CN101059515A - Use of phage chip as protein chip or gene chip - Google Patents
Use of phage chip as protein chip or gene chip Download PDFInfo
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Abstract
The invention discloses an application for using bacteriophage chip as protein chip or gene chip, which selects bacteriophage peptide base or builds a bacteriophage display base, to obtain the bacteriophage clone of special external peptide (or antigen epitope), uses automatic or non-automatic method, to directly couple the bacteriophage with different external peptides according to preset array mode to one chip, to obtain the bacteriophage chip. The inventive chip can be a protein chip for showing external peptide, used to check, select and identify the special combine material in the antigen, ligand or natural or artificial compound in the samples, or the gene chip for cloning relative genes, to check the gene. The inventive bacteriophage chip has low cost, simple preparation, high stability, high specificity and long service life.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to the preparation and the application thereof of bacteriophage chip, particularly show the preparation and the application thereof of the bacteriophage biochip of exogenous antigen peptide.The present invention will show that the different bacteriophages of exogenous peptide is fixed on the chip slapper base according to the array way that designs, obtain the bacteriophage chip, this chip both can be used as the protein chip of showing exogenous peptide, be used for detection, evaluation and the screening of specificity junction mixture among the various sample antibody of human body or animal used as test, part and natural or the synthetic material, also can be used as genetic chip, be used for detection, evaluation and the screening of specific gene.
Background technology
Biochip mainly is meant by little processing and microelectric technique and makes up the miniature organism chemical analysis system at the solid chip surface, with realize to tissue, cell, protein, nucleic acid, carbohydrate and the other biological component of viable organism carry out accurately, the detection of quick, large information capacity.Common biochip is divided three classes: the first kind is a micro-array chip, comprise genetic chip (Gene chip, DNA chip, DNAmicroarray), protein chip (Protein chip), cell chip and organization chip (cell chip andtissue chip, cell microarray and tissue microarray); Second class is micro-fluidic chip (microfluid) (belonging to active chip), comprises all kinds of specimen preparation chips, polymerase chain reaction (PCR) chip, capillary electrophoresis chip and chromatogram chip etc.; The 3rd class is integrated analytic system or title chip lab (Lab-on-a-chip) based on biochip.It is the up-to-date successes achieved in research in fields such as set molecule biology, immunology, semiconductor, microelectronics, laser, chemical dye in the forward position of one new and high technology, it uses the field of almost having contained all biotechnologys such as biology, medical science, pharmacy, agronomy, environmental protection, legal medical expert, hygiene.The unexpected emergence of biochip technology and the huge commercial value that will bring thereof and application prospect have caused the extensive concern and the attention of countries in the world, are becoming 21 century one of the most potential biological industry.The most ripe, and widely used at present is genetic chip, still, realizes this and executor because protein is the real function of various biological phenomenas, and therefore, protein chip will have more wide application prospect.But the preparation how high flux obtains, the various protein molecule of purifying is used for protein chip still exists many technical bottlenecks and the technology barrier that is difficult to overcome.In addition,, protein is coupled to the space structure that might destroy protein on the chip, makes the protein loss of activity because protein has comparatively complicated spatial structure feature.Especially for molecular weight＞10
5The protein that dalton and structure are complicated, difficulty is bigger; Moreover in the time of on the same chip slapper base in protein molecule coupling road that a large amount of physics are different with chemical property, the treatment technology of chip slapper base also is essential problem and the technological difficulties of considering.Just because of this, with physicochemical property are basic identical and the antibody coupling that biologically active is different is lower in the technical difficulty that same chip slapper base prepares the protein antibody chip, and outside the development rapidly, the development of protein-chip still faces many technical matterss.Therefore, the novel biochip of research and development is extremely important.
1985, Smith at first proposed phage display (phage display) technology forefathers on to the basis of filobactivirus molecular biology research.Its ultimate principle is by utilizing chemosynthesis, random mutation, enzyme to cut the dna fragmentation of setting up coded polypeptide, albumen or antibody library with methods such as PCR, exogenous dna fragment is inserted among phage encoded protein gene PIII or the PVIII, make the expression product fusion of exogenous dna fragment correspondence in the coat protein of bacteriophage, form fusion, be presented on phage surface, collect then this recombinant phage in the host bacterium the amplification supernatant and make up.This technology is incorporated into such one of filobactivirus dexterously with the phenotype of protein molecule and genotype and is convenient to it is carried out on the carrier of a series of biochemistry and genetic manipulation, thereby make fusion be illustrated in the outside of phage particle, the DNA that coding merges residue then is positioned at phage particle inside.This physics between display protein and its coding DNA is got in touch and is made us to screen a large amount of protein variant strains by a kind of simple " biological elutriation ", by further Function Identification obtain can with the phage clone of target molecule specific bond.Just concerning one to one according to phage clone institute's displayed polypeptides or albumen and corresponding encoded gene can be clearly and the genotype and the phenotype characteristics of the corresponding part of target molecule.And each albumen can both connect by its corresponding dna sequence dna.
In recent years, Zhu Shenggeng leader's seminar is by the screening to phage antibody library, the bacteriophage of showing different single-chain antibodies is coupled on the silicon chip, preparation phage antibody chip (phage antibodychip) is used for the identification and the proteomics research (Hong Long of specific antigen, Liao Wei, Wei Fang, Zhao new life, Zhu Shenggeng. be used to discern the phage antibody chip of different cell protein groups. Acta PhySico-Chimica Sinica .2004; 20 (10): 1182-1185; Liao Wei, Hong Long, Wei Fang, Zhu Shenggeng, Zhao new life. the display systems of utilization improves the phage antibody chip. Acta PhySico-Chimica Sinica .2005,21 (5): 508-511; Cen Xiaodong, WANG WENJUAN, Zhao new life, Wang Xuan, love and respect one's elder brother in Shen, Tan Taochao, Bi Qun, Zhu Shenggeng. the identification of high density phage antibody chip pair cell surface protein. Acta PhySico-Chimica Sinica .2006,22 (7): 777～779).Although, the phage antibody chip, be different from traditional protein antibody chip, utilize the alternative traditional monoclonal antibody of the bacteriophage of showing antibody fragment to prepare the phage antibody chip and have a lot of advantages: 1. by affine elutriation phage antibody library, acquisition need not immune animal at the phage antibody of various antigens; 2. obtain phage antibody by the monoclonal amplification, avoided the monoclonal antibody preparation and the purge process of complex steps; 3. direct interference (Arnaud, the M.C. of chip surface antagonist determining area have been avoided; Gazarian, T.; Rodriguez, Y.P.; Gazarian, K.; Sakanyan, V.Proteomics, 2004,4:1959.Cekaite, L.; Haug, O.; Myklebost, O.; Aldrin, M.; Ostenstad, B.; Holden, M.; Frigessi, A.; Hovig, E.; Sioud, M.Proteomics, 2004,4:2572.).But, the phage antibody chip is the same with the protein antibody chip, also can only be used for detecting and the screening specific antigen, can not be used for detection, evaluation and the screening of specificity junction mixture among the various sample antibody of human body or animal used as test, part and natural or the synthetic material, can not be used for detection, evaluation and the screening of specific gene as genetic chip.Therefore, development can be used for the detection and the screening bacteriophage chip of specific antibody, part, still is significant and real necessity.
Phage peptide library is a very important branch of display technique of bacteriophage.Nineteen ninety Scatt (Scott JK, Smith GP.Searching for peptide ligands with an epitopelibrary.Science, 1990; 249 (1): 386-390.) design and set up phage random peptide library (phage random peptide library).This phage peptide library is by inserting the gene order of different small peptides among the phage single-chain DNA, the N end of the surperficial coat protein of the external source random series polypeptide of expression and filobactivirus is merged form fusion with certain space conformation, be presented in phage surface, and can be used as the simulating peptide (minotope) of any target epitope.Although peptide section sequence at random and natural molecule that phage random peptide library is showed there is no similarity, still have function or the antigenicity similar with natural molecule.As filtering out the little peptide of simulation ring-type (U.S.Patent No.5 the small peptide storehouse at random from phage surface, 835,382), though the sequence of its sequence and natural EPO does not also have similarity, but it can combine with EPO acceptor high affinity really, and the function of simulation hematopoietin (EPO).For a long time, the phage peptide library technology is because of its peculiar advantage in the analysis of epitope, in the research of gene vaccine, polypeptide vaccine and recombinant vaccine, bringing into play enormous function (Matthew A.S, Deborah S, Kenichi M, et al.HSV-1 amplicon peptidedisplay vector.J Vir Meth.2002; 107:71-79).At present, the diversity at random of phage random peptide library can reach 10
7～10
9Small peptide length develops into 9 peptides, 15 peptides by 6 initial peptides, 7 peptides, up to 38 peptides, 40 peptides, phage random peptide library is one of peptide storehouse most widely used in the present phage display peptide library (Ming-juan Zhang, Jun Yang, Zhuo-ren Lu.A New OuabainConjugated Peptide Was Found From Phage Displayed Peptide Library.Am J Hypert.2004,17:619-623; Zhang Mingjuan, Yang Jun, Lv Zhuoren. the screening of phage peptide library. clinical examination magazine, 2002; 20 (4): 247-248).
Summary of the invention
The object of the present invention is to provide a kind of new purposes of bacteriophage chip, the bacteriophage that particularly will show different exogenous peptides is directly by people or unartificial method, array pattern according to design in advance is coupled to preparation bacteriophage chip on the same chip slapper base, this bacteriophage chip both can be used as protein chip, be used for detection, evaluation and the screening of specificity junction mixture among the various sample antibody of human body or animal used as test, part and natural or the synthetic material, also can be used as genetic chip, be used for detection, evaluation and the screening of gene.
For realizing above-mentioned task, the present invention adopts display technique of bacteriophage, screening phage peptide library or structure phage display storehouse, obtain to show the phage clone of specificity exogenous peptide (or epitope), utilize automatic or non-automatic mode, array pattern according to design will show that the bacteriophage of different exogenous peptides directly is coupled on the same chip slapper base, obtain the bacteriophage chip.This chip both can be used as the protein chip of showing exogenous peptide, be used for detection, screening and the evaluation of specificity junction mixture among the various sample antibody of human body and animal used as test, part and natural or the synthetic material, also can be used as the genetic chip of clone's related gene, be used for detection, evaluation and the screening of gene.
Novelty part of the present invention is: the antibody that 1. screens phage peptide library can be directly from the patient, need not express, the purifying corresponding antigens, do not need immune animal to prepare antibody yet, do not knowing in advance to diagnose with under the situation of target antigen, use the patients serum from the antigenic determinant storehouse, to filter out the bacteriophage of showing the specific antigen determinant, this bacteriophage can only with patients serum's antibody response, and can not with normal person's seroreaction, thereby set up the specificity diagnostic method of disease; 2. utilize display technique of bacteriophage, by affine elutriation to phage random peptide library or antigenic storehouse, can obtain to show the bacteriophage of relative specific exogenous peptide (epitope), by simple monoclonal amplification, can obtain a large amount of recombinant phages by high flux, simultaneously, phage particle is stable, be easy to long preservation, avoided complex steps and the protein expression of complexity and the degraded of purge process and protein; 3. because all show that the bacteriophage of different exogenous peptides all has identical genetic background, thereby be fixed on the chip slapper base by identical coupling mode, at utmost reduce the intractability of chip slapper base, be beneficial to the optimization of bacteriophage chip preparation condition and the control of standardized operation and chip quality; 4. adopt the bacteriophage of showing different exogenous peptides to substitute traditional protein molecular and prepare biochip, avoided the direct interference of the antagonism protein molecular of chip surface, the architectural feature and the biologically active that have kept the display protein molecule to the full extent, reduce the space steric effect of combination between the Ag-Ab (receptor-ligand), increased substantially the sensitivity and the signal to noise ratio (S/N ratio) of bacteriophage chip; 5. display technique of bacteriophage is incorporated into this microbe carrier of filobactivirus with the phenotype and the genotype of protein molecule, with each peptide species or protein with the formal representation of fusion and be illustrated in phage surface, simultaneously its genetic code is integrated in phage genome, thereby both can be used as the protein chip of showing exogenous peptide, be used for detection, screening and the evaluation of specificity junction mixture among the various sample antibody of human body and animal used as test, part and natural or the synthetic material, also can be used as the genetic chip of clone's related gene, be used for genetic test.
Therefore, the present invention has broad application prospects and market.
Description of drawings
Fig. 1 shows the bacteriophage chip detection mouse anti HPVl6L1VLP antiserum of anti-HPVl6 L1 epitope mimic peptide.
The present invention is described in further detail below in conjunction with embodiment that accompanying drawing and inventor provide.
Embodiment
The present invention utilizes display technique of bacteriophage, by screening phage peptide library or bacteriophage antigenic storehouse, obtain to show the phage clone of specificity exogenous peptide (or epitope), to show that the bacteriophage of different exogenous peptides is directly by artificial or unartificial method, array pattern according to design in advance is coupled on the same chip slapper base and prepares biochip, might become the diverse new bio chip of a class and traditional genetic chip and protein chip---bacteriophage chip (phage chip or phagemicroarray), this chip both can be used as the protein chip of showing exogenous peptide, be used for the various sample antibody of human body or animal used as test, the detection of specificity junction mixture among part and natural or the synthetic material, identify and screening, also can be used as genetic chip, be used for the detection of gene, identify and screening.
1. show the screening and the evaluation of the phage clone of specificity exogenous peptide or epitope
1.1 adopt specific antibody or serum (can from patient or animal used as test), the albumen or the non-protein matter of perhaps various natural or synthetic.Method by simple " absorption-wash-out-amplification ", screening phage random peptide library (phage random peptide library), picking monoclonal bacteriophage, through extraction, order-checking and the homology analysis of amplification, DNA, the phage clone preservation of definite displaying specificity exogenous peptide or epitope is standby, and (method sees Ph.D.-12 for details
TMPhage DisplayPeptide Library Kit instructions).
1.2 obtain the target protein gene order by PCR equimolecular clone technology, be inserted into phage display system (as strand filobactivirus display systems, bacteriophage lambda display systems, T4 phage display system etc.), make up phage display storehouse (concrete grammar is referring to " molecular cloning ").Conventional method prepares the antiserum or the antibody of this albumen, method by " absorption-wash-out-amplification ", screen the phage display storehouse (phage display library) of above-mentioned structure, picking monoclonal bacteriophage, perhaps direct picking monoclonal bacteriophage at random, and, determine to show that the phage clone of specific antigen epi-position is preserved standby (method is the same) through extraction, order-checking and the homology analysis of amplification, DNA.
2. the preparation of bacteriophage chip:
Adopt artificial or unartificial method the displaying specificity exogenous peptide of above-mentioned acquisition or the bacteriophage of epitope to be coupled on the different chip slapper bases according to the array pattern that designs with relevant device, as different microballons such as microslide, silicon chip, ceramics, nylon membrane, pvdf membrane, NC film, polyacrylamide gel or magnetic bead, agaroses, can obtain the bacteriophage chip.Simultaneously, said chip sheet base can pass through or without different activation processing such as aldehyde radicalization, sulfhydrylation, amination or epoxy radicals, be beneficial to bacteriophage is presented in microslide on the chip slapper base in various possible modes surface.
3. the application of bacteriophage chip:
3.1 bacteriophage chip provided by the present invention can be used as the protein chip of showing exogenous peptide or epitope, be used for detection, screening and the evaluation of ligands specific among the various sample antibody of human body or animal used as test, part and natural or the synthetic material or bond, concrete grammar is as follows:
3.1.1 the application of bacteriophage chip in antibody test:
(1) conventional method is sealed the bacteriophage chip for preparing, add dilution good human body or animal used as test antiserum, antibody, body fluid or tissue extract, 4 ℃ are spent the night or incubated at room, adopt the flushing of PBS damping fluid, the second antibody that adds fluorescence or non-fluorescent label, hatch for 37 ℃, put into the chip reading apparatus after the flushing of PBS damping fluid and carry out interpretation of result.
(2) carry out fluorescence or non-fluorescent label according to conventional method to containing detected antibody.The bacteriophage chip that sealing prepares, containing that adding is diluted according to a certain percentage is labeled antibody, and 4 ℃ are spent the night or incubated at room, adopt the flushing of PBS damping fluid, hatch for 37 ℃, put into the chip reading apparatus after the flushing of PBS damping fluid and carry out interpretation of result.
3.1.2 the application of bacteriophage chip in part detects:
To ligands specific or bond among the human body that contains detected part or experimental animals serum, body fluid, tissue extract and natural or the synthetic material, carry out fluorescence or non-fluorescent label according to conventional method.The bacteriophage chip that conventional method sealing prepares adds the extract that is labeled part that contains of dilution according to a certain percentage, 4 ℃ spend the night or 37 ℃ hatch, put into the chip reading apparatus after the flushing of PBS damping fluid and carry out interpretation of result.
3.1.3 the application of bacteriophage chip in ligand screening
(1) conventional method is sealed the bacteriophage chip for preparing, ligands specific or bond among the human body that contains detected part of adding dilution according to a certain percentage or experimental animals serum, body fluid, tissue extract and natural or the synthetic material, 4 ℃ are spent the night or incubated at room or 37 ℃ are hatched, the flushing of PBS damping fluid, adopt surface-enhanced laser desorb/ionization-flight time mass spectrum (SELDI-TOF-MS) technology, carry out ligand screening.
(2) conventional method is sealed the bacteriophage superbead chip (phage Bead Array) for preparing, ligands specific or bond among the human body that contains detected part of adding dilution according to a certain percentage or experimental animals serum, body fluid, tissue extract and natural or the synthetic material, 4 ℃ are spent the night or incubated at room or 37 ℃ are hatched, the flushing of PBS damping fluid, conventional method is separated microballon, carries out ligand screening.
3.2 bacteriophage chip provided by the present invention can be used as the genetic chip of integrating target gene, is used for detection, evaluation and the screening of human body or animal used as test blood, body fluid, secretion, tissue extract or synthetic gene (as DNA or RNA).
3.2.1 be used for detection, the evaluation of human body or animal used as test blood, body fluid, secretion, tissue extract or synthetic gene (as DNA or RNA).
Conventional method extraction human body or animal used as test blood, body fluid, secretion, tissue extract or synthetic gene are (as DNA or RNA.If behind the mRNA, reverse transcription is cDNA, then cDNA is cut into the long fragment of 200～600bp, and screening cDNA3 ' holds, and obtains the cDNA of q.s with the method for pcr amplification).Adopting radio-labeled technology or fluorescence labeling technology to carry out the mark of gene, the detection of genetic chip is carried out in mass spectrophotometry or chemiluminescence etc.
Below be the embodiment that the inventor provides, need to prove that following embodiment only is used for understanding, describing the present invention, the present invention is not limited to these embodiment.
The plasmid that adopts among the embodiment, phage random peptide library and main agents are as follows:
Phage random dodecapeptide storehouse (Ph.D.12
TMPhage Display Peptide Library) and E.coli ER2738 (Cat.8110S) available from New England Biolabs company; Protein G MagneticBeads is available from New England BioLabs company; HPV-16 L1 monoclonal antibody is available from Neomarker company; HRP-IgG is available from DAKO company; Cy3 dUTP/Cy5 dUTP is available from U.S. Amersham Pharmacia company.
The total RNA extraction reagent box available from the German Qiagen .Gen III Microarray Spotter of company chip point sample instrument available from Amersham Pharmacia company; Constant temperature hybridization case is available from Shellab company; Chip scanner is available from Amersham Pharmacia company; Polymerase chain reaction (PCR) instrument is available from MJResearch company; ScanArray 3000 laser confocal scanning instrument, General Scanning Inc; PTC-100 pcr amplification instrument, MJ Research Inc; UV-2000 ultraviolet transmission analyser, the sky, Shanghai can Science and Technology Ltd..
The configuration of all ingredients that relates in the present embodiment and method are referring to " molecular cloning ", " ProBondTM purification system instructions ", " the New England BioLabs Ph.D.-12 of company
TMPhageDisplay Peptide Library Kit instructions " etc.
Embodiment 1: the preparation of bacteriophage chip
1. show screening and the evaluation of the bacteriophage of exogenous peptide or epitope
Method 1: the screening of phage random peptide library and evaluation
(1) serum, antibody, body fluid or the tissue extract of conventional method collection and separation human body or animal used as test.
(2) " biological elutriation " method screening phage random peptide library: get the phage peptide library (1.0 * 10 after amplification and the dilution
11Pfu) and the normal rabbit igg solution of 300ng to 2ml in conjunction with in the liquid, 37 ℃, 50rpm is hatched in vibration, and 1h adsorbs in advance, adds the magnetic bead 50 μ l of Protein G mark, 37 ℃, after 50rpm 20min is hatched in vibration, magnetic force absorption 2min, get supernatant, with 1/6 volume 2.5M PEG/NaCl precipitation, 200 μ lTBS dissolving, join 2ml and contain the human body of 300ng antibody or the serum of animal used as test, antibody, body fluid or tissue extraction in conjunction with in the liquid, 37 ℃, hatch 1h, the magnetic bead 50 μ l that add the Protein G mark, 37 ℃, 50rpm is hatched in vibration, 20min, magnetic force absorption 2min, TBST (TBS+0.1%Tween-20) flushing, 5min * 3 time, add 100 μ l 0.2Mglycine-HCl (pH2.2), 1mg/mlBSA jiggles 10min, magnetic force absorption 5min, collect eluent, add 1M Tris-HCl (pH9.1) neutralization, detect phage titre, leave and take 20 μ l and be used for preserving, all the other eluents are joined in the 20ml ER2738 nutrient solution (having reached exponential phase), at 37 ℃, 230rpm is hatched 4.5h.1/6 volume 2.5M PEG/NaCl precipitation, 200 μ l TBS dissolving is the bacteriophage of having increased, detects phage titre, 4 ℃ of preservations.Get equivalent bacteriophage (1.0 * 10
11Pfu) repeat above-mentioned screening process 3 times, (method sees Ph.D.-12 for details to detect the phage titre of the 3rd eluent
TMPhage Display Peptide Library Kit instructions).
(3) fast purifying bacteriophage sequencing template and order-checking: with aseptic peg wood, dip in the blue bacteriophage bacterial plaque of getting in the double-deck agar double dish of measuring the 3rd eluent phage titre at random, input contains in the ER2738 nutrient solution that 5ml reached exponential phase, at 37 ℃, concuss is hatched 230rpm, 4.5h.Get supernatant and centrifugal once more after centrifugal, get 80% supernatant, (method sees Ph.D.-12 for details to NaI method fast purifying bacteriophage sequencing template
TMPhage Display Peptide Library Kit instructions), agarose gel electrophoresis detects, the promise bio tech ltd order-checking of Shanghai China.
(40) sequential analysis: adopt the Blastn of NCBI and Blastp to analyze, and determine to show the phage clone of specificity exogenous peptide or epitope through homology analysis.
Method 2: the structure in phage display storehouse and screening
(1) structure in specific proteins phage display storehouse: obtain the target protein gene order by PCR equimolecular clone technology, be inserted into strand filobactivirus display systems, make up phage display storehouse (concrete grammar is referring to " molecular cloning ").
(2) screening of phage clone: conventional method prepares the antiserum or the antibody of this albumen, method by " absorption-wash-out-amplification ", screen the phage display storehouse (phagedisplay library) of above-mentioned structure, picking monoclonal bacteriophage, perhaps direct picking monoclonal bacteriophage at random, through extraction, order-checking and the homology analysis of amplification, DNA, determine to show the phage clone preservation standby (method is the same) of specific antigen epi-position.
2. the preparation of bacteriophage chip
Adopt artificial or unartificial method the displaying specificity exogenous peptide of above-mentioned acquisition or the bacteriophage of epitope to be coupled on the different chip slapper bases according to the array pattern that designs with relevant device, as different microballons such as microslide, silicon chip, ceramics, nylon membrane, pvdf membrane, NC film, polyacrylamide gel or magnetic bead, agaroses, can obtain the bacteriophage chip.Simultaneously, said chip sheet base can pass through or without different activation processing such as aldehyde radicalization, sulfhydrylation, amination or epoxy radicals, be beneficial to bacteriophage is presented in microslide on the chip slapper base in various possible modes surface.Method is as follows: adopt the chip point sample instrument that the solution 1 μ l of the bacteriophage of the displaying specificity exogenous peptide of above-mentioned process evaluation is coupled on the NC film according to the array pattern that designs in advance, and establish negative control.
Embodiment 2: the application of bacteriophage chip in antibody test
The bacteriophage chip that method 1:5% skimmed milk room temperature sealing 2h prepares, the test sample to be checked that adds dilution in 1: 500, this incubated at room 2h, TBST (TBS that contains 0.5%Tween-20) washes, 10min * 3, the goat anti-rabbit igg incubated at room 20min of the HRP mark of dilution in 1: 1000, TBST washes 10min * 3, the DAB colour developing.Can detect the specific antibody (Fig. 1) in the sample.
Method 2: the antibody in the conventional method separating sample, adopt the Cy3 fluorescent marker method to carry out the mark (Cy3 fluorescent labeling reagent box is available from Pharmacia company) of antibody.5% skimmed milk room temperature sealing bacteriophage chip, the test sample to be checked of adding Cy3 mark, this incubated at room 2h, TBST (TBS that contains 0.5%Tween-20) washes, 10min * 3, fluorescent scanning instrument sentence read result.
Embodiment 3: the application of bacteriophage chip in part detects
Adopt part in conventional method preparation, the separating sample, adopt the Cy3 fluorescent marker method to carry out holoprotein mark (Cy3 fluorescent labeling reagent box is available from Pharmacia company).5% skimmed milk room temperature sealing bacteriophage chip, the test sample to be checked of adding Cy3 mark, this incubated at room 2h, TBST (TBS that contains 0.5%Tween-20) washes, 10min * 3, fluorescent scanning instrument sentence read result.
Embodiment 4: the application of bacteriophage chip in ligand screening
Method 1: the bacteriophage chip that the conventional method sealing prepares, ligands specific or bond among the human body that contains detected part of adding dilution according to a certain percentage or experimental animals serum, body fluid, tissue extract and natural or the synthetic material, 4 ℃ are spent the night or incubated at room or 37 ℃ are hatched, the flushing of PBS damping fluid, adopt surface-enhanced laser desorb/ionization-flight time mass spectrum (SELDI-TOF-MS) technology, carry out ligand screening.
Method 2: the bacteriophage superbead chip (phage Bead Array) that the conventional method sealing prepares, ligands specific or bond among the human body that contains detected part of adding dilution according to a certain percentage or experimental animals serum, body fluid, tissue extract and natural or the synthetic material, 4 ℃ are spent the night or incubated at room or 37 ℃ are hatched, the flushing of PBS damping fluid, conventional method is separated microballon, carries out ligand screening.
Embodiment 5: the application of bacteriophage chip in genetic test
Conventional method is separated human body or the stripped flesh tissue sample of animal used as test, is cut into a plurality of 1cm
3Fritter, rinsing sample in RNase-Free 0.9% physiological saline, behind the mRNA, reverse transcription is cDNA in the extraction sample, then cDNA is cut into the long fragment of 50～600bp, screening cDNA3 ' holds, and uses the cDNA of the method acquisition q.s of pcr amplification.
With reference to Draghici method (Draghici S, Kulaeva O, HoffB, Petrov A, Shams S, Tainsky MA.Noise sampling method:an ANOVA approach allowing robustselection of differentially regulated genes measured by DNA microarrays.Bioinformatics.2003; 19:1348-1359.) row cDNA single-stranded probe and purifying (Cy3 fluorescent labeling reagent box is available from Pharmacia company).With Cy3 dUTP mark normal structure mRNA, with Cy5dUTP marker samples tissue mRNA. concrete grammar is: the cDNA clone solution of getting 60 times of dilutions is as template, add primer and 10 * reaction buffer, 5 μ l, the dATP of 10nmol, dCTP, the dTTP of dGTP and 5nmol, Cy3-dUTP 1 μ l and the 3U Tag enzyme of 1mmol/L, adding water accent final volume is 50 μ l.Adopt the thin walled tube of 0.2ml, by 94 ℃, 5min, (94 ℃, 40s; 55 ℃, 40s; 72 ℃, 90s) * 30,72 ℃, the amplification of the amplification program of 5min obtains the purpose band, uses PCR purification kit purifying amplified production then, and product finally is dissolved in 5 an amount of * SSC, and in the 0.2%SDS damping fluid, final concentration is about 50ng/ μ l.
The fluorescence content of certification mark.Detect 260nm with spectrophotometer, 550nm, the A value during 650nm calculates fluorescence content then.The probe that mark is good (Cy3/Cy5) is dissolved in the hybridization buffer of 30 μ L by the equivalent mixing.Place 95 ℃ of distilled waters to bathe sex change 5min respectively chip and hybridization mixed probe, mixed probe is added on the bacteriophage chip immediately, place 65 ℃ of hybridization of wet box 4h, put 2 * SSC then, wash 5min in the 0.1%SDS solution, 0.1 * SSC washes 5min in the 0.1%SDS solution, wash several times 0.1 swing in * SSC the solution, dry.ScanArray3000 laser confocal scanning instrument scans under proper condition and detects laggard line data analysis of its hybridization signal and processing.
Claims (8)
1. the bacteriophage chip is used for the application of detection, evaluation and the screening of specificity junction mixture among human body or the various sample antibody of animal used as test, part and natural or the synthetic material as protein chip, or is used for the application of specific gene detection as genetic chip.
2. application as claimed in claim 1 is characterized in that, the preparation that is used for the bacteriophage chip can obtain by following approach:
Adopt specific antibody or part method by conventional absorption, wash-out, amplification, the screening phage random peptide library, picking monoclonal bacteriophage, and, determine to show the phage clone of specificity exogenous peptide or epitope through extraction, order-checking and the homology analysis of amplification, DNA;
Adopt display technique of bacteriophage, by making up the phage display storehouse of showing specific proteins fragment or small peptide, picking monoclonal bacteriophage, and through extraction, order-checking and the homology analysis of amplification, DNA, determine to show the specificity exogenous peptide or bacteriophage.
3, application as claimed in claim 1 is characterized in that, the bacteriophage of showing different exogenous peptides is coupled on the chip slapper base according to the array pattern that designs in advance by artificial or unartificial method, makes the bacteriophage chip.
4, application as claimed in claim 1, it is characterized in that, described bacteriophage chip slapper base is microslide, silicon chip, ceramics, nylon membrane, pvdf membrane, NC film or polyacrylamide gel, perhaps magnetic bead, the different microballon of agarose, and said chip sheet base through or without the different activation processing of aldehyde radicalization, sulfhydrylation, amination or epoxy radicalsization.
5, application as claimed in claim 1 is characterized in that, comprises 1 or 1 above phage clone on the described bacteriophage chip at least.
6, application as claimed in claim 1 is characterized in that, described bacteriophage chip is used for detection, evaluation and the screening of human body or animal used as test blood, body fluid, secretion, tissue extract specific antibody or part.
7, application as claimed in claim 1 is characterized in that, described bacteriophage chip is used for detection, evaluation and the screening of natural or synthetic material's ligands specific or bond.
8, application as claimed in claim 1 is characterized in that, described bacteriophage chip is used for detection, evaluation and the screening of human body or animal used as test blood, body fluid, secretion, tissue extract or synthetic gene.
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| CN2007100178358A CN101059515B (en) | 2007-05-14 | 2007-05-14 | Use of phage chip as protein chip or gene chip |
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| CN2007100178358A CN101059515B (en) | 2007-05-14 | 2007-05-14 | Use of phage chip as protein chip or gene chip |
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| CN101059515B CN101059515B (en) | 2012-02-01 |
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| CN2007100178358A Expired - Fee Related CN101059515B (en) | 2007-05-14 | 2007-05-14 | Use of phage chip as protein chip or gene chip |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102305853A (en) * | 2011-07-20 | 2012-01-04 | 龙岩九健生物芯片技术研究所 | Method for showing antigen clone biological marker of phage |
| CN102466729A (en) * | 2010-11-05 | 2012-05-23 | 北京工业大学 | Method for screening tumor specificity target and targeting ligand based on tissue chip |
| CN108802381A (en) * | 2018-06-13 | 2018-11-13 | 河南中泽生物工程有限公司 | Bovine viral diarrhea virus differentiates test strip and preparation method thereof |
| CN113176310A (en) * | 2021-03-18 | 2021-07-27 | 厦门大学 | Automatic phage panning platform based on digital microfluidic technology |
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| CN106636068B (en) * | 2016-12-28 | 2019-06-07 | 浙江大学 | A kind of screening technique of fibroin affinity peptide |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004089069A (en) * | 2002-08-30 | 2004-03-25 | Mitsubishi Chemicals Corp | Method for producing multicopy displaying phage |
| CN1217000C (en) * | 2003-07-30 | 2005-08-31 | 中国药科大学 | Exhibiting method of preparing antibody by antigen utilizing bacteriophage exhibiting technique |
| US7858323B2 (en) * | 2004-06-09 | 2010-12-28 | The Regents Of The University Of Michigan | Phage microarray profiling of the humoral response to disease |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102466729A (en) * | 2010-11-05 | 2012-05-23 | 北京工业大学 | Method for screening tumor specificity target and targeting ligand based on tissue chip |
| CN102466729B (en) * | 2010-11-05 | 2015-06-17 | 北京工业大学 | Method for screening tumor specificity target and targeting ligand based on tissue chip |
| CN102305853A (en) * | 2011-07-20 | 2012-01-04 | 龙岩九健生物芯片技术研究所 | Method for showing antigen clone biological marker of phage |
| CN102305853B (en) * | 2011-07-20 | 2013-11-06 | 龙岩九健生物芯片技术研究所 | Method for showing antigen clone biological marker of phage |
| CN108802381A (en) * | 2018-06-13 | 2018-11-13 | 河南中泽生物工程有限公司 | Bovine viral diarrhea virus differentiates test strip and preparation method thereof |
| CN113176310A (en) * | 2021-03-18 | 2021-07-27 | 厦门大学 | Automatic phage panning platform based on digital microfluidic technology |
| CN113176310B (en) * | 2021-03-18 | 2023-10-20 | 厦门大学 | Automatic phage panning platform based on digital microfluidic technology |
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| CN101059515B (en) | 2012-02-01 |
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