerase gene, e.g., an Arabinose promoter. The transformant contains the recombinant expression plasmid as described above. The invention provides a method for fermentation production of polypeptide. The method comprises a step of culturing any transformant as described in the specification. According to the invention, cost for current induced recombinant expression inducers is lowered.