137 results about "T7 RNA polymerase" patented technology

The invention relates to a T7 RNA polymerase mutant and application thereof. Specifically, the invention discloses a T7 RNA polymerase mutant, and the polymerase mutant has at least one amino acid mutation at sites corresponding to a wild type T7 RNA polymerase and selected from the following groups: the 44th site, the 45th site, the 46th site, the 172nd site, the 389th site, the 425th site, the 441st site, the 632th site, the 643th site, the 754th site and the 789th site; the serial number of the mutation site is based on the sequence shown in SEQ ID NO: 1, the mutation leads to obvious reduction of a byproduct dsRNA generated in the process of enzyme synthesis of mRNA, and meanwhile, the generation of RNA aggregates is reduced.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. Wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, the enzyme specific activity of the T7 RNA polymerase variant is remarkably improved compared with that of the wild type T7 RNA polymerase, the addition amount of the polymerase in the in-vitro transcription reaction process can be reduced, and the cost is saved; in addition, the mutant also effectively reduces the generation of incomplete fragment impurities, and the mRNA integrity is significantly improved to 99%. The invention further provides a method for preparing RNA through in-vitro transcription.

The present disclosure provides T7 RNA polymerase variants, the use of which increases transcription efficiency and improves the quality and yield of the enzyme. Also provided are polynucleotides encoding the T7 RNA polymerase variants, host cells capable of expressing the T7 RNA polymerase variants, and methods of using the T7 RNA polymerase variants for high-efficiency transcription.

Provided is a variant of an RNA polymerase, the variant being characterized in that an amino acid corresponding to arginine at position 386 in the amino acid sequence of a wild-type T7 RNA polymerase represented by SEQ ID NO: 1 is substituted with an aromatic amino acid.

The invention discloses a preparation method of high-purity circular RNA (Ribonucleic Acid). The preparation method comprises the following steps: constructing a cyclization skeleton sequence with self-splicing activity; the cyclization skeleton sequence comprises the following modular elements from the 5'end to the 3 'end: a T7 promoter, a 5'-terminal homologous fragment, a 3 'intron-exon fragment, a 5'-internal homologous fragment, a 5 '-terminal spacer sequence, an IRES sequence, a target fragment, a 3'-terminal spacer sequence, a 3 '-internal homologous fragment, an exon fragment-5' intron, a 3 '-terminal homologous fragment and an Xba I endonuclease site; inserting the cyclized skeleton sequence into a pUC57 vector, and carrying out plasmid amplification; an Xba I incision enzyme linearization vector is utilized to obtain an in-vitro amplification template; the method comprises the following steps: carrying out an in-vitro amplification reaction by using dsRNA-free T7RNA polymerase to obtain dsRNA-free precursor RNA; cyclizing the precursor RNA to obtain crude circular RNA; treating the crude circular RNA by utilizing poly (A) or poly (U) polymerase, and adding poly (A) or poly (U) long tails to linear RNA in the crude circular RNA; and purifying by using an affinity chromatography column to obtain the high-purity circular RNA.

The invention relates to the technical production field of metabolic engineering and genetic engineering, and particularly provides a genetically engineered bacterium for producing 2 '-deoxyuridine as well as a construction method and application of the genetically engineered bacterium. According to the strain, deoxyuridine kinase tdk is knocked out on an E.coli W3110 genome, and T7 RNA polymerase is integrated; an aspartate transaminase gene aspC, a glutamine synthetase gene glnA, a 5 '-deoxynucleotidase gene yfbR and a uridine monophosphate kinase gene pyrH are subjected to overexpression; the method comprises the following steps: introducing a ribonucleoside diphosphate reductase gene nrdBCA from Escherichia phage T4 in a heterologous series connection manner; the CTP synthase gene pyrG * of Corynebacterium glutamicum is introduced in a heterologous mode, and the CTP synthase gene pyrG * of Corynebacterium glutamicum is introduced in a heterologous mode. The safe, efficient and low-cost production of the 2 '-deoxyuridine is realized.

The invention discloses a T7 RNA polymerase mutant capable of being used for preparing mRNA vaccines and improving thermal stability and transcription performance and a preparation method of the T7 RNA polymerase mutant, relates to the technical field of biology, and particularly discloses a T7 RNA polymerase mutant containing mutation sites K8G, H27K, A70N, K180D and S192P / S192G relative to wild type T7 RNA polymerase. The amino acid sequence of the wild type T7 RNA polymerase is as shown in SEQ ID NO: 1. Compared with wild type T7 RNA polymerase, the T7 RNA polymerase has higher specific enzyme activity and heat stability, the heat resistance of one mutant is improved, a large number of transcription products can still be obtained at 45 DEG C, and transcription by-products of the other mutant are reduced. The mutant T7 RNA polymerase disclosed by the invention has a good application prospect in the fields of RNA vaccines and medicines, in-vitro transcription, gene editing and the like.

The invention provides a kind of RNA polymerase variants and application thereof, the variants have improved catalytic activity compared with wild type T7 RNA polymerase, and the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process can be increased. In addition, the disclosure also provides a method for preparing RNA by using the variant, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

The invention relates to a T7RNA polymerase mutant for synthesizing single-stranded DNA as well as a screening method and application of the T7RNA polymerase mutant, and belongs to the technical field of enzyme engineering. The invention provides a method for directed evolution and screening of a T7RNA polymerase mutant, which comprises the following steps: knocking out exonuclease and mismatch repair protein from a chassis strain genome, knocking in single-stranded annealing protein, resistance gene containing deletion mutation and a single-stranded DNA synthesis template, and constructing to obtain a genetically engineered bacterium for screening; a T7RNA polymerase mutant library is constructed, recombinant plasmids with the mutant library are transferred into engineering bacteria, T7RNA polymerase mutants synthesize single-stranded DNA to repair deletion mutation of resistance genes, the transformed engineering bacteria can grow on a resistance plate, the T7RNA polymerase mutants obtained through multiple rounds of screening can efficiently synthesize the single-stranded DNA through in-vitro verification, and the T7RNA polymerase mutant can be used for preparing the single-stranded DNA. A brand new tool is provided for synthesis of single-stranded DNA, and the method has the potential of being applied to the fields of gene editing, evolutionary engineering and the like.

The present invention belongs to the field of genetic engineering technology and discloses a plasmid-free genetically engineered bacterium that can efficiently synthesize γ-aminobutyric acid from scratch using a cheap carbon source as a substrate, a method and an application thereof. The engineered bacterium is based on wild-type E. coli MG1655, overexpresses the T7 RNA polymerase gene; deletes the genes gabT and puuE; overexpresses gadC and gad bm , gdh, gltA, pyc, and ppc genes; and a growth-coupled promoter was used to dynamically regulate the expression of the sucA and argA genes in the GABA production pathway in Escherichia coli. All genetic manipulations in this engineered bacterium were performed within the genome, leaving no plasmid residues and requiring no antibiotics or inducers. The resulting strain exhibited stable production performance and a simple fermentation process. Using the engineered bacterium under a staged pH control process for 38 hours, the yield of γ-aminobutyric acid reached 35.4 g / L, demonstrating promising industrial application potential.

The invention discloses a modular fluorescent RNA aptamer biosensor system, and relates to the field of medicine. Comprising a probe module, an enzyme system, a dye and a reaction buffer solution, the probe module comprises a promoter probe P and a reporter probe R, the enzyme system comprises SplintR ligase and T7RNA polymerase, and the reaction buffer solution comprises a transcription buffer solution and a SplintR ligase buffer solution. The promoter probe P comprises a T7 promoter sequence and a 5 '-phosphorylated upstream recognition region, and the length of the promoter probe P is 10nt. According to the present invention, the ligase-assisted probe assembly and the T7RNA polymerase-mediated fluorescent RNA aptamer transcription are integrated, the simultaneous detection of the multiple circRNA can be achieved without the complex probe labeling, the femtomole-level sensitivity and the excellent single base mutation distinguishing ability in the complex sample are provided, and the real-time fluorescence instrument and the portable device are adapted.

The invention discloses a T7 RNA (Ribonucleic Acid) polymerase mutant, and the T7 RNA polymerase mutant comprises any one or more of the following amino acid mutation sites: W797L, P657K, Y846R, M306K, N601E, P476E, S606V, K642G, P742G, I117L, Q786L, S430P, L446F or T375K, and the T7 RNA polymerase mutant has RNA polymerase activity. The invention also discloses an application of the T7 RNA polymerase mutant in in-vitro transcription, non-coding RNA synthesis, RNA drug synthesis, in-vivo protein expression or cell-free protein expression.

The present application relates to T7 RNA polymerase variants. The present invention provides engineered T7 RNA polymerase variants and compositions thereof. These variants have been evolved for selective incorporation of symmetrically capped GTP analogs relative to GTP at the initiation of in vitro transcription. The invention also provides methods of using the variants provided herein. The invention also provides an application of the composition provided by the invention.

The invention provides a T7RNA polymerase variant and application thereof, the amino acid sequence of the T7RNA polymerase variant comprises substitution or deletion of at least one of the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437 and D438. Compared with wild type T7RNA polymerase, the variant has improved catalytic activity, and can improve the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process. In addition, the invention also provides a method for preparing RNA by using the variant, and more capped mRNA can be obtained by adopting the method for preparing RNA molecules.

The invention provides a kind of RNA polymerase variants and application thereof, the variants have improved catalytic activity compared with wild type T7RNA polymerase, and the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process can be increased. In addition, the disclosure also provides a method for preparing RNA by using the variant, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

Provided are T7 RNA polymerase variants and the use thereof. The T7 RNA polymerase variants may reduce dsRNA byproducts produced, e. g. during an IVT reaction, while retaining RNAP activity.

The invention provides a preparation method of thermal-stability T7RNA polymerase and the thermal-stability T7RNA polymerase, belongs to the field of biology, and can solve the problems that an existing T7RNA polymerase preparation process is complex, the expression culture period is long, multi-step purification treatment is needed, liquid change treatment needs to be carried out independently before storage, and the production cost is high. The method comprises the following steps: transferring plasmids containing a T7RNA polymerase gene sequence into expression host bacteria M15, culturing, screening, cloning, adding glycerol for strain preservation, and establishing a strain three-level library; the method comprises the following steps: taking glycerol bacteria, carrying out streak resuscitation on a flat plate, selecting monoclone, inoculating the monoclone into an LB culture medium, culturing, carrying out enlarged culture, carrying out IPTG induced expression, centrifugally collecting thalli, cleaning, adding a lysis buffer solution, resuspending, carrying out ultrasonic cell disruption, centrifuging, and collecting supernate; purifying by nickel column affinity chromatography, removing impure protein by an impure washing buffer solution, eluting target protein, purifying by matching with anions and cations, and collecting the purified protein; and mixing the purified protein with a storage buffer solution, and subpackaging and storing. The method can be applied to rapid preparation of the thermal-stability T7RNA polymerase.

The invention provides a microRNA fluorescent biosensor for quantitative detection of colorectal cancer. The microRNA fluorescent biosensor comprises a microRNA padlock probe for colorectal cancer, diethyl pyrocarbonate treatment water, T4DNA ligase, T7RNA polymerase, an rNTPs mixture, DNA endonuclease, a signal probe, a biological buffer solution and a Simply P total RNA extraction kit. According to the invention, simultaneous detection of a plurality of miRNAs (miR-21, miR-17, miR-31 and miR-92a) related to colorectal cancer can be realized; the adopted fluorescent probe has universal capability and can be applied to other gene detection by changing a target object recognition domain; a promoter is not needed to start transcription, so that the experimental design and operation are simplified.

The invention relates to a T7RNA polymerase-guided bacillus subtilis cytosine base editor and application thereof, and belongs to the technical field of gene editing. A T7RNAP guided cytosine base editor is developed in bacillus subtilis, four kinds of cytidine deaminase from different sources are selected to be fused with T7RNAP, the cytidine deaminase is fused at the N end of the T7RNAP, and a fusion body with the highest mutation activity is obtained through screening; the cytosine base editor is used for evolving a global transcriptional regulatory factor CodY, in-situ evolution of genome protein is achieved, and the expression level of bovine-derived beta-lactoglobulin (beta-Lg) is improved. According to the cytosine base editor, the operable range of the cytosine base editor on a genome is widened, multi-site base (C-T) editing can be realized, and an editing window is large.

The invention relates to the technical field of solid tumor immunotherapy, in particular to an IL-2mRNA multichannel delivery method for solid tumor immunotherapy, which comprises the following steps: designing a DNA template, using a plasmid containing an IL-2 gene sequence such as human IL-2cDNA, adding a T7 promoter, a 5'untranslated region UTR and a 3 'UTR at two ends to enhance translation efficiency, carrying out in-vitro transcription through T7 RNA polymerase, and carrying out in-vitro transcription through T7 RNA polymerase. And adding a 5 '-end cap analogue such as CleanCapAG to realize co-transcription cap adding, and then preparing lipid nanoparticle LNP encapsulation and carrying out surface modification. According to the IL-2mRNA multi-channel delivery method for solid tumor immunotherapy, through multi-channel delivery design, targeting optimization and combined treatment cooperation, the toxicity problem of a traditional IL-2 therapy and the drug resistance of MHC-I defective tumors are remarkably solved, meanwhile, efficient conversion is achieved through existing equipment, IL-2 expression can be accurately regulated and controlled, the immune microenvironment can be remodeled, and the immunotherapy effect is good. Meanwhile, the system is compatible with various treatment means such as chemotherapy, radiotherapy and immune checkpoint inhibitors.

The invention relates to a T7RNA polymerase mutant and application thereof. Compared with a wild type T7RNA polymerase, the T7RNA polymerase mutant has the following mutations: P266L, S430P, N433T, S633P, Y639F, V689Q, V783M, F849I and F880Y, and the T7RNA polymerase mutant has the following mutations: P266L, S430P, N433T, S633P, Y639F, V689Q, V783M, F849I and F880Y. Or, on the basis of the mutation, at least one of the following mutations: S43Y, G47A and S641A can also exist. The T7RNA polymerase mutant provided by the invention can be used for identifying and doping one or more nucleotide substrates in rNTPs, 2 '-F-dNTPs and dNTPs, so that a single-stranded DNA molecule, a fluorinated RNA molecule and a perfluorinated DNA molecule are efficiently synthesized in vitro.

The invention discloses an Escherichia coli recombinant bacterium capable of efficiently expressing dsRNA, the Escherichia coli recombinant bacterium is an rnc gene defect type TG1 strain, and the following plasmids are transferred: a first plasmid comprising a T7 RNA polymerase expression module, a second plasmid comprising a dsRNA polymerase expression module, a third plasmid comprising a dsRNA polymerase expression module, and a fourth plasmid comprising a dsRNA polymerase expression module; and a second plasmid comprising a dsRNA expression module. The invention also discloses a method for producing the dsRNA. The invention also discloses an application of the recombinant escherichia coli in preparation of nucleic acid pesticides. The invention provides a novel rnc gene defect type escherichia coli recombinant bacterium for dsRNA production in the field of nucleic acid pesticides. The rnc knockout of the strain has no obvious influence on the growth rate of the strain, and the strain has great industrial fermentation potential. According to the recombinant bacterium, a double-plasmid expression system is adopted, and meanwhile, the dsRNA expression module structure of double T7 terminators is utilized to optimize and improve the expression quantity and purity of dsRNA synthesis.

The invention relates to the technical field of bioengineering, in particular to a preparation method and application of an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No: 1 and is named as L8. The preparation method of the antibacterial peptide L8 comprises the following steps: S1, obtaining an L8 target gene segment; connecting an L8 target gene segment with a pLC vector to obtain a connection product; transforming the connection product into an escherichia coli DH5-alpha competent cell, and screening positive clones to obtain a pLC-L8 recombinant plasmid; s2, taking the pLC-L8 recombinant plasmid as a substrate, and carrying out cell-free protein synthesis reaction to obtain the antibacterial peptide L8, the cell-free protein synthesis system comprises 175 mM of potassium glutamate, 10 mM of ammonium glutamate, 2.7 mM of potassium oxalate, 33 mM of PEP, 38 mM of compound amino acid, 10 mM of magnesium glutamate, 2% (w / v) PEG-8000, 5 nM of pLC-L8 recombinant plasmid, 11.25 mM of lactose, 33.33% (v / v) of crude extract, 100 mg / mL of escherichia coli tRNA and 2.5 nM of T7 RNA polymerase. The antibacterial peptide L8 is obtained by optimizing the antibacterial peptide LRGG, and compared with the LRGG, the antibacterial peptide L8 subjected to cell-free protein synthesis reaction expression has better antibacterial activity and biological safety.

The present application relates to the field of genetic engineering technology, and specifically, to a method for preparing a transcription reaction solution, a locked nucleic acid-modified RNA, and an application of a mutated T7 RNA polymerase. A transcription reaction solution for preparing locked nucleic acid-modified RNA comprises a DNA template, a mutated T7 RNA polymerase, LNA-NTPs, and a buffer; wherein the amino acid sequence of the mutated T7 RNA polymerase is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7. The present application can significantly improve the transcription yield of locked nucleic acid-modified RNA by adding the above-mentioned mutated T7 RNA polymerase to the transcription reaction solution for synthesizing locked nucleic acid-modified RNA, and the operation is simple, which is conducive to industrial production.

To provide T7 RNA polymerase variants.SOLUTION: The present invention provides engineered T7 RNA polymerase variants and a composition thereof. The variants have been devised in order to selectively incorporate a symmetrically capped GTP analogue exceeding GTP in starting in-vitro transcription. The present invention further provides a method for using a variant provided in the specification. The present invention further provides a use of a composition provided in the specification. The engineered RNA polymerase variant can include at least one substitution or substitution set in a polypeptide sequence.SELECTED DRAWING: None

The present application provides a class of T7 RNA polymerase variants and applications thereof, wherein the amino acid sequence of the T7 RNA polymerase variant comprises at least one substitution or deletion selected from the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437, D438. The variant has improved catalytic activity relative to the wild-type T7 RNA polymerase, and can increase the capping rate of mRNA products during in vitro transcription (co-transcription capping). In addition, the present application also provides a method for preparing RNA using such variants, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) detection system integrating target chain amplification and crRNA synthesis into one tube, and belongs to the technical field of biosensing and molecular diagnosis. The system comprises an annular probe; the annular probe is amplified into double strands by phi29 DNA polymerase after being combined with a target, T7 RNA polymerase transcription is started to generate pre-crRNA, then the pre-crRNA is processed into mature crRNA by Cas12a, an ssDNA amplification product is recognized, an FQ reporter probe is cut, and a fluorescence signal is generated; wherein the sequence of the annular probe comprises a sequence complementary to a target chain, a single-chain T7 promoter sequence and a DNA sequence corresponding to a crRNA repetitive sequence. The detection platform integrates nucleic acid amplification and crRNA synthesis, real one-tube and one-step detection is realized, gRNA does not need to be additionally added, and the detection platform has the advantages of high sensitivity, high specificity, portability in operation and the like.

The present application relates to the technical field of genetic engineering, in particular to a T7-293T cell and application of the T7-293T cell in bovine parainfluenza virus type 3 infectious clone rescued virus, and the T7-293T cell provided by the present application has the preservation number of CGMCC No.46335.The T7-293T cell provided by the present application is a 293T cell stably expressing T7 RNA polymerase, and has the advantages of high transfection efficiency and cheap transfection reagent.Further, the present application establishes a method for bovine parainfluenza virus type 3 infectious clone rescued virus based on the T7-293T cell, constructs three genes of NP, P and L of bovine parainfluenza virus type 3 into the same helper plasmid, and co-transfects the T7-293T cell with the full-length gene plasmid of the virus, so as to avoid the low efficiency of co-transfecting four plasmids, and further improve the success rate of bovine parainfluenza virus type 3 infectious clone rescued virus.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. According to the present invention, the wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, and the capping rate of the RNA product can be substantially improved compared with the wild type T7 RNA polymerase;

The application belongs to the technical field of biological detection, and particularly relates to a visual detection kit for specifically diagnosing Theileria annulata under constant temperature conditions; the kit comprises RPA reaction products, crRNA, Cas13a protein, RNase inhibitor, reporter molecule, T7 RNA polymerase, NTP and NTT, the RPA reaction products are obtained by amplification of a primer pair shown in SEQ ID NO. 4 and 5, and the sequence of the crRNA is shown in SEQ ID NO. 1; the method is that the RPA reaction products, the crRNA, the Cas13a protein, the RNase inhibitor, the reporter molecule, the T7 RNA polymerase, the NTP and the NTT are mixed and incubated at an isothermal temperature; the incubation product is transferred into a detection buffer, inserted into a test strip, and the result is observed after incubation. The detection method has high sensitivity and high specificity, and can be used for specific detection of Theileria annulata.