40 results about "T4 bacteriophage" patented technology

Disclosed are adenovirus vectors comprising a ROBO4 enhancer / promoter operatively linked to a transgene. Also disclosed are adenovirus vectors comprising a chimeric AD5-T4 phage fibritin shaft, a trimerization domain displaying a myeloid cell-binding peptide (MBP), and a ROBO4 enhancer promoter operatively linked to a transgene. Also disclosed are methods of expressing a transgene in an endothelial cell in vivo, comprising administering to a mammal an adenovirus comprising a ROBO4 enhancer / promoter operatively linked to a transgene. Also disclosed are uses of the adenoviral vectors, including mobilization of granulocytes, monocytes and lymphocytes from bone marrow, mobilization of cancer cells in vivo, selective targeting of endothelial cells, and cancer treatment methods.

The invention discloses an adenovirus multi-fluorescent quantitative PCR (polymerase chain reaction) detection kit, comprising PCR liquid, a primer probe combination, a positive quality control sample, and a negative quality control sample. The adenovirus multi-fluorescent quantitative PCR detection kit also comprises liquid for nucleic acid extraction and an inner reference substance for nucleic acid extraction, wherein the inner reference substance is inactivated T4 bacteriophage; the primer probe combination comprises a primer probe (general: SEQ ID NO4-6; subtype SEQ ID NO7-9) for adenovirus nucleic acid and a primer probe (SEQ ID NO1-3) for the inner reference substance; the 5' terminals of the SEQ ID NO3, 6, 9 are connected to a fluorescent report group; and the 3' terminals are connected to a fluorescent quenching group. A using method of the kit is also disclosed by the invention. Viral nucleic acid extraction is carried out by the liquid for nucleic acid extraction before the fluorescent quantitative PCR detection; and the inner reference substance is added in the extraction process of the viral nucleic acid. The kit and the using method thereof disclosed by the invention have the characteristics of being convenient to use, and accurate in detection result; all adenoviruses can be detected; and B and E subgroups can be parted.

The present invention discloses one kind of recombinant analogous human collagen and its biological synthesis process. The recombinant analogous human collagen consists of one collagen region and one non-collagen region. The non-collagen region in the carboxyl group end of the collagen region has one amino acid sequence originated the minor fibrin of T4 bacteriophage and as shown in SEQ ID No. 3, and the collagen region has the amino acid sequence shown in SEQ ID No. 2. The recombinant analogous human collagen has relatively high heat stability, high hydrophilicity, high biocompatibility, heat denaturation temperature and heat decomposition temperature higher than that of natural collagen, and stable structure. It may be applied widely in biomedicine material, tissue engineering, cosmetics, food and other fields.

The invention discloses a method for preparing iron nano-magnetic particles by taking a T4 phage as a template in the field of magnetic recording materials. In the method, the T4 phage obtained by enrichment and the solution of iron chloride are incubated, centrifuged and reduced by utilizing intrinsic structural characteristics of an organism and a molecular recognition function thereof to obtain iron nano-particles which have small particle sizes and high dispersion degrees and are regularly distributed on the capsid surface of the T4 phage, and the prepared iron nano-particles have the characteristics of nonreversible magnetization process, high coercive force and the like, so that the iron nano-particles prepared by the method have wide application prospect in the field of high-intensity magnetic recording materials. Moreover, in the method, a biological nano-structure which exists in nature is directly taken as the template, so that the method has simple preparation processes, moderate reaction conditions and environment friendliness. Since a biological template has the characteristics of self-producing capability, high appearance repeatability and the like, large scale production is easy to realize.

The invention belongs to the field of feed additives, and in particular relates to a pemix containing T4 lysozyme from T4 bacteriophage. The T4 lysozyme from the T4 bacteriophage replaces the traditional animal antibiotics, and the antibacterial growth-promoting feed premix containing the T4 lysozyme is prepared. A method for preparing the premix comprises the following steps of: centrifuging T4 lysozyme basic solution to remove impurities, performing membrane concentration, refining and purifying by using cation exchange resin, preparing protease powder by using a low-temperature spray drying method, screening the protease powder, adding auxiliary materials, and uniformly mixing at a high speed by a mixer to prepare the T4 lysozyme premix. The added auxiliary materials comprise soya beanmeal, bran, wheatmeal, wheat middling, bentonite, CaCO3, and zeolite. Experimental results show that the premix containing the T4 lysozyme has the effect of treating piglet diarrhea, which is obviously superior to that of a premix containing lysozyme from chicken egg albumin; and experimental results for feeding meat chickens and carp show that the premix containing the T4 lysozyme has the weightincreasing effect and feed utilization rate which are slightly superior to those of the premix containing lysozyme from chicken egg albumin.

The invention discloses a kind of foot-and-mouth disease genetic engineering vaccine and its the preparing method.Its structure expression is: Entire virus genetic engineering vaccine: Phage T4-Soc-Hoc-FMDV-P1; Asian unit genetic engineering vaccine: Phage T4-Soc-Hoc-FMDV-F3.The preparing method is: Takes O blood serum foot-and-mouth disease virus poisonous, extracts total gene group RNA; From blood serum poisonous DNA measure foreword to synthesizes two DNA nucleic acid probe;takes the probe as the directing thing, carries on Rt-pcr chain type DNA expand increase response, obtains the viral granule P1gene;gene recombinate the P1gene and the T4bacteriophage material particle, pRH-Soc, obtains T4bacteriophage conformity material particle pRH-Soc-P1;homology reorganizes pRH-Soc-P1with the T4bacteriophage carrier phage T4- í¸ Soc & í¸ Hoc, then enters in the backwoods coli E.coli.CR63host mycelium, obtains this invention genetic engineering vaccine phage T4-Soc-FMDV-P1.compares this invention with the deactivation entire viral vaccine, it does not have to fight fire, maintains the entire viral antigenicity but not have the infection.may inject, also may take orally, convenient, quick, the immunity effect is good.

The invention discloses an Escherichia coli expression vector capable of controlling the self-cracking of a host bacterium, which belongs to the technical field of microbes and gene engineering. In the invention, the composition of the codon of a lysozyme gene in a T4 phage and a restriction endonuclease identification site are optimized, and the optimized T4 lysozyme gene mutation lyMu and a modified Escherichia coli expression vector are recombined to form a recombinant expression vector pEly. The expression vector pEly can be used for inducing expression of an exogenous gene, and when the exogenous gene is induced to express, T4 lysozyme is expressed. The cell undergoing induction expression is treated by ethylene diamine tetraacetic acid (EDTA) solution and then self-cracks, so that the product of expression in the cell is released. The expression vector obtained by implementing the invention can realize the express of various exogenous proteins in Escherichia coli hosts, the host bacteria release the products of expression after undergoing EDTA treatment, and thus, the recovery of the products of the expression can be promoted. The expression vector has an application prospect in fields of enzymic preparation production, polypeptide medicine production and the like.

The invention discloses a preparation method of a T4 bacteriophage supported precious metal nano-particle catalyst. In the method, by utilizing inherent structural characteristics and molecular recognition function of living organisms, the precious metal nano-particle catalyst which is configured regularly on the capsid surface of a T4 bacteriophage and has uniform particles and high dispersion can be obtained through the steps of adding the T4 bacteriophage obtained by enrichment into a precious metal salt solution, hatching, centrifuging, and reducing. The method can control the size and the distribution of the precious metal nano-particles in a highly efficient mode, and greatly improves the electro-catalysis activity of the precious metal nano-particles. Meanwhile, by directly taking a biological nano-structure existing in the nature as a carrier, the method avoids complex preparation process of conventional carriers, thereby reducing production cost. Because the biological carriers have the characteristics of self-production, high feature repeatability and the like, the method is easy to realize large-scale production.

Compositions and methods comprising bacteriophages are provided. In particular, the present invention includes novel and customized T4 bacteriophages uniquely designed for effective antigen and foreign particle presentation. The present invention also provides in vitro methods for the making of customized T4 bacteriophages. The compositions and methods of the present invention may be used for effective vaccine delivery systems.

The invention provides a kit for identifying foot-and-mouth disease and immunity of an entry animal. The kit comprises three pre-coated ELISA (Enzyme Linked Immunosorbent Assay) microwell plates, a goat anti-swine antibody-horseradish peroxidase conjugate, a washing solution, a substrate, a color developing agent, a sample diluent, a stop solution, a positive reference substance and a negative reference substance, wherein the three pre-coated ELISA microwell plates are respectively pre-coated by a foot-and-mouth disease 3ABC antigen under multi-copy showing on the surface attached by T4 phage, a foot-and-mouth disease 3B antigen under multi-copy showing on the surface attached by T4 phage, and a foot-and-mouth disease 3D antigen under multi-copy showing on the surface attached by T4 phage; the washing solution is 0.15mol / L of PBS (Phosphate Buffer Solution) with pH (Potential Of Hydrogen) of 7.2; the substrate is 0.1% of TMB (Tetramethyl Benzidine) solution; the color developing agent is 0.015% of H2O2; the sample diluent comprises 0.1% of bovine serum albumin and 0.1% of sodium azide which serves as a protective agent; the stop solution is 2mol / L of H2SO4; the positive reference substance is diluted by 500 times and adopts 0.1% of sodium azide as the protective agent; and the negative reference substance is diluted by 500 times and adopts 0.1% of sodium azide as the protective agent.

The invention provides an isothermal nucleic acid amplification reaction reagent. The isothermal nucleic acid amplification reaction reagent is characterized by comprising components as follows: 100-800 mM of a Tris-HCl buffer solution, 10-150 mM of sodium chloride, 10-150 mM of potassium chloride, 10-50 mM of magnesium chloride, 5-15 mM of dithiothreitol, 5%-20% of polyvinylpyrrolidone, 10-20 mM of ATP (adenosine triphosphate), 1-5 mM of dNPTs, 10-50 mM of phosphoenolpyruvate, 500-1,500 ng / mu l of pyruvate kinase, 10-500 ng / mu l of BSA (bovine serum albumin), 25-200 pmol of each primer in a primer group, 50-200 ng / mu l of T4 bacteriophage DNA helicase gp41 protein, 100-500 ng / mu l of streptomyces coelicolor recA protein, 200-1,000 ng / mu l of single-strand binding protein and 50-200 ng / mu l of escherichia coli DNA polymerase I. The invention further provides an isothermal nucleic acid amplification method. According to the isothermal nucleic acid amplification reaction reagent and the isothermal nucleic acid amplification method, nucleic acid amplification under the isothermal condition at the lower temperature is realized, and a traditional nucleic acid amplification reaction process is greatly simplified.

Techniques from two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, are developed to construct new plague vaccines. The NH2-terminal β-strand of F1 of Yersinia pestis is transplanted to the COOH-terminus of F1 of Yersinia pestis and the NH2-terminus sequence flanking the β-strand of F1 of Yersinia pestis is duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 is fused to the V antigen of Yersinia pestis to thereby form a fusion protein F1mut-V mutant, which produces a completely soluble monomer. The fusion protein F1mut-V is then arrayed on phage T4 nanoparticles via a small outer capsid protein, Soc, from a T4 phage or a T4-related phage. Both the soluble and T4 decorated F1mut-V provided approximately 100% protection to mice and rats against pneumonic plague evoked by high doses of Yersinia pestis CO92.

Techniques from two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, are developed to construct new plague vaccines. The NH2-terminal β-strand of F1 of Yersinia pestis is transplanted to the COOH-terminus of F1 of Yersinia pestis and the NH2-terminus sequence flanking the β-strand of F1 of Yersinia pestis is duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 is fused to the V antigen of Yersinia pestis to thereby form a fusion protein F1mut-V mutant, which produces a completely soluble monomer. The fusion protein F1mut-V is then arrayed on phage T4 nanoparticles via a small outer capsid protein, Soc, from a T4 phage or a T4-related phage. Both the soluble and T4 decorated F1mut-V provided approximately 100% protection to mice and rats against pneumonic plague evoked by high doses of Yersinia pestis CO92.

The invention discloses a method for showing a foreign protein macromolecule on the surface of a T4 bacteriophage by using an intracellular synchronous expression method. The method for showing the foreign protein macromolecule on the surface of the T4 bacteriophage comprises the following steps that an expression plasmid of recombinant protein fused by Hoc (Highly-antigenic Outer Capsid) protein or Soc (Small Outer Capsid) protein with a natural T4 bacteriophage control area and foreign protein is constructed; the expression plasmid of the foreign recombinant protein is transformed into an escherichia coli cell; the escherichia coli cell containing the expression plasmid of the foreign recombinant protein is infected by an Hoc protein and Soc protein deleted T4 bacteriophage virus strain; T4 bacteriophage particles generated in the infection step and showing the foreign recombinant protein are separated; and the foreign recombinant protein shown on capsid of the obtained T4 bacteriophage particles is detected. The method does not require overexpression of the foreign recombinant protein, avoids a technical difficulty that large-amount overexpression of the foreign recombinant protein is required in an in-vitro showing method, does not require separation or purification of the foreign recombinant protein, and avoids a time-consuming and expensive protein separation and purification process.

The invention relates to expression and purification of T4 bacteriophage polynucleotide kinase (T4 PNK) and particularly relates to soluble expression and a purification method of the T4 PNK, belonging to the field of biochemistry. According to the invention, a T4 PNK gene is cloned into a pGEX vector through vector construction, the T4 polynucleotide kinase and GST undergo fused expression under the condition of low temperature, then, the protein expression level reaches 15%, and the solubility reaches 95%.

The invention relates to an emulational teaching model for demonstrating how a bacteriophage invades bacteria, in particular to a teaching demonstration model for a bacteriophage invading bacteria. The teaching demonstration model comprises a T4 bacteriophage model and an escherichia coli thallus model. The T4 bacteriophage model is composed of a protein head module and a hollow protein tail module, and a bacteriophage DNA molecule module is included in the protein head module. The escherichia coli thallus model is composed of simulative flagellums, a capsule, a cell wall module, a cell membrane module, an escherichia coli DNA molecule module, a ribosome module and a cytoplasm module, small holes are formed in the capsule, the cell membrane and the cell wall of the model, and therefore the NDA molecule module of the bacteriophage model can move into the escherichia coli thallus model conveniently. Multiple rails facilitating the moving of all the modules and a baffle hiding the protein shell of the model are arranged on the base plate of the whole model and the cytoplasm module.

The invention is directed to a novel recombinant T4 phage particle expressing a HOC and / or SOC Helicobacter pylori and / or SCLC fusion peptide as well as methods for their preparation and methods of use in compositions and kits.

The invention provides a preparation method of a phage display-expressing circovirus antigen vaccine. The preparation method comprises the following steps: extracting porcine circovirus II type (PCV2) RNA, carrying out inverse transcription, carrying out a polymerase chain reaction (PCR) for amplification so as to obtain porcine II type circovirus ORF2 gene's open reading frame fragments, directionally inserting the fragments into a T4 phage; obtaining a T4 phage with the ORF2 gene by an immunoscreening method; purifying and infecting X-Blue ST1(CCTCC M 2014397) host cell again; carrying out fermental cultivation to obtain the X-Blue ST1 host cell, and carrying out inducible expression and inactivation and taking a culture as an antigen; carrying out ELISA calibration of antigen valence, and carrying out double dilution on the antigen with normal saline to the final concentration of 107U / ml; and a vaccine adjuvant is added according to the ratio of 1:2.5, so as to obtain the vaccine. According to the invention, the phage is utilized to express the porcine circovirus II type antigen and the vaccine is prepared. Thus, the virulence enhancement risk of an attenuated vaccine is avoided. In addition, fermental cultivation is beneficial to large-scale industrial production. Cost of the vaccine provided by the invention is far lower than that of a vaccine produced by a cell culture method.

The invention discloses a method for connecting DNA fragments through T4 bacteriophage DNA topoisomerase. The method comprises the steps that primers are designed, wherein the primers are designed for at least two DNA fragments to be connected respectively; 2, the fragments are amplified, wherein the corresponding DNA fragments to be connected are used as templates, the primers of the DNA fragments are subjected to PCR amplification to obtain amplified products, and the amplified products are purified; 3, the fragments are connected. According to the DNA fragment connection method, T4 bacteriophage DNA topoisomerase I is used for achieving the functions of enzyme digestion and connection at the same time, the method has the advantages of being rapider and more efficient compared with a traditional method that a restriction enzyme is utilized at first for producing matched tail ends for the DNA fragments, T4 ligase is utilized for connecting fragments after purification, sequences inside the fragments to be connected in the method will not influence connection, and defects of a digestion step in the traditional method are eliminated.

The invention provides a PCR enhancer composition. The PCR enhancer composition comprises any one of T4 phage gene 32 coding protein, trehalose and nonidet P-40. The invention further provides a droplet-type reverse transcription digital PCR solution which is characterized by comprising the PCR enhancer composition. The PCR enhancer composition comprises the T4 phage gene 32 coding protein, the trehalose and the nonidet P-40, and preferably, the PCR solution contains 0.05-1 mg / mL T4 phage gene 32 coding protein, 0.1-2 mol / L trehalose and 0.02-1% w / v nonidet P-40. The invention also relates toa kit containing the PCR solution and a method for a droplet-type reverse transcription digital PCR by using the PCR solution. The PCR solution can be used for improving the amplification efficiency of the positive droplet-type reverse transcription digital PCR, generation of false negative is reduced, and accurate quantitative analysis of nucleic acid target molecules is facilitated.

The invention relates to a method for efficiently expressing hypersensitive protein by using a T4 phage display technology. The method comprises the following steps: (1) sequentially connecting an encoding gene of the hypersensitive protein with TEV protease and a T4 bacteriophage Hoc protein gene segment to prepare a fusion recombinant protein encoding gene, wherein the encoding region of the hypersensitive protein is located at the upstream of a TEV-Hoc fusion protein gene and having consistent expression cassettes; (2) transferring a coding gene of the fusion recombinant protein into a pUC18 plasmid; (3) transferring the plasmid into an escherichia coli host; (4) infecting the Escherichia coli host by using Hoc-T4 bacteriophage deleted by Hoc protein, and culturing the host to express and display the T4 bacteriophage of the fusion recombinant protein; (5) separating the T4 bacteriophage showing the fusion recombinant protein; and (6) separating the fusion recombinant protein from the T4 bacteriophage, and carrying out TEV protease digestion to obtain the hypersensitive protein.

The invention discloses a method for showing a foreign protein macromolecule on the surface of a T4 bacteriophage by using an intracellular synchronous expression method. The method for showing the foreign protein macromolecule on the surface of the T4 bacteriophage comprises the following steps that an expression plasmid of recombinant protein fused by Hoc (Highly-antigenic Outer Capsid) protein or Soc (Small Outer Capsid) protein with a natural T4 bacteriophage control area and foreign protein is constructed; the expression plasmid of the foreign recombinant protein is transformed into an escherichia coli cell; the escherichia coli cell containing the expression plasmid of the foreign recombinant protein is infected by an Hoc protein and Soc protein deleted T4 bacteriophage virus strain; T4 bacteriophage particles generated in the infection step and showing the foreign recombinant protein are separated; and the foreign recombinant protein shown on capsid of the obtained T4 bacteriophage particles is detected. The method does not require overexpression of the foreign recombinant protein, avoids a technical difficulty that large-amount overexpression of the foreign recombinant protein is required in an in-vitro showing method, does not require separation or purification of the foreign recombinant protein, and avoids a time-consuming and expensive protein separation and purification process.

The invention discloses a preparation method of a T4 bacteriophage supported precious metal nano-particle catalyst. In the method, by utilizing inherent structural characteristics and molecular recognition function of living organisms, the precious metal nano-particle catalyst which is configured regularly on the capsid surface of a T4 bacteriophage and has uniform particles and high dispersion can be obtained through the steps of adding the T4 bacteriophage obtained by enrichment into a precious metal salt solution, hatching, centrifuging, and reducing. The method can control the size and the distribution of the precious metal nano-particles in a highly efficient mode, and greatly improves the electro-catalysis activity of the precious metal nano-particles. Meanwhile, by directly taking a biological nano-structure existing in the nature as a carrier, the method avoids complex preparation process of conventional carriers, thereby reducing production cost. Because the biological carriers have the characteristics of self-production, high feature repeatability and the like, the method is easy to realize large-scale production.

The invention provides a method for preparing high-purity cryptoprotein. The method comprises the steps of (1) sequentially connecting a coding gene of a target protein with a TEV protease and a T4 phage Soc protein gene fragment to prepare a fusion recombinant protein coding gene; (2) transferring the gene encoding the fusion recombinant protein into a plasmid; (3) transferring the plasmid into abacterium coli host; (4) infecting the bacterium coli host with a Soc-deficient Soc-T4 phage, and cultivating the host to express the T4 phage displaying the fusion recombinant protein; (5) isolatingthe T4 phage displaying the fusion recombinant protein; (6) isolating the fusion recombinant protein from the T4 phage, and obtaining the target protein by TEV protease digestion. The method can obtain the high-purity cryptoprotein easily and quickly, an obtained cryptoprotein solution can be directly used as an agricultural antibacterial bacteriostatic agent to achieve the effect of promoting plant growth, and the method has a good application prospect.

The invention relates to a method for efficiently expressing hypersensitive protein by using a T4 phage display technology. The method comprises the following steps: (1) sequentially connecting an encoding gene of the hypersensitive protein with TEV protease and a T4 bacteriophage Soc protein gene segment to prepare a fusion recombinant protein encoding gene, wherein a encoding region of the hypersensitive protein is located at the upstream of a TEV-Soc fusion protein gene and having consistent expression cassettes; (2) transferring the coding gene of the fusion recombinant protein into a pUC18 plasmid; (3) transferring the plasmid into an escherichia coli host; (4) infecting the escherichia coli host by using Soc-T4 bacteriophage without Soc protein, and culturing the T4 bacteriophage ofwhich the host expresses and displays the fusion recombinant protein; (5) separating the T4 bacteriophage showing the fusion recombinant protein; and (6) separating the fusion recombinant protein fromthe T4 bacteriophage, and carrying out TEV protease digestion to obtain the hypersensitive protein.

The invention discloses a method for improving the titer of a T4 bacteriophage through cooperation of a high-voltage electrostatic field and 1,3-diglyceride and collagen peptide. The titer of the T4 bacteriophage is improved by adding the 1,3-diglyceride and the collagen peptide and culturing the T4 bacteriophage under the high-voltage electrostatic field. The T4 bacteriophage cultured by the invention improves the bacteriophage effect on Escherichia coli AMC 198, is expected to replace antibiotics, has a wider application prospect in the field of bacteriophages, and has important significance in prevention and treatment of bacterial pollution in the fields of clinic, food and agriculture.

The invention provides a chemiluminescence biosensor for detecting 5-hydroxymethylcytosine, a detection method and application thereof, and belongs to the technical field of molecular detection. The chemiluminescent biosensor of 5-hydroxymethylcytosine includes T4 bacteriophage β-glucosyltransferase, uridine diphosphate glucose, sodium periodate, aldehyde group reaction probe ARP, terminal transferase, hemoglobin and luminol solution. The hydroxymethylcytosine-glycosylation, periodate oxidation, biotinylation-isothermal signal amplification strategy designed in the present invention does not need to change the reaction temperature, nor does it require a special labeled nucleic acid probe or a specific template for signal amplification , which can detect 5hmC of any sequence in genomic DNA and does not involve isotopic labels or specific antibodies, eliminating radioactive hazards and misinterpretation of genome-wide mapping data in antibody-based experiments. This enables genome-wide analysis of 5hmC in a limited number of biological and clinical samples.

The present invention relates to a recombined T4 bacteriophage for expressing cholecystokinin gene, the recombined T4 bacteriophage has chicken cholecystokinin 33 peptide gene or nucleotide acid in series. This invention offers chicken cholecystokinin 33 peptide or amino acid residue sequence in series, utilizes recombined T4 bacteriophage to carry active immunity technology and application for animal.