94 results about "T7 RNA polymerase" patented technology

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 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 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 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.

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 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.

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 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.

Provided is an RNA polymerase mutant in which an amino acid residue selected from the group consisting of glutamic acid at position 48 and arginine at position 50 in the amino acid sequence of a wild-type T7 RNA polymerase set forth in SEQ ID NO: 1 is substituted with an aliphatic amino acid residue.

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 application discloses a recombinant Escherichia coli for efficiently expressing dsRNA, which is a TG1 strain with gene defects and into which the following plasmids are introduced: a first plasmid comprising a T7 RNA polymerase expression module; and a second plasmid comprising a dsRNA expression module. rnc The application also discloses a method for producing dsRNA, and application of the recombinant Escherichia coli in preparing nucleic acid pesticides. rnc The application provides a new recombinant Escherichia coli with gene defects for producing dsRNA in the field of nucleic acid pesticides. rnc The knockout has no significant influence on the growth rate of the recombinant Escherichia coli, and the recombinant Escherichia coli has great industrial fermentation potential. The recombinant Escherichia coli adopts a double-plasmid expression system, and the dsRNA expression module structure with double T7 terminators is used to improve the expression amount and purity of dsRNA synthesis.

The present invention discloses a recombinant Vibrio natriegens and a method for promoting the secretion and expression of exogenous proteins. The recombinant Vibrio natriegens is constructed by using the Vibrio natriegens with the preservation number of CICC 10908 as the chassis cell, knocking out the deoxyribonuclease gene on its genome, and inserting a combined expression cassette of T7 RNA polymerase and aminoglycoside adenyltransferase. The recombinant plasmid carrying the exogenous protein is transformed into the above-mentioned recombinant Vibrio natriegens, and the seed liquid is inoculated into the fermentation medium. After culturing to the mid-logarithmic growth phase, IPTG and NaCl are added, and then the culture is continued to obtain a fermentation broth containing the exogenous protein. The present invention solves the problem of secreting and expressing exogenous proteins by Vibrio natriegens at the fermentation level for the first time, with a short culture time, low cost, and certain universality, providing technical guidance for the research of Vibrio natriegens engineering bacteria in the fields of protein engineering and fermentation engineering.

A T7 RNA polymerase mutant that reduces transcriptional byproducts, its preparation method, and its applications are disclosed, relating to the field of biotechnology. This T7 RNA polymerase mutant has a single mutation site, E242L, compared to the wild-type T7 RNA polymerase. Compared to existing multi-site mutations, this method is not only simpler to operate, but the single-point mutation is also more conducive to the study of functional mechanisms and subsequent precise regulation of function. This T7 RNA polymerase mutant exhibits better thermostability, still yielding a large amount of transcription product at 45℃, and significantly reduces transcriptional byproducts.

This disclosure provides a class of RNA polymerase variants and their uses. These variants exhibit improved catalytic activity compared to wild-type T7 RNA polymerase, increasing the capping rate of mRNA products during in vitro transcription (co-transcriptional capping). Furthermore, this disclosure provides methods for preparing RNA using these variants. Using this method to prepare RNA molecules, a higher amount of capped mRNA can be obtained.

The present invention discloses a T7 RNA polymerase mutant with high thermal stability and its application, relating to the field of biotechnology. The mutants include C125A, C216L, Q269M, C347L, M401K, W422F, Y457L, G464M, A468F, F481W, Q505A, C510E, C515P, V567P, V687E, M696A, A703T, H772K, P780K, R792M, H799G, K801G, E830G, C839N, P865L and / or A881F; the amino acid sequences of the mutants are shown in SEQ ID NO. 3 and SEQ ID NO. 28, respectively. Methods for selecting the mutation sites, expressing the mutants, measuring the melting temperature, and measuring the activity of the mutants are also disclosed. This mutant can undergo transcription reactions at high temperatures, reducing byproducts in the preparation of mRNA vaccines and drugs, and is suitable for isothermal nucleic acid amplification techniques. This invention utilizes deep learning to predict amino acid mutation sites, improve protein thermal stability, and rapidly screen for variants with high-temperature activity and stability. The new site has a significant effect on improving stability.

This disclosure provides T7 RNA polymerase mutants generated using the large language program EVOLVE-Pro. EVOLVE-Pro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with enhanced properties.

The invention discloses a T7 RNA polymerase mutant with high thermal stability and application of the T7 RNA polymerase mutant, and relates to the technical field of biology, the T7 RNA polymerase mutant comprises mutants C125A, C216L, Q269M, C347L, M401K, W422F, Y457L, G464M, A468F, F481W, Q505A, C510E, C515P, V567P, V687E, M696A, A703T, H772K, P780K, R792M, H799G, K801G, E830G, C839N, P865L or / and A881F; amino acid sequences of the amino acid sequences are respectively shown as SEQ ID NO. 3 to SEQ ID NO. 28. The invention discloses a mutation site selection method, a mutation site expression method, a melting temperature measurement method and an activity measurement method. The mutant can be subjected to transcription reaction at high temperature so as to reduce by-products generated in preparation of mRNA vaccines and drugs and is suitable for a nucleic acid isothermal amplification technology. According to the invention, mutation sites of amino acids are predicted by using deep learning, the thermal stability of proteins is improved, and variants with high-temperature activity and stability are rapidly screened out. The new point location has a good stability improvement effect.

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

The invention provides a method for rescuing a novel measles recombinant coronavirus. The pGEM3Zf (+)-T7RNAP plasmid constructed by the invention can be subjected to in-vitro reverse transcription processing to obtain T7RNA polymerase mRNA (messenger Ribonucleic Acid). The method comprises the following steps: by taking an MRC-5 cell as a cell matrix, co-transfecting the MRC-5 cell by using a pT7-MVEZ-COV2S.B.1.617.2 plasmid constructed by the invention and a helper plasmid in a physical electric shock manner, and simultaneously transfecting T7RNA polymerase mRNA (messenger ribonucleic acid). T7RNA polymerase mRNA translates and expresses T7RNA polymerase in an MRC-5 cell, the T7RNA polymerase further drives a T7 promoter, transcription of a full-length plasmid containing all genetic information of the virus and expression of N, P and L genes are started, virus packaging and proliferation are completed in a host cell, and the paramyxovirus with infection activity is obtained.

The invention relates to the technical field of protein engineering, and discloses a T7 RNA polymerase mutant as well as a preparation method and application thereof. The T7 RNA polymerase mutant provided by the invention is a protein as shown in A1) or A2): A1) relative to a wild type T7 RNA polymerase, the T7 RNA polymerase mutant comprises an amino acid substitution site A124E / G47S; the amino acid sequence of the wild type T7 RNA polymerase is as shown in SEQ ID NO: 1; a2) is a fusion protein obtained by connecting a label to the N end or / and the C end of the amino acid sequence in A1). Compared with wild type T7 RNA polymerase, the T7 RNA polymerase mutant has the advantage of low yield of by-products.

The invention relates to T7RNA polymerase and a preparation method thereof. The preparation method comprises the following steps: carrying out affinity chromatography on a crude enzyme solution containing T7RNA polymerase to obtain a first eluent; carrying out ion exchange chromatography on the first eluent to obtain a second eluent; the pH value of an elution buffer solution for hydrophobic chromatography is 7.5-8.5, and the elution buffer solution for hydrophobic chromatography comprises a buffer system, 0.4 M-0. 6M elution salt and 5% (v / v) glycerol. In the preparation method of the T7RNA polymerase, the crude enzyme containing the T7RNA polymerase is subjected to affinity chromatography, ion exchange chromatography and hydrophobic chromatography in sequence, and a specific elution buffer solution is adopted in the hydrophobic chromatography process, so that most impurities can be removed, and the T7RNA polymerase with relatively high purity and relatively low endotoxin residue is obtained.

Provided are a T7 RNA polymerase mutant capable of not generating an immunogenic by-product and having high transcriptional activity, and the use thereof, belonging to the technical field of biology. The amino acid sequence of the T7 RNA polymerase mutant is as shown in SEQ ID NO: 2. The T7 RNA polymerase mutant which is constructed by transformation of an original T7 RNA polymerase generates much less immunogenic RNAs than wild-type T7 RNA polymerases during a transcription phase, thus simplifying nucleic acid production processes, reducing the content of immunogenic by-products generated during the nucleic acid production processes, and shortening the time for synthesizing nucleic acids. The T7 RNA polymerase mutant has wide application prospects in the fields of RNA vaccines and drugs, in-vitro transcription, gene editing, virus RNA probe detection, etc.

The invention relates to a plasmid-free gene editing system based on T7RNA polymerase and application of the plasmid-free gene editing system, and belongs to the technical field of gene editing. The plasmid-free gene editing system constructed by the invention comprises a T7 RNA polymerase, a Cas12a nuclease, a selection marker gene and a short double-stranded DNA expression cassette, wherein the short double-stranded DNA expression cassette sequentially comprises a T7 promoter, a first forward repetitive sequence, a spacer sequence and a second forward repetitive sequence. According to the plasmid-free gene editing system, T7RNA polymerase is expressed in host bacteria, short double-stranded DNA is transcribed to generate guide RNA, Cas12a nuclease is driven to achieve rapid and efficient gene editing, the problems that in the prior art, linear fragment transformation is complex and tedious, and part of strains lack genetic plasmids are solved, iterative editing and multi-site editing are supported at the same time, and the plasmid-free gene editing system is suitable for large-scale popularization and application. And a brand-new convenient tool is provided for gene editing of non-model fungi.

The present application relates to the field of genetic engineering and bioengineering, and particularly relates to a genetically engineered surfactin-producing bacterium and application thereof.The genome of the genetically engineered bacterium comprises the following: an expression frame of T7 RNA polymerase; an expression frame of a surfactin synthesis gene cluster srfAABCD; and an expression frame of a long-chain fatty acid-coenzyme A ligase gene lcfA; the expression frame of the surfactin synthesis gene cluster srfAABCD and the expression frame of the long-chain fatty acid-coenzyme A ligase gene lcfA further comprise a T7 promoter.The present application realizes efficient synthesis of surfactin by Bacillus subtilis, and the final shake flask yield reaches more than 19.3 g / L, which has great industrial application potential.