62 results about "Point mutant" patented technology

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 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 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.

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 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 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 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.

The invention relates to a single-point mutant and a combined mutant based on wild type gsh enzyme and a preparation method for producing glutathione by using the single-point mutant and the combined mutant. According to the method, the synthesis efficiency of GSH is improved, and the production cost of related biopharmaceutical products is expected to be remarkably reduced.

The invention discloses a cutinase mutant and application thereof in degradation of polyethylene glycol terephthalate, and belongs to the field of environmental science. According to the present invention, BhrPETase derived from bacteria HR29 and LC cutinase of a metagenome in plant compost are modified to obtain mutants, the mutants are subjected to single-point or multi-point mutation near the substrate binding site of the BhrPETase or the LC cutinase, and the 184th site and the 156th site are subjected to single-point mutation or combined mutation to construct six mutants; compared with a wild type enzyme, the six mutants (M1-M6) have the advantages that the enzyme activity and the PET degradation efficiency are obviously improved, and the industrial prospect is good.

The invention discloses a point mutation gene specificity loop-mediated isothermal amplification detection method, and belongs to the technical field of point mutation gene specificity detection. Synthesizing a wild type primer group and a mutant type primer group aiming at the target point mutant gene, wherein each of the wild type primer group and the mutant type primer group comprises a forward inner primer FIP and a backward inner primer BIP; the first basic group at the 5'end of the forward inner primer FIP is complementary to the basic group of a template to be detected at a target mutation site, and the first basic group at the 5 'end of the backward inner primer BIP is the same as the basic group of the template to be detected at the target mutation site; introducing a basic group which is not matched with the basic group of the target detection gene to a second basic group at the 5'end of the forward inner primer FIP and the backward inner primer BIP; taking a template containing a target point mutation gene as a to-be-detected template, and performing LAMP amplification reaction by using the wild type primer group to obtain an amplification reaction result. The detection sensitivity is not sacrificed while the specificity is improved, and stable detection of low-abundance point mutation genes is realized by optimizing the primer binding efficiency and amplification kinetics.

The point mutation rate detection method according to the present invention is used in a multiplex ligation-dependent probe amplification (MLPA) measurement, and includes: a measurement step of measuring at least an intensity SMT among the intensity SMT of a mutation-derived signal as a fluorescence signal emitted from a mutation site of a sample and an intensity SWT of a wild-type derived signal as a fluorescence signal emitted from a site of the sample other than the mutation site, using an electrophoresis device; and a rate calculation step of calculating a rate of the intensity SMT to a reference value having a higher intensity than the SMT. In the point mutation rate detection method, an upper limit of a dynamic range of measurement for the fluorescence signal of the electrophoresis device is equal to or more than a predetermined value.

The invention belongs to the technical field of enzyme engineering and food biology, and particularly relates to an FTase enzyme two-site mutant and application thereof. The two-point mutant 142P-242K is successfully constructed through rational design and directional transformation, the enzyme activity of the two-point mutant 142P-242K is improved by about 8 times compared with that of an original enzyme, and the synergistic enhancement effect is remarkable; in a 500 g / L sucrose substrate, the conversion rate of the fructo-oligosaccharide reaches 52%, the yield is 267.3 g / L, and the product is mainly GF2 and better meets the application requirements of prebiotics; the mutant provided by the invention has the advantages of mild optimum conditions, capability of activating enzyme activity, easiness in industrial amplification and cost reduction. The invention provides a key technology for high-efficiency and low-cost production of functional oligosaccharides, and has remarkable application prospects and economic benefits.

The invention discloses a lipase mutant and application thereof, and belongs to the field of gene engineering and enzyme engineering. On the basis of staphylococcus epidermidis-derived lipase, two single-point mutants A86K and Q125L and a two-point combined mutant A86K-Q125L are obtained through screening, the reaction specificity to isoamyl alcohol is improved by the mutants, the reaction specificity to ethanol is reduced by the mutants, and the three mutants can catalyze esterification reaction under the condition of high water activity. The mutants provided by the invention are more suitable for industrial production than natural lipase, especially in the field of fusel oil removal of Baijiu, and have very huge application prospects and industrial values.

The invention belongs to the technical field of biology, and particularly relates to a single-point mutation Phi29 DNA polymerase and a recombinant expression system thereof. The Phi29 DNA polymerase mutant provided by the invention comprises five single-point mutants at any one single-point mutation site in T204E, K208E, R236E, R415G and K478D on an amino acid sequence shown as SEQ ID NO.2. Compared with a wild type Phi29 DNA polymerase, the Phi29 DNA polymerase mutant has the advantage that the thermal stability of the Phi29 DNA polymerase mutant at 40-70 DEG C is better. The Phi29 DNA polymerase mutant obtained by the construction method provided by the invention has better thermal stability, can show higher thermal stability when being used for DNA amplification at a higher temperature, and has higher application potential in the fields of molecular biology research, clinical diagnosis and the like.

The invention relates to the technical field of genetic engineering and protein engineering modification, in particular to a xylanase mutant. The mutant is a single-point mutant provided on the basis of wild type xylanase Xyn, and the specific activity of the mutant is generally improved by 12.4%-63.4%; wherein the specific activity of the xylanase mutant containing I128E single-point mutation is highest and reaches 1273.9 U / mg, so that the production cost of the xylanase is reduced, and wide application of the xylanase mutant in the field of feeds is promoted.

The invention discloses a PET hydrolase mutant with improved thermal stability and application thereof, and belongs to the field of enzyme engineering. Starting from HotPETase, research is carried out based on deep learning design, enzyme engineering optimization and application. 10 to-be-verified single-point mutants capable of improving the enzyme performance are selected through model prediction in combination with manual evaluation, and then experimental screening verification is carried out. The method comprises the following steps: comprehensively selecting better single-point mutants and combined mutants, and evaluating by taking an amorphous PET (Polyethylene Terephthalate) film (with the crystallinity of 6.7%) as a substrate to obtain dominant mutants. Candidate mutants are efficiently expressed and purified based on cSAT 2.0, the enzyme activity of the optimal mutant Y63F is equivalent to that of HotPETase, and the thermal stability of the optimal mutant Y63F is 1.5 times that of HotPETase after the optimal mutant Y63F is subjected to heat treatment at 65 DEG C for 2 hours. The optimal mutant Y63F is subjected to a protein melting temperature (Tm) test, and the result shows that the Tm is increased by 2.5 DEG C.