31 results about "Site specificity" patented technology

The present invention provides in vivo systems in which activity of a biological cleavage enzyme, such as a site-specific recombinase, a homing endonuclease, or an intein, is linked to cell viability and therefore can be selected. The invention further provides methods of making cells in which the activity of a biological cleavage enzyme is linked to viability, as well as methods of identifying new biological cleavage enzymes, including enzymes having altered site specificity, using such cells.

A binary plasma nanoparticle site specificity self-assembling method belongs to the technical field of material chemistry. The method comprises the following steps of: synthetising gold nanoparticles; synthetising gold nanorods; decorating DNA1 by using the gold nanoparticles; decorating DNA2 by using the gold nanorods; forming a DNA composite connecting arm; and assembling the gold nanoparticles and the gold nanorods. According to the invention, two important plasma nanoparticles, i.e. the gold nanoparticles and the gold nanorods are selected to prepare a discrete binary self-assembling body. The gold nanorods have space geometry and chemical anisotropy, so that the physical properties and the chemical properties of the gold nanorods in different space sites are different. Therefore, the technical method for controlling the space site specificity of binary gold nanomaterials is very important. The method provided by the invention has the advantage that a binary gold nanomaterial assembling body with uniform and controllable structure and sites as well as high yield is prepared.

A method for preparing hydro / organo gelators from disaccharide sugars by biocatalysis and their use in enzyme-triggered drug delivery. Controlled delivery of an anti-inflammatory, chemopreventive drug is achieved by an enzyme-triggered drug release mechanism via degradation of encapsulated hydrogels. The hydro- and organo-gelators are synthesized in high yields from renewable resources by using a regioselective enzyme catalysis and a known chemopreventive and anti-inflammatory drug, curcumin, is encapsulated in the gel matrix and released by enzyme triggered delivery. The release of the drug occurs at the physiological temperature and control of the drug release rate is achieved by manipulating the enzyme concentration and temperature. The by-products formed after the gel degradation clearly demonstrated the site specificity of degradation of the gelator by enzyme catalysis. The present invention has applications in developing cost effective, controlled drug delivery vehicles from renewable resources, with a potential impact on pharmaceutical research and molecular design and delivery strategies.

The invention relates to a multiplex ligation-dependent probe amplification kit for detecting hereditary deafness susceptibility gene mutation. The multiplex ligation-dependent probe amplification kit comprises a detection probe for detecting mutation site specificity, a universal primer, a positive DNA template, a DNA ligase, a connection buffer solution, a hybridization solution and a PCR reaction reagent, wherein the detection probe is composed of a pair of short probe and long probe and corresponds to one mutation site. The method is high in sensitivity and is accurate in result, and compared with the traditional detection technology, the kit can be used for completing high-throughput screening of multiple key hereditary deafness gene mutation diagnosis at one time, and has the advantages of high economical efficiency, high efficiency and high accuracy.

The present invention provides in vivo systems in which activity of a biological cleavage enzyme, such as a site-specific recombinase, a homing endonuclease, or an intein, is linked to cell viability and therefore can be selected. The invention further provides methods of making cells in which the activity of a biological cleavage enzyme is linked to viability, as well as methods of identifying new biological cleavage enzymes, including enzymes having altered site specificity, using such cells.

The invention discloses a programmable multi-site specificity transcription inhibition system and the application thereof. The programmable transcription inhibition system with the multi-site specificity comprises (A) and (B) as follows: (A) site specificity transcription inhibition protein FndCpf1 (D917A), or DNA or RNA capable of expressing the FndCpf1 (D917A); (B) programmable sgRNA which consists of 19 to 36nt of repetitive sequences and variable 16 to 31nt of spacer sequences which are alternately arrayed in sequence from the end 5' to the end 3' and targets multiple sites, or a DNA circular plasmid or a DNA linear fragment capable of transcribing the programmable sgRNA. According to the programmable multi-site specificity transcription inhibition system, a gene transcription regulation and control tool which is efficient, has site specificity and can target multiple sites at the same time is obtained.

A preparation process of bar code-type gene chip is to fix oligonucleotide segments with specific base sequence onto the surface of substrate in microarray mode. The forward primer upstream sequence with mononucleotide site specificity to be detected is made to complement and pair with bar code, the middle is the limited enzyme incising site, and the downstream sequence is made to pair with the target sequence. There is a biotin or a fluorescent group mark in the 5'-end, and a nucleotide incapable of pairing with target sequence is introduced to the place of 1-4 nucleotides apart from the 3'-end upstream through PCR, purification, enzyme incising and hybrid elution with the bar code gene chip. The bar code base sequence has composition independent on target nucleic acid to be detected and has detected segment length within 30 nucleotides, and this reduces the cross hybridizing and error hybridizing.

The invention relates to a method of determining the binding site specificity of an analyte to a ligand having at least two different binding sites, which method comprises immobilizing the ligand to a solid support, mixing the analyte with a reversibly binding reference analyte which has a dissociation behaviour that differs significantly from that of the analyte, contacting the mixture with the ligand to determine dissociation characteristics for the mixture, and determining therefrom the binding site specificity of the analyte in relation to the reference analyte. The invention also relates to an analytical system for carrying out the method, and to a computer program, computer program product and computer system for performing the method.

The invention provides a rapid and accurate fluorescent PCR based method for detecting 1494del6 polymorphism of a TYMS gene. Aiming at two alleles corresponding to 1494del6 polymorphism of the TYMS gene, the method respectively designs specific primers of the alleles, and an SYBR green technology is combined, so that after the fluorescent PCR, specific genotype of 1494del6 polymorphism locus of the sample can be accurately determined according to an amplification curve. The specific primers designed by the invention have the advanategs of high site specificity, high detection sensitivity and material and time saving.

A method of characterizing the protein O-GlcNAcylation site-specificity of an antibody. A method of detecting or quantitating the expression of site-specific O-GlcNAcylated proteins expressed in cells and biological samples. A method of diagnosing cancer in a host based on the cellular expression of site-specific O-GlcNAcylated proteins. A method of screening anti-cancer compounds according to their ability to increase a level O-GlcNAcylation of oncogene or tumor suppressor proteins. Methods of treating cancer in an animal host by administering compounds that increase a level of O-GlcNAcylated c-myc or p53 in cancer cells. A method of distinguishing subclasses of pancreatic cancer according to the sensitivity of pancreatic cancer cells to an imidazole derivative, and a method of personalized pancreatic cancer treatment delivered according to the sensitivity subclasses.

The invention discloses a recombinant S1 protein of a novel mutant strain of a porcine epidemic diarrhea virus and a subunit vaccine of the recombinant S1 protein. The protein is obtained by the steps of cloning a fusion gene fragment PE(delta III)-S1(m)-KDEL3 containing a KDEL3 gene sequence, a gene sequence of III-region-deleted pseudomonas aeruginosa exotoxin A and an S1(m) gene sequence to a baculovirus vector to obtain a recombinant vector; carrying out recombinant vector site specificity transposition on a seroconversion DH10Bac competent cell of the recombinant vector to obtain a recombinant bacmid; transfecting the recombinant bacmid to an Sf9 cell under the mediation of a liposome to obtain a recombinant Sf9 cell; expressing by using the recombinant Sf9 cell. According to the invention, a PE(delta III)-S1(m)-KDEL3 fusion protein is firstly expressed by using a baculovirus expression system on the basis of optimizing the predilection of a codon of mammalian with S1 genes, so that the recombinant baculovirus for efficiently and accurately expressing the fusion protein is obtained. Proved by IFA and Western blot, the recombinant baculovirus can be used for accurately expressing the fusion protein, and the fusion protein has favorable reactionogenicity.

The invention discloses a detection method based on a protein substrate and application thereof. The detection method includes following steps: (1) preparing the protein substrate having a recognition site of a to-be-detected protease; (2) linking a detection component onto the recognition site of the to-be-detected protease of the protein substrate to form a site-specificity-marked protein substrate; (3) performing interaction to the site-specificity-marked protein substrate with the to-be-detected protease; and (4) detecting the interaction of the site-specificity-marked protein substrate with the to-be-detected protease according to the change of a signal of a specificity mark. The method can be used for screening compounds to achieve high throughput screening of drugs, thereby increasing development of the drugs.

The invention relates to an Escherichia coli MG1655 bacterial strain lacking a sahn gene, a construction method and application. The construction method comprises the following steps that a linear DNA fragment of which the middle part is a selection marker gene and the two ends are homologous sequences is utilized, by means of the effect of a Red recombinase, a homologous recombination between the homologous sequences and a desired gene occurs, and thus a target gene is replaced with the marker gene; in order to eliminate the trace of the selection marker gene, special sites which can be recognized by a Cre site specificity recombinase are added on the both sides of the selection marker gene; and the selection marker gene is deleted through the effect of the site specificity recombinase, and the Escherichia coli MG1655 bacterial strain lacking the sahn gene, namely MG1655 delta sahn, is obtained. The invention further provides the application of the bacterial strain. The quorum sensing characteristic of the mutant strain delta sahn is utilized as a contrast, and a novel type inhibitor which is 1-((4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl)-5-(1H-1,2,3-triazo1-4-yl)piperidin-3-ol is screened, so that the growth and splitting ability of pathogenic bacterium is reduced.

The invention discloses a KASP molecular marker for detecting a rice blast resistance broad-spectrum gene Pil and application. The molecular marker comprises an SNP locus 1 and an SNP locus 2; the SNPlocus 1 is located at the base 30654770 of a rice chromosome 11, and the molecular marker polymorphism is A / T; the SNP locus 2 is located at the base 30655545 of the rice chromosome 11, and the molecular marker polymorphism is C / G. The molecular marker is high in Pi1 gene site specificity, an application method of the SNP molecular marker is convenient to implement, the detection result is accurate and reliable, the marker can be used for identification and assisted breeding of the rice Pi1 gene, and the problems that traditional breeding is low in efficiency and long in cycle can be solved.

The invention provides a method for synthesizing (nitroalkynyl)benzene compounds shown in formula II, comprising: adding phenylacetylene compounds shown in formula I as materials, along with a nitrifying agent, into an organic solvent, hermetically reacting at 20-50 DEG C, performing TLC (thin layer chromatography) tracing until the reaction is over, and post-treating the reacted liquid to obtain (nitroalkynyl)benzene compounds shown in the formula II. The nitrifying method of the invention has the advantage of good nitrifying site specificity, nitrifying is carried out only on alkynyl, no nitrification products are produced on benzene rings, the reaction process is good in safety, environment-friendly and good in substrate adaptability, and it is possible to provide alkynyl nitrifying for various substituents.

Methods and compositions are provided for altering the DNA recognition and cleavage characteristics of an endonuclease without prior knowledge of the endonuclease's three-dimensional structure and / or amino acid residues responsible for activity and / or specificity. Methods include subjecting a mutagenized endonuclease gene library to a genetic selection in prokaryotic cells which tolerate the expression of mutated endonuclease and where the endonuclease is active and determining the altered recognition-site specificity for the endonuclease.

The invention provides a quick human HLA-B*5801 genotype detection kit and method. The kit mainly comprises universal primers, a universal fluorescent probe, ligation probes, ligation reaction and polymerization reaction mixed liquid, enzyme mixed liquid, a negative control and a positive control. Single nucleotide polymorphic site specificity is controlled through novel heatproof ligase reaction,only nucleotides matched with the ligation probes complete ligation reaction and start next polymerase chain reaction, and HLA-B*5801 genotype can be detected through the universal florescent probe;if the nucleotides are not matched with the ligation probes, ligation cannot be completed, and reaction is terminated. All detection processes can be completed in a same reaction system, and high specificity and high sensitivity in HLA-B*5801 genotype detection are achieved as compared with those of a traditional method.

The invention discloses a method of utilizing a SSR label to identify a Ping'ou hybridized hazel species. An SSR label is combined with a capillary electrophoresis technology to establish a Ping'ou hybridized hazel species identification method based on four pairs of EST-SSR labels. For the first time, in domestic, the SSR amplification data of 43 Ping'ou hybridized hazel species (lines) widely planted in China is reported; a comparable data result is provided for the identification of unknown species; the provided method has the advantages of co-dominance, good repeatability, strong site specificity, and digitalization of amplification strips, and the method is universal, convenient and effective.

A method of characterizing the protein O-GlcNAcylation site-specificity of an antibody. A method of detecting or quantitating the expression of site-specific O-GlcNAcylated proteins expressed in cells and biological samples. A method of diagnosing cancer in a host based on the cellular expression of site-specific O-GlcNAcylated proteins. A method of screening anti-cancer compounds according to their ability to increase a level O-GlcNAcylation of oncogene or tumor suppressor proteins. Methods of treating cancer in an animal host by administering compounds that increase a level of O-GlcNAcylated c-myc or p53 in cancer cells. A method of distinguishing subclasses of pancreatic cancer according to the sensitivity of pancreatic cancer cells to an imidazole derivative, and a method of personalized pancreatic cancer treatment delivered according to the sensitivity subclasses.

The invention provides a rapid and accurate fluorescent PCR based method for detecting 1494del6 polymorphism of a TYMS gene. Aiming at two alleles corresponding to 1494del6 polymorphism of the TYMS gene, the method respectively designs specific primers of the alleles, and an SYBR green technology is combined, so that after the fluorescent PCR, specific genotype of 1494del6 polymorphism locus of the sample can be accurately determined according to an amplification curve. The specific primers designed by the invention have the advanategs of high site specificity, high detection sensitivity and material and time saving.

The invention discloses a recombinant S1 protein of a novel mutant strain of a porcine epidemic diarrhea virus and a subunit vaccine of the recombinant S1 protein. The protein is obtained by the steps of cloning a fusion gene fragment PE(delta III)-S1(m)-KDEL3 containing a KDEL3 gene sequence, a gene sequence of III-region-deleted pseudomonas aeruginosa exotoxin A and an S1(m) gene sequence to a baculovirus vector to obtain a recombinant vector; carrying out recombinant vector site specificity transposition on a seroconversion DH10Bac competent cell of the recombinant vector to obtain a recombinant bacmid; transfecting the recombinant bacmid to an Sf9 cell under the mediation of a liposome to obtain a recombinant Sf9 cell; expressing by using the recombinant Sf9 cell. According to the invention, a PE(delta III)-S1(m)-KDEL3 fusion protein is firstly expressed by using a baculovirus expression system on the basis of optimizing the predilection of a codon of mammalian with S1 genes, so that the recombinant baculovirus for efficiently and accurately expressing the fusion protein is obtained. Proved by IFA and Western blot, the recombinant baculovirus can be used for accurately expressing the fusion protein, and the fusion protein has favorable reactionogenicity.

A preparation process of bar code-type gene chip is to fix oligonucleotide segments with specific base sequence onto the surface of substrate in microarray mode. The forward primer upstream sequence with mononucleotide site specificity to be detected is made to complement and pair with bar code, the middle is the limited enzyme incising site, and the downstream sequence is made to pair with the target sequence. There is a biotin or a fluorescent group mark in the 5'-end, and a nucleotide incapable of pairing with target sequence is introduced to the place of 1-4 nucleotides apart from the 3'-end upstream through PCR, purification, enzyme incising and hybrid elution with the bar code gene chip. The bar code base sequence has composition independent on target nucleic acid to be detected and has detected segment length within 30 nucleotides, and this reduces the cross hybridizing and error hybridizing.

The invention belongs to the technical field of molecular biology, and particularly relates to a novel in vitro screening method of ribozyme and ribozyme obtained through screening by the method and capable of catalyzing RNA shearing activity. According to the in vitro screening method, toxoprotein ibsC is used as a report gene, ribozyme obtained through in vitro transcription controls the expression of toxicity protein on the shearing effect of mRNA corresponding to the ibsC so as to influence cell survival, and therefore, active ribozyme is obtained through screening. According to the method, a complicated in vitro screening step is not needed, so that influence of an inactive RNA sequence on the screening success rate can be reduced to the largest extent, and a simple efficient common intra-cellular screening platform is provided for ribozyme evolution. The effect manner of the ribozyme obtained through screening with a substrate is mainly an intermolecular shearing manner, and is suitable for catalysis and shearing of site specificity of target substrate molecules outside cells, in prokaryotic cells and in eukaryotic cells, and expression reduction on selected genes is realized.

The invention discloses an FLP-containing pBBR1MCS-2 recombinant plasmid and a method for modifying a zymomonas mobilis genome DNA. The plasmid is constructed by the step of: inserting a segment Pgap-FLP into pBBR1MCS-2 through C1aI and XbaI sites to obtain pBBR1MCS-2-Pgap-FLP, wherein the Pgap-FLP is a nucleotide sequence shown in SEQ ID NO:1 in a sequence table. The plasmid can be conveniently transferred in an electroporation manner to enter zymomonas mobilis, and can be conveniently removed. By combining with an FRT-FLP site specificity recombinant system from saccharomyces cerevisiae, the invention provides a rapid, efficient, convenient and stable method for modifying zymomonas mobilis genome DNA. The invention provides a method for modifying DNAs at multiple sites of the zymomonas mobilis genome by circularly utilizing a selective marker gene.

The invention belongs to the genetic engineering field, especially to a site-specific recombinase, a preparation method thereof and applications. A phi C31 site-specific recombinase is an effective tool for mammalian cells genetic operation, but its mediated integration site specificity and integration efficiency on mammalian cells await further improvement. Thus the invention provides a phi C13 site-specific recombinase mutant capable of identifying a genome MpsL1 site with specificity and having higher activity. Thereby the site-specific recombinase of the invention can be an effective tool for the genetic engineering operation, especially the gene fixed-point insertion and recombination, and open up a novel path for researching the gene function analysis and the gene therapy.

Methods and compositions are provided for altering the DNA recognition and cleavage characteristics of an endonuclease without prior knowledge of the endonuclease's three-dimensional structure and / or amino acid residues responsible for activity and / or specificity. Methods include subjecting a mutagenized endonuclease gene library to a genetic selection in prokaryotic cells which tolerate the expression of mutated endonuclease and where the endonuclease is active and determining the altered recognition-site specificity for the endonuclease.

The invention provides a method for synthesizing (nitroalkynyl)benzene compounds shown in formula II, comprising: adding phenylacetylene compounds shown in formula I as materials, along with a nitrifying agent, into an organic solvent, hermetically reacting at 20-50 DEG C, performing TLC (thin layer chromatography) tracing until the reaction is over, and post-treating the reacted liquid to obtain (nitroalkynyl)benzene compounds shown in the formula II. The nitrifying method of the invention has the advantage of good nitrifying site specificity, nitrifying is carried out only on alkynyl, no nitrification products are produced on benzene rings, the reaction process is good in safety, environment-friendly and good in substrate adaptability, and it is possible to provide alkynyl nitrifying for various substituents.

The invention belongs to the genetic engineering field, especially to a site-specific recombinase, a preparation method thereof and applications. A phi C31 site-specific recombinase is an effective tool for mammalian cells genetic operation, but its mediated integration site specificity and integration efficiency on mammalian cells await further improvement. Thus the invention provides a phi C13 site-specific recombinase mutant capable of identifying a genome MpsL1 site with specificity and having higher activity. Thereby the site-specific recombinase of the invention can be an effective toolfor the genetic engineering operation, especially the gene fixed-point insertion and recombination, and open up a novel path for researching the gene function analysis and the gene therapy.

The invention relates to an Escherichia coli MG1655 bacterial strain lacking a sahn gene, a construction method and application. The construction method comprises the following steps that a linear DNA fragment of which the middle part is a selection marker gene and the two ends are homologous sequences is utilized, by means of the effect of a Red recombinase, a homologous recombination between the homologous sequences and a desired gene occurs, and thus a target gene is replaced with the marker gene; in order to eliminate the trace of the selection marker gene, special sites which can be recognized by a Cre site specificity recombinase are added on the both sides of the selection marker gene; and the selection marker gene is deleted through the effect of the site specificity recombinase, and the Escherichia coli MG1655 bacterial strain lacking the sahn gene, namely MG1655 delta sahn, is obtained. The invention further provides the application of the bacterial strain. The quorum sensing characteristic of the mutant strain delta sahn is utilized as a contrast, and a novel type inhibitor which is 1-((4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methyl)-5-(1H-1,2,3-triazo1-4-yl)piperidin-3-ol is screened, so that the growth and splitting ability of pathogenic bacterium is reduced.