165 results about "Restriction enzyme" patented technology

The invention provides a high-stability recombinant XVII type collagen as well as a construction method and application thereof, and belongs to the technical field of bioengineering. According to the invention, an amino acid sequence of natural human (XVII) type collagen is selected as a basic unit and repeated for multiple times to obtain recombinant (XVII) type collagen; in the design process of the recombinant XVII type collagen, an unstable GXY triad is avoided, potential matrix metalloproteinase restriction enzyme cutting sites and glycosylation sites are abandoned, a KGD active sequence is reserved, and the probability of containing sites which are easy to degrade can be reduced; a sequence fragment of the recombinant XVII type collagen is derived from a full-length sequence of a natural collagen gene, has excellent solubility, biological activity and high stability, and can be kept stable in a high-temperature storage environment and under the condition of avoiding alkalinity; the product can be used as a raw material in the production process of medicines, medical equipment, biological materials or cosmetics, and has good practicability.

The invention discloses a cathepsin B activated fluorescent probe which comprises a dicyanoisosulfone fluorescent report unit and a cathepsin B specific restriction enzyme cutting site GFLG peptide chain. When CTSB specifically cuts a GFLG peptide chain, a DCO molecule with an amino group with relatively strong electron donating ability is generated, so that the fluorescence emission behavior of the probe is rapidly activated through a one-step enzyme catalysis reaction. The invention also provides a preparation method of the cathepsin B activated fluorescent probe. The invention also provides a nano particle for packaging the cathepsin B activated fluorescent probe, and the nano particle is simple in preparation process and mild in reaction condition. The cathepsin B activated fluorescent probe is prepared into nanoparticles, and the nanoparticles can realize accumulation of the material in tumor tissues and accurate imaging in apoptotic tumor cells by enhancing the permeation retention effect, have better biocompatibility and can be used for detecting the apoptotic tumor cells.

The invention belongs to the technical field of yeast protein processing, and particularly relates to a method for improving the gas-liquid interface property of yeast protein. The method comprises the following steps: dispersing yeast protein in water to obtain a yeast protein dispersion liquid; adjusting the temperature and the pH value of the yeast protein dispersion liquid, stabilizing for a period of time, then carrying out restriction enzyme hydrolysis, and centrifuging after enzyme deactivation to obtain supernate. The surface tension of the yeast protein obtained by the method is reduced, the foaming ability and the foam stability are remarkably improved, and bubbles generated by whipping are small and uniform. And the method is green and efficient, and is beneficial to full utilization of high-quality protein resources.

The invention provides a preparation method, application and the like of large intestine expression recombinant A-type botulinum toxin (BONT / A). The recombinant BONT / A nucleic acid expression cassette comprises a tag, a restriction enzyme cutting site and a BONT / A nucleic acid sequence, and an exogenous protease recognition site is not introduced between a BONT / A light chain and a BONT / A heavy chain. The invention also provides a recombinant vector containing the nucleic acid expression cassette, a recombinant bacterium, a coded and expressed protein and the like, the prepared recombinant BONT / A does not introduce exogenous amino acid or only introduces two amino acids at one position, and the consistency of the recombinant BONT / A with the natural A-type botulinum toxin is ensured to the greatest extent. Through novel molecular design and preparation process, the recombinant reBONT / A with higher toxicity and low immunogenicity is efficiently obtained, and wide industrial application is facilitated.

The invention belongs to the field of molecular biological detection, and particularly relates to an FBXO32 gene SNP (Single Nucleotide Polymorphism) marker for rapidly detecting economic traits of Tibetan chicken as well as a detection method and application. It is found for the first time that G / A mutation at the 8916th site of the Tibetan chicken FBXO32 gene is closely related to whether the gene has an Hpa II restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention belongs to the field of molecular biological detection, and particularly relates to a PLS3 gene SNP marker for rapidly detecting economic traits of Tibetan chicken, a detection method and application. It is found for the first time that G / A mutation at the 28469th site of the Tibetan chicken PLS3 gene is closely related to whether the gene has a BstZ17 I restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention relates to the technical field of aquaculture fish vaccines, in particular to a fusion gene of grass carp NPM1a and II type reovirus VP38, recombinant protein, a composition and application. The nucleotide sequence of the fusion gene is as shown in SEQ ID NO: 1. The fusion gene is obtained by connecting grass carp NPM1a and II type reovirus VP38 through a linker sequence, a BamH restriction enzyme cutting site is added to the 5'end of the fusion sequence, and an XhoI restriction enzyme cutting site is added to the 3 'end of the fusion sequence; the sequence of the linker is as shown in SEQ ID NO: 2. The subunit vaccine of the fusion protein of the grass carp NPM1a and the II type reovirus VP38 is prepared, and indole-3-lactic acid is used as an immunopotentiator in a combined manner, so that the technical bottlenecks of low protection rate, poor intestinal mucosal barrier repairability and the like of the existing vaccine are overcome.

The invention belongs to the technical field of biology, and discloses a wheat bionic peptide and a biosynthesis method and application thereof, and the method comprises the following steps: carrying out amino acid sequence analysis on a protein sequence of a target wheat peptide, and determining a target nucleotide sequence corresponding to a coded same protein sequence according to codon degeneracy; optimizing the target nucleotide sequence according to password preference of escherichia coli to obtain a wheat bionic peptide nucleotide sequence; treating and combining the obtained wheat bionic peptide nucleotide sequence and a vector plasmid through restriction enzyme to construct a recombinant plasmid; transferring the recombinant plasmid into host bacteria, performing fermentation culture to induce protein expression, separating protein, and purifying to obtain the wheat biomimetic peptide. The wheat biomimetic peptide is obtained in a biosynthesis mode, and compared with plant extraction and chemical synthesis, the cost is reduced, and meanwhile, the safety of the product is guaranteed.

The invention provides high-stability recombinant I-type collagen and application thereof, and belongs to the technical field of collagen. The invention aims to avoid potential restriction enzyme cutting sites and unstable sequence fragments during sequence design through directional selection of the existing amino acid sequence, so that the amino acid sequence can be efficiently expressed in pichia pastoris, and the recombinant I-type collagen obtained after purification has very high stability; the recombinant I-type collagen gives consideration to factors such as related biological active sites, hydrophilicity and the like, has related biological activity while being relatively high in stability, and has very good practicability.

The invention discloses a method for simultaneously determining gene point mutation and DNA methylation based on a CRISPR / Cas12a system. The method is characterized by comprising four steps of enzyme digestion, recognition, conversion and detection, wherein in the enzyme digestion step, methylation-sensitive restriction endonuclease (MSRE) is selected according to a target site sequence, unmethylated DNA is not hydrolyzed by MSRE and is kept complete, methylated DNA is hydrolyzed and broken under the action of MSRE, and the broken 3'end can be used as a primer P1 to trigger SDA1 amplification; wherein in the recognition step, a Flap primer and an Invading primer are designed according to a point mutation sequence, and an enzyme digestion product generated under the action of FEN1 enzyme can be used as a primer P2 to trigger SDA2 amplification; wherein in the conversion step, two different SDA amplification templates are designed according to sequences of P1 and P2, and amplification of SDA1 and SDA2 is triggered by enzyme digestion products P1 and P2 respectively, so that DNA methylation and gene mutation information are quickly converted into two different amplification products T1 and T2; wherein in the detection step, crRNA1 and crRNA2 are respectively designed as guide chains of a CRISPR / Cas12a reaction system according to sequences of T1 and T2, and synchronous detection of gene point mutation and DNA methylation is realized through'strong 'and'weak' of fluorescence signals. Based on a CRISPR / Cas12a system, the method solves the problem that a traditional detection method cannot realize simultaneous detection of gene point mutation and DNA methylation, can be used for scientific research analysis and clinical detection, and has a wide market prospect.

The invention discloses a micro-ring plasmid synthesis method, and belongs to the technical field of gene editing. Two pairs of primers are designed, eukaryotic expression plasmids containing selection markers and target fragments are used as templates for PCR amplification to obtain two target fragments, vector plasmids loxp-R6K with specific sequences and the target fragments form recombinant plasmids, the recombinant plasmids contain sequences such as the target fragments, restriction enzyme cutting sites and R6K replication start sites, and the recombinant plasmids are used for preparing the recombinant plasmids. The recombinant plasmid is transformed in Escherichia coli GT115 competent cells, the recombinant plasmid is verified to have the recombination efficiency of 81% after bacterial colony PCR amplification, a correctly cloned seed solution is screened and subjected to induced expression to form a bacterial solution containing a screening-label-free micro-plasmid, the micro-ring plasmid is separated and purified, and the screening-label-free micro-ring plasmid is obtained. The sequence of the microring plasmid only contains a target fragment and an R6K replication start site, the accuracy of the size sequence of the microring plasmid in a bacterial colony cultured and cloned by the microring plasmid is as high as 90%, and the microring plasmid can be efficiently and stably obtained.

The invention discloses a matrix metalloproteinase-responsive multifunctional bionic ferritin nano material as well as a preparation method and application thereof. The method comprises the following steps: modifying melittin on the surface of a ferritin nanoparticle through a genetic engineering method, connecting by using a linker containing a matrix metalloproteinase 9 (MMP-2 / 9) restriction enzyme cutting site, expressing the obtained recombinant ferritin (SEQ ID NO: 6) in escherichia coli, and self-assembling to form the enzyme response functionalized bionic nanoparticle. According to the method, melittin can be connected to ferritin nanoparticles in series, the toxicity of melittin is remarkably reduced, and the melittin responds to matrix metalloproteinase in a tumor microenvironment so as to kill tumor cells; meanwhile, intravenous administration of melittin can be realized, and the melittin injection has a good treatment effect on in-situ and metastatic tumors.

The invention relates to the technical field of agriculture, and particularly provides a CAPS molecular marker of a rice salt-tolerant ST4 promoter region and application of the CAPS molecular marker. The method comprises the following steps: selecting leaves of rice to extract genome DNA (Deoxyribonucleic Acid) of the tissues, and then respectively carrying out PCR (Polymerase Chain Reaction) amplification by taking the DNA of the tissues as a template and taking corresponding sequences of ST4-PRO-F1 and ST4-PRO-R1 as specific primers to obtain amplified fragments; carrying out enzyme digestion on the amplified fragment by using Eco81I restriction enzyme, and carrying out electrophoresis after enzyme digestion; after the ST4 promoter fragment is subjected to electrophoresis, a single plant with only one 2162bp main band is a salt-tolerant single plant, and a single plant with 1366bp and 784bp bands appearing in electrophoresis is a salt-sensitive single plant. According to the invention, the rice variety improved by utilizing the wild rice ST4 gene can be rapidly bred, the current situations of much field work and large workload caused by carrying out a large amount of salt tolerance identification by utilizing a population material can be improved, and the method has important theoretical and practical significance for breeding a new variety of rice with salt tolerance character.

The invention relates to a method for detecting deletion in ITR to be detected contained in an AAV vector to be detected. The method comprises the following steps: cutting a fragment containing a sequence of the ITR to be detected from the AAV vector to be detected by using restriction enzyme, detecting the size of the fragment of an enzyme digestion product, and determining whether deletion exists in the ITR to be detected according to the size of the fragment.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The invention relates to the technical field of molecular biology, in particular to a methylation marker for diagnosis of diabetic nephropathy. Specifically, the methylation marker comprises a P site of a CAT gene promoter region and / or an E site of a CAT gene exon 1 region. Based on a detection system combining methylation sensitive restriction enzyme with quantitative PCR, the kit has the advantages of simplicity and convenience in operation, good repeatability, accuracy in quantification and easiness in standardization, and is suitable for low-invasive samples such as peripheral blood. By effectively distinguishing pure type 2 diabetes patients from diabetic nephropathy combined patients, a reliable technical means is provided for risk stratification, early intervention and dynamic monitoring of diabetic people, and the method has good clinical application prospects and popularization value.

Methods for the processing and analysis of blood samples obtained with blood collection tubes that reduce contamination of cfDNA by genomic DNA but that inhibit digestion by methylation-sensitive and / or methylation-dependent restriction enzymes.

The present disclosure describes methods, kits, and systems for digesting polyribonucleotides. The methods involve selectively forming an oligonucleotide (e.g., DNA: RNA or RNA: RNA) duplex with a single-stranded target RNA, and then selectively cleaving the target RNA into smaller fragments using a sequence-specific nuclease that acts only on the RNA within the duplex. Additional sequence-specific ribonucleases can be used to provide additional cleavage of the target RNA at a predetermined site. By forming double strands to increase the availability of nuclease enzymes that can be used to cleave the single-stranded target RNA and to selectively control the location at which the target RNA is cleaved, the target RNA can be digested into fragments within a controllable size range that are optimal for polynucleotide analysis, such as by liquid chromatography and mass spectrometry.

The invention discloses a dCAPS molecular marker primer linked with a brassica napus saline-alkaline tolerance gene BnaC03.RBGB3 and application of the dCAPS molecular marker primer. The primer comprises a forward primer and a reverse primer, and through PCR amplification and specific restriction enzyme digestion treatment, two genotypes of high resistance and high sensitivity of brassica napus saline-alkaline tolerance can be distinguished on agarose gel electrophoresis; the high-resistance genotype is represented by two electrophoretic bands (195bp and 23bp), and the high-sensitivity genotype is a band which cannot be digested by enzyme. The method disclosed by the invention is simple and convenient to operate and reliable in result, can be used for rapidly screening and identifying the saline-alkaline-tolerant variety of the brassica napus, and provides a powerful tool for breeding the saline-alkaline-tolerant variety of the brassica napus. By implementing the technology, not only is the breeding efficiency improved, but also a new thought and strategy are provided for molecular marker-assisted breeding of saline-alkaline tolerance of crops, and development and application of a saline-alkaline tolerance breeding technology of brassica napus and even other crops are promoted.

The invention discloses a dCAPS molecular marker for identifying sweet potato pulp color characters and application of the dCAPS molecular marker, and belongs to the technical field of sweet potato breeding. According to the dCAPS molecular marker provided by the invention, on the basis of SNP variation sites, mismatched bases are introduced to form restriction endonuclease restriction enzyme cutting sites, the sweet potato pulp color can be judged by utilizing PCR-restriction enzyme cutting-polyacrylamide gel electrophoretic separation, and then sweet potato varieties with high and low carotenoid contents can be identified according to the sweet potato pulp color. The method has important guiding significance and application value for assisting sweet potato carotenoid content related molecular breeding research.

The present disclosure provides a method for detecting the methylation and mutation status of a DNA sample, specifically, a method for simultaneously detecting the methylation and mutation status of DNA molecules in a sample, the method comprising treating the sample with a methylation-sensitive restriction enzyme, performing hybrid capture on amplification products of the restriction enzyme-treated sample and a sample that has not been subjected to enzyme cleavage treatment with a probe set including a mutation capture probe and a methylation capture probe, and sequencing the captured products.

[Problem] To provide: an adapter for binding to polynucleotide fragments that is used for preparing a genome-wide gRNA library for genome editing systems; a gRNA library that is obtained by using the adapter; and uses of the gRNA library. [Solution] An adapter having a structure described in A). A) A structure having i) to iii). i) A polynucleotide chain having sequences i)-1 to i)-3. i)-1: Sequence corresponding to the forward primer. i)-2: Sequence of the recognition site of a restriction enzyme capable of excising a gRNA spacer sequence from the 5' end of a polynucleotide fragment bound to the adapter. i)-3: Sequence of the recognition site of a restriction enzyme capable of cleaving an adapter having the structure of A) from the polynucleotide fragment. ii) A polynucleotide chain having the sequence complementary to i). iii) A single-stranded polynucleotide chain for formation of a loop connecting the 5' end of i) and the 3' end of ii) of the double strand resulting from the binding of i) and ii).

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The invention relates to the technical field of biological pharmacy, and discloses a messenger ribonucleic acid sequence design method and device, computing equipment and a storage medium. According to the method, on the basis that a given 5 'UTR sequence serves as initial input, new codons are added one by one through a growth method, the overall-length minimum free energy and codon use efficiency of the whole sequence are calculated, scored and sorted, an elimination method is used for excluding sequences containing restriction enzyme cutting sites / poor properties / poorer scores, and finally, the sequences containing the restriction enzyme cutting sites / poor properties / poorer scores are obtained. And selecting representative sequences by using a cluster clustering search method to increase diversity, and finally obtaining the sequences for recommendation synthesis. The invention provides an mRNA sequence optimization design method in an environment with 5 'UTR and 3' UTR, the full-length minimum free energy of an mRNA sequence and the codon use efficiency of a CDS region are considered, the two targets can be optimized at the same time, the stability can be optimized from the perspective of overall molecules, the expressibility of the CDS region is optimized at the same time, and the method has the advantages of being simple in structure, convenient to operate and the like. And the stability and expressibility of the mRNA sequence are ensured.

The invention provides a method and a device for simulating a pulsed field gel electrophoresis band based on a sequencing technology. The method for simulating the pulsed field gel electrophoresis band based on the sequencing technology comprises the following steps: acquiring nanopore sequencing data of a biological sample to be analyzed, and assembling based on the nanopore sequencing data to obtain a genome sequence; according to the sequence characteristics of the restriction endonuclease, identifying a restriction enzyme cutting site corresponding to the restriction endonuclease from the genome sequence; when the restriction enzyme cutting site is the restriction enzyme cutting site subjected to methylation modification, removing the restriction enzyme cutting site to obtain a residual restriction enzyme cutting site; calculating the distance difference between two adjacent restriction enzyme cutting sites according to all residual restriction enzyme cutting sites, and taking the distance difference as the molecular weight of a simulated band; aiming at the molecular weight of each simulated strip, determining a strip position corresponding to the molecular weight; and drawing a simulated pulse field gel electrophoresis band result according to the position of each band and the molecular weight corresponding to the band position so as to accurately simulate the band result.

The present disclosure provides methods and compositions for preparing DNA molecules useful for determining the methylation status of selected regions of a DNA molecule, which utilize methylation-sensitive restriction enzymes and typically involve the amplification or selective enrichment of one or more target regions.

The invention discloses a high-efficiency expression method of porcine interleukin 22 (pIL-22) and application of the pIL-22 in resisting PEDV (porcine epidemic diarrhea virus) infection, and belongs to the technical field of biology. The high-efficiency expression method comprises the following steps: inserting a swine-derived interleukin 22 gene (SEQ ID NO: 1) into a pcDNA3.1 (+) vector restriction enzyme cutting site BamH I / Xho I to obtain a recombinant vector, mixing the recombinant vector and a transfection reagent, transfecting HEK-293 cells, collecting a cell suspension, ultrasonically crushing, filtering with a filter membrane to obtain a target protein, and finally purifying with a Protein A chromatographic column to obtain the swine-derived interleukin 22. (1) by adopting a mammalian cell expression system and a culture mode of suspension cells, not only can high-activity pIL-22 be expressed, but also the expression efficiency of the pIL-22 is greatly improved, and important guarantee and reference basis are provided for developing novel anti-PEDV drugs;

The invention belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to a preparation method of Jingzhaotoxin polypeptide IX (JZTX-IX) and application of Jingzhaotoxin polypeptide IX in treatment of diabetes mellitus. A chilobrachys jingzhao toxin polypeptide IX coding gene, a 6 * His tag nucleotide sequence, a thrombin restriction enzyme cutting site nucleotide sequence and an MBP hydrotropy tag nucleotide sequence are connected to a prokaryotic expression vector for prokaryotic expression, and the expressed polypeptide is purified to obtain the chilobrachys jingzhao toxin polypeptide IX protein. The target polypeptide with high purity, complete disulfide bond folding and uniform conformation is obtained, so that the high purity and biological activity of the target polypeptide are ensured. On the basis, the treatment activity of the JZTX-IX on diabetes mellitus especially type II diabetes mellitus is found and verified, and the JZTX-IX can remarkably reduce fasting blood glucose of mice with the type II diabetes mellitus, improve glucose tolerance and insulin sensitivity, relieve polydipsia and polyuria symptoms and promote insulin secretion and islet cell proliferation.

The invention discloses a high-efficiency expression and purification method of 5 '-3' exonuclease, which comprises the following steps: based on a Bac-to-Bac system, adding a tag and a TEV restriction enzyme cutting site to optimized 5 '-3' exonuclease Xrn1, then connecting the 5 '-3' exonuclease Xrn1 to a multiple cloning site behind a Polyhedrin promoter of a pFastBac expression vector to prepare expression baculovirus plasmids, and carrying out purification on the expression baculovirus plasmids to obtain the 5 '-3' exonuclease. Then transfecting the recombinant baculovirus particles into insect cells to generate recombinant baculovirus particles; finally, the 5 '-3' exonuclease Xrn1 is obtained through nickel column affinity chromatography, Heparin affinity chromatography and gel filtration chromatography purification, the invention provides a more economical and practical technical route for synthesizing the 5 '-3' exonuclease Xrn1, and a technical foundation is laid for large-scale production of the 5 '-3' exonuclease.

To provide a preparation method of a unit DNA composition in which the mole number of a plurality of unit DNA are arrayed better, and a manufacturing method of a DNA connected body.SOLUTION: A preparation method of a unit DNA composition has: a process of preparing solution including a plurality of unit DNA, to which an additional sequence is connected, for each kind of the unit DNA; a process of, after preparation of each solution, measuring the concentration of the unit DNA in each solution in the state where an additional sequence is connected to the unit DNA, dispensing each solution on the basis of its result, and making the mole numbers of the unit DNA in each solution become closer to the same with each other. A manufacturing method of a DNA connected body has: a process of preparing a unit DNA composition; a process of preparing a vector DNA; a process of removing each additional sequence from the unit DNA in which an additional sequence in solution after preparation is connected by using restriction enzyme; and a process of connecting the vector DNA and each unit DNA with each other after the removal process.SELECTED DRAWING: None