CN1512176A - Protein chip and its preparing method and use - Google Patents
Protein chip and its preparing method and use Download PDFInfo
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- CN1512176A CN1512176A CNA021597626A CN02159762A CN1512176A CN 1512176 A CN1512176 A CN 1512176A CN A021597626 A CNA021597626 A CN A021597626A CN 02159762 A CN02159762 A CN 02159762A CN 1512176 A CN1512176 A CN 1512176A
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Abstract
The present invention relates to marketable high-density protein chip in completely new concept and comprising substrate layer, specific affinity layer and biological macromolecular layer. The protein chip may have protein, antibody or artificial short peptide dots over 1000-100000 in 10 sq cm. The present invention also relates to the process of forming high density precursor protein chip with gene transcription segment capable of performing in-situ geen transcription and translation with chip preparing instrument, and then preparing high density protein chip with no-cell protein expression system. The present invention promotes the practical use and commercialization of protein chip technology and will find its wide application in clinical diagnosis, medical research, medicine development and inspection, protein block research and other fields.
Description
Technical field
The present invention relates to a kind of protein-chip and its production and application, relate in particular to the acellular outer synthetic method of aleuroplast settle at one go of exsomatizing prepare protein-chip with and in clinical diagnosis, medical research, new drug development and field related application such as check, proteome research.
Background technology
The genome of and other a large amount of species determined in human genome DNA's sequence is by under the background of progressively rapid test, extremely urgent task is the function that efficient, quick and practical method of development one cover or technology are studied the coded protein of genome at present, with the comprehensive complete understanding of each bioprocess and approach in the acquisition pair cell.Little array technology has been given play to it as a kind of method efficiently, comprehensively and efficiently at present and has distinctively been obtained effect a large amount of, panel data fast in fields such as research gene expression, gene mutation and gene transcription regulations.Because protein than mRNA further, study a large amount of protein so research and develop the little array technology of protein at present, even the function of whole protein group has become the biological forward position of extensive and back genome on the realization gene function.Protein biochip technology is used a large amount of mature DNA chip technologies, has developed forms such as the SELDI chip that to comprise low-density metal surface, highdensity three dimensional gel sheet chip, the cylindric chip of high density.(Zhu H.et al.Science.2001 293:2101-5) has reported the work that how prepares protein group chip and application thereof in the high density smooth, glass surface as document 1.How protein efficiently being fixed in solid surface is another important problem of making protein-chip.The solid surface that exists comprises that other also have surfaces such as cellulose nitrate, metal or Poly-L-lysine covering with the PDMS or the glass surface of nickelous, epoxy radicals or aldehyde radical activation at present.
Yet obstacle maximum in the protein-chip field is the high investment of fund, the long period of research and development and the high complexity of technology thereof.The method of present employing comprises the preparation of gene clone, active somatic cell protein expression, extensive high-level efficiency protein purification and protein-chip and uses this four part.At present this four part all is that substep carries out separately, and the enforcement in each step all requires special equipment and manpower, the product in each step also write down categorizedly and preserve.One of wherein Eukaryotic gene clone needs to consume great amount of manpower and material resources, room and time; Protein expression and extensive purification not only consume great amount of manpower and material resources, also must grasp technical skill, at present have only very few several laboratories to accomplish in the world, in addition, the storage life of protein also is less than 6 months in-80 degrees centigrade of refrigerators; And the manufacturing process of protein-chip is also because the instability of protein self, must be under the condition of strictness control repeatedly debugging could realize that in case the more important thing is and make protein-chip, this chip not only is difficult to preservation (＜7 days), and can't transport, more can not form product.
Therefore, in the protein-chip field, as a large amount of Eukaryotic genes of low-cost clone how, efficiently obtain the practicability of multiple proteins and protein-chip and the bottleneck that commercialization is still this field.Existent method also can't adapt to the research work of research complicated protein group as human at present.And how protein-chip is brought into play more effective effect and is still waiting further breakthrough in clinical diagnosis, biomedicine and fundamental research.
Summary of the invention
The objective of the invention is to overcome the instability of protein self, must be under the condition of strictness control repeatedly debugging could realize that in case the more important thing is and make protein-chip, this chip not only is difficult to preservation (＜7 days), and can't transport, more can not form the defective of product; And overcome the traditional preparation process method must be first clone gene express fragment, through the defective of numerous and diverse step of cell inner expression, synthetic protein purifying; Thereby provide a kind of holding time long, practicability and commercial protein-chip and application thereof.And provide a kind of manufacturing process simple, need not to utilize the protein-chip preparation method of active somatic cell expressing protein and loaded down with trivial details purification step.
Protein-chip provided by the invention comprises: a substrate, and affine layer is arranged on the substrate, and on the affine layer bio-macromolecule layer is arranged, wherein bio-macromolecule layer is the encoding gene fragment, or the product rete of encoding gene fragment.
Used substrate is conventional bio-chip substrate material; It can be the solid composite material of glass, quartz or surface coating; The solid composite material of this surface coating is pottery, plastics, graphite or the polymerization silane (as dimethylsilane) of metal-coated membrane (as gold, silver, copper, zinc) preferably.Protein-chip of the present invention, its substrate layer structure can be level and smooth shape (substrate surface are a continuous plane), shown in Figure 1A; Also can be micropore shape (substrate surface be provided with separate to be provided with and the cavity of seepage mutually not, this cavity can form separating device by substrate material itself and obtain, as Figure 1B, cavity also can be pasted the grid-like glue material of one deck and obtain on substrate), the diameter of cavity or length can arrive the hundreds of micron for several nanometers as required; The substrate layer structure also can be island-projection shape (substrate surface for the protrusion of column) one by one, shown in Fig. 1 C.When the cavity of micropore core sheet is 0.1nm～3nm, be referred to as capillary micropore core sheet, shown in Fig. 1 D; Detect when micropore shape, island-projection shape are particularly useful for different sample with capillary micropore core sheet, usefulness person can adopt suitable chip according to its needs.
The substrate layer surface must be activated through chemical modification, to reach the fixedly purpose of affinity layer.When making substrate, can obtain the hydroxyl of surface activation by simple acid-alkali treatment, to be used for the affinity layer of covalent bond silane compound with glass or quartz material; When making substrate, can adopt metal coatings such as gold, silver, copper, zinc to realize surface activation, to reach the purpose of covalently bound sulfydryl alkyl compound unimolecular film with materials such as pottery, plastics, graphite or poly dimethyl silane.
Described protein-chip, its on-chip affine layer is a kind of and unimolecular film albumen mass-energy high special affinity, this unimolecular film is an organic long-chain, covalent bond has affinity molecule (see among Fig. 3 shown in the label 4) on it, affinity molecule can be biotin, avidin (Avidin), antibody variable region, glutathione, polyacrylamide gel, Ago-Gel or sequestrant, these sequestrant chelated metal ions Ni
2+, Fe
3+, Cu
2+, Ga
2+Or Ca
2+Deng, as EDTA, EGTA, NTA (tetradentatenitrolotriacetic acid), IDA (iminodiacetic acid) or TCE (tris (carboxymethyl) ethylenediamine), three imido acetoacetates (tridentate iminodiacetic acid) etc.These dissociation constants (Kd) are 10
-7Above molecule can with affinity labelling on synthetic protein strong bonded very optionally, thereby realize the purpose of high special ground fixing protein, the high background of having avoided non-specific binding to cause.And because the affinity labelling on the different coding gene outcome always occurs at same position, albumen mass-energy is fixed in chip surface with on all four spatial orientation by special affinity labelling, thereby has kept the natural structure phase of biomacromolecule to greatest extent.
Bio-macromolecule layer on the protein-chip of the present invention, it is affine layer is the encoding gene fragment, or the product of encoding gene fragment.The encoding gene fragment is selected RNA or DNA in an embodiment for use, and the RNA that selects for use can be the RNA of mRNA, tRNA and synthetic etc., and the DNA that selects for use can be the DNA of cDNA, pcr amplification product, synthetic and the nucleotide fragments in the genome etc.The target gene that increases the mRNA that also can adopt reverse PCR (RT-PCR) technology directly to extract in biological tissue is expressed fragment.This encoding gene is not limited to and is used for synthetic protein, also but design template is used for synthesizing manually small peptide (Random peptide) at random, antibody random variation district (described) as Phage display technology, or contain the manually small peptide or the protein at random of other protein combination region of variability (described) as the Affibody technology.
The present invention further provides a kind of preparation method of described protein-chip, comprised the steps:
(2) processing of substrate surface and activation: clean solid substrate, and substrate surface is activated the unimolecular film that makes its covalently bound affine layer.The substrate layer surface must be activated through chemical modification, to reach the fixedly purpose of affinity layer, when making substrate with glass or quartz material, can be obtained the hydroxyl of surface activation by simple acid-alkali treatment, to be used for covalent bond affinity layer; When making substrate, can adopt metal coatings such as gold, silver, copper, zinc to realize surface activation, to reach the purpose of covalently bound sulfydryl alkyl compound unimolecular film with materials such as pottery, plastics, graphite or poly dimethyl silane.
(3) preparation of bio-macromolecule layer: gene amplification product is added on the substrate that is combined with affine layer that above-mentioned steps (1) obtains, makes the protein-chip that bio-macromolecule layer is the encoding gene fragment.
Protein-chip obtained by this method is a kind of precursor protein matter chip, must utilize as follows when in use acellular in-vitro transcription-translation system from DNA to mRNA, mRNA free protein in protein, the solution promptly settles at one go and finishes to this series reaction of surperficial fixing protein with change in same reaction system a step and finish.This using method comprises that specifically step (3) is the translation process (referring to Fig. 3) of gene amplification product:
1) volume ratio (20～200): (1～10): 1 with in-vitro transcription translation reaction liquid, and 100mM magnesium acetate and 1mM methionine mix;
2) bio-macromolecule layer that the present invention is made is that the protein-chip of encoding gene fragment is immersed in above-mentioned steps 1) in the mixed liquor made, 30 ℃ of incubations 1 to 3 hour, the biochemical reaction of realization from the encoding gene fragment to protein, and finish conversion simultaneously from free protein to surperficial fixing protein.
3) with buffer solution for cleaning step 2) protein-chip that obtains, the protein-chip that makes can use or refrigerate stand-by.
Described use step is further preferably in step 2) in protein-chip is immersed in after the mixed liquor that step 1) makes, also comprise the bubble of removing chip surface with concussion or ultrasonic washing instrument.
This invention also can form 1000 to 100000 protein spots, i.e. high-density protein matter chip or the little array of high-density protein matter in the solid surface of 2cm * 5cm.
The invention further relates to the purposes of described protein-chip in clinical diagnosis, medical research, new drug development and fields such as check, proteome research.For example, this protein-chip can be used for sketching the contours little to organelle, cell, arrive various body fluid, tissue, organ, system greatly, even the protein of whole biology composition profile, thereby find the special biomarker of disease, to be used for early clinical diagnosis to various malignant diseases; And for example, this protein-chip can be used for finding organizing or the protein of disease specifically expressing, thereby promotes the research of preclinical medicine; And for example, this protein-chip can be used for new drug development and check, by research, can find which medicine micromolecule has and to reach the purpose that suppresses pathology with certain disease or the effect of cancer associated protein, thereby find new drug protein and the interphase interaction of medicine micromolecule; And for example, this protein-chip can be used for the fundamental research of protein group, protein-chip can be used as experiment porch and studies interaction between protein-protein, protein-DNA, protein-RNA, protein-fat, protein-antibody, protein-small peptide, the protein-polysaccharide, protein-chip also can be used as the downstream substrate that experiment porch is studied the posttranscriptional modification of eukaryotic protein and sought various enzymes, thereby provides abundant experimental basis for the network chart that sketches the contours the various biological approaches in the biosome.
It is supporting with combining of downstream experiment that the present invention has expanded protein-chip, as can be with the detection means research protein and other intermolecular constant that combines and dissociate of surface plasma body resonant vibration (SPR) on the protein-chip that covers at surface metal, can't measure multiple proteins and other intermolecular combination or the difficult problem of dissociation constant on a large scale synchronously thereby solved in the past.Also can be on the micropore protein-chip that surface metal covers to determine protein bound on which kind of molecule and the protein-chip in conjunction with mass spectral detection means, thereby shorten sense cycle greatly, improve detection efficiency.
Protein-chip of the present invention is compared with existing protein-chip, has following advantage:
(1) protein-chip provided by the invention is a kind of precursor protein matter chip, in the high density that has kept existing protein-chip, high-efficiency characteristics, its protein array density can reach 1000-100000 protein, antibody or artificial small peptide and put per 10 square centimeters.The present invention only needs the coded sequence of coding target protein or small peptide is made the precursor chip, realize then original position protein transcribe, synthetic, the clone gene active somatic cell that has omitted classic method is fully expressed synthetic protein and is prepared the brand-new preparation method that settles at one go of four big steps of protein-chip with the chip preparing instrument.This method has solved protein effectively and has been difficult for preserving and the problem of degrading in time.The user can be converted into fresh protein-chip by precursor protein matter chip as required at any time, farthest avoided the protein problem of unstable, this precursor chip can be preserved (～5 years) and transportation under room temperature, promoted the practicability and the commercialization of protein-chip greatly.
(2) provided by the invention by substrate layer, the protein-chip that special affinity layer and encoding gene slice layer constitute, saved the manpower of a large amount of consumption at clone's eukaryotic gene extron, material resources, expense and time, and saved the big quantity space of preserving these clones, also omitted with active somatic cell marking protein and complicated protein purification step, effectively avoided owing to preserve the possibility that many covers are cloned the cross pollution that brings, and solved the inhomogenous situation of protein output that the growth differences owing to cell line causes, and then eliminated the greatest differences that is fixed on the albumen quality on the chip surface that causes thus, thereby greatly reduce the experimental error that causes because of the difference that is fixed on the chip surface protein quality and quantity.
(3) protein-chip of the present invention has adopted the affinity layer of tool high degree of specificity, can make the encoding gene product be fixed in chip surface with on all four spatial orientation by special affinity labelling, thereby has kept the natural structure phase of biomolecule to greatest extent.The template that is fixed in the surface can be used for biomolecule such as synthetic protein, small peptide, antibody variable region, and the present invention has greatly improved the dirigibility of protein-chip in practical application.
(4) it is supporting with combining of various downstreams experiment that the present invention has further expanded protein-chip, as can be with the detection means research protein and other intermolecular constant that combines and dissociate of surface plasma body resonant vibration (SPR) on the protein-chip that covers at surface metal, can't measure protein and other intermolecular combination and the difficult problem of the constant that dissociates on a large scale synchronously thereby solved in the past; This protein-chip can be studied protein bound on which kind of molecule and the protein-chip in conjunction with mass spectral detection means, thereby has shortened sense cycle greatly, has improved detection efficiency.
(5) the present invention has also expanded the Application for Field such as mensuration of protein-chip in the determining of drug screening, the evaluation of pesticide effectiveness, pharmaceutical target albumen, drug side-effect, finds and popularizes the fundamental research of protein group and lay a solid foundation for realizing real-time clinical monitoring, new drug.
Description of drawings
Figure 1A is the structure sectional view of level and smooth substrate protein-chip
Figure 1B is the structure sectional view of microporous substrate protein-chip
Fig. 1 C is the structure sectional view of island-projection substrate protein-chip
Fig. 1 D is the structure sectional view of capillary microporous substrate protein-chip
Wherein, 1 is that substrate 2 is bio-macromolecule layer for affinity layer 3
I represents to amplify
Fig. 2 is the processing of substrate surface among the embodiment 1
Wherein 1 is that substrate 2 is the affinity layer for metal coating 3
S1 is that plated film step S2 is in conjunction with affine layer step
Fig. 3 is equipped with protein-chip for settling legal system at one go
Wherein 1 is that substrate 2 is the affinity layer for metal coating 3
4 is that affinity molecule 5 is affinity labeling for protein 6
Embodiment
Embodiment 1: prepare protein-chip on high density island-projection glass substrate
Step 1: the processing of substrate surface and activation (referring to shown in Figure 2)
1) with pure water high density island-projection graphite substrate is washed three times, with 100% ethanol microslide is washed three times again.
2) in the vacuum evaporation platform, graphite substrate to be plated is placed on the sample stage, plating metal place the evaporation boat in, under the fusing point of gold in 10
-2Pa is plated on gold on the surface of graphite substrate.
3) graphite substrate that plating is good places and contains 10-sulfydryl decane-NTA (Ca
2+) solution in, at room temperature reaction more than three hours, obtain being connected with the substrate of affine layer.
4) use phosphate buffer (PBS) on shaking table, to clean above-mentioned 3 again) substrate that obtains three times.
5) clean five times with 70% ethanol, clean three times with 95% ethanol again, clean three times with 100% ethanol at last.The substrate that is connected with affine layer that makes is preserved under dry free from dust condition.
Step 2: preparation island-projection precursor protein matter chip (referring to shown in Figure 3)
1) be stored in that the amplification gene product melts in room temperature in-80 ℃ of refrigerators, and in 3000rpm centrifugal 5 minutes.
2) the island-projection substrate that is connected with affine layer for preparing in the step 1 is arranged on the workbench of chip synthesizer, 384 orifice plates (seeing embodiment 4) that fill gene outcome are placed on the raw material platform of machine intimate.
3) the input preparation settles the various parameters of precursor protein matter chip at one go on the control computer of chip synthesizer, and start-up routine.
4) take off the chip synthesizer and finish printing precursor protein matter chip, under dry free from dust condition, preserve chip.
Step 3: prepare the island-projection protein-chip from island-projection precursor protein matter chip
1) 80: 2: 1 ratio is mixed in-vitro transcription translation system, 100mM magnesium acetate and 1mM methionine.
2) the mixing object point of previous step being made with the chip synthesizer (about 0.3 receive liter) on the island-projection on the precursor chip of step 2 preparation; Perhaps, this precursor chip surface is placed step 3 1) potpourri made, remove bubble with concussion or ultrasonic washing instrument then.
3) go on foot the precursor chip that has added potpourri with second and place in the closed container of preserving moisture, cultivated 1 to 3 hour on shaking table in 30 ℃, thereby make the island-projection protein-chip.
4) use phosphate buffer (PBS) on shaking table, to clean the island-projection protein-chip totally three to five times at last.The island-projection protein-chip that makes can use immediately, also can be stored in 4 ℃ of refrigerators stand-by
As described in step 3, chip surface in the unimolecular film with affinity can be finished the three-step reaction of the past body protein chip to protein-chip a step, i.e. genetic transcription from the dna profiling to mRNA reaction, gene translation from the mRNA template to protein reacts, and is fixed to the process of chip surface by the affine micromolecule on the affine layer of its affinity labeling specific bond that connects from the synthetic protein that is in free state.Entire reaction can be under 30 ℃ of conditions of preserving moisture incubation several hours, after the preparation of protein-chip is finished in washing.
Embodiment 2: prepare protein-chip on the high density microporous substrate
Step 1: the processing of chip surface and activation
1) water washes high density micropore glass substrate (cavity is that diameter is 7 microns a circular hole) flushing three times three times with microslide with 100% ethanol again.
2) glass substrate is placed the NaOH solution soaked overnight of 0.2M, clean 3 times with high purity water then, again with absolute ethyl alcohol flushing one time.
3) glass substrate after will cleaning places and contains (CH
3O)
3-Si-(CH
2)
20In the solution of-biotin, more than three hours, obtain being connected with the substrate of affine layer at room temperature reaction.
4) use phosphate buffer (PBS) on shaking table, to clean above-mentioned 3 again) substrate that obtains three to five times.
5) clean five times with 70% ethanol, clean three times with 95% ethanol again, clean three times with 100% ethanol at last.The substrate that is connected with affine layer that makes is preserved under dry free from dust condition.
Step 2: preparation high density micropore precursor protein matter chip
Identical with step 2 among the embodiment 1.
Step 3: prepare high density micropore protein-chip from high density micropore precursor protein matter chip
1) 20: 8: 1 ratio is mixed in-vitro transcription translation system, 100mM magnesium acetate and 1mM methionine.
2) potpourri of the first step being made with the chip synthesizer is sub-packed in the micropore on the high density micropore precursor chip among the embodiment 5 (about every hole 0.3 receive liter); Perhaps, the potpourri that places the first step to make high density micropore precursor chip surface is removed bubble in the micropore with concussion or ultrasonic washing instrument then.
3) go on foot the precursor chip that has added potpourri with second and place in the closed container of preserving moisture, on shaking table, cultivated 1 to 3 hour in 30 degrees centigrade, thus high density micropore spike protein matter chip.
4) use phosphate buffer (PBS) on shaking table, to clean the island-projection protein-chip three to five times at last.The high density micropore protein-chip that makes can use immediately, also can be stored in 4 ℃ of refrigerators stand-by.
Embodiment 3: prepare protein-chip on level and smooth substrate
Step 1: the processing of chip surface and activation
1) water washes level and smooth shape quartz substrate flushing three times three times with microslide with 100% ethanol again.
2) quartz substrate places the HCl solution soaked overnight of 0.2M, cleans 3 times with high purity water then, again with absolute ethyl alcohol flushing one time.
3) quartz substrate after will cleaning places and contains (CH
3O)-Si-(CH
2)
22In the solution of-glutathione, more than three hours, obtain being connected with the substrate of affine layer at room temperature reaction.
4) use phosphate buffer (PBS) on shaking table, to clean above-mentioned 3 again) substrate totally three to five times.
5) clean five times with 70% ethanol, clean three times with 95% ethanol again, clean three times with 100% ethanol at last.The substrate that is connected with affine layer that makes is preserved under dry free from dust condition.
Step 2: the level and smooth precursor protein matter of preparation high density chip
Identical with step 2 among the embodiment 1.
Step 3: prepare the level and smooth protein-chip of high density from the level and smooth precursor protein matter of high density chip
1) 80: 5: 1 ratio is mixed in-vitro transcription translation system, 100mM magnesium acetate and 1mM methionine.
2) potpourri that places the first step to make the level and smooth precursor chip surface of high density covers slide, removes bubble with concussion or ultrasonic washing instrument then.
3) go on foot the precursor chip that has added potpourri with second and place in the closed container of preserving moisture, on shaking table, cultivated 1 to 3 hour in 30 degrees centigrade, thus high density micropore spike protein matter chip.
4) use phosphate buffer (PBS) on shaking table, to clean the island-projection protein-chip three to five times at last.The high density micropore protein-chip that makes can use immediately, also can be stored in 4 ℃ of refrigerators stand-by.
Embodiment 4: prepare protein-chip on the capillary microporous substrate
Step 1: the processing of chip surface and activation
1) water washes the dimethylsilane substrate (cavity is that diameter is the circular hole of 2 nanometers) of capillary micropore shape three times, with 100% ethanol microslide is washed three times again.
2) in the vacuum evaporation platform, dimethylsilane substrate to be plated is placed on the sample stage, plating metal copper place the evaporation boat in, under the fusing point of copper in 10
-2Pa is plated on copper on the surface of graphite substrate.
3) the dimethylsilane substrate that has plated copper places and contains HS-(CH
2)
22In the solution of-avidin, more than three hours, obtain being connected with the substrate of affine layer at room temperature reaction.
4) use phosphate buffer (PBS) substrate three to five times on shaking table again.
5) clean five times with 70% ethanol, clean three times with 95% ethanol again, clean three times with 100% ethanol at last.The substrate that is connected with affine layer that makes is preserved under dry free from dust condition.
Step 2: step 2 is identical among preparation high density level and smooth precursor protein matter chip and the embodiment 1.
Step 3: prepare the level and smooth protein-chip of high density from the level and smooth precursor protein matter of high density chip
1) 100: 10: 1 ratio is mixed in-vitro transcription translation system, 100mM magnesium acetate and 1mM methionine.
2) the level and smooth precursor chip surface of the high density potpourri that places the first step to make covers slide, then with shaking or ultrasonic washing instrument is removed bubble.
3) go on foot the precursor chip that has added potpourri with second and place in the closed container of preserving moisture, on shaking table, cultivated 1 to 3 hour in 30 degrees centigrade, thus high density micropore spike protein matter chip.
4) use phosphate buffer (PBS) on shaking table, to clean the island-projection protein-chip three to five times at last.The high density micropore protein-chip that makes can use immediately, also can be stored in 4 ℃ of refrigerators stand-by.
Embodiment 5: the preparation of gene template
1) gets 10 gram biological tissues and grind, add 20 milliliters of RNA extracts rapidly, in being incubated 30 minutes on ice with liquid nitrogen.
2) press the description operation of the mRNA extraction agent box of Promega company, can obtain about 5 microgram mRNA.
3) above-mentioned gained mRNA is diluted to 2 nanograms/microlitre, gets 1 microlitre again, add in the 45 mixed in advance microlitre RT-PCR reaction mixtures, add the 4ul gene specific primer again, place PCR reaction instrument to react as the RT-PCR reaction template.
4) get 5 microlitre volumetric reaction products as gel detection, the PEG precipitation method or the ammonium sulfate precipitation method of surplus DNA available standards are removed unreacted primer, and according to the gel detection result DNA product of amplification are dissolved in the 3xSSC damping fluid again with 0.1 microgram/microlitre.
5) the PCR product is sub-packed in 384 orifice plates, and is stored in-80 ℃ of refrigerators stand-by.
Embodiment 6: protein-chip is used for the discovery of drug target protein
1) protein-chip with prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) the compound rapamycin (Rapamycin) that will have biotin labeled synthetic is got 120 microlitre diluted chemical compound liquid and is added on the protein-chip surface with the phosphate buffer dilution, covers with cover glass then, avoids bubble as far as possible.
3) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
5) get 200 microlitres and dilute and good be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible by the fluorescently-labeled streptavidin of Cy-3 (Streptavidin) solution.
6) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
7) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
8) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.
9) with laser chip scanner scanning protein-chip to obtain data, the protein that can combine with the medicine micromolecule can show the fluorescence bright spot on scintigram.The title of these protein can check in database, for example, found with brewer's yeast in protein Frp1 have the mankind's of high homology the protein of unknown function.
Embodiment 7: protein-chip is used for new drug and finds
1) protein-chip of prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) library of compounds that will have biotin labeled synthetic dilutes with phosphate buffer, gets 120 microlitre diluted chemical compound liquid and is added on the protein-chip surface, covers with cover glass then, avoids bubble as far as possible.Protein-chip places the environment of preserving moisture incubation one hour under room temperature.
3) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
4) get 200 microlitres and dilute and good be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible by the fluorescently-labeled Streptavidin solution of Cy-3.
5) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
6) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
7) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.Which can combine with drug molecule with definite protein with laser chip scanner scanning protein-chip.
8) protein that can combine with drug molecule from new optional step 7 prepares the micropore protein-chip, repeat above-mentioned experimental procedure, then reacted chip is placed efficient mass spectrometer to identify one by one and the structure of the drug molecule of protein bound, reach the purpose that new drug is found.
Embodiment 8: protein-chip is used to confirm side effects of pharmaceutical drugs
1) protein-chip of prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) will have fluorescently-labeled medicine micromolecule and dilute, and get 120 microlitre dilutions and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible with phosphate buffer.
3) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
5) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
6) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.
7) with laser chip scanner scanning protein-chip to obtain data, the protein that can combine with the medicine micromolecule can show the fluorescence bright spot on scintigram.The identity of these protein can check in database.
8), can confirm except expection and protein that this medicine combines, to also have other which protein to combine, these additional proteins promptly constitute this side effects of pharmaceutical drugs and compose at once with this medicine by analyzing data.By analyzing the function of these additional proteins, can more in depth understand the mechanism of these side effects of pharmaceutical drugs.
Embodiment 9: protein-chip is used to discern new biomarker
1) protein-chip of prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) use the protein of Cy-3 fluorescence labeling and Cy-5 fluorescence labeling difference mark respectively from cancerous tissue and normal structure extraction, after removing unnecessary fluorescence labeling, mix and with phosphate buffer diluted protein matter potpourri, get 120 microlitre mixture diluted liquid and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
3) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
5) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.
6) under two frequencies, scan protein-chip obtaining data with the laser chip scanner, can on scintigram, show the green fluorescence bright spot with the protein on the chip of protein bound in the cancerous tissue; Can on scintigram, show the red fluorescence bright spot with the protein on the chip of protein bound in the normal structure.The identity of these protein can check in database.
7) by the ratio of the shown red green of each protein spots on the comparable chip, can determine can specific recognition the albumen of extraordinary expressed protein or other biomacromolecule in the cancerous tissue, these protein promptly can be used as the biomarker of early-stage cancer clinical diagnosis.
Embodiment 10: protein-chip is used for the clinical diagnosis (is example with the hepatitis detection) of infectious disease
1) protein-chip of prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.Be loaded with 12 kinds of antibody discerning first, second, third, fourth, the scorching antigen of viral hepatitis type E on this protein-chip.Each protein-chip is divided into 30 districts, can test 20 patients and negative, positive control simultaneously.
2) get each 0.5ml of blood sample of 20 patients and a healthy people, left the heart 5 minutes in 3000, take out serum 500vl, and rare with phosphate buffer.Get 120 microlitre mixture diluted liquid and be added to respectively in each zone on protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
3) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
5) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.The chip phosphate buffer that submerges is diluted in the damping fluid of the good goat antibody that has the fluorescently-labeled anti-human IgG of Cy3 room temperature incubation one hour.
6) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
7) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.
8) with laser chip scanner scanning protein-chip to obtain data, be tested and appraised the positive classification that the patients serum presents on protein-chip, can determine the type of the hepatitis virus of patient infection, thereby reach the purpose of diagnosis.
Embodiment 11: protein-chip is used for the antibody of confirming that the allergic disease human serum is relevant with allergy
1) protein-chip of bright preparation cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.Be loaded with 30 kinds of known anaphylactogens (allergen) on this protein-chip.Each protein-chip is divided into 30 districts, can test 20 patients and negative, positive control simultaneously.
2) get each 0.5ml of blood sample of 20 patients and a healthy people, left the heart 5 minutes in 3000, take out serum 500vl, and rare with phosphate buffer.Get 120 microlitre mixture diluted liquid and be added to respectively in each zone on protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
3) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
5) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.The chip phosphate buffer that submerges is diluted in the damping fluid of the good goat antibody that has the fluorescently-labeled anti human IgE of Cy3 room temperature incubation one hour.
6) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
7) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.
8) with laser chip scanner scanning protein-chip to obtain data, be tested and appraised the positive classification that the patients serum presents on protein-chip, can determine to cause patient to produce irritated type, thereby reach the purpose of diagnosis.
Embodiment 12: protein-chip is used to confirm the substrate (is example with the phosphokinase) of enzyme
1) protein-chip of preparation cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.Be loaded with 1000 kinds of mankind's protein on this protein-chip.
2) phosphokinase (CD2) albumen of getting the people of purifying uses phosphate buffer rare to 1 nanogram/microlitre.Get 120 microlitre mixture diluted liquid and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
3) protein-chip places the environment of preserving moisture 30 degree incubation half an hour down.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
5) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.The chip phosphate buffer that submerges is diluted in the damping fluid of the good goat antibody that has the fluorescently-labeled anti-phosphorylated tyrosine of Cy3 room temperature incubation one hour.
6) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
7) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.
8) with laser chip scanner scanning protein-chip to obtain data, the protein that those are positive on the chip i.e. the substrate of this phosphokinase (CD2).
Embodiment 13: protein-chip is used to screen specific antibody (is example with the il-1)
1) selects bacteriophage to present carrier cloning artificial recombination fragment, present vector library to obtain 108 bacteriophage.The library is divided into 10 of homogeneous
4Part, use the pcr amplified dna template then, prepare precursor protein matter chip with point sample instrument at last.
2) method that adopts embodiment 2 to set forth prepares protein-chip.
3) protein-chip of prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
4) with biotin labeling kit mark il-1, after removing unreacted biotin, with the good albumen of phosphate buffer dilution mark.
5) get the good albumen of 120 microlitre marks and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
6) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
7) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
8) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.To obtain data, the artificial antibody group that can combine with labelled protein can show the fluorescence bright spot on scintigram with laser chip scanner scanning protein-chip.
9) repeating step 1 can further be determined special anti-il-1 artificial antibody to step 8.
Embodiment 14: protein-chip is used to study the interaction between protein-protein
10) protein-chip of prepared fresh cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
11) with biotin labeling kit mark protein to be studied, after removing unreacted biotin, with the good albumen of phosphate buffer dilution mark.
12) get the good albumen of 120 microlitre marks and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
13) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
14) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
15) get 200 microlitres and dilute and good be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible by the fluorescently-labeled Streptavidin solution of Cy-3.
16) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
17) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
18) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.To obtain data, the protein that can combine with labelled protein can show the fluorescence bright spot on scintigram with laser chip scanner scanning protein-chip.The title of these protein can check in database.
Embodiment 15: protein-chip is used to study the interaction between protein-DNA
1) protein-chip of bright preparation cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) with biotin labeling kit mark dna molecular to be studied, after removing unreacted biotin, with the good DNA of phosphate buffer dilution mark.
3) get the good DNA of 120 microlitre marks and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
4) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
5) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
6) get 200 microlitres and dilute and good be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible by the fluorescently-labeled Streptavidin solution of Cy-3.
7) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
8) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
9) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.To obtain data, the protein that can combine with marker DNA can show the fluorescence bright spot on scintigram with laser chip scanner scanning protein-chip.The title of these protein can check in database.
Embodiment 16: protein-chip is used to study the interactional combination between protein-biomacromolecule and the constant that dissociates
1) protein-chip of bright preparation cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) protein-chip places the SPR instrument, gets the good biomacromolecule of 120 microlitre fluorescent labellings and is added to the protein-chip surface, simultaneously with the binding curve between the protein fixing on SPR instrument observation biomacromolecule and the chip.
3) after question response is finished, with the biomacromolecule of the cleaning buffer solution elution of bound protein on chip that contains detergent, simultaneously with the dissociation curve between the protein fixing on SPR instrument observation biomacromolecule and the chip.
4), can on a large scale, efficiently measure the constant that combines and dissociate between the protein fixing on each biomacromolecule and the chip by data analysis.
Embodiment 17: protein-chip is used to study the interaction between protein-lipid molecule
1) protein-chip of bright preparation cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) get the good lipid molecule of 120 microlitre fluorescence labelings and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
3) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
4) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
5) protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.To obtain data, the protein that can combine with the mark polysaccharide can show the fluorescence bright spot on scintigram with laser chip scanner scanning protein-chip.The title of these protein can check in database.
Embodiment 18: protein-chip is used to study the interaction between protein-polysaccharide
1) protein-chip of preparation cleans totally three to five times with the large volume cleaning buffer solution on shaking table, and is stand-by.
2) biotin labeling kit mark polysaccharide to be studied is after removing unreacted biotin, with the good albumen of phosphate buffer dilution mark.
3) get the good polysaccharide of 120 microlitre marks and be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible.
4) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
5) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.
6) get 200 microlitres and dilute and good be added to the protein-chip surface, cover with cover glass then, avoid bubble as far as possible by the fluorescently-labeled Streptavidin solution of Cy-3.
7) protein-chip places the environment of preserving moisture incubation one hour under room temperature.
8) protein-chip that whole cover glass is covered is not in the large volume cleaning buffer solution, and it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes.Not in the large volume ultrapure water, it is inferior to give a baby a bath on the third day after its birth at the shaking table supernatant, each 15 minutes with whole protein-chip.
Protein-chip hydro-extractor centrifugal 5 minutes dryings under 3000 speed of changeing.To obtain data, the protein that can combine with the mark polysaccharide can show the fluorescence bright spot on scintigram with laser chip scanner scanning protein-chip.The title of these protein can check in database.
Claims (13)
1. protein-chip comprises: substrate, and on-chip affine layer, and the bio-macromolecule layer on the affine layer, it is characterized in that: described bio-macromolecule layer is the encoding gene fragment, or the product rete of encoding gene fragment.
2. by the described protein-chip of claim 1, it is characterized in that described substrate comprises: the solid composite material of glass, quartz or surface coating.
3. by the described protein-chip of claim 2, it is characterized in that the solid composite material of described surface coating comprises: the pottery of metal-coated membrane, plastics, graphite or polymerization silane.
4. by claim 1 or 2 or 3 described protein-chips, it is characterized in that described substrate is level and smooth shape, has capillary micropore shape or an island-projection shape.
5. by the described protein-chip of claim 1, it is characterized in that described affine layer is in conjunction with biotin, avidin, antibody variable region, polyacrylamide gel, Ago-Gel or the sequestrant unimolecule rete as affinity molecule.
6. the preparation method of the described protein-chip of claim 1 comprises the steps:
(1) processing of substrate surface and activation: clean solid substrate, and substrate surface is activated the unimolecular film that makes its covalently bound affine layer;
(2) preparation of bio-macromolecule layer: gene amplification product is added on the substrate that is combined with affine layer that above-mentioned steps (1) obtains, makes the protein-chip that bio-macromolecule layer is the encoding gene fragment.
7. by the preparation method of the described protein-chip of claim 6, it is characterized in that also comprising the translation process of step (3) gene amplification product:
1) (20～200) by volume: (1～10): 1 with in-vitro transcription translation reaction liquid, and 100mM magnesium acetate and 1mM methionine mix;
2) protein-chip that claim 10 is made is immersed in above-mentioned steps 1) in the mixed liquor made, 30 ℃ of incubations 1 to 3 hour, the biochemical reaction of realization from the encoding gene fragment to protein, and finish conversion simultaneously from free protein to surperficial fixing protein;
3) with buffer solution for cleaning step 2) protein-chip that obtains, the protein-chip that makes can use or refrigerate stand-by.
8. by the described protein-chip of claim 6, the product rete that it is characterized in that the encoding gene fragment is artificial random fragment, small peptide, protein or antibody.
9. by the described protein-chip of claim 6, it is characterized in that the encoding gene fragment is RNA or DNA.
10. by the preparation method of the described protein-chip of claim 6, it is characterized in that in the described step (1) substrate activated and comprise glass or quartz substrate surface with acid or alkali treatment or plated film.
11. by the described method of producing protein of claim 7, it is characterized in that described step 2) in protein-chip be immersed in also comprise after the mixed liquor that step 1) makes with shaking or ultrasonic washing instrument is removed the bubble of chip surface.
12. the application of the described protein-chip of claim 1 in screening of medicaments target protein, screening new drug, proteome research, analysis drug side-effect and clinical diagnosis.
13. be included as the affirmation of antibody relevant in clinical diagnosis, people's cardiovascular disease, early diagnosis of cancer or the allergic disease human serum of infectious disease by the application of the described protein-chip of claim 12 in clinical diagnosis with allergy.
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| CN101813701A (en) * | 2010-04-13 | 2010-08-25 | 无锡中美亿芯生物科技有限公司 | Method for preparing protein chip by in-situ synthesis |
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| CN109239182A (en) * | 2018-09-04 | 2019-01-18 | 南京林业大学 | A method of with cellulase in-situ modification gold chip |
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