77 results about "Point mutant" patented technology

The invention belongs to the technical field of biology, and particularly provides reverse transcriptase UCRT vZ mutants with improved thermal stability, the reverse transcriptase UCRT vZ mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild type reverse transcriptase UCRT vZ, the half-life period of the mutants is longer at 65 DEG C; the double-mutant has a better effect, and the half-life period of the double-mutant is about four times that of the wild reverse transcriptase UCRT vZ. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) at a higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides a reverse transcriptase UCRT v6 mutant with improved thermal stability, the reverse transcriptase UCRT v6 mutant comprises four single-point mutants, six double mutants and four three-point mutants, and compared with wild type reverse transcriptase UCRT v6, the half-life period of the mutant is longer at 65 DEG C; the three mutants are better in effect, and the half-life period of the three mutants is about 5 times that of the wild reverse transcriptase UCRT v6. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT v6 mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribonucleic Acid) at higher temperature, and has higher application potential.

The present invention relates to a fusion protein that produces point mutations in cells, its preparation and use. Specifically, the fusion protein provided by the present invention contains a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity, or is formed by a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity. The present invention also relates to the coding sequence of the fusion protein, a polynucleotide sequence containing the coding sequence, a nucleic acid construct containing the polynucleotide sequence, a corresponding host cell, a method for producing point mutations in a cell, and a kit, etc. By using the present invention, it is possible to achieve site-directed mutagenesis while obtaining high mutation efficiency and a variety of mutation combinations in a specific gene region.

The invention belongs to the technical field of biology, and particularly provides reverse transcriptase UCRT vL mutants with improved thermal stability, the reverse transcriptase UCRT vL mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild type reverse transcriptase UCRT vL, the half-life period of the mutants is longer at 65 DEG C; the three mutants are better in effect, and the half-life period of the three mutants is about three times that of the wild type reverse transcriptase UCRT. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vL mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) at higher temperature, and has higher application potential.

The present invention relates to the field of bioengineering technology and provides a sucC mutant and its application in the fermentative production of L-valine. By performing point mutations on the sucC gene, the present invention reduces the enzyme expression activity, balances the metabolic flux problem between the TCA cycle for cell growth and product accumulation, and thus improves the valine yield. Further, through experimental screening, a recombinant strain capable of significantly increasing the valine yield is obtained. From the fermentation results, it can be seen that this method can significantly increase the valine yield, from the original 1.3 g / L to 1.61 g / L, with a 23.8% increase in yield, providing strong strain support for further industrialization.

The invention relates to the technical field of gene engineering, in particular to a kit for constructing a TBX19 p.Thr187Ala point mutation mouse model as well as a construction method and application of the kit. The kit provided by the invention comprises gRNA (guide ribonucleic acid) of which the nucleotide sequence is shown as SEQ ID NO.1 and single-stranded Oligo donor DNA (deoxyribonucleic acid) of which the nucleotide sequence is shown as SEQ ID NO.3. The invention also provides a kit for detecting the gRNA. According to the kit provided by the invention, a TBX19 p.Thr187Ala point mutation mouse model can be constructed, and the mouse model can be used as an animal model for studying the occurrence and development course of a hypothalamus-pituitary-adrenal axis function weakening and acrenocorticotropin (ACTH) deficiency model, so as to study the molecular and cellular mechanism of the TBX19 gene influencing animal (mouse) behaviors. The animal model can also be used as an animal model for research and development of adrenocorticotropin deficiency drugs.

The invention belongs to the technical field of bioengineering, and discloses a glycosyl transferase mutant which is obtained by manufacturing at least one point mutation with an amino acid sequence as shown in SEQ ID NO.1, mutated genes are cloned to corresponding carrier plasmids and transformed into escherichia coli for expression, and the obtained enzyme can be applied to production of stevioside D. The invention further discloses a preparation method of the glycosyl transferase mutant. The obtained glycosyl transferase mutant has the characteristics of high efficiency and stability, and can be coupled with sucrose synthase to efficiently prepare stevioside D.

The invention relates to the technical field of gene engineering, and discloses an alpha-L-rhamnosidase mutant and application thereof. According to the present invention, single-point or multi-point mutation of sites such as N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A and L176A is performed on the wild type alpha-L-rhamnosidase, such that the relative enzyme activity of the alpha-L-rhamnosidase is improved, and the efficiency of converting icariin to generate icariside I is improved. Especially, single-point mutants N325A and V337G have excellent enzymatic characteristics, when the icariin is catalyzed, the mutants N325A and V337G can specifically convert a substrate to generate icariside I, and no other by-products are generated.

The present invention relates to the field of bioengineering technology and provides a ppc mutant and its application in the fermentative production of L-valine. By performing point mutations on the ppc gene, the expression activity of phosphoenolpyruvate carboxylase is reduced, the competition of the bypass pathway is weakened, the supply of pyruvate precursors is enhanced, and the production capacity and biomass of the valine engineering strain are further improved. It can be seen from the fermentation results that this method can significantly increase the yield of valine, from the original 1.3 g / L to 1.49 g / L, with a 14.6% increase in yield. Therefore, the present invention has application value.

Various embodiments relate generally to the field of nucleic acid amplification and detection, in particular loop-mediated isothermal nucleic acid amplification and the detection of amplicons using designed detection probes. Moreover, various embodiments also relate to methods and kits for determining the presence or quantity of point mutations in a target nucleic acid molecule in a sample using loop-mediated isothermal amplification, which may be used for identifying genetic variants. In one aspect, the detection probe is a single-stranded probe comprising a nucleotide sequence fully complementary to a nucleotide sequence of the probe binding site comprising the point mutation. In another aspect, the detection probe comprising a nucleotide complementary to the point mutation at the penultimate base relative to the 3′ end of the detection probe.

The invention relates to a gene point mutation mouse model construction method. The method comprises the following steps: designing sgRNA and ssODN by taking T274M mutation on Kcnq2Exon6 as a target spot; the method comprises the following steps: selecting a female mouse at a set week age as an ovum donor, injecting a certain dose of serum gonadotropin and human chorionic gonadotropin into the abdominal cavity of the female mouse, mating with a breeding male mouse, and collecting fertilized ova of the mated female mouse; injecting Cas9 protein, ssODN and sgRNA into the pronucleus of the fertilized ovum through a micro-injection technology, transplanting the fertilized ovum after injection into the oviduct pot belly of the female rat to be pregnant, and obtaining a newborn rat after the female rat to be pregnant is delivered; according to a Kcnq2 gene point mutation site, a pair of identification primers F1 and R1 are designed at the two ends of the mutation site respectively, and a newborn mouse is identified, so that a Kcnq2T274M / + point mutation mouse with T274M mutation successfully knocked in is screened out. The Kcnq2T274M / + point mutation mouse editing method is simple in preparation and high in repeatability, and a larger number of Kcnq2T274M / + point mutation mice can be obtained while the editing efficiency is improved.

The invention relates to the field of gene engineering, in particular to a proximity marker enzyme mutant and application thereof. The proximity marker enzyme mutant takes ProtA-Turbo as a starting sequence, and one or more sites in K71, K240, K335, K345 and K351 are mutated into non-polar hydrophobic amino acid, polar neutral amino acid, acidic amino acid, basic amino acid or non-natural amino acid. The proximity marker enzyme mutant overcomes the influence of self biotinylation on the proximity marker activity, can continuously and stably mark the proximity proteome of the target protein on the immobilized cell, and obviously improves the sensitivity of identifying the proximity interacting protein of the target protein.

The invention belongs to the field of molecular genetics, and particularly relates to a method for regulating and controlling the length or width of corn leaves. The invention provides a method for regulating and controlling the length or width of a corn leaf through precise point mutation.

The invention belongs to the technical field of animal model construction, and particularly relates to a mouse model based on alpha-synuclein mutation and a construction method and application thereof. The mouse Snca gene is subjected to site-specific modification and mutates at the site S129A, S129D or Y125F, an alpha-syn mutation animal model which does not need exogenous induction and can naturally develop typical PD pathology and behavior phenotypes is constructed, and the limitation of an existing model in the aspects of pathology reduction degree and experiment controllability is broken through.

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, the reverse transcriptase UCRT vZ mutant includes four single-point mutants, six double mutants and four triple mutants, compared with a wild-type reverse transcriptase UCRT vZ, the half-life of the mutants is longer at 65 DEG C, the effect of the double mutants is better, and the half-life is about 4 times that of the wild-type reverse transcriptase UCRT vZ. The application further provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vZ mutant has higher thermal stability and great application potential.

The invention belongs to the technical field of bioengineering, and particularly relates to a mutant of sucrose amylase and application of the mutant in alpha-arbutin synthesis. According to the method, a single-point mutant and a multiple mutant are obtained by carrying out site-specific treatment on an amylosucrase sequence as shown in SEQ ID NO.1. Compared with a wild enzyme, the enzyme activity of the mutants is improved to different extents, the enzyme activity of part of the mutants is gradually improved along with increase or superposition of mutation sites, and the synergistic effect of the mutation sites is shown. In all multiple mutants, the activity of a five-site heavy mutant V5 (A284V / D386P / L330 / V92E / A323R) is improved most remarkably, the enzyme activity of the mutant is improved by 6.5 times compared with that of a wild enzyme, the thermal stability is also obviously improved, and the half-life period at 45 DEG C is 1.37 times that of the wild enzyme. The mutant can be applied to synthesis of alpha-arbutin, and the conversion rate is increased by 15.6%, so that the mutant has a good industrial application prospect.

Provided are high-specific-activity cellulase mutants and use thereof. Compared to the wild-type cellulase, the specific activity of the provided mutants that comprise single mutation sites P14A, P19T, S75A, S109N, H118P, M123I, M123L, I130L, I130Q, Y167E, Y167F, Y167W, A176S, A176F, D177S, and L192I, respectively, under neutral conditions is generally increased by 10.4% to 76.1%. The single-point mutant comprising I130L has the highest specific activity, which reaches 183.64 U / mg. The production costs of the cellulase mutants are significantly lower, which facilitates the wide use thereof.

The invention relates to the technical field of genetic engineering and protein modification, in particular to a high-temperature-resistant xylanase mutant and application thereof. The invention provides a mutant comprising mutation sites selected from I24T, G25N, V27Y, T41V, S65N / Q and N160A / M / R on the basis of wild type xylanase PT. The heat resistance of the xylanase mutant is remarkably improved, and after the xylanase mutant is treated at 95 DEG C for 5 min, the enzyme activity residual rate is improved by 13.1%-23.2% compared with that of a wild type; wherein the xylanase single-point mutant containing S65N single-point mutation has the strongest heat resistance, the enzyme activity residual rate is as high as 82.9%, and an unexpected technical effect is achieved.

The present invention relates to a use of a substance comprising point-mutated FoxM1 protein and peptide for inhibiting growth, migration and invasion of cancer cells. In addition, it relates to a use of a substance comprising point-mutated FoxM1 protein and peptide for inhibiting malignancy in a tumor microenvironment by inhibiting polarization into tumor-associated macrophages in a tumor microenvironment. It relates to a use as an apoptotic inducer using the action of sensitively inhibiting cancer cell death in metastatic cancer cells as well as general solid cancers.

The invention belongs to the technical field of gene mutation detection, and particularly relates to an isothermal amplification method and a kit for detecting EGFR-L858R and EGFR-T790M point mutation. One purpose of the invention is to provide a kit for detecting EGFR-L858R and EGFR-T790M point mutation. The kit comprises a capture probe sequence as shown in SEQ ID NO.5 and a signal probe sequence as shown in SEQ ID NO.6, wherein the capture probe sequence is used for detecting L858R point mutation; a capture probe sequence as shown in SEQ ID NO.10 and a signal probe sequence as shown in SEQ ID NO.11, which are used for detecting T790M point mutation. According to the technical scheme, PdAgBP is taken as a solid-phase immobilized substrate, EXPAR isothermal amplification and nucleic acid site LNA padlock are taken as a dual signal amplification mechanism, and biosensors based on BPE-ECL are respectively constructed for detection of point mutation EGFR L858R and EGFR T790M.

The present invention belongs to the technical field of animal modeling. Particularly disclosed are a PKHD1L1 gene point mutation rat model, a construction method therefor, and a detection method therefor. In the present invention, a murine P.L867S is knocked into the PKHD1L1 gene on the basis of a CRISPR / Cas9 system, and a point mutation is performed to change corresponding bases from TTA to TCA, such that a PKHD1L1 point mutation rat is constructed and obtained. The rat model can be further applied in various research scenarios such as epilepsy pathogenesis, design of a novel anti-epileptic drug and the like. The present invention provides use of a method for detecting cortical excitability abnormity in detecting a phenotype of an epilepsy animal model. In the present invention, after the animal model is constructed and obtained, a somatosensory evoked potential method is further adopted to detect the presence of a phenotype of cortical excitability abnormity and thereby confirm whether the model is successfully constructed or not. The present invention can be used for detecting cortical excitability abnormity, and can also be used for verifying the effectiveness of a related drug or treatment.

This invention discloses a base editor, a base editing method, and its applications. First, the invention constructs highly efficient targeting sites unrestricted by PAM sequences through point mutation. A random insertion library is constructed using a transposase system, and proteins capable of cytosine base editing in mammals are screened using a BFP-GFP reporter system. Further point mutations are performed on deaminases to screen for highly efficient cytosine base editors unrestricted by PAM. The optimized base editing method of this invention achieves an efficiency of over 70%, with some sites reaching over 90%, demonstrating significant application value in basic life sciences, medicine, and other fields.

The invention relates to an SNP molecular marker related to WSSV tolerance in a procambarus clarkia Nrf2 gene and application of the SNP molecular marker. According to the invention, 10 SNP mutation sites are found in a 5'terminal regulation region of the Nrf2 gene of the procambarus clarkia by combining whole genome re-sequencing data of a laboratory, and two SNP molecular markers related to WSSV tolerance characters are screened out through transcriptional activity detection of a point mutation luciferase reporter vector of the PcNrf2 gene, so that the breeding process of the procambarus clarkia tolerant WSSV variety is accelerated.

The present application relates to the technical field of genetic engineering and protein modification, and particularly relates to a cellulase mutant with improved specific activity and application thereof. The cellulase mutant has higher specific activity under the condition of 50 DEG C. Compared with the wild type, the specific activity of the cellulase mutant provided by the present application, which respectively contains N60Q, L78V, A92E, N124Q, M125I, Q169E, Q187R, S230G, D239A single-point mutations, is generally increased by 8.6%-35.7% under the condition of 50 DEG C. Among them, the specific activity of the L78V single-point mutant and the N124Q single-point mutant is increased by 30% and 35.7% respectively, thereby facilitating the wide application of the cellulase in the textile field.

The invention relates to the technical field of cell engineering, in particular to a cell strain causing pathogenic splicing mutation of a female HA patient and a construction method of the cell strain. A construction method of the T point mutation cell strain comprises the following steps: designing an sgRNA sequence based on a mutation site of a target gene F8; the method comprises the following steps: based on an sgRNA sequence and a CRISPR / Cas9 gene editing technology, constructing a plurality of pX458-sgRNA plasmids by taking a pX458 plasmid as a vector; the method comprises the following steps: constructing an LA-pMD-18T-pGK-puro-mcherry-RA donor plasmid by taking a pMD-18T-pGK-puro-mcherry plasmid as a vector on the basis of an sgRNA (small guide ribonucleic acid) sequence and a pX458-sgRNA plasmid; the preparation method comprises the following steps: simultaneously transfecting a pX458-sgRNA plasmid and an LA-pMD-18T-pGK-puro-mcherry-RA donor plasmid into a cell, and carrying out drug screening, so as to obtain a stable cell strain. In addition, the invention also provides application of the mutant cell strain. The discovery of the novel pathogenic splicing mutation not only enriches an F8 gene mutation database, but also provides a new strategy for precise treatment of female HA patients in the future.

The invention belongs to the technical field of bioengineering, and particularly relates to an enzyme activity improving mutant of ergothioneine precursor synthetase and application of the enzyme activity improving mutant. According to the present invention, starting from the ergothioneine sulfoxide synthetase CtEgtB from the thermophilic photoacid bacillus thermophilus, based on Apla-Fold 3.0 structure prediction and artificial evaluation, five single-site mutants and combined mutants capable of potentially improving the enzyme activity are selected, and the enzyme activity experiment test is performed; according to the invention, beneficial mutation sites are screened, and the enzyme activity of the optimal mutant is 4.5 times that of an original enzyme. The optimal mutant crude enzyme is applied to a biological enzyme method, 2.3 g / L of ergothioneine sulfoxide is synthesized within 6 hours, and the method can be applied to industrial production of ergothioneine.

The invention belongs to the field of gene recombination engineering, and particularly relates to a CRISPR / Cas targeting vector for C.658660del AAG conditional point mutation of an Mmachc gene and a construction method of the CRISPR / Cas targeting vector. The vector comprises: (1) a VB157 skeleton plasmid, wherein the VB157 skeleton plasmid comprises an ampicillin resistance expression cassette and a kanamycin resistance expression cassette; (2) a fragment A (5'arm) and a fragment E (3 'arm) which are obtained by amplification from a C57BL / 6JCya mouse BAC clone or wild type (WT) genome; (3) a segment B containing a wild type mouse Mmachc gene; (4) obtaining a c.658660delAAG mutation fragment under the combined action of a fragment C and a fragment D which are positioned on loxP sites on two sides of exon 4 of the Mmachc gene and amplification primers of the fragment C and the fragment D; and (5) an F fragment of a 6xSV40pA sequence located at the upstream of the second loxP site. According to the CRISPR / Cas targeting vector for the C.658660 del AAG conditional point mutation of the Mmachc gene and the construction method of the CRISPR / Cas targeting vector, the CRISPR / Cas targeting vector for the C.658660 del AAG conditional point mutation of the Mmachc gene can be accurately constructed.

To provide nucleic acid sequences for targeting one or more point mutations in the p53 gene.SOLUTION: In particular, the sites of the point mutations in p53 are selected from the group consisting of R249, R248, R273 and R175. Also disclosed herein is a method for treating cancer in a subject, the method comprising administering to the subject one or more of the nucleic acid sequences disclosed herein.SELECTED DRAWING: None

The application provides a formic acid dehydrogenase mutant, which is mutant I or mutant II; the mutant I is a single-point mutant based on wild-type formic acid dehydrogenase and is selected from one of R69H, K89E, R213H, R323H and K326E; the mutant II is a double-point mutant based on R69H and is selected from one of R69H / K89E, R69H / R213H, R69H / R323H and R69H / K326E. The application also provides a coding gene of the mutant and a recombinant expression vector and an engineering bacterium containing the gene. Meanwhile, the application also provides a preparation method of the mutant and an application of formic acid hydrogen production. The application obtains a mutant with high activity and substrate resistance by rational site-directed mutagenesis on wild-type formic acid dehydrogenase, significantly improves the catalytic activity and substrate resistance of the enzyme, improves the formic acid biological hydrogen production efficiency, reduces the production cost and is beneficial to industrial application.

The present application relates to the technical field of genetic engineering and protein modification, and particularly relates to a high specific activity cellulase mutant and application thereof. The mutant provided by the present application comprises substitution of amino acids at at least one position selected from the group consisting of 44, 84, 109, 123, 178, 183, 194, 225 and 234. The specific activity of the mutant at 50 DEG C is generally increased by 7.1% to 38%; wherein the specific activity of the S44N single-point mutant, the I123L single-point mutant and the L194I single-point mutant is respectively increased by 26.8%, 25.7% and 38%, thereby facilitating the wide application of the cellulase in the textile field.