54 results about "Gene splicing" patented technology

The invention discloses a recombinant protein for diagnosing bovine tuberculosis, which is obtained by a method comprising the following steps: extracting the genome DNA of mycobacterium tuberculosisvar. bovis as a template of a PCR reaction to amplify out CFP10 and ESAT6 genetic fragments; using a gene splicing technique to amplify out a target gene CFP10-ESAT6 fusion gene; cloning and transforming the target gene to obtain a recombinant fungus, and inducing and expressing the recombinant fungus according to a conventional method, and purifying an expression product to obtain the recombinantprotein. The recombinant protein and a diagnostic reagent provided by the invention are used for diagnosing the bovine tuberculosis to obtain the advantages of low cost, high speed, high sensitivityand specificity.

The invention relates to an ELISA diagnosis kit for swine fever virus. In the invention, by gene splicing technology, the swine fever virus E2 is jointed with secretion signal peptide sequence and purification label at two sides and is inserted into the autographa californica nucleopolyhedrosis virus expression vector so as to construct recombinant autographa californica nucleopolyhedrosis virus CGMCC No.2671 and to obtain coated ELISA swine fever virus Shimen Strain E2 gene soluble expression antigen. The kit of the invention is rapid, sensitive, and specific to the diagnosis and the immune evaluation of swine fever virus, and the diagnosis can be completed once within 2 hours.

The invention discloses a construction method of recombinant Yarrowia lipolytica for synthesis of xylitol and a strain thereof. Yarrowia lipolytica is used as a synthetic chassis. Through the improvement means of metabolic engineering, the yeast undergoes gene splicing, genes for synthesizing xylitol from glucose, fructose, glycerol and starch as carbon sources are introduced, and metabolic pathways of synthesis by-products are blocked, so that recombinant Yarrowia lipolytica can synthesize xylitol from glucose, fructose, glycerol and starch as carbon sources by fermentation, and an engineering strain for synthesizing xylitol from carbon sources such as glucose and the like is obtained. After fermentation, xylitol crystals are obtained by separation of a bacterial liquid, ion exchange of aclarified fermentation liquid, decolorization, concentration, crystallization and other processes. The construction method of the engineering yeast for synthesizing xylitol from carbon sources such as glucose and the Yarrowia lipolytica strain obtained by the method and for synthesizing xylitol from carbon sources such as glucose can simplify existing methods for chemically synthesizing xylitol and have better application value.

The invention relates to the technical field of gene engineering, in particular to novel broad-spectrum chimeric lysin BGS-PlySb and an encoding gene and application thereof. The novel broad-spectrum chimeric lysin BGS-PlySb has an amino acid sequence shown as in SEQ ID NO. 1; the encoding gene of the novel broad-spectrum chimeric lysin BGS-PlySb has a nucleotide sequence shown as in SEQ ID NO. 2. A novel chimeric lysin is constructed by means of gene splicing and is suitable for killing various species of Streptococcus and Staphylococcus, particularly various species of Enterococcus, and Staphylococcus aureus, as well as Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus mutans, Streptococcus iniae and other species; the novel broad-spectrum chimeric lysin BGS-PlySb has good stability and is insensitive to high-concentration NaCl, recombinant protein GBS-PlySb is well expressible in Escherichia coli BL21 (DE3), and a high dose of GBS-PlySb is free of significant cytotoxicity. Therefore, the novel broad-spectrum chimeric lysin BGS-PlySb is applicable independently to or as an additive to the control of various species of Streptococcus and the treatment of infections caused by these species, and has a promising application prospect.

Disclosed are systems and methods for simultaneous detection of DNA and RNA genetic alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient without physically separating RNA from DNA. The systems and methods are similarly applicable to the simultaneous detection of DNA and RNA genetic alterations in solid tissues comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof. The present method utilized a barcoding method for analysis. The streamlined methods improve the simplicity, quantification accuracy and detection sensitivity and specificity of non-invasive detections of biomarkers.

Disclosed are systems and methods for detecting genetic alterations comprising androgen receptor gene splice variants (AR-Vs), mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient. The systems and methods are similarly applicable to the detection of gene alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof of other genes of interest. The streamlined methods improve the consistency and simplicity of non-invasive detections of biomarkers.

The invention discloses a method for excavating a low-abundance expression gene and system identification splicing variants by using a reverse transcription technique combining nested PCR (Polymerase Chain Reaction) with gene-specific primers (GSP), and application thereof. The method is based on reverse transcription PCR of 3'GSP1, gradient PCR is utilized to search for the best annealing temperature of each gene for each primer pair combination, and whether it is a new splicing variant is judged on the basis of GSP nested PCR sequencing and sequence analysis. The method is simple and easy to implement, has the advantages of favorable repetitiveness, high stability and strong reliability, greatly saves the experimental cost, can be applied to excavation of eucaryote low-abundance expression gene under different physiopathological states, and identification and detection of high/low-abundance expression gene splicing variants, and can be used for excavating low-abundance expression gene and identifying and detecting splicing variants of high expression gene and low-abundance expression gene. The method can be used for detecting both known splicing variants of targeted genes and unknown splicing variants of genes, and has wide application prospects.

This invention discloses a lysin that can kill many species of Streptococci. A new lysin, ClyR, was constructed by the gene splicing method. The ClyR can effectively kill different species of Streptococci, including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus mutans, Streptococcus equi, and various Enterococci and Staphylococcus aureus. ClyR shows good stability and is not sensitive to EDTA and high concentration of NaCl. Moreover, the ClyR is active in a wide range of pH and maintains high activity in pH 5-11. Recombinant protein ClyR is well expressed in E. coli stain BL21 (DE3). High doses of ClyR showed no apparent toxicity in mice. Furthermore, administration of 0.8 mg per mouse once is able to completely protect the mouse infected with lethal doses of Group B Streptococci. The ClyR can be used alone or in combination with different forms of reagents and solutions, for the control of a variety of Streptococci and for the treatment of infections caused by these bacteria. It has a broad application prospect.

The invention relates to a preparation method of an artificial antibacterial peptide PR39-R1T and application thereof. Amino acid sequences of the artificial antibacterial peptide PR39-R1T are designed artificially, and escherichia coli preference codons are adopted for designing and coding the amino acid sequences of the artificial antibacterial peptide; an overlap region amplification gene splicing method is adopted for synthesizing target genes to construct a prokaryotic expression vector, and the prokaryotic expression vector is converted into escherichia coli BL21 or Rosetta for induction expression; enzyme digestion and nickel ion affinity chromatographic purification are performed on an expression product through enterokinase, and then the artificial antibacterial peptide PR39-R1T can be obtained. The antibacterial peptide has the high broad-spectrum antibacterial activity, can inhibit gram-positive bacteria and gram-negative bacteria and does not have the hemolytic activity. Therefore, the artificial antibacterial peptide can be used as a novel antibacterial agent, has the good application prospect on the aspects of food corrosion prevention, disease treatment and the like, and has the developing potential. The preparation method is high in expression efficiency, simple in separation and purification, low in production cost, good in stability and capable of being applied to large scale production.

The invention discloses an enterovirus 71 capsid protein 3 recombinant antigen belonging to the technical field of virus detection and an application thereof. The gene nucleotide sequence of the recombinant antigen is showed in a sequence table SEQ ID NO: 1. The gene splicing technology is adopted, and through the annealing extending polymerase chain reaction (PCR) technology, a VP3 gene for codon optimization escherichia coli is obtained, the optimization VP3 gene is efficiently expressed in escherichia coli, and a corresponding recombined EV71-VP3 gene is obtained through an affinity chromatography method in purification mode. The obtained EV71-VP3 antigen can have a specific immune response with serum of patients of hand-foot-and-mouth disease, and the recombinant antigen can improve detection sensitivity of hand-foot-and-mouth disease by combining with EV71-VP1 for use and can be used for clinical diagnosis of EV71 acute infection.

The invention provides a mini-gene splicing reporter plasmid and construction method and application thereof. A sequence of the mini-gene splicing reporter plasmid is as shown in a sequence table <400>1. Three non-continuous sequences on a DMD gene are obtained through PCR (polymerase chain reaction) and inserted to pcDNA 3.1 respectively to construct the mini-gene splicing reporter plasmid. The mini-gene splicing reporter plasmid can be used for identifying and analyzing splicing regulation elements in exons and introns and detecting whether micromutation (point mutation and microdeletion/repetition) affects splicing of the exons or not.

The invention relates to the field of gene engineering, in particular to a method related to gene cloning. The method for quickly amplifying target genes from a genome DNA comprises the following steps of: (1) preparing the genome DNA; (2) designing amplification primers of gene exons; (3) carrying out exon amplification for respectively amplifying each exon; and (4) amplifying gene segments. In the invention, by using a special primer design method, required exons are firstly amplified from the genome and then purified and mixed, and the primers at both ends are used for amplifying the required target genes in a Touch-down program. The gene splicing method avoids the preparation of RNA and a process of synthesizing cDNA by reverse transcription, thus the gene cloning can be quickly and accurately finished within several hours. Meanwhile, any cDNA sequence the sequence information of which is known can be cloned by applying the method, and the amplification length is not limited.

The invention relates to a repeated module gene-splicing method, which includes the following steps: (1) two PCR primers are utilized to amplify target gene; (2) the PCR primers and plasmid pET-LIC are cut by double restriction enzymes, segments are connected with carrier, and after competent cells are transformed, plasmid pET-TS is obtained; (3) the plasmid pET-TS is cut by double restriction enzymes, and plasmid segments are recovered, and are then connected with the target gene segments to transform, so that plasmid pET-TS2 is obtained; (4) the plasmid pET-TS2 is cut by double restriction enzymes, and plasmid segments are recovered, and are then connected with the target gene segments to transform, so that plasmid pET-TS3 is obtained; (5) the plasmid and target gene PCR product are cut by double restriction enzymes, and the same method is utilized to continuously insert repeated segments. The invention repetitively splices a plurality of repeated module genes, moreover, no restriction enzyme cutting site residues are left between the modules, thereby the seamless connection between the repeated modules is truly realized, and moreover, splicing is directional.

The invention discloses a method for using polyethyleneimine to strengthen sensitivity and specificity of PCR gene splicing by overlap extension. The method comprises the steps of designing an HU-Beta primer sequence which needs to be synthesized, and obtaining a blended overlapping oligonucleotides template, wherein every sequence contains 25bp overlapping oligonucleotides template so as to cover the entire DNA(200-1500 basic group) sequence; adding DNA polymerase, head and tail primer, and the solution of blended overlapping oligonucleotides which is used as a template in the last step into into PEI ,the volume of which should be 10<-5>-10<-13> that of the DNA polymerase, head and tail primer, and the solution of blended overlapping oligonucleotides; carrying out repeated circulation according to three steps of melting, annealing, extending of conventional PCR, synthesizing and amplifying the target nucleic acid segment. The method for using polyethyleneimine to strengthen sensitivity and specificity of gene splicing by overlap extension can specifically and efficiently synthesize and amplify the target nucleic acid sequences.

The invention provides a gene splicing site identification model constructing method based on a particle swarm optimization twin support vector machine. Firstly splicing site identification is used asa two-class machine learning identification problem, and furthermore identification is finished through splicing sequence characteristic next to a site. Secondly, for aiming at a problem of relatively difficult twin support vector machine parameter setting in a class identification process, a particle swarm algorithm is used for parameter optimization of the twin support vector machine, thereby further improving the identification performance of the splicing site and preventing parameter selecting blindness. An experiment result verifies feasibility of the method and can effectively improve splicing site identification rate and accuracy.

The invention discloses a construction method of engineering bacteria producing beta-hydroxy-beta-methyl butyric acid, which includes the following steps: by taking yarrowia lipolytica as chassis cells, interrupting and inactivating five genes involved in fatty acid degradation and metabolism in the genome of yarrowia lipolytica, i.e. pox2, pox3, hbd, hcd and faa1, to obtain a gene-splicing strain; and integrating genes of LiuC, AibA and AibB from myxococcus xanthus, and a TesB gene from Escherichia coli into the genome of the gene-splitting strain to obtain the engineering bacteria producingbeta-hydroxy-beta-methyl butyric acid. The engineering bacteria constructed by the invention can ferment and synthesize the target product HMB with glucose as a carbon source, and the maximum yield can reach 10g/L.

The invention discloses a U1-snRNA for repairing TPP1 gene splicing defects and a vector containing a base sequence for expressing the U1-snRNA. The base sequence for expressing the U1-snRNA sequentially comprises a U1-snRNA initial basic group, a sequence complemented with a TPP1 gene Exon12 donor splicing site or a downstream intron sequence target and a U1-snRNA downstream sequence, and the U1-snRNA downstream sequence is as shown in SEQ ID NO.20. The U1-snRNA is from a nucleic acid molecule prepared according to a TPP1 gene donor splicing site or donor splicing site downstream intron sequence. By the aid of a U1-snRNA technique, U1-snRNA of a target gene serves as a target medicine, and repairing is implemented in the splicing process of Pre-mRNA, so that aberrant mRNA expression caused by splicing site mutation is effectively restrained. The U1-snRNA has the advantages of high specificity, high efficiency and small side effect, can make up for the deficiency of current treatment means of diseases caused by aberrant splicing and possibly serves as a new method for treating specific types of diseases in the near future.

The invention discloses a murine IL-27 recombinant protein eukaryotic expression vector and a construction method. The construction process comprises the following steps that primers are designed to amplify the cDNA (complementary deoxyribonucleic acid) sequence of encoding mouse EBI3 amino acids 1-228 and the cDNA sequence of encoding mouse p28 amino acids 29-234; and then an integrated DNA fragment is obtained by an SOEPCR (gene splicing by overlap extension polymerase chain reaction) method. The fragment is connected with a pcDNA3.1 + vector to obtain a murine IL-27 recombinant protein eukaryotic expression vector pSZ12. EBI3 and p28 are connected by the pre-designed amino acid encoding sequence. The vector constructed by the invention can not only effectively express a heterodimer IL-27 formed by EBI3 and p28, but also contain 6 * histidine encoding sequences to greatly facilitate the purification of recombinant protein. The key fragment can be cloned to other vectors so as to facilitate the application of protein expression in different cases, and an effective tool is provided for the study of the IL-27 function and the gene therapy method.

The invention discloses a gene and a polypeptide sequence of polyphenol oxidase YHL21. The polyphenol oxidase is confirmed by gene splicing and high-throughput screening of enzyme performance, and has the advantages of wide temperature-resistant range, strong high-temperature catalytic performance, easy membrane immobilization and the like, wherein functional enzyme is constructed by a single peptide fragment. The invention also discloses a method for carrying out recombinant expression and secretion production on the polyphenol oxidase YHL21 in a bacillus subtilis strain 168, and a method for immobilizing the recombinant polyphenol oxidase on the surface of a membrane by utilizing a polyvinylidene fluoride (PVDF) membrane, and the membrane enzyme composite system can stably and efficiently degrade organic matters such as phenol, parachlorophenol and the like in an aqueous solution. Enzyme engineering optimization is carried out through gene splicing, polyphenol oxidase capable of efficiently degrading phenolic pollutants is obtained, recombinant secretory expression is carried out through bacillus subtilis, enzyme immobilization is carried out through PVDF, and the catalytic efficiency of enzyme is improved.