31 results about "Site specificity" patented technology

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.

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.

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 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.

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.