18 results about "Point mutant" patented technology

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.

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 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 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.

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 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 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 application provides a construction method and application of a CYP1A1 double-site mutant mouse model, and belongs to the technical field of genetic engineering. Specifically, the double-site mutant mouse model provided by the application is a CYP1A1 p.L118R and CYP1A1 p.R245P double-site mutant mouse model, which is obtained by the following method: S1: designing gRNA and a donor vector; S2: microinjecting Cas9 mRNA, gRNA and the donor vector into a fertilized egg; S3: obtaining F0 generation mice; S4: mating the positive F0 generation mice with wild-type mice to obtain F1 generation mice; S5: interbreeding the positive F1 generation mice to obtain F2 generation mice; and S6: screening the F2 generation mice to obtain the double-site mutant mouse model. The application can solve the technical problems of a complex preparation process and a low success rate of a double-site mutant mouse model far apart.

The present application relates to the technical field of genetic engineering and protein engineering, and particularly relates to a xylanase mutant. The mutant is a single-point mutant provided based on wild-type xylanase Xyn, and the specific activity of the mutant is generally increased by 12.4%-63.4%. The xylanase mutant containing the I128E single-point mutation has the highest specific activity of 1273.9 U / mg, which is conducive to reducing the production cost of the enzyme and promoting the wide application of the enzyme in the feed field.

The application discloses a construction method and application of a Clock gene site-directed mutant mouse model, and belongs to the technical field of basic medical animal models. The construction method comprises the following steps: gRNA vector design and construction, synthesis of a Donor Oligo sequence, co-injection of the gRNA vector, a Cas9 vector and the synthesized Donor Oligo into a fertilized egg to obtain F0 generation transgenic mice; and crossbreeding of the F0 generation mice with wild type mice to obtain F1 generation transgenic mice. Phenotype analysis on the F1 generation transgenic mice shows that the Clock gene site-directed mutant mouse exhibits an age progression heart failure phenotype, which is basically consistent with heart failure caused by aging in a natural physiological state, and therefore, the mouse can be used as a model of chronic heart failure disease, and is expected to be used for in-depth research on the pathogenesis based on the model, thereby providing technical support for prevention and treatment of the disease.

The application discloses a spinosyn polyketide synthase acyltransferase mutant and application thereof, relates to the field of bioengineering, and comprises a first mutant, a second mutant or a third mutant; the amino acid sequences are shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 respectively. The application discloses a coding gene, a recombination carrier and a recombination bacteria of the mutant and application of the mutant in production of spinosyn A with high purity and single component. The application discloses a soluble expression method of the acyltransferase domain SpnAT8 protein of the spinosyn polyketide synthase module eight, which comprises culturing bacteria, eluting the target protein and further purifying the target protein by using fast protein liquid chromatography. The four-point mutant provided by the application has the activity of recognizing malonyl-CoA restored to 19.5% compared with the wild type, and almost loses the activity of recognizing methylmalonyl-CoA.

This invention discloses a thermostable L-tryptophan oxidase mutant, belonging to the field of biotechnology. This invention analyzes information that improves enzyme thermostability from an evolutionary perspective, integrates and analyzes sequences of the thermostable reverse transcriptase family, and combines bioinformatics and crystallography methods to obtain a novel L-tryptophan oxidase mutant with high stability. The thermostable L-tryptophan oxidase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type L-tryptophan oxidase, both single-point mutants and combined mutants have longer half-lives at 50°C; especially the combined mutants, which exhibit a synergistic effect of the thermostability of the single-point mutants, with a half-life approximately 8 times that of the wild type. The thermostable L-tryptophan oxidase mutants provided by this invention have excellent catalytic activity and promising application prospects.

The invention discloses a construction method and application of a glucomannan 4-beta-mannoyltransferase 9 mutant, and belongs to the technical field of variation or genetic engineering. The mutant disclosed by the invention is based on a wild type sequence as shown in SEQ ID NO.1, and comprises mutation at sites of Y327W, Y327F, R326D, R326E, F532W, F322W and R326K. A key catalytic area is simulated and recognized through a three-dimensional structure, a high-catalytic-activity mutant is obtained through experimental verification, and the effects of R326D, F532W and a two-site mutant F532W / R326D are remarkable. The yield of the dendrobium glucomannan synthesized by using an engineering strain of the mutant reaches 457.5 mg / L, which is obviously superior to that of a wild type, so that the mutant has higher industrial application potential.

The application discloses a method and a kit for realizing high-sensitivity and multiplex detection of bladder cancer specific point mutation, and the kit comprises sequences of SEQ ID NO.1-SEQ ID NO.9; wherein: the primer of SEQ ID NO.1, the mutant primer of SEQ ID NO.2 and the wild type probe of SEQ ID NO.3 are used for high-sensitivity detection of FGFR3 S249C mutation; the primer of SEQ ID NO.4, the mutant primer of SEQ ID NO.5 and the wild type probe of SEQ ID NO.6 are used for high-sensitivity detection of PIK3CA E545K mutation; the primer of SEQ ID NO.7, the primer of SEQ ID NO.8 and the probe of SEQ ID NO.9 are used for detection of a target ACTB reference gene. The application realizes selective and efficient amplification of a mutant template through a wild type fluorescent probe and a mutant primer. The method has a detection capacity of 3 / 10,000 for a low-abundance mutation.