WO2017008380A1 - 低丰度差异蛋白的分离方法及其应用 - Google Patents

低丰度差异蛋白的分离方法及其应用 Download PDF

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WO2017008380A1
WO2017008380A1 PCT/CN2015/088825 CN2015088825W WO2017008380A1 WO 2017008380 A1 WO2017008380 A1 WO 2017008380A1 CN 2015088825 W CN2015088825 W CN 2015088825W WO 2017008380 A1 WO2017008380 A1 WO 2017008380A1
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abundance
protein
low
sample
column
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French (fr)
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张贯京
李荣秀
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深圳市华科安测信息技术有限公司
深圳市贝沃德克生物技术研究院有限公司
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Publication of WO2017008380A1 publication Critical patent/WO2017008380A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the technical field of protein detection, and particularly relates to a method for separating low-abundance difference proteins and an application thereof.
  • biomarker molecules most of which are less abundant proteins.
  • These low-abundance proteins are found in very low concentrations in plasma, and are often found in high-abundance proteins such as albumin, making it difficult to detect in the early detection of disease latency, greatly delaying the discovery of disease. Therefore, it is of great medical significance to distinguish the plasma proteins in normal and abnormal physiological conditions and find the corresponding biomarker molecules to assist in the early detection of the disease.
  • Lipoprotein if there are too many proteins, the identification of protein spots is also difficult; secondly, the high-abundance protein in plasma will mask the micro-proteins that have a very important biological activity, although various affinity methods have been reported. Such as CB dye medium, antibody medium, etc. to remove high-abundance proteins, but the protein can be removed is limited, and can not remove most of the house-keeping protein and normal functional proteins; third, biomarkers related to abnormal physiological state Proteins are not limited to low-abundance proteins, and some of the more abundant proteins are also reported in disease reports, so using only high abundance removal can lead to inaccurate results.
  • the inventors of the present invention earlier proposed in 2010, CN102430176A, a method for enriching differential proteins between a protein to be tested and a control protein, thereby increasing the concentration of the protein or polypeptide to be tested.
  • the technically realized process uses the standard control protein as an antigen to immunize, and prepares an immunoglobulin capable of binding to at least one protein or polypeptide in the control sample (in fact, a primary antibody produced by using a standard control protein as an antigen, a polyclonal antibody, and then the immunoglobulin is used as a chromatography column for adsorbing a large amount of high-abundance protein in the sample to be tested, so that the flow-through component is an enriched and concentrated protein to be tested.
  • Low abundance difference protein is used.
  • the antibody was prepared by immunization using a normal control protein.
  • the control protein itself was a normal protein, and the low-abundance protein isolated and concentrated was only a potential marker protein newly emerging or up-regulated, and could not be further. It is directly determined that the expression of these proteins is a down-regulated protein in abnormal plasma or an up-regulated protein in normal plasma. It is necessary to further carry out a large number of comparative tests in comparison, and the accuracy is reduced after the complicated steps are increased, and the accuracy is improved.
  • the workload has caused deficiencies in the implementation of the above methods.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for separating and separating low-abundance differential proteins enriched in low-abundance down-regulated proteins in abnormal plasma and applications thereof.
  • a method for separating low-abundance differential proteins comprising the following steps:
  • the healthy control protein sample is immunized as an antigen to prepare a high-abundance protein polyclonal antibody; and the high-abundance protein multi-antibody is used as an affinity ligand to prepare a high-abundance affinity chromatography column;
  • the patient control protein sample is subjected to the column on the high-abundance affinity chromatography column, and the flow-through component is collected;
  • the flow-through component is concentrated and then immunized as an antigen to prepare a low-abundance protein polyclonal antibody; and the low-abundance protein polyclonal antibody is used as an affinity ligand for immobilization and solid phase matrix preparation for low-abundance affinity chromatography. column;
  • the histone sample to be tested is subjected to the column on the low-abundance affinity chromatography column, and the eluted fraction is collected.
  • the invention provides a method for separating the low-abundance difference protein in the detection of the low-abundance differentially expressed protein based on the above-mentioned method for separating the low-abundance difference protein.
  • the method and application of the low-abundance difference protein of the present invention are based on the original method, and the protein of the healthy group is first immunized to prepare a high-abundance multi-antibody capable of filtering a large number of background high-abundance difference proteins. Then, the high-abundance protein chromatography column is used to prepare a high-abundance protein chromatography column to remove the background high-abundance protein in the differentially expressed protein sample of the abnormal patient, and the collected flow-through component is a low-abundance differential expression of the abnormal patient.
  • the flow-through component is concentrated and then used as an antigen for a second immunization to obtain a low-abundance differentially expressed polyclonal antibody, and then the low-abundance differentially expressed polyclonal antibody is used as a ligand to further prepare a low-yield protein.
  • the chromatographic column of the differential protein can directly separate the down-regulated protein in the non-normal plasma or the up-regulated protein in the normal plasma.
  • the whole process can directly separate the up-regulated protein in normal plasma at a time, which reduces the complexity of the separation process, improves the separation and detection efficiency, and the final elution component directly targets the differentially expressed low-abundance protein. Higher specificity and specificity.
  • FIG. 1 is a photograph of silver staining by SDS-PAGE electrophoresis using the eluate obtained by the above steps in the plasma of normal human plasma and another liver cancer patient, respectively, according to an embodiment of the present invention.
  • the present invention provides a method for separating low-abundance differential proteins, including the steps summarized as follows:
  • the high-abundance protein polyclonal antibody obtained in step S20 is used as an affinity ligand, and is fixed on a solid phase carrier of the chromatography column to prepare a high-abundance affinity chromatography column;
  • the patient control protein sample is subjected to a high-abundance affinity chromatography column prepared in step S30, and the flow-through liquid is collected;
  • step S50 after the flow through the step S40 is concentrated, and then re-immunized as an antigen to prepare a low-abundance protein polyclonal antibody;
  • the low-abundance protein polyclonal antibody obtained in step S50 is used as an affinity ligand, and is fixed on a solid phase carrier of the chromatography column to prepare a low-abundance affinity chromatography column;
  • the histone sample to be tested is subjected to the column on the low-abundance affinity chromatography column prepared in step S60, and the eluted fraction is collected.
  • the method for isolating the low-abundance difference protein of the present invention is based on the original method, and firstly immunizing with the protein of the healthy group to prepare a high-abundance multi-antibody capable of filtering a large amount of background high-abundance difference protein, and then The high-abundance protein chromatography column was used to remove the background high-abundance protein in the differentially expressed protein samples of abnormal patients, and the collected flow-through components were low-abundance proteins differentially expressed in abnormal patients.
  • the flow-through component is concentrated and then used as an antigen for the second immunization to obtain a polyclonal antibody with a low-abundance differentially expressed protein, and then the low-abundance is further prepared by using the low-abundance differentially expressed protein polyclonal antibody as a ligand.
  • the differential protein chromatography column can directly separate the down-regulated protein in the non-normal plasma or the up-regulated protein in the normal plasma. Therefore, the above operation process of the invention is adopted as a whole, which reduces the complexity of the separation process, improves the separation and detection efficiency, and the final elution component directly targets the differentially expressed low-abundance protein, and has higher specificity and Targeted.
  • step S10 a protein obtained from a healthy normal person and a protein obtained from a diseased patient are respectively used as a source of a high-abundance protein and a low-abundance difference protein, respectively, in the patient
  • the types and amounts of low-abundance difference proteins in the obtained proteins are significantly different from the normal standards, and it is easier to directly reflect the expression of low-abundance differential proteins.
  • the affinity ligands for the low-abundance protein in the sample to be tested are directly prepared by using the two controls as a sample, and the direct isolation of the up-regulated protein in the normal plasma is achieved.
  • the subsequent processing is as follows:
  • control protein sample is used as an antigen to immunize rabbits (also can be other immune bodies such as rats), and immune polyclonal antibodies are prepared by immunization; most proteins (essentially large amounts of high-abundance proteins) are immune during immunization.
  • the prepared polyclonal antibody against high-abundance protein is passed through High-abundance protein polyclonal antibodies to remove background high-abundance proteins.
  • step S30 the high-abundance protein polyclonal antibody prepared in step S20 is used as an affinity ligand, and is fixed to a solid phase carrier to prepare a column for specifically adsorbing high-abundance protein; the solid phase carrier is used as a carrier for chromatography.
  • the type of glucan gel or the like is selected; the manner in which the affinity ligand is fixedly bound to the solid phase carrier can be achieved by a usual method such as covalent crosslinking.
  • Step S40 After preparing the high-abundance affinity chromatography column, the high-yield chromatography column is used to perform column treatment on the abnormal patient protein sample, and the affinity medium of the chromatography is aimed at the polyclonal process.
  • the antibody eliminates the system of high-abundance protein, so a large amount of high-abundance protein in the column protein sample will be adsorbed on the column, and the components in the obtained flow-through component will be differentially expressed in most abnormal patients. Low abundance protein.
  • the flow through component can be re-columned to maximize the removal of background proteins.
  • the abnormal patient protein control sample in order to reduce or reduce the non-specific adsorption of proteins in the high-abundance column and the sample, the abnormal patient protein control sample is passed through an empty column containing only the solid phase matrix and no ligand. Column treatment, such that many impurities or non-specific adsorption factors in the abnormal patient protein control sample will be initially filtered to enhance the affinity specificity of the high-abundance protein chromatography column.
  • step S50 step S40 is collected to concentrate the flow-through liquid mainly containing the low-abundance difference protein of the abnormal patient, and then the concentrated protein is again used as the antigen for the second immunization, thereby preparing the differential protein for low abundance.
  • Polyclonal antibody due to the low amount of differential protein expression, the amount of protein expression can be increased, so that the amount of protein control samples in abnormal patients can be increased until the collected low-concentration samples are collected.
  • the amount of difference protein can satisfy the use requirements of the present invention.
  • the second immunization with the low-abundance protein in the step S50 needs to be carried out by using the immune body different from the step S20.
  • the first immunization in the step S30 is performed by the rabbit, and the re-immunization of the step S40 can be performed. It was carried out using rats.
  • step S50 By the immunization of step S50, a polyclonal antibody specific for a low-abundance differentially expressed protein can be obtained, and then steps S60 and S70 can finally pass the affinity and specificity of the low-abundance difference protein possessed by the polyclonal antibody.
  • the same differential proteins in the protein sample to be tested are separated.
  • the process of implementation is still a method similar to the above-mentioned high-abundance chromatography column, and the low-abundance differential protein polyclonal antibody obtained by the second immunization in step S50 is used as an affinity ligand, and the value is fixed in a covalent or non-covalent manner.
  • an affinity chromatography column for the low-abundance protein is prepared; then the protein sample to be tested is treated with the low-abundance affinity chromatography column, and the low-abundance difference protein in the protein sample to be tested can be The adsorption of the column, after elution with the eluent can directly obtain the up-regulated protein which is more abundant in the normal plasma.
  • step S70 since the content of the high-abundance protein in the protein sample to be tested itself has an absolute advantage, a strong competitive advantage may be generated in the adsorption, so in order to further improve the low abundance in the implementation.
  • the specificity and the competitive disadvantage of reducing low-abundance proteins so it is possible to preliminarily analyze the histone samples to be tested by gradient centrifugation before the column is passed, and to remove some molecular weights by gradient centrifugation. Or it is a large amount of high-abundance protein, which is more conducive to improving the specificity of low-abundance protein affinity adsorption.
  • the histone sample to be tested may also be subjected to a column using a blank column that does not contain a low-abundance protein polyclonal antibody.
  • the column is subjected to a blank column which is not coupled with a polyclonal antibody and contains only a solid phase matrix.
  • the object of the present invention is to use it as a background, factors which cause non-specific binding, and small particle fragments and the like are retained in the column.
  • the blank chromatography column containing only the column matrix can be used to initially remove background interference, and further enhance the specific adsorption effect of the multi-resistance column on the sample to be tested.
  • the affinity chromatography column may be further washed with a buffer to remove the protein which is not retained and retained in the interstitial space, thereby reducing the background interference.
  • the buffer used for washing may be any buffer which does not interfere with the antigen-antibody binding reaction.
  • the elution solution of imidazole or the like is finally used for elution, and the low-abundance protein in the sample to be tested can be isolated.
  • the eluate is then concentrated, silver stained by SDS-PAGE, Western-blot After analysis and mass spectrometry, low-abundance difference proteins can be quickly displayed.
  • the above-mentioned overall process is based on the fact that the low-abundance protein species and the amount of differential expression are relatively small. Therefore, after finally performing the column purification as described above, the eluted fraction obtained after elution is analyzed by LTQ-MASS.
  • the process previously employed by the inventors used a high-abundance protein to prepare a specific polyclonal antibody for chromatographic collection of the flow-through solution, while the flow-through solution contained a large amount of protein present in normal plasma, such as Apo. E, Hemopexin, Sex hormone-binding globulin, Apo B-100 and Haptoglobin-related Protein, etc.; these proteins appear in the flow through solution because the concentration of these proteins in plasma is greatly increased under various abnormal physiological conditions, exceeding the polyclonal antibody on the affinity adsorption medium.
  • the ratio is such that it cannot be completely adsorbed; therefore, the final flow through solution requires further separation and purification for alignment and mass spectrometry.
  • the adsorption process is specific adsorption
  • the finally collected eluate is basically a specific protein specifically adsorbed, and substantially does not contain the above high-abundance protein, and can directly perform alignment and mass spectrometry.
  • the above patients can be selected according to different types of different diseases, such as lymphoma, melanoma, gastric cancer, rectal cancer, bacterial infection, blood cancer, breast cancer, cervical cancer, colon cancer, which are generally easy to miss early diagnosis.
  • the control patient protein sample is obtained from a blood sample of a patient diagnosed as having the above-mentioned disease, and after the blood sample is collected, the plasma after separating the blood cells is centrifuged as a sample.
  • the amount of the substrate used for preparing the chromatography column should be adjusted to be slightly above the saturation level, maintaining the volume with the polyclonal antibody matrix, and the non-specific background is reduced as much as possible.
  • the capture amount of the corresponding antigen by the polyclonal antibody is related to the flow rate, and the column capacity is more effective when the flow rate is slower, because the coupled antibody has more time to bind to the antigen.
  • the mechanical pressure during loading should be as small as possible and avoid excessive contact with air.
  • the chromatographic medium in the chromatography column preferably adopts Sepharose CL in the present invention.
  • 4B as a solid phase matrix for the column, in the use of the invention, Sepharose CL Compared with other kinds of substrates, 4B has better porosity, which can increase the capacity of adsorption and avoid the shortage of target protein due to the competition of high abundance proteins in the sample to be tested.
  • the method for isolating the above-described low-abundance difference protein of the present invention is still difficult to perform and the method for antibody adsorption of high-abundance protein immunity is compared with the conventional proteomic 2D electrophoresis difference, and the present invention uses a low-abundance protein as a direct method.
  • the target is immunoaffined for affinity adsorption, and gradually weakens and reduces the competition of high-abundance proteins and background interference in all steps and processes, thereby improving the ability of low-abundance protein separation and enrichment, and finally obtaining specificity. Strong and relatively complete low-abundance differentially expressed proteins; providing better biological means for biomarker discovery and related monitoring.
  • the present invention further proposes the application of the above-described method for separating low-abundance differential proteins in the detection of low-abundance differential proteins, and the above-mentioned separation method can directly enrich low-abundance difference proteins. Can be used directly for subsequent CCD detection analysis, mass spectrometry, etc.
  • the separation process can be completed in one step according to the above method of the present invention, and the overall accuracy is good.
  • the biomarkers of the low-abundance difference proteins related to liver cancer are analyzed for the purpose of analysis. Therefore, the obtained protein samples are obtained from the blood samples of the already diagnosed liver cancer patients and normal humans, and the specific steps are as follows: get on:
  • step S21' taking the healthy human plasma sample 2mL in step S10', and 2 Mol Freund's complete adjuvant mixing, ultrasonic emulsification completely; three rabbits were subcutaneously injected, and blood samples were taken from the rabbit's ear root vein;
  • the rabbit blood sample taken in step S22' is subjected to an ELISA test to determine the specific adsorption of the polyclonal antibody obtained in the protein solution obtained in S22'; when the ELISA test detects the blood titer of the immunized rabbit reaches 104 ⁇ At 105 hours, the rabbits were sacrificed to take blood. If the ELISA test results are not up to standard, then the rabbits are subjected to enhanced immunization by repeating step S22' until the standard is met, and the rabbits are sacrificed for blood collection.
  • a rabbit can bleed 100 ⁇ 120 mL; put the obtained blood into a container pre-filled with anticoagulant, mix well, centrifuge at 1000 rpm for 4 min at 4 ° C, and take out the supernatant as plasma;
  • step S25' 10 mL is taken out from the rabbit plasma of step S24' to extract a high-abundance protein polyclonal antibody;
  • Sepharose CL 4B medium obtained from Shanghai source leaf biological CAS No.61970-08-9;
  • Sepharose CL 4B medium of step S31' using deionized water 500 Wash in mL, drain to a wet cake (settlement volume of about 45 mL), transfer to a 200 mL beaker; suspend the gel in 100 mL of deionized water and gently stir with a magnetic stir bar;
  • the filtrate was neutralized with unreacted CNBr with 500 mL of 0.1 M FeSO4 and poured into a waste tank;
  • step S34' with 0.1M at pH 8.5 NaHCO3 was dialyzed from the eluted protein solution prepared in step S25' (i.e., the high abundance polyclonal antibody isolated in the immunization step), mixed with the medium, uniformly mixed for 35 hours at 4 ° C; and added with ethanolamine 0.5 Mol, shake at 5 ° C for 5 h; wash the medium with pH 7.0, 0.01 M in PBS buffer, and install the column.
  • S41' takes 20 ⁇ L of the patient control plasma sample of step S10', and adds 980 ⁇ l of 10 mM.
  • step S42' takes a Sepharose CL 4B medium to prepare a blank chromatography column, and the sample to be tested prepared in step S41' is 10 After 5% PBS equilibrated with a blank column, and washed with PBS for 3 volumes to collect the flow through;
  • S51' will pass all the plasma samples of the abnormal patient through the flow through the column, and after all collection (if the amount is insufficient, the amount of the plasma sample of the abnormal patient can be further increased), and concentrated by the dialysis bag;
  • step S52' then refers to step S21' ⁇
  • the low-abundance differentially expressed protein in the plasma of the patient obtained by concentrating in step S51' is used as an antigen, and the rat is re-immunized as an immunized body, and finally the polyclonal antibody of the low-abundance protein is finally extracted;
  • a low-abundance affinity chromatography column using the low-abundance protein polyclonal antibody as an affinity ligand is prepared by the polyclonal antibody of the low-abundance protein prepared in the step S52';
  • the plasma protein sample to be tested is preliminarily removed by gradient centrifugation to remove some high-abundance proteins
  • step S72' a sample of the plasma protein to be tested with a high abundance protein is initially removed in step S71', using Sepharose CL 4B medium preparation A blank chromatography column is passed through the column to remove background interference and collect flow through;
  • the low-abundance affinity chromatography column after the step S73' is passed through the column is eluted with an imidazole eluate to collect the eluted fraction.
  • step S74' is collected into the eluate, i.e., there is a low abundance differentially expressed protein to be prepared.
  • the following process continues in the present invention:
  • step S80' then adds the eluate obtained in step S74' to a 1/4 volume of 50% TCA solution, and after 4 hours at 4 ° C in the refrigerator, at 4 Centrifuge at 12000 rpm for 30 min at ° C to take a precipitate;
  • the above precipitated sample was added to 1 mL, pre-cooled acetone at -20 ° C, 4 ° C, 12000 rpm, centrifuged 10 Min, discard the supernatant (repeated 3 times); vacuum dry, store in a refrigerator at 4 ° C for use.
  • test sample 1 and sample 2 plasmas of patients who have been diagnosed as liver cancer and normal healthy human plasma are selected as test sample 1 and sample 2 to be tested, respectively, for treatment, and finally the eluate is collected. Then perform SDS-PAGE electrophoresis silver staining, followed by Western-blot LTQ-MASS analysis, the results of the comparison are shown in the attached figure.
  • 1 is a film obtained by SDS-PAGE electrophoresis silver staining using the plasma of normal human plasma and another liver cancer patient as the test protein, respectively, according to the above-mentioned steps; the bands 1 to 3 in FIG.
  • the bands 4 ⁇ 6 are the eluted proteins of the liver cancer patients after affinity adsorption by polyclonal antibody; the bands 1 ⁇ 6 of the electrophoretic film are obviously It can be seen that the number of proteins in normal human bands is significantly less than that in non-normal human bands. Moreover, the number of proteins stained in the strips 4 ⁇ 6 is 2, and only 1 in the strips 1-3, and the dragging of the strips 4 ⁇ 6 is obvious because of the differential expression of more proteins. of.
  • the proteins expressed by normal humans and liver cancer patients are different, and the specific related functions may not be accurate, and further tracking and analysis are needed, but the difference between acetonitrile detection is Can be determined.
  • the low-abundance differentially expressed proteins obtained by the method of the present invention cannot be obtained by proteomics such as conventional 2D electrophoresis; and the low abundance can be directly isolated and prepared in one time.

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Abstract

一种低丰度差异蛋白的分离方法，包括：获取健康对照组蛋白样品、患者对照组蛋白样品和待测组蛋白样品；将健康对照组蛋白样品作为抗原进行免疫，制备高丰度蛋白多抗，并以该高丰度蛋白多抗为亲和配体制备高丰度亲和层析柱；将患者对照组蛋白样品于高丰度亲和层析柱过柱，收集流穿组份；将流穿组分浓缩后作为抗原进行再次免疫，制备低丰度蛋白多抗；并以低丰度蛋白多抗作为亲和配体制备低丰度亲和层析柱；将待测组蛋白样品于低丰度亲和层析柱进行过柱，并收集洗脱组分。该过程可以一次直接分离得到正常血浆中含量较多的上调蛋白，降低了分离过程的繁复性，而且直接针对差异表达的低丰度蛋白，具有更高的特异性和针对性。

Description

低丰度差异蛋白的分离方法及其应用
技术领域
本发明属于蛋白检测技术领域，具体涉及一种低丰度差异蛋白的分离方法及其应用。
背景技术
经过对所有疾病潜伏和发病过程的研究已经证实，潜在的疾病或潜伏期的生理状态都已经找到了很好的和生理状态相关的生物标记分子，这些生物标记分子，大多是丰度较低的蛋白。这些低丰度蛋白在血浆中，都只有很低的浓度，且经常会被白蛋白等高丰度蛋白，致使在疾病潜伏的前期检测中难以被检测出，大大延迟了疾病的发现时间。所以，差异性的比较正常及非正常生理状态下的血浆蛋白，并找出相应的生物标记分子，从而辅助疾病的更早发现具有重要的医学意义。
然而基于上述目的，要差异性的比较正常及非正常生理状态下的血浆蛋白，并找出相应的生物标记分子，采用目前蛋白质组学的常规检测手段，存在着比较大的困难。原因在于寻找生物标记蛋白的过程中，要先消除高丰度蛋白对整个系统的掩盖，使很低浓度的蛋白可以被检测到。目前通常的方法是将所有的蛋白显示在2D电泳胶上，然后对比不同样本之间的差异蛋白；而其中，首先2D电泳并不能显示出极度酸性或者碱性的蛋白，以及疏水性较大的脂蛋白；如果蛋白质过多，蛋白斑点的识别也存在着较大的困难；其次血浆中的高丰度蛋白会掩盖真正具有很重要生物活性作用的微量蛋白，虽然已经有报导各种亲和方法如CB染料介质、抗体介质等来去除高丰度蛋白，但能去除的蛋白还是很有限，并不能去除大部分house-keeping蛋白和正常功能蛋白；第三，和非正常生理状态相关的生物标记蛋白并不局限于低丰度蛋白，有些丰度较高的蛋白也在疾病报道中出现，因此所以仅仅使用高丰度去除也会导致结果不准确。
基于上述不足，本案的发明人早先于2010年在CN102430176A号专利中提出了一种对待测蛋白和对照蛋白之间的差异蛋白进行富集，从而提升待测蛋白或者多肽浓度的方法。其技术实现的过程采用将标准对照蛋白作为抗原进行免疫，制备得到能够与该对照样品中的至少一种蛋白或多肽结合的免疫球蛋白（其实就是以标准对照蛋白作为抗原产生的一抗抗体，属于多克隆抗体），然后用该免疫球蛋白制成亲和介质的层析柱，用来吸附待测样品中的大量高丰度蛋白，从而流穿组分即为富集浓缩的待测蛋白中的低丰度差异蛋白。
但是这一早先的做法中，采用正常对照蛋白通过免疫制备得到抗体，对照蛋白本身采用的是正常蛋白，所分离和浓缩得到的低丰度蛋白只是新出现或上调的潜在标志蛋白，而无法进一步直接确定这些蛋白的表达是属于非正常血浆中的下调蛋白或者是正常血浆中含量较多的上调蛋白，需要进一步的在采用对比进行大量的对比检测，增加繁复步骤之后降低了准确性，提升了工作量，使得上述方法实施中存在不足。
发明内容
本发明实施的目的在于克服现有技术的上述不足，提供一种能够直接分离富集非正常血浆中的低丰度下调蛋白的低丰度差异蛋白的分离方法及其应用。
为了实现上述发明目的，本发明实施例的技术方案如下：
一种低丰度差异蛋白的分离方法，包括如下步骤：
获取健康对照组蛋白样品和患者对照组蛋白样品和待测组蛋白样品；
将健康对照组蛋白样品作为抗原进行免疫，制备高丰度蛋白多抗；并以所述高丰度蛋白多抗为亲和配体与固相基质制备高丰度亲和层析柱；
将患者对照组蛋白样品于所述高丰度亲和层析柱进行过柱，并收集流穿组份；
将所述流穿组分浓缩后作为抗原进行免疫，制备低丰度蛋白多抗；并以所述低丰度蛋白多抗作为亲和配体固定与固相基质制备低丰度亲和层析柱；
将待测组蛋白样品于所述低丰度亲和层析柱进行过柱，并收集洗脱组分。
本发明在上述低丰度差异蛋白的分离方法的基础上，还提出一种上述低丰度差异蛋白的分离方法在低丰度差异表达蛋白检测上的应用。
本发明的低丰度差异蛋白的分离方法和应用，在原先方法的基础上，先用健康组的蛋白进行一次免疫，制备能用来滤除大量背景高丰度差异蛋白的高丰度多抗，然后用该高丰度蛋白多抗制备高丰度蛋白层析柱去除非正常患者差异表达蛋白样品中的背景高丰度蛋白，收集的流穿组分即为非正常患者差异表达的低丰度蛋白；然后将该流穿组分浓缩之后作为抗原去进行第二次免疫，得到低丰度差异表达的多抗，之后再用该低丰度差异表达的多抗作为配体进一步制备低丰度差异蛋白的层析柱，便可以直接一次分离待测组蛋白样品中属于非正常血浆中的下调蛋白或者是属于正常血浆中含量较多的上调蛋白。整体过程可以一次直接分离得到正常血浆中含量较多的上调蛋白，降低了分离过程的繁复性，提升了分离和检测效率，而且最后的洗脱组分直接针对差异表达的低丰度蛋白，具有更高的特异性和针对性。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1为本发明实施例分别采用正常人血浆和另一名肝癌患者血浆分别作为待测蛋白按照上述步骤检测获得的洗脱液进行SDS-PAGE电泳银染的图片。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实例提出一种低丰度差异蛋白的分离方法，包括步骤概括如下：
S10，获取健康对照组蛋白样品、患者对照组蛋白样品和待测组蛋白样品；
S20，将健康对照组蛋白样品作为抗原进行第一次免疫，制备高丰度蛋白多抗；
S30，将步骤S20获得的高丰度蛋白多抗作为亲和配体，固定至层析柱的固相载体上，制备高丰度亲和层析柱；
S40，将患者对照组蛋白样品用步骤S30制备的高丰度亲和层析柱进行过柱，收集流穿液；
S50，将步骤S40收集的流穿液进行浓缩之后，作为抗原进行再次免疫，制备低丰度蛋白多抗；
S60，将步骤S50获得的低丰度蛋白多抗作为亲和配体，固定至层析柱的固相载体上，制备低丰度亲和层析柱；
S70，将待测组蛋白样品于步骤S60所制备的所述低丰度亲和层析柱进行过柱，并收集洗脱组分。
本发明的低丰度差异蛋白的分离方法，在原先方法的基础上，先用健康组的蛋白进行一次免疫，制备能用来滤除大量背景高丰度差异蛋白的高丰度多抗，然后用该高丰度蛋白多抗制备高丰度蛋白层析柱去除非正常患者差异表达蛋白样品中的背景高丰度蛋白，收集的流穿组分即为非正常患者差异表达的低丰度蛋白；然后将该流穿组分浓缩之后作为抗原去进行第二次免疫，得到低丰度差异表达蛋白的多抗，之后用该低丰度差异表达蛋白的多抗作为配体进一步制备低丰度差异蛋白的层析柱，便可以直接一次分离待测组蛋白样品中属于非正常血浆中的下调蛋白或者是属于正常血浆中含量较多的上调蛋白。从而整体采用本发明的上述操作过程进行，降低了分离过程的繁复性，提升了分离和检测效率，而且最后的洗脱组分直接针对差异表达的低丰度蛋白，具有更高的特异性和针对性。
其中，本发明的上述过程，在步骤S10中采用从健康正常人获取的蛋白、和患病的患者获取的蛋白这两者分别作为高丰度蛋白和低丰度差异蛋白的来源，在该患者获取的蛋白中低丰度差异蛋白的种类和量是明显不同于正常标准的，更加易于直接反映低丰度差异蛋白的表达情况。获取之后用该两种对照作为样品直接制备出针对对待测样品中低丰度蛋白的亲和配体，达到一次直接分离得到正常血浆中含量较多的上调蛋白的目的。具体，之后的处理如下：
为了提升分离低丰度蛋白的特异性和针对性，避免之后层析分离中的高丰度背景蛋白的干扰，需要进一步降低高丰度蛋白的竞争性抑制，所以在步骤S20中本发明将健康对照组蛋白样品作为抗原免疫家兔（也可以是大鼠等其他的免疫体），通过免疫制备得到免疫的多克隆抗体；免疫过程中大部分蛋白（基本为大量的高丰度蛋白）产生免疫由于；仅有小部分蛋白（基本为低丰度蛋白）因为本身结构的原因或者蛋白含量过低的原因，不能发生免疫反应；所以制备得到的针对于高丰度蛋白的多克隆抗体，通过该高丰度蛋白多克隆抗体来去除背景高丰度蛋白。
进一步，步骤S30将步骤S20制备得到的高丰度蛋白多抗作为亲和配体，固定至固相载体制备特异性吸附高丰度蛋白的层析柱；固相载体作为层析的载体，可以选择葡聚糖凝胶等类型进行；亲和配体与固相载体固定结合的方式可以采用共价交联等通常方式实现。
步骤S40在制备得到高丰度亲和层析柱之后，用该高丰度层析柱对非正常患者蛋白样品进行过柱处理，过柱的过程中由于层析的亲和介质是针对多克隆抗体剔除高丰度蛋白的系统，所以此次过柱蛋白样品中的大量高丰度蛋白会被吸附在层析柱上，得到的流穿组分中的成分即绝大多数非正常患者差异表达的低丰度蛋白。当然，在这一过程中为了尽量消除或者减少高丰度在流穿液中的量，可以将流穿液组分再次进行过柱，以尽量提升对背景蛋白的去除。同时，在实施的过程中，为了减少或者降低高丰度层析柱与样品中蛋白的非特异性吸附，将非正常患者蛋白对照样品用只含有固相基质而不含有配体的空柱子先过柱处理，这样非正常患者蛋白对照样品中的很多杂质或者非特异性吸附的因素会被初步滤除，提升高丰度蛋白层析柱亲和的特异性。
然后，步骤S50将步骤S40收集到主要是含有非正常患者低丰度差异蛋白的流穿液进行浓缩，然后将浓缩的蛋白再次作为抗原进行第二次免疫，制备得到针对于低丰度差异蛋白的多克隆抗体。其中，需要指出的是，由于低丰度差异蛋白表达的量较少，所以为了能使其达到免疫的量，实施中可以加大非正常患者蛋白对照样品的量，直至所收集到的低丰度差异蛋白的量能满足本发明的使用要求。同时，本步骤S50中用低丰度蛋白进行的第二次免疫需要采用与步骤S20不同的免疫体进行，比如步骤S30中的第一次免疫采用家兔进行，该步骤S40的再次免疫便可以采用大鼠进行。
通过步骤S50的免疫即能得到特异性针对低丰度差异表达蛋白的多抗，之后步骤S60和S70通过该多抗所具有的低丰度差异蛋白的亲和性和特异性，便能最终将待测蛋白样品中同类差异蛋白进行分离。实施的过程仍然是类似上述高丰度层析柱的方法，将步骤S50进行第二次免疫得到的低丰度差异蛋白多抗作为亲和配体，共价或者非共价的方式固定值固相载体上，制备针对低丰度蛋白的亲和层析柱；然后用该低丰度亲和层析柱处理待测蛋白样品，那么待测蛋白样品中的低丰度差异蛋白便能被该层析柱吸附，吸附之后用洗脱液进行洗脱便能直接得到待测样品属于正常血浆中含量较多的上调蛋白。
当然，在步骤S70的实施过程中，由于本身待测蛋白样品中高丰度蛋白的含量占到绝对优势，可能在吸附中会产生较强的竞争性优势，所以在实施中为了进一步提升低丰度的特异性、以及降低低丰度蛋白的竞争性劣势，因此可以采用在过柱之前将待测组蛋白样品先用梯度离心的方式进行初步处理，通过梯度离心的方式便可以初步除掉一些分子量或者是量大高丰度蛋白，从而更加利于提升低丰度蛋白亲和吸附的特异性。
当然，在待测组蛋白样品进行过柱的过程中，同样也是为了避免非特异性吸附，也可以采用将待测组蛋白样品用不含有低丰度蛋白多抗的空白层析柱进行过柱，采用该未偶联有多克隆抗体只含有固相基质的空白柱子进行过柱，本发明的目的是将其作为背景，可以引起非特异性结合的因素、以及小颗粒碎片等等被阻留在该只含有柱基质的空白层析柱上，从而实现初步去除背景干扰，更进一步提升多抗层析柱对于待测样品的特异性吸附的效果。
同时在上述步骤S70中，待测样品进行过柱处理之后，可以进一步用缓冲液对亲和层析柱进行洗涤，以去除没有结合而滞留在间质空隙内的蛋白，降低本底干扰。用于洗涤的缓冲液可以采用任何不干扰抗原-抗体结合反应的缓冲液都可以。
吸附之后最后再采用咪唑等洗脱液进行洗脱，即可分离得到待测样品中的低丰度蛋白。然后对洗脱液进行浓缩、SDS-PAGE电泳银染、Western-blot 分析、质谱之后便能将低丰度差异蛋白很快地显示出来。上述整体的过程基于本身差异表达的低丰度蛋白种类和量都比较少，因此最终按照上述实施过柱纯化之后，之后再进行洗脱得到的洗脱组分，用LTQ-MASS分析后，大致上仅需要从剩余的几十个蛋白质中比较不同生理状态的差异功能蛋白质；这大大减少了蛋白质差异显示的工作量，跳过了繁复的2D电泳，在疾病的早期检测和生物标记分子的发现检测领域有着较大的潜力。同时过柱吸附和洗脱的过程不需要多次重复，耗时和工作量大大缩短。
发明人早先采用的过程用高丰度蛋白制备特异性多抗进行层析收集流穿液，而流穿液中含有很多在正常血浆中存在的大量蛋白，如Apo E，Hemopexin，Sex hormone-binding globulin，Apo B-100和Haptoglobin-related protein等；这些蛋白之所以会在流穿溶液中出现，是因为在各种不同的非正常生理状态下，这些蛋白在血浆中的浓度大大增加，超过了多克隆抗体在亲和吸附介质上的比例，使得不能被完全吸附；因此最终的流穿液还需要进一步进行分离和纯化才能进行比对和质谱。而本发明中吸附的过程中都是特异性吸附，最终收集到的洗脱液，基本上都是特异性吸附的指定蛋白，基本上不含有上述高丰度蛋白，可以直接进行比对和质谱。
以上患者的可以根据不同的研究不同疾病的类型进行选择，可以选择比如一般容易错过早期诊断的淋巴癌、黑色素瘤、胃癌、直肠癌、细菌感染、血液病变、乳腺癌、宫颈癌、结肠癌、食道癌、淋巴瘤、白血病、肝癌、肺癌、非小细胞淋巴瘤、间皮瘤、多发性硬化、神经系统感染等等。对照患者蛋白样品采用从确诊为患有上述疾病的患者血样中进行获取，采集完血样后离心处理分离血细胞之后的血浆作为样品即可。
用本发明的亲和层析柱纯化的过程中，制备层析柱所用基质的量须调整到略高于其饱和水平，保持带有多克隆抗体基质的体积，使非特异性本底尽可能降低。而多克隆抗体对对应的抗原的捕获量与流速有关，流速较慢时柱容量更有效，因为偶联的抗体就有更多时间与抗原结合。同时，上样时的机械性压力应尽可能小，并避免过度与空气接触。
基于上述实施过程中，层析柱中的层析介质本发明中优选采用Sepharose CL 4B作为层析柱的固相基质，在本发明的使用中，Sepharose CL 4B相比其他种类的基质，具有比较好的多孔性，可以增加吸附的容量，避免因为待测样品中的高丰度蛋白的竞争性造成目的蛋白不足的情形。
本发明的上述低丰度差异蛋白的分离方法，相比通常的蛋白质组学的2D电泳差异性比较仍然难以进行以及高丰度蛋白免疫的抗体吸附的方法，本发明以低丰度蛋白作为直接目标进行免疫后亲和吸附，并且在所有的步骤和过程中逐步削弱和降低高丰度蛋白的竞争性以及背景性干扰，进而提升低丰度蛋白分离和富集的能力，最终获得特异性较强且种类较为完全的低丰度差异表达蛋白；为生物标记物的发现和相关的监测提供了更优的生物学手段。
所以基于相关的监测和检测的目的，本发明进一步提出上述低丰度差异蛋白的分离方法在低丰度差异蛋白检测中的应用，通过上述分离的方法可以直接富集到低丰度差异蛋白，可以直接用于后续的CCD检测分析，质谱等等。分离的过程按照本发明的上述方法一次即可完成，而且整体准确性较好。
为使本发明的上述实施的技术细节和过程方法能更易于本领域技术人员的理解和实施参考，同时凸显出本发明方法相比现有2D电泳以及高丰度蛋白免疫的抗体吸附的进步性，以下通过具体的实施例和实际分析数据进行举例说明。
实施例1
在该实施例1中以分析研究肝癌相关的低丰度差异蛋白的生物标记物为目的，因此采用获取的蛋白样品分别从已经确诊的肝癌患者和正常人血样中获得，具体步骤实施步骤参考如下进行：
S10’，分别获取健康人血浆蛋白样品、肝癌患者的血浆蛋白样品、以及待测蛋白血浆样品（以上血浆为将获取的血液样品添加抗凝剂并后进行离心处理，弃去细胞沉淀后的上层黄色清液）。
S21’，取步骤S10’中健康人血浆样品2mL，与2 ml弗氏完全佐剂混合，超声波乳化完全；对3只家兔进行皮下多点注射，并于家兔耳根静脉取血样保存；
S22’，加强免疫：两周后同样再次制取2mL步骤S21’中获得的蛋白溶液，与2 ml弗氏不完全佐剂混合，超声波乳化完全；对3只家兔进行皮下多点注射，并于家兔耳根静脉取血样保存；
S23’，将步骤S22’中取的家兔血样用ELISA实验测定生成的多克隆抗体对S22’中获得的蛋白溶液的特异性吸附情况；当ELISA试验检测免疫的家兔血滴度达到104~105时，处死家兔取血。如果ELISA试验检测结果达不到标准，那么继续重复步骤S22’对家兔进行增强免疫，直至达到符合标准时，处死家兔取血。
S24’，从步骤S23’中获得的家兔血样中分离血浆，一般一只家兔可放血100~120 mL；将取得的血液放入预先装有抗凝剂的容器，充分混匀后，1000 rpm 4℃离心5 min，取出上清即为血浆；
S25’，从步骤S24’的兔血浆中取出10mL，提取高丰度蛋白多克隆抗体；
用pH7.0的PBS缓冲液稀释5倍待用；取出Protein G柱（1ml），先用pH7.0的PBS缓冲液冲洗，直至蛋白核酸检测仪显示的数据达到基线；取出pH7.0的PBS缓冲液稀释的兔血浆溶液，以0.5 ml/min的流速上Protein G柱，上样完成后用pH 7.0的PBS缓冲液冲洗至基线，收集流穿液；当然，这一个步骤中收集的流穿液中也可能还含有尚未被吸附的多克隆抗体，所以该流穿液可以重复多次过柱，以降低多克隆抗体的浪费；
用pH2.5Gly-HCl缓冲液冲洗介质，接收洗脱蛋白溶液，即为针对高丰度蛋白的多克隆抗体，然后立即调节pH值至中性；测定A280nm以及A260nm，估算蛋白浓度；对洗脱蛋白溶液进行SDS-PAGE分析。
S30’，制备多克隆抗体亲和柱：
S31’，获取Sepharose CL 4B介质（购自上海源叶生物CAS No.61970-08-9）；
S32’，然后将步骤S31’的Sepharose CL 4B介质，用去离子水500 mL清洗，抽干成湿饼状（沉降体积为约45 mL），转移到200 mL烧杯中；凝胶悬浮在100 mL去离子水中，用磁力搅拌棒轻轻搅拌；
S33’，取4.2 g CNBr溶解在50 ml HPLC级乙腈中，加入到凝胶悬液中，放入pH探头，用20%的NaOH维持反应混合物的pH在11.0，整个系统处于冰浴中，温度维持0℃；反应10~15 min时，检查CNBr是否全部溶解。此时，NaOH消耗速度将减少；pH稳定在11.0后，反应混合物倾入预冷的砂芯玻璃漏斗中，用1L冰冷的去离子水和500mL冰冷的偶联缓冲掖（pH8.5，0.1M NaHCO3）快速地洗涤凝胶，由于活化载体的不稳定性，凝胶必须立即偶联蛋白质或配基；
滤出液用500 mL0.1 M FeSO4中和没反应的CNBr，倒入废液缸；
S34’，用pH 8.5的0.1M NaHCO3透析从步骤S25’制备获得的洗脱蛋白溶液（即免疫步骤中分离的高丰度多抗），并与介质混合，4℃环境下均匀混合35h；加入乙醇胺0.5 ml，4℃环境下振荡5 h；用pH 7.0，0.01 M的PBS缓冲液清洗介质后，装柱。
S40’上样过柱：
S41’将步骤S10’的患者对照血浆样品取20µL，加入980 µl 10 mM PBS；使用公式：C（mg/ml）=1.45ｘA280nm-0.74ｘA260nm估算样品的蛋白浓度；根据估算的浓度取约含1 mg待测样品备用；
S42’取Sepharose CL 4B介质制备一空白层析柱，将步骤S41’制备的待测样品上10 mM的PBS平衡过的空白层析柱后，并用PBS清洗3个体积收集流穿液；
S43’将步骤S42’中用空白层析柱初步过滤之后收集的流穿液，上10mM的PBS平衡过的步骤S34’制备的层析柱；并用PBS冲洗介质3~4个柱体积，接收各流穿溶液（该流穿液可以再次进行过柱，并重复2~3次）。
S51’将全部的非正常患者的血浆样品全部过柱之后的流穿液，全部收集后（如果量不够可以进一步加大非正常患者的血浆样品的量），用透析袋浓缩；
S52’然后参照步骤S21’~ S25’的过程，将步骤S51’浓缩得到的病患血浆中的低丰度差异表达蛋白作为抗原，以大鼠为免疫体进行再次免疫，同样最后提取低丰度蛋白的多克隆抗体；
S60’，将步骤S52’制备的低丰度蛋白的多克隆抗体，参照步骤S30’的全部过程制备以该低丰度蛋白多克隆抗体为亲和配体的低丰度亲和层析柱；
S71’，将待测血浆蛋白样品用梯度离心沉降的方式初步简单去除一些高丰度蛋白之后备用；
S72’，将步骤S71’中初步去除了一些高丰度蛋白的待测血浆蛋白样品，用Sepharose CL 4B介质制备一空白层析柱过柱，去除背景干扰，收集流穿液；
S73’，将步骤S72’中空白过柱之后的流穿液，上10mM的PBS平衡过的步骤S60制备的低丰度亲和层析柱，并用PBS冲洗介质3~4个柱体积；
S74’，将步骤S73’过柱之后的低丰度亲和层析柱用咪唑洗脱液进行洗脱，收集洗脱组分。
基本上步骤S74’收集到洗脱液之中即存在所要制备的低丰度差异表达蛋白。为了验证这一结果，本发明中继续进行下述过程：
S80’然后将步骤S74’中获得的洗脱液加入1/4倍体积的50%TCA溶液，4℃冰箱过夜后，于4 ℃下12000 rpm离心30min，取沉淀；
上述沉淀样品加入1mL，-20℃预冷丙酮清洗，4℃，12000 rpm，离心10 min，弃上清（重复3次）后；真空干燥，保存于4℃冰箱待用。
为了突出对比肝癌患者和正常人蛋白差异表达的情况，分别同时选取已经确诊为肝癌患者血浆和正常健康人血浆分别作为待测样本1和待测样本2，进行处理，最后收集洗脱液。然后进行SDS-PAGE电泳银染，后Western-blot 、LTQ-MASS分析，比对的结果参见附图所示。其中附图1为分别采用正常人血浆和另一名肝癌患者血浆分别作为待测蛋白按照上述步骤检测获得的洗脱液进行SDS-PAGE电泳银染的胶片；图1中的条带1~3为正常人血浆经过多克隆抗体亲和吸附后的洗脱蛋白，条带4~6为肝癌患者血浆经过多克隆抗体亲和吸附后的洗脱蛋白；从电泳胶片的条带1~6明显可以看出，正常人条带的蛋白数量明显少于非正常人条带的蛋白量。而且胶条4~6中蛋白的染色明显的蛋白数量为2条、胶条1~3只有1条，并且胶条4~6的拖带现象比较明显，是因为存在较多的蛋白差异表达所造成的。
进一步进行LTQ-MASS分析的结果如下表：
表1 正常人血浆经过多克隆抗体亲和吸附后的洗脱蛋白
Group number Protein name 备注
正常血浆蛋白 Gene_Symbol=TF Serotransferrin precursor 高丰度蛋白残留
正常血浆蛋白 Gene_Symbol=ALB Uncharacterized protein ALB 高丰度蛋白残留
正常血浆蛋白 Gene_Symbol=FGA Isoform 1 of Fibrinogen alpha chain precursor 高丰度蛋白残留
正常血浆蛋白 Gene_Symbol=IGHG1 Putative uncharacterized protein DKFZp686N02209 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGHM FLJ00385 protein (Fragment) 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGHG2 Putative uncharacterized protein DKFZp686I04196 (Fragment) 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=GH1;CSH1;CSH2 Isoform 1 of Somatotropin precursor 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=PTPRK Protein tyrosine phosphatase, receptor type, K 免疫球蛋白片段
正常细胞功能蛋白 Gene_Symbol=ELL RNA polymerase II elongation factor ELL 　
正常细胞功能蛋白 Gene_Symbol=RPS27A;UBC;UBB ubiquitin and ribosomal protein S27a precursor 　
正常细胞功能蛋白 Gene_Symbol=ARMC3 Isoform 2 of Armadillo repeat-containing protein 3 　
正常细胞功能蛋白 Gene_Symbol=PDE4A Isoform 3 of cAMP-specific 3',5'-cyclic phosphodiesterase 4A 　
正常细胞功能蛋白 Gene_Symbol=- Lamin-like protein in HindIII repetitive element derived DNA, 3' end (Fragment) 　
正常细胞功能蛋白 Gene_Symbol=SLC22A11 Isoform 1 of Solute carrier family 22 member 11 　
正常细胞功能蛋白 Gene_Symbol=ZCCHC11 Isoform 1 of Zinc finger CCHC domain-containing protein 11 　
正常细胞功能蛋白 Gene_Symbol=GRHL1 Uncharacterized protein GRHL1 　
正常细胞功能蛋白 Gene_Symbol=B3GALT6 B3GALT6 protein (Fragment) 　
正常细胞功能蛋白 Gene_Symbol=- Chromosome 1 open reading frame 113 　
新蛋白 Gene_Symbol=RP5-1054A22.3 Novel protein 新蛋白
新蛋白 Gene_Symbol=DKFZp686D0972 hypothetical protein LOC345651 假设蛋白
新蛋白 Gene_Symbol=Hypothetical protein DKFZp686O01196 假设蛋白
新蛋白 Gene_Symbol=Hypothetical protein DKFZp686P15220 假设蛋白
新蛋白 Gene_Symbol=45 kD Keratin type II protein 假设蛋白
表2 肝癌患者血浆经过多克隆抗体亲和吸附后的洗脱蛋白
Group Protein name 备注
正常血浆蛋白 Gene_Symbol=ALB Uncharacterized protein ALB 残留的高丰度蛋白
正常血浆蛋白 Gene_Symbol=LOC653879 similar to Complement C3 precursor 残留的高丰度蛋白
正常血浆蛋白 Gene_Symbol=A2M Alpha-2-macroglobulin precursor 残留的高丰度蛋白
正常血浆蛋白 Gene_Symbol=IGHG2 Putative uncharacterized protein DKFZp686I04196 (Fragment) 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGHG1 Putative uncharacterized protein DKFZp686P15220 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGHM FLJ00385 protein (Fragment) 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGKV1-5 IGKV1-5 protein 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGKV1-5 IGKV1-5 protein 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGL@ IGL@ protein 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGKV3-20 Ig kappa chain V-III region HAH precursor 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=- 42 kDa protein 免疫球蛋白片段
正常血浆蛋白 Gene_Symbol=IGHG1 Putative uncharacterized protein DKFZp686O01196 食道肿瘤组织中提取的cDNA编码的免疫球蛋白重链
肝癌相关差异蛋白 Gene_Symbol=KRT13 31 kDa protein I型细胞角蛋白
肝癌相关差异蛋白 Gene_Symbol=- 45 kDa protein II型细胞角蛋白
肝癌相关差异蛋白 Gene_Symbol= Human protein IPI00018320/ IPI00001434 Isoform 2 of Protocadherin-11 Y-linked/ Protocadherin beta-14, associated with placenta
肝癌相关差异蛋白 Gene_Symbol=Hypothetical protein DKFZp686O01196 Similar to immunoglobulin heaven chain; the cDNA was cloned from esophagus tumor tissue
肝癌相关差异蛋白 Gene_Symbol=Hypothetical protein DKFZp686P15220 Similar to immunoglobulin heaven chain; the cDNA was cloned from rectum tumor tissue
肝癌相关差异蛋白 Gene_Symbol=45 kD Keratin type II protein Specificity of expression of cytokeratin proteins is retained in malignant tumors
肝癌相关差异蛋白 Gene_Symbol=Human protein IPI00018320 Isoform 2 of Protocadherin-11 Y-linked, and it was reported to influence the apoptotic sensitivity prostate cancer cell
　　　　　　　　　　　　　　　　　　　　　　　　　　
　
从上述差异蛋白的比对结果，正常人和肝癌患者表达的蛋白是存在差异的，具体的相关的功能上可能还不一定准确，有待于更进一步的跟踪和分析，但是乙腈检测到存在差异是可以确定。而且从上述还可以看出本发明的方法得到的低丰度差异表达蛋白，这些是采用常规的2D电泳等蛋白质组学手段无法获取的；而且可以直接一次性便能分离和制备得到低丰度差异表达蛋白的种类和数量，进一步进行层析的流穿液进行LTQ-MASS分析的结果可以看出，检出的正常血浆蛋白和正常细胞功能蛋白的种类和数量减少了，说明本发明方法对于高丰度蛋白的背景干扰去除的效果更好，对低丰度差异表达蛋白具有更高的特异性和针对性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包括在本发明的保护范围之内。

Claims (11)

  1. 一种低丰度差异蛋白的分离方法，其特征在于，包括如下步骤：
    获取健康对照组蛋白样品、患者对照组蛋白样品和待测组蛋白样品；
    将健康对照组蛋白样品作为抗原进行免疫，制备高丰度蛋白多抗；并以所述高丰度蛋白多抗为亲和配体与固相基质制备高丰度亲和层析柱；
    将患者对照组蛋白样品于所述高丰度亲和层析柱进行过柱，并收集流穿组份；
    将所述流穿组分浓缩后作为抗原进行再次免疫，制备低丰度蛋白多抗；并以所述低丰度蛋白多抗作为亲和配体固定与固相基质制备低丰度亲和层析柱；
    将待测组蛋白样品于所述低丰度亲和层析柱进行过柱，并收集洗脱组分。
  2. 如权利要求1所述的低丰度差异蛋白的分离方法，其特征在于，将待测组蛋白样品于所述低丰度亲和层析柱进行过柱，并收集洗脱组分步骤之前，还包括：
    将所述待测组蛋白样品用不含低丰度蛋白多抗为亲和配体的空白层析柱进行过柱。
  3. 如权利要求1所述的低丰度差异蛋白的分离方法，其特征在于，所述固相基质为Sepharose CL 4B。
  4. 如权利要求1所述的低丰度差异蛋白的分离方法，其特征在于，将患者对照组蛋白样品于所述高丰度亲和层析柱进行过柱，并收集流穿组份步骤之前，还包括：
    将所述患者对照组蛋白样品进行梯度沉降处理。
  5. 如权利要求1所述的低丰度差异蛋白的分离方法，其特征在于，将待测组蛋白样品于所述低丰度亲和层析柱进行过柱，并收集洗脱组分步骤之后，还包括：
    将所述洗脱组分进行浓缩、电泳、Western-blot分析和质谱处理。
  6. 如权利要求1所述的低丰度差异蛋白的分离方法，其特征在于，将患者对照组蛋白样品于所述高丰度亲和层析柱进行过柱，并收集流穿组份步骤之前，还包括：
    将所述患者对照组蛋白样品用不含高丰度蛋白多抗为亲和配体的空白层析柱进行过柱。
  7. 如权利要求6所述的低丰度差异蛋白的分离方法，其特征在于，将待测组蛋白样品于所述低丰度亲和层析柱进行过柱，并收集洗脱组分步骤之前，还包括：
    将所述待测组蛋白样品用不含低丰度蛋白多抗为亲和配体的空白层析柱进行过柱。
  8. 如权利要求6所述的低丰度差异蛋白的分离方法，其特征在于，所述固相基质为Sepharose CL 4B。
  9. 如权利要求6所述的低丰度差异蛋白的分离方法，其特征在于，将患者对照组蛋白样品于所述高丰度亲和层析柱进行过柱，并收集流穿组份步骤之前，还包括：
    将所述患者对照组蛋白样品进行梯度沉降处理。
  10. 如权利要求6所述的低丰度差异蛋白的分离方法，其特征在于，将待测组蛋白样品于所述低丰度亲和层析柱进行过柱，并收集洗脱组分步骤之后，还包括：
    将所述洗脱组分进行浓缩、电泳、Western-blot分析和质谱处理。
  11. 如 权利要求1所述的低丰度差异蛋白的分离方法在低丰度差异表达蛋白检测上的应用。
PCT/CN2015/088825 2015-07-10 2015-09-02 低丰度差异蛋白的分离方法及其应用 WO2017008380A1 (zh)

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