652 results about "Point mutation" patented technology

The invention discloses polyketide synthase or a polyketide synthase mutant and application thereof, and belongs to the technical field of gene engineering. The polyketide synthase is AspiPKS4, AspiPKS5, AspiPKS6 and AspiPKS7 from cyathea spinulosa, FhPKS2, FhPKS3, FhPKS4, FhPKS5 and FhPKS6 from ficus himalayana, and CrPKS from floating fern. The mutant is obtained by carrying out single point mutation on the second amino acid of AspiPKS7 or deleting an N-terminal redundant sequence. The polyketide synthase or the mutant is used for replacing NnHisspS in an original FBP luminescence system, and the biological self-luminescence level can be remarkably improved. The invention further provides a simplified and improved FBP system which comprises the polyketide synthase or the polyketide synthase mutant, CPH, H3H and Luz, and the application convenience and the application luminescence capacity of the FBP system are improved.

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

The invention discloses a hemoglobin-resistant Taq DNA polymerase mutant and a construction method thereof, and relates to the field of biology, and the hemoglobin-resistant Taq DNA polymerase mutant is characterized in that a nucleotide sequence for coding the Taq DNA polymerase is shown as SEQ ID NO.1, and the mutant comprises at least one mutation or all mutations selected from S623D and E721A sites. On the basis of natural Taq DNA polymerase, the molecular structure of the Taq DNA polymerase is modified through rational design and combination with a site-specific mutagenesis biotechnology, so that the Taq DNA polymerase mutant is more suitable for PCR amplification of samples containing hemoglobin and the like than a wild type, and the result judgment accuracy of a sample containing a hemoglobin inhibitor is more facilitated.

The invention discloses a high-efficiency lysine decarboxylase mutant and an application of the high-efficiency lysine decarboxylase mutant in synthesis of pentamethylene diamine. A series of mutants including a single mutant (such as N218G, T222V, C244P and the like), a double mutant and a three mutant are obtained by carrying out multi-site mutation on wild type lysine decarboxylase (SEQ ID NO.2) from Klebsiella grimontii. The catalytic efficiency of the mutant is remarkably improved, and the activity of the mutant is improved by 5.0-10.2 times compared with that of a wild type enzyme. According to the method disclosed by the invention, the substrate conversion rate of 99% or above can be realized within 2 hours through an optimized process of constructing an expression vector and a genetically engineered bacterium, inducing expression of lysine decarboxylase and synthesizing 1, 5-pentanediamine by utilizing a whole-cell catalysis technology. Compared with a traditional chemical method and a natural enzyme method, the method disclosed by the invention has the advantages of simplified process, high catalytic efficiency, low production cost and the like, and is suitable for producing bio-based 1, 5-pentamethylene diamine and nylon 56 salt.

The present disclosure relates in some aspects to methods for analyzing target nucleic acids and their spatial locations in a biological sample. In some aspects, the presence / absence, amount, and / or identity of variant sequences (e.g., single nucleotide variations such as SNPs or point mutations) in a plurality of target nucleic acids in a cell or tissue sample are analyzed in situ in the sample. Also provided are compositions and kits for use in accordance with the methods.

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an alpha-2, 3-sialyltransferase mutant and application thereof. The a-2, 3-sialyltransferase mutants V175N, P198A, G173D, D174Q, F196K and A315H are obtained by performing site-specific mutagenesis on six amino acids in an a-2, 3-sialyltransferase amino acid sequence coded by a gene derived from Campylobacter jejuni (OH4384), the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3'-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the P198A, the G173D, the D174Q, the F196K and the A315H, the optimal recombinant microbial strain 3 '-SL-09 (V175N / P198A / D174Q) is obtained through combination of multiple mutation sites, the yield can reach 5.91 g / L, the bottleneck problem of low catalytic efficiency of sialyltransferase is effectively solved, and an enzyme element with excellent performance is provided for high-efficiency and low-cost biological manufacturing of 3'-SL.

The present invention provides a 4-vinylguaiacol oxygenase mutant for synthesizing vanillin, and an engineered bacterium thereof. The 4-vinylguaiacol oxygenase mutant is an enzyme having the sequence as shown in SEQ ID NO. 1, which exhibits a double-site mutation of D27P / T216M, D27P / M351F, D27P / Y156D, M351F / Y156D, T216M / Y156D or T216M / M351F, or a single-site mutation of T216M, T216L, S280M, G307P or M351F, and can be used for catalyzing 4-vinylguaiacol to generate vanillin.

The invention belongs to the technical field of biological pharmacy, and particularly relates to influenza virus-like particles for chimeric expression of new coronavirus RBD protein as well as a preparation method and application of the influenza virus-like particles. The virus-like particle is prepared by embedding fusion protein on the surface of influenza virus M1, and the fusion protein comprises an HA2 segment of HA protein of influenza virus, an extracellular functional region of M2e matrix protein, a conserved region short peptide of nucleoprotein, and RBD tandem protein with mutated five sites of 417, 452, 478, 484 and 501 of new coronavirus; the virus-like particles can be used for preventing specific influenza and new crowns; the preparation method of the virus-like particles is simple and low in cost, the virus-like particles are good in immunogenicity and cross protectiveness, the preparation requirements of large-scale influenza vaccines can be met, the biological safety is high, and the immunity of an organism to various influenza viruses and new coronal viruses can be stimulated.

Disclosed is a Taq DNA polymerase mutant. Compared to a wild-type Taq DNA polymerase, the Taq DNA polymerase mutant has a mutation at one or more of amino acid positions 116, 439, and 468, wherein the amino acid sequence of the wild-type Taq DNA polymerase is set forth in SEQ ID NO: 1. Compared to the wild-type Taq DNA polymerase, the Taq DNA polymerase mutant of the present invention has higher enzyme activity than the wild-type enzyme; both single-site mutants and multi-site mutants show improved tolerance to corn leaf supernatant, mouse serum, and / or heparin sodium, and thus can be used for direct rapid amplification of samples.

The invention discloses a dihydrodaidzein reductase mutant and application of the dihydrodaidzein reductase mutant in synthesis of (3S, 4R)-tetrahydrodaidzein, and belongs to the technical field of biology. According to the mutant, wild-type dihydrodaidzein reductase DHDR is taken as a parent, single-point mutation or combined mutation is carried out, and a DHDR mutant is obtained; the parent site of the single point mutation is one of the 120th site, the 122nd site, the 170th site, the 178th site, the 214th site, the 219th site, the 220th site or the 220th site; the combined mutation adopts two modes: one mode is that a single-point mutation DHDR mutant is taken as a parent, mutation is carried out, and a combined mutation mutant I is obtained; and 2, taking the DHDR combined mutation mutant I as a parent, and performing mutation to obtain a combined mutation mutant II. The DHDR mutant disclosed by the invention is used for synthesizing (3S, 4R)-tetrahydrodaidzein by reducing dihydrodaidzein, and asymmetric reduction with high activity and high stereoselectivity can be realized.

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for creating a corn dwarfing material and application of the corn dwarfing material. The invention discloses a method for creating a corn dwarfing material, which specifically comprises the following steps: carrying out specific key site editing on a target gene ZmGA20ox3 and / or ZmGA20ox5 in corn by using a genetic engineering means, and carrying out specific amino acid site mutation on a protein coded by the target gene ZmGA20ox3 and / or ZmGA20ox5 subjected to specific key site editing, and further cultivating and screening to obtain the corn dwarfing material. The method provided by the invention can be used for cultivating transgenic corn plants with different plant heights and other agronomic traits, and meanwhile, a new breeding technical scheme is provided for creating dwarf corn, reducing the height of a corn ear, reducing the included angle of corn plant leaves and increasing the corn yield; crops can be more densely planted in agriculture, so that the yield of the crops in unit area is improved, and the method has important breeding significance.

The invention provides a nucleotide mutation site prediction model construction and disease-related point mutation identification method. Specifically, the invention provides a deep learning model-fused nucleotide mutation site prediction model construction method and a disease-related point mutation identification method. According to the method, DNA point mutation and RNA point mutation can be recognized from transcriptome sequencing data in a high-sensitivity and high-specificity mode, and basic data is provided for explaining mutation generation mechanisms and functions on the whole transcriptome and genome level.

The invention discloses a method for biosynthesizing epigallocatechin gallate glucoside, and belongs to the technical field of biocatalytic synthesis. According to the method, a sucrose phosphorylase mutant is adopted as a catalyst, the sucrose phosphorylase mutant is subjected to single-point mutation or combined mutation on the basis of an amino acid sequence SEQ ID NO.1, and the enzyme activity of the sucrose phosphorylase is improved through mutation; the mutant is utilized to realize efficient catalytic synthesis of epigallocatechin gallate glucoside, and in the whole catalytic process, the synthesis method is mild in condition, simple to operate, short in reaction time and high in catalytic efficiency, and has a good application prospect.

The invention belongs to the technical field of biosynthesis, and particularly relates to an engineering bacterium for preparing stevioside through de novo fermentation as well as a preparation method and application of the engineering bacterium. According to the invention, mevalonate kinase in the MVA pathway is mutated and optimized. The method comprises the following steps: carrying out point mutation on mevalonate kinase MvK of a wild type source, constructing a stevioside synthesis route in an escherichia coli host, and comparing the influence of MvK mutation on the yield of stevioside, so as to determine a Q160L mutant, and applying the Q160L mutant to the stevioside production route to realize efficient production of stevioside.

The invention relates to the technical field of molecular biology, in particular to a qPCR (quantitative polymerase chain reaction) detection kit for detecting cis-trans mutation and a use method of the qPCR detection kit. The invention provides a primer pair which is used for detecting a first mutation site and / or a second mutation site of a target gene and comprises a universal primer F matched with the upstream of the first mutation site, a downstream primer R-BW matched with a wild type sequence of the second mutation site and a downstream primer R-BM matched with a mutation type sequence of the second mutation site, the fluorescent probe P-AW is matched with the upstream of the first mutation site, the fluorescent probe P-AM is matched with the mutant sequence of the first mutation site, and the fluorescent groups of the fluorescent probe P-AW and the fluorescent probe P-AM are different. According to the reaction system disclosed by the invention, the MGB fluorescent probe and the ARMS primer are combined, and mutation information and a cis-trans relationship can be judged according to the existence of a fluorescent signal. Compared with an existing detection method, the method for detecting the cis-trans relation through fluorescent quantitative PCR is proposed for the first time, detection can be completed within 2 h, specificity is good, the cost advantage is achieved, operation is easy and convenient, and clinical operation is convenient.

The invention discloses a biosensor responding to glutaric acid concentration, and belongs to the technical field of biochemical engineering. According to the invention, regulatory protein and promoter elements are constructed on PSC101 series plasmids to obtain the biosensor responding to glutaric acid concentration. By performing point mutation on a-10 region and a-35 region of the response promoter PgcdH and optimizing expression of regulatory protein through the promoter and RBS engineering, the response sensitivity is improved, the response range is widened, and the application prospect in the field of glutaric acid high-yield strain screening is very wide.

To improve allele discrimination, the inventors adapted the system combining Cas13a detection sensitivity with allele-specific PCR amplification to propose CASPER (Cas13a Allele-Specific PCR Enzyme Recognition) as a new versatile, easy-to-implement, and highly sensitive method to detect low-frequency of sequence variant. CASPER enabled specific and sensitive detection of KRASG12D with low DNA input such as DNA extracted from patient's pancreatic ultrasound-guided fine-needle aspiration fluids. CASPER is easy to implement and a versatile reliable method virtually adaptable to any point mutation.

The invention discloses a method for creating new dwarf tomato germplasm by EMS mutagenesis, which comprises the following steps: selecting new dwarf tomato seeds with full grains and no damage, soaking in 84 disinfectant fluid with the ratio of 1: 1, and washing with tap water for 5 times; the dwarf tomato seeds in the step (S1) are soaked in an EMS-phosphoric acid buffer solution with the concentration being 0.4%-2% and subjected to vibration treatment in a dark place for 6-36 h; detoxicating the mutagenized seeds in the step (S2), pouring out an EMS solution, washing twice with an antidote, and washing with tap water for 2 hours; (S3) accelerating germination of the detoxified seeds in the step (S3), accelerating germination in a dark incubator, picking out the germinated seeds for planting, and observing and counting mutagenesis effects; and (S4) sowing the seeds collected in the step (S4) in the whole growth period. The EMS mutagenesis is high in point mutation frequency and relatively less in chromosome distortion, and the method has the advantages that the operation is simple and convenient, the breeding period is short, and new genes and new characters which are difficult to generate by a traditional breeding method can be generated.

The invention provides an efficient double-base editing system without PAM (Pulse Amplitude Modulation) limitation. According to the technology, different point mutations are introduced into SpRY, the SpRY is fused with efficient adenine deaminase and cytosine deaminase to construct a double-base editor, and efficient PAM-limitation-free double-base editing is achieved in the rice protoplast. According to the plant basic group editing method, efficient and PAM-less double basic group editing can be achieved, and the plant basic group editing technology is wider in target range, higher in efficiency and higher in double basic group editing product proportion.

The invention relates to a detection kit for combined methylmalonemia, which is a cb1C type kit and comprises a specific primer pair and a probe combination for detecting at least one mutation site of MMACHC, the mutation site is selected from c.609Ggt; a is c.567 dupT, c.658660delAAG, c.482Ggt, and c.482Ggt; a, c.1Agt; g, c, 80Agt; g, c, 217Cgt; 315Cgt, T, c.315Cgt; g and c.394 Cgt; t). The MMACHC gene hotspot mutation region detection kit based on the real-time fluorescent quantitative PCR technology can detect multiple mutation sites on the MMACHC gene at the same time, detection of three genotypes of wild type, homozygous mutant type and heterozygous mutant type on each mutation site is completed, the gene detection requirements of clinical combined type MMA are effectively met, the detection time is shortened, and the detection efficiency is improved. The mutation site coverage rate of CblC defective methylmalonic acidmia combined with homocysteinemia on the MMACHC gene is increased to 93.28%, mutation hot spots of most people are covered, and various types of mutation conditions in hot spot mutation areas can be rapidly, accurately and sensitively detected.

The invention relates to a carbonyl reductase mutant based on cosubstrate catalytic ability optimization and application thereof, the mutant is constructed on the basis of a carbonyl reductase BaSDRX amino acid sequence as shown in SEQ ID No.2, the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.1 in a sequence table, and the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.2 in the sequence table. The mutant is single-point mutation or multi-point combined mutation on the basis of an amino acid sequence of carbonyl reductase BaSDRX as shown in SEQ ID No.2: threonine Thr133 at the 133 site, arginine Arg137 at the 137 site and glutamic acid Glu142 at the 142 site; when the mutant enzyme or a recombinant cell containing the mutant enzyme is applied to synthesis of (S)-1-(2, 6-difluorophenyl) ethanol through asymmetric reduction of 2, 6-difluoroacetophenone, high catalytic activity and high stereoselectivity are achieved, and high-optical-purity (S)-1-(2, 6-difluorophenyl) ethanol (eet; therefore, the method has a good industrial application and development prospect.

The invention discloses an acid protease mutant PepA4 with improved heat resistance and application thereof, and relates to the technical field of gene engineering and enzyme engineering. The amino acid sequence of the acid protease mutant PepA4 is obtained by carrying out D67A, E103D, E304K and S368L point mutation on an amino acid sequence as shown in SEQ ID NO: 1, and the amino acid sequence of the acid protease mutant PepA4 is as shown in SEQ ID NO: 2. The high-temperature-resistant acid protease disclosed by the invention can still maintain the enzyme activity of 80% or above at 75 DEG C for 5 minutes. The acid protease provided by the invention has better acid resistance, and can be widely applied to food processing, leather processing and feed processing industries.

The invention provides a primer probe set for digital PCR detection of non-small cell lung cancer EGFR mutation and application thereof, and relates to the technical field of digital PCR detection, the primer probe set comprises a primer pair and a probe for specific detection of EGFR gene exon 19 deletion mutation (19del) and exon 21 L858R point mutation, and the 19del mutation probe comprises a plurality of sequences to cover common subtypes. The primer probe group can be combined with a microfluidic digital PCR platform for use, a sample is divided into tens of thousands of microdroplets for amplification, and FAM, HEX, ROX and CY5 fluorescence channel signals are detected to realize absolute quantification. The system provided by the invention has high sensitivity and excellent linearity and precision, is particularly suitable for detecting extremely-low-frequency EGFR mutation in body fluid such as plasma, and has important application value in individualized diagnosis and treatment, curative effect monitoring and drug resistance evaluation of non-small cell lung cancer.

The invention discloses a preparation method of anthraquinone drugs, which comprises the following steps: by taking CCTCC NO: M 2017842 cercospora JNU001 as an original strain, knocking out a polyketide synthase CTB1 gene on the genome of the original strain, then carrying out single-point mutation on a BTG5 gene to obtain a cercospora JNU001 delta CTB1 / BTG5-T318C mutant strain, and fermenting the mutant strain to obtain six anthraquinone drugs. According to the present invention, the aureospora sp. Strain is modified through the gene editing strategy to produce the anthraquinone drug, the prepared anthraquinone drug has characteristics of various production types, simple separation and low cost, and the application of the anthraquinone drug is substantially expanded.

The invention discloses a self-assembled ferritin nano antigen particle and application thereof in preparation of a duck hepatitis A vaccine. The VP1 part in the duck hepatitis A virus P1 protein is connected with the N end of a self-assembled ferritin nanoparticle subunit through a connecting sequence to obtain the fusion protein, and the amino acid sequence of the fusion protein is shown as SEQ ID NO.1; in order to improve the expression quantity or expression efficiency of the fusion protein in bombyx mori, a nucleotide sequence of a coding gene of the fusion protein is optimized and modified according to the codon preference of bombyx mori, and an optimized gene sequence with the nucleotide sequence shown as SEQ ID NO.2 is obtained; the fusion protein is subjected to single-site or multi-site mutation, the mutant with obviously improved titer is obtained through screening, and the soluble expression quantity of the mutant is obviously improved. According to the invention, a silkworm or insect cell eukaryotic expression system is used for expressing recombinant protein, and an expression product causes a neutralizing anti-duck hepatitis A virus antibody.

The invention relates to an islet repair ICK mode oral polypeptide optimization system taking VDAC1 as a target based on ESM2 and a genetic algorithm, and the system comprises a mutation module which is used for carrying out mutation and recombination by adopting the genetic algorithm according to original protein sequence data to generate an initial population; the prediction module is used for respectively inputting the protein sequence data of the initial population into a protein dimer dissociation energy prediction model, a protein toxicity prediction model and a protein cell penetrability prediction model to obtain a dissociation energy prediction value delta G, a toxicity probability value T and a cell membrane passing transmission probability Ed; and the optimization module is used for acquiring the optimal individual performance of the highest fitness score to output the Pareto optimal sequence index, and recording the hotspot residue distribution and the site mutation frequency at the same time. On the basis of keeping the biological activity of the original sequence, the key drug properties are improved in a breakthrough manner.

The invention discloses a cellobiose 2-epimerase mutant as well as a gene, an expression vector, recombinant bacteria, a preparation method and application thereof. The mutant is obtained by performing the following point mutation on an amino acid sequence as shown in SEQ ID No.1: S12A, S24A, G131A, G176A, G224A, K46R, K69R, K72R, K91R, K103R, K201R, K219R, K223R, K241R, K263R, K266R, K273R, K284R, K325R, K335R, K349R and K387R. The invention further discloses a preparation method of the mutant. The invention further discloses a gene segment, a plasmid and a recombinant bacterium for recombinant expression of the mutant, and the gene segment, the plasmid and the recombinant bacterium are used for recombinant expression preparation of the mutant. The mutant can be used for continuously and stably catalyzing and preparing lactulose at high temperature, is relatively good in thermal stability and has a relatively good industrial application prospect.

The application discloses a method for transforming any polypeptide sequence into an antibacterial peptide, and belongs to the technical field of bioinformatics. The application develops a hypergraph neural network predictor for predicting the antibacterial activity score of a polypeptide, performs fine-tuning training on a pre-trained protein language model to capture antibacterial peptide sequence features, and trains a strategy network for selecting a to-be-mutated amino acid site in a polypeptide sequence. For any starting polypeptide sequence, the strategy network is first used to select a to-be-mutated amino acid site, then the fine-tuned protein language model is used to give the amino acid type after mutation of the site, and then the hypergraph neural network predictor is used to predict the antibacterial activity score of the sequence after mutation. The process is iteratively executed until an ideal antibacterial peptide sequence output is obtained. The method has the ability to transform any polypeptide sequence into an antibacterial peptide, and the antibacterial activity score of the transformed polypeptide is greatly improved, and can be applied to the actual development of antibacterial drugs, thereby laying a foundation for solving drug-resistant bacterial infections.

The invention discloses application of novel reverse transcriptase and related fusion protein of the novel reverse transcriptase in plant-guided editing. Candidate RT protein is obtained through systematic mining from a public database, novel reverse transcriptase cl1 with activity is screened out in combination with a fluorescence report system, and a guided editing system suitable for plants is constructed. The system tests in wheat protoplast, and verifies the editing activity of cl1 to a plurality of endogenous targets. Further combining with protein structure prediction and rational design point mutation optimization, the cl1 mutant with higher editing efficiency is obtained, and the method can be applied to accurate and effective editing of plant genomes and plant breeding and improvement.

The invention discloses a reverse transcriptase, wherein a coding sequence of the reverse transcriptase is derived from a Rattus norvegicus genome. The protein sequence of the protein comprises a protein sequence of a wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus, or a protein sequence which is subjected to engineering modification on the basis of the wild type Rattus norveicus. Furthermore, the protein sequence of the gene is as shown in any one of SEQ No.1-55. In a rice genome site and a human HEK 293T cell genome site, the system shows efficient editing efficiency of a guide editor. The invention provides a series of efficient reverse transcriptase components which can be carried on a guide editing system, important bottom-layer technical support is provided for research of point mutation in a genome, creation of a special site disease model and correction of genetic mutation sites, and the reverse transcriptase component has a wide application prospect in the field of precision medicine and precision agriculture breeding.