163 results about "In vitro recombination" patented technology

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

The present invention relates generally to compositions and methods for enhancing recombinational cloning of nucleic acid molecules. In particular, the invention relates to compositions comprising one or more ribosomal proteins and one or more additional protein components required for recombinational cloning. More particularly, the invention relates to such compositions wherein the ribosomal proteins are one or more E. coli ribosomal proteins, still more particularly wherein the ribosomal proteins are selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L20, L21, and L23 through L34, and most particularly S20, L27, and S15. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and / or DNA segments. The invention also relates to isolated nucleic acid molecules produced by the methods of the invention, to vectors comprising such nucleic acid molecules, and to host cells comprising such nucleic acid molecules and vectors.

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Such methods, systems, and compositions for seamless nucleic acid assembly include those for in vitro recombination cloning, single-stranded hierarchal DNA assembly, or overlap extension PCR without primer removal.

The present invention relates to a method for producing a recombinant polynucleotides comprising the steps of generating a pool of unidirectional single-stranded polynucleotide fragments from two or more homologous double-stranded polynucleotides, conducting a polymerization process comprising multi-cyclic extension reactions using the unidirectional single-stranded polynucleotide fragments as templates and specific oligonucleotides as primers to obtain recombinant polynucleotides, and conducting a polymerase chain reaction using at least one primer to amplify the recombinant polynucleotides; and a method for constructing a recombinant DNA library comprising the steps of inserting the recombinant polynucleotide prepared by the above method into a vector and transforming an expression cell with said vector containing the recombinant polynucleotide to obtain a plurality of mutant clones. The method of the present invention can be used for in vitro recombination of homologous polynucleotides and the directed molecular evolution.

The invention relates to an invitro recombinant expression technology and functional identification of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) gene. Cf-PGRP is provided with an amino acid sequence in a SEQ ID NO.1. The Cf-PGRP is obtained by the invitro recombinant expression technology, and the recombinant protein has broad sterilizing effect, obvious killing effect on Gram positive bacteria and the certain antibacterial effect on Gram negative bacteria and potential application value in aspects of developing broad antibacterial medicines, immune reinforcing agent, feed additive, and the like.

The invention relates to a Fenneropenaeus chinensis antimicrobial protein gene and the recombination expression and the application. It has the amino acid sequence of SEQ ID No.2 and basic group sequence of SEQ ID No.1 in sequence table. It selects the carrier of the antimicrobial protein pCR T7 / NT TOPO TA and host bacterium BL21(DE3)pLysS that could high efficiently express recombination protein. Aimed at the feature of plural disulfide bonds, using the oxidation reduction system made of oxidized form of glutathione and glutathione in separating and purification and protein recombination process, the recombination protein with antimicrobial ability would be gained. The protein has strong inhibition to the growth of gram positivebacteria, and some inhibition to gram negativebacteria.

The invention relates to a high-specific activity L-glutamate oxidase gene multisite mutant, and a preparation method and application thereof. The nucleotide sequence of the mutant is shown as SEQ ID No 64; the amino acid sequence of encoded protein is shown as SEQ ID No 65. A molecular in vitro recombination technique is used for screening high-specific activity L-glutamate oxidase gene; through vector building, enzyme activity screening, multisite mutation techniques and the like, the high-specific activity L-glutamate oxidase gene multisite mutant is obtained through preparation. The multisite mutant contains 9 different mutation sites on the same gene, so that the oxidation specificity and the specific activity of the multisite mutant on L-glutamic acid are greatly improved; the multisite mutant can be used for detecting the content of the L-glutamic acid in food.

The invention relates to the technologies of clam ferroprotein gene cloning and in vitro recombinant expression in molecular biology. In the invention, the expression sequence tag (EST) technology and rapid amplification of cDNA ends (RACE) 3' and 5' are utilized to clone ferroprotein gene cDNA with the total length of 798bp from a clam larva, the cloned ferroprotein gene contains an open reading frame with the length of 513bp, 171 amino acids are coded, the length of a non-coding region 5' is 119bp, the length of a non-coding region 3' is 154bp, a tailing signal is contained, and the gene plays an important role in formation of a shell during development of the clam larva. In the invention, the in vitro prokaryotic recombinant expression technology is utilized to obtain recombinant clam ferroprotein; and the ferroprotein has antioxidant activity, can be used for regulating and controlling formation and growth of shells of shellfish larvae such as clams and the like, and can be applicable to development regulation and immunological enhancement in the shellfish larva production process.

The invention relates to the technical field of molecular biology, in particular to eriocheir sinensis Crustin-1 gene clone and a recombination expression technology thereof. Crustin-1 gene of which the cDNA span is 821bp is cloned from eriocheir sinensis by using an expressed sequence tag (EST) technology and a 3' and 5' end rapid augmentation technology (RACE) and comprises a 315bp open reading frame, polyadenylic acid tailing signals and polyadenylic acid tails, 104 amino acids are coded, the length of a 5' non-coding region is 239bp, the length of a 3' non-coding region is 267bp, and the Crustin-1gene play an important role in the immune defense aspect of the eriocheir sinensis. The invention obtains eriocheir sinensis Crustin-1 protein by using the in-vitro recombination expression technology, the recombination protein has stronger bacteriostatic activity for gram-positive bacteria, the minimal inhibitory concentrations of the recombination protein for micrococcus luteus, bacillus subtilis, micrococcus tetragenus and bacillus thuringiensis are 0.12 mu m, 0.23 mu m, 0.46 mu m and 0.12 mu m respectively, and the recombination protein does not have obvious inhibiting function. The invention can lay the foundation for the disease control of the eriocheir sinensis, the gene assistant breeding and the development of feed additives.

This invention discloses a new preparation for the heatproof lactobionic enzyme, which belongs to the biology technique filed. This invention includes the following steps: constructing the heatproof lactobionic enzyme gene onto the baculoviral vector to obtain the recombining baculoviral vector; through internal or external combining, conforming the heatproof lactobionic enzyme gene on the baculoviral gene group to obtain the recombining baculoviral; using the obtained recombining baculoviral to infect the insect host; culturing the infected insect hose to do the heatproof lactobionic enzyme expression; collecting and purifying the expression product. This invention can produce cheap heatproof l lactobionic enzyme with high efficient and large scale, and the product can be used in foodstuff and feedstuff production field extensively.

The invention discloses a dynein mosaic type recombinant human type-B adenovirus and a preparation method thereof. The skeleton of the dynein mosaic type recombinant human type-B adenovirus is a human type-B adenovirus genome, and a base sequence which encodes a receptor binding domain of dynein is a base sequence which encodes a corresponding domain of a human type-C adenovirus. By a molecular cloning method, Ad5-knob gene fragments are cloned and replaced to recombinant shuttle plasmids, in-vitro recombinant on the Ad5-knob gene fragments and a recombinant human type-3 adenovirus genome is realized, obtained knob gene fragments are replaced into type-5 recombinant human type-3 adenovirus genome, and therefore, dynein mosaic type recombinant human type-3 adenovirus rAd3-FK5 is obtained. The dynein mosaic type recombinant human type-3 adenovirus rAd3-FK5 can be infected with mouse primitive epithelial cells and golden hamster lung and kidney primitive cells in vitro, and the infection efficiency of the dynein mosaic type recombinant human type-3 adenovirus rAd3-FK5 is close to that of Ad5, and is much higher than that of a parent strain rAd3E, in golden hamster cells, significant copying exists, and the dynein mosaic type recombinant human type-B adenovirus can be used for small animal model research of human type-3 adenovirus vaccines and antiviral drug evaluation.

The invention belongs to the field of biological medicinal preparation and relates to a preparation technology and an application of a natural recombinant nanostructured lipid carrier. The specifically-refereed nanoparticle is natural recombinant lipoprotein drug-loaded nanoparticle and is prepared by in vitro recombination of an apolipoprotein mixture and lipid which are obtained by extraction and purification of a blood plasma ingredient IV and a medicament. The recombination process is realized by a film dispersion method, an emulsification-evaporation method or an emulsification-evaporation improved method. The technical problem to be solved in the invention is the extraction and purification method of apolipoprotein and lipid mixture and the preparation technology of the natural recombinant lipoprotein drug-loaded nanoparticle. Preparation conditions are mild, and costs are low. The natural recombinant lipoprotein nanoparticle provided by the invention has remarkable advantages of spheroidic structure, high bionic performance, high penetrability, efficient carrying performance, tumor targeting and the like. The invention provides property evaluation and application of the above natural recombinant lipoprotein nanoparticle. Normal saline, or a phosphate buffer or 5% glucose solution is added to dissolve the nanoparticle, and the dissolved nanoparticle can be used to treat diseases in a mode of intravenous injection, intramuscular injection, oral medication or cutaneous penetration. The technology provided by the invention is simple and low-cost and is easy for industrial production.

The invention belongs to the technical field of molecular biology, and in particular relates to a recombinant protein of a bay scallop peptidoglycan recognition protein as well as preparation and application thereof. Recombinant protein of a peptidoglycan recognition protein (AiPGRP) gene is as shown in an amino acid sequence in a sequence table SEQ ID NO.1. Specifically, bay scallop AiPGRP recombinant protein is obtained by utilizing an in vitro-recombination expression technology, and bacteria-agglomeration activity and antibacterial activity of the recombinant protein of the bay scallop AiPGRP gene are disclosed. The recombinant protein obtained in the invention can be utilized for developing an antibacterial feed additive and a novel immunoenhancer.

The invention belongs to the field of biochemistry, and discloses an antibacterial protein LYS-ABP and an antibacterial protein MBP-LYS-ABP having an LYS-ABP protein N terminal added with MBP, and preparation methods thereof, wherein the antibacterial protein LYS-ABP and the antibacterial protein MBP-LYS-ABP have no stimulation to human bodies and have good antibacterial effect. The antibacterial protein LYS-ABP and the antibacterial protein MBP-LYS-ABP have no stimulation to the human bodies and have good antibacterial effect, the antibacterial effect is better than single muramidases and antibacterial peptide proteins, and the biological activity stability is good. The antibacterial protein MBP-LYS-ABP is obtained by in-vitro recombinant expression, and is easy to prepare and relatively low in cost; and the antibacterial protein MBP-LYS-ABP has high soluble expression yield and is suitable for large-scale preparation. The antibacterial protein LYS-ABP is obtained by enzyme digestion of a recombinant expression-prepared MBP-LYS-ABP fusion protein with thrombin, and is easy to prepare and relatively low in cost.

The invention discloses a coding gene of haemaphysalis qinghaiensis aquaporins (AQPs) and an application of the coding gene. The coding gene of the AQPs can be used for preparing novel anti-tick medicines and anti-tick vaccines. A sequence of the coding gene of the AQPs is shown as SEQ ID NO:1. The invention also provides specific primers cloned by the coding gene of the AQPs, wherein sequences ofthe specific primers are shown as SEQ ID NO:2 and SEQ ID NO:3; the invention also provides a method for cloning the coding gene of the AQPs, and the cloned gene product can be also applied to in-vitro recombinant protein expression and antibody preparation; and an obtained antibody can be used for preparing the novel anti-tick medicines and the anti-tick vaccines. The invention has important theoretical significance an potential application value on prevention and control of ticks. The coding gene of the haemaphysalis qinghaiensis aquaporins (AQPs) and the cloning method of the coding gene provided by the invention are applicable to systematic researches of subtype, varieties, expression distributin, protein structures, biological functions and the like of the AQPs.

The invention relates to an Eriocheir sinensis anti-lipopolysaccharide factor gene cloning and in vitro recombinant expression technology of molecular biology, and the Eriocheir sinensis anti-lipopolysaccharide factor gene has the base sequence in a sequence table of SEQ ID No: 1. The Eriocheir sinensis anti-lipopolysaccharide factor is studied by utilizing a constructed Eriocheir sinensis cDNA library and adopting the RACE technology and the in vitro recombinant expression technology, the cDNA sequence of the anti-lipopolysaccharide factor is cloned from the Eriocheir sinensis for the first time, the gene has the sequence which is indicated in the sequence table, and the prokaryotic recombinant technology is utilized for expressing a recombinant protein with stronger bactericidal activity.

The invention belongs to the field of molecular biology and the technical field of genetic engineering, and in particular discloses apostichopus japonicus peptidoglycan recognition protein with sterilization activity as well as a preparation method and application of the apostichopus japonicus peptidoglycan recognition protein. The apostichopus japonicus peptidoglycan recognition protein AJ-PGRP is obtained for the first time by utilizing an in-vitro recombination expression technology; the apostichopus japonicus peptidoglycan recognition protein (AJ-PGRP) is shown as an amino acid sequence in a sequence table of SEQ ID NO. 1. The recombinant protein has agglutination effect on staphylococcus aureus and escherichia coli, and has inhibition effect on the staphylococcus aureus, the escherichia coli, vibro splendidus and bacillus subtilis, and has potential application value in the aspects of developing novel broad-spectrum antibacterial drugs, feed additives and the like.

The invention relates to a cynoglossus semilaevis female specificity CSW3 protein as well as a gene and an application thereof, which belong to the technical field of functional gene selection and application in an aquatic biological technology. The amino acid sequence of the cynoglossus semilaevis female specificity CSW3 protein is SEQ ID NO. 1; a nucleotide sequence for encoding the CSW3 gene of the CSW3 protein is SEQ ID NO.2. By adopting the in-vitro recombination cynoglossus semilaevis female specificity CSW3 protein of the CSW3 gene, the expression level of female relevant gene foxl2 can be obviously increased, and the biological activity for stimulating the female relevant gene expression can be realized; meanwhile, the biological activity for reducing the male relevant gene sox9 and amh expression level can be realized. After a CSW3 gene recombination product is used as a feed additive, the development of the female gonad can be stimulated, and the ratio of the female fish can be increased, so that the cynoglossus semilaevis female specificity CSW3 protein has the application potential on controlling the gender of the cynoglossus semilaevis and increasing the ratio of the female fish fries.

The invention discloses an in-vitro method for evaluating a repair function by reconstructing a normal human body three-dimensional skin model. According to the method, an in-vitro recombined three-dimensional normal human skin model is utilized, SDS or a mechanical method is adopted to cause damage to the skin, the repairing effect of a tested substance on the damaged skin is evaluated by analyzing the recovery situation after damage or the intensity of inflammatory reaction, and the method specifically comprises the following steps: (1) preparation of a damage model, (2) restoration, (3) measurement of evaluation indexes, and (4) result analysis. The method has the advantages of high flux, repeatability, high correlation with people, multi-index evaluation and the like, and can be used for quickly evaluating various tested objects.

The invention provides a novel gene, which is separated from cynoglossus semilaevis and related to immune. The gene is subjected to in-vitro recombinant expression to obtain a recombinant expression product, and the antipathogen activities and functions of the recombinant expression product are identified to provide a gene resource and technical method for cynoglossus semilaevis disease control, green feed additives and antibacterial agents. The gene codes the following proteins: (a) protein with an amino acid sequence of SEQ ID NO:2; (b) protein including amino acids from the 117th-255th bits in (a) and has functions of (a); and (c) protein which is substituted, deleted or added by one or more amino acids in the amino acid sequence of (a) or (b) and has the protein activity in (a). The gene is closely related to immune resistance, the gene expression quantity of the gene is related to pathogenic stimulation, and the in-vitro recombinant protein of the gene can remarkably inhibit the proliferation of gram-negative bacteria, positive bacteria and viruses, so that the gene has application potentials in cynoglossus semilaevis disease control, green feed additive and antibacterial agents.

The invention discloses a naphthalene detecting biological cell sensor, as well as a preparing method and a use method thereof. A gene recombination cell is used as the naphthalene detecting biological cell sensor, a salicylic acid detecting light sensing cell is used as a host cell of the gene recombination cell, and a plasmid of the gene recombination cell is recombined by a naphthalene degrading gene and a vector plasmid. The naphthalene detecting biological cell sensor can immediately emit light at the presence of naphthalene and has the advantages of great selectivity, high flexibility, high analysis speed, simplicity in operation, low instrument price and capability of carrying out continuous and stable online detection in a complicated system. The invention further provides a preparing method for the cell sensor. Adopted construction conditions are mild, the method is controllable, simple and feasible, the in-vitro recombination of a macro-molecular degrading fragment is finished by a Gibson cloning technology, and an important means is provided for the detection of a specific pollution substrate.

The invention discloses a pelteobagrus fulvidraco beta defensin gene, and beta defensin antibacterial peptide and application thereof. An in vitro recombinant expression technology is used for successfully expressing a pelteobagrus fulvidraco beta defensin recombinant protein PET-32a-Pf_BD, and antibacterial activity identification results show that the pelteobagrus fulvidraco beta defensin gene has a certain bacteriostasis for staphylococcus aureus in gram-positive bacterium, escherichia coli in gram-negative bacterium, aeromonas hydrophila, edwardsiella ictaluri and flavobacterium columnare. Therefore, a foundation is laid for application of the defensin recombinant protein, used as a broad-spectrum antibacterial drug, in aquaculture.

The invention belongs to the technical field of molecular biology and particularly relates to application of crassostrea gigas C-type agglutinin-4 (CgCLec-4).According to the method, an in vitro recombination expression technology is utilized, recombination protein of a carbohydrate recognition domain (CRD) of crassostrea gigas CgCLec-4 is obtained, and application of crassostrea gigas C-type agglutinin-4 (CgCLec-4) recombination protein to preparation of a bacteriostatic agent or a fodder additive is obtained.The obtained recombination protein can be used for development of an antibacterial fodder additive or novel immunopotentiator.

The invention relates to an expression technology of Chlamys farreri serine protease enzyme inhibitor CfKZSPI gene clone and in-vitro reconstitution. Chlamys farreri serine protease enzyme inhibitor CfKZSPI gene has motif sequence in the sequence table SEQ ID No.1. The invention takes use of the constructed Chlamys farreri cDNA library and adopts RACE technology and in-vitro reconstitution expression technology to study Chlamys farreri serine protease enzyme inhibitor CfKZSPI, and the full-length sequence of cDNA of serine protease enzyme inhibitor CfKZSPI is cloned from Chlamys farreri, and the gene has the sequence provided in the sequence list; and pronucleus reconstitution technology is used to express the recombinant protein with strong trypsin inhibition activity.

The invention discloses an eriocheir sinensis HSP90 gene clone and application thereof. The gene has a base sequence as shown in SEQ ID NO.1. The protein encoded by the gene has an amino acid sequence as shown in SEQ ID NO.2. The HSP90 gene provided by the invention is firstly obtained from eriocheir sinensis hepatopancreas by amplification, can be used for in vitro recombination expression and gene transfer of the HSP90 gene, and lays a foundation for developing medicine products, selected genetic breeding and improving, and enhancing stress resistance to high temperature, low temperature, heavy metal ion exceeding, pathogenic bacterial infection and the like.

Methods are disclosed for assembling a plurality of double-stranded DNA fragments into DNA molecules in a single in vitro recombination reaction comprising contacting the plurality of double-stranded DNA fragments with a bacterial extract derived from a RecA deficient bacterial strain so as to assemble the plurality of DNA fragments into DNA molecules.