CN110244047B - Lung cancer serum diagnosis marker and application thereof, and separation and identification method of soluble protein related to lung cancer - Google Patents

Lung cancer serum diagnosis marker and application thereof, and separation and identification method of soluble protein related to lung cancer Download PDF

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CN110244047B
CN110244047B CN201910496459.8A CN201910496459A CN110244047B CN 110244047 B CN110244047 B CN 110244047B CN 201910496459 A CN201910496459 A CN 201910496459A CN 110244047 B CN110244047 B CN 110244047B
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代玉梅
蓝姣利
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Guangzhou Women and Childrens Medical Center
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Abstract

The invention relates to a lung cancer serum diagnosis marker and application thereof, and a separation and identification method of lung cancer related soluble proteins. The lung cancer serum diagnostic marker comprises at least one of expressing up-regulated full-length soluble hMSH3 protein, expressing down-regulated full-length soluble hMSH6 protein and expressing down-regulated soluble hMSH6 protein fragments with molecular weight of 45KD-50 KD. The invention creatively researches and analyzes lung cancer patients by further exploring the effect and significance of hMSH2 and the soluble forms of the interaction proteins in the V gamma 9 delta 2T cell-mediated cancer immune escape, utilizes the technical means such as serum protein extraction, immunoblotting and/or co-immunoprecipitation and the like to determine the existence forms and the expression differences of the soluble hMSH2, the hMSH3 and the hMSH6 in serum, and screens, separates, purifies and identifies the soluble forms of other proteins which interact with the soluble hMSH2, thereby providing technical support for further exploring the effect of the hMSH2 and the interaction proteins in the V gamma 9 delta 2T cell-mediated lung cancer natural immunity.

Description

Lung cancer serum diagnosis marker and application thereof, and separation and identification method of soluble protein related to lung cancer
Technical Field
The invention relates to the field of molecular biological detection, in particular to a lung cancer serum diagnosis marker and application thereof, and a separation and identification method of lung cancer related soluble proteins.
Background
Lung cancer is currently one of the cancers with the highest mortality rate in humans. According to global cancer statistics data in 2018, the total annual incidence number of the cancer is 209 ten thousand, and the total annual incidence number of the cancer accounts for 11.6 percent of the total cancer cases; the total annual death count is 176 ten thousand, accounting for 18.4% of the total cancer death count. In the past 30 years, the incidence rate and death rate of lung cancer in China are rapidly increased, and the total incidence rate of lung cancer in China reaches 73 ten thousand cases only in 2015.
The conventional treatment means for lung cancer at present comprise operation treatment, radioactive treatment, chemical drug treatment and the like. Common side effects of the traditional therapies, such as tumor metastasis caused by surgery, adverse reactions such as nausea, vomiting and alopecia accompanied by radiotherapy and chemotherapy, and extremely low five-year survival rate, promote the generation and development of an immunoadoptive therapy strategy based on activation of tumor-associated antigens (or ligands). Wherein, the V gamma 9 delta 2T cells can directly identify and bind antigen in a non-limiting way by MHC (major histocompatibility complex, histocompatibility complex), and can generate a large amount of IFN-gamma after ligand and receptor activation, and the NK cells are easy to be activated and amplified in vitro and in vivo and the like, and become the afterward of tumor immunity adoptive treatment.
In view of the problems of large side effect, high mortality rate and the like in the prior art, the early diagnosis of lung cancer has important significance for preventing and treating lung cancer.
Disclosure of Invention
Based on the above, it is necessary to provide a lung cancer serum diagnosis marker and application thereof, and a separation and identification method of lung cancer related soluble proteins, which are used for guiding the diagnosis practice of lung cancer, and widening the range of the diagnosis method and the treatment method of lung cancer.
A lung cancer serum diagnostic marker comprising at least one of a full length soluble hMSH3 protein having a molecular weight of 130KD up-regulated relative to the expression of healthy human serum, a full length soluble hMSH6 protein having a molecular weight of 160KD down-regulated, and a soluble hMSH6 protein fragment having a molecular weight of 45KD-50KD down-regulated.
In one embodiment, the lung cancer serum diagnostic marker further comprises a full length soluble Hsp70 protein having a molecular weight of 70KD that is down-regulated relative to the expression of healthy human serum.
The application of the lung cancer serum diagnosis marker in any embodiment in preparing lung cancer diagnosis reagent, diagnosis chip, diagnosis kit or detection equipment.
A lung cancer serum diagnosis chip, on which an antibody corresponding to the lung cancer serum diagnosis marker of any one of the above embodiments is immobilized, wherein the antibody can specifically bind to the corresponding marker.
In one embodiment, the antibody is a monoclonal antibody.
A method for separating and identifying soluble protein related to lung cancer comprises the following steps:
extracting serum proteins from a serum sample of a lung cancer patient;
the related antibodies of hMSH2 protein, hMSH3 protein and hMSH6 protein are used for separating and identifying the extracted serum proteins by using an immunoblotting method and/or a co-immunoprecipitation method.
In one embodiment, the method for separating and identifying soluble protein related to lung cancer further comprises the step of removing albumin and immunoglobulin in serum proteins after extracting the serum proteins in the serum sample of the lung cancer patient, and the subsequent separating and identifying is to separate and identify the serum proteins from which the albumin and the immunoglobulin are removed.
In one embodiment, the method for isolating and identifying soluble proteins associated with lung cancer further comprises the step of detecting the concentration of serum proteins depleted of albumin and immunoglobulin using BCA protein concentration detection.
In one embodiment, the presence and expression differences of the protein of interest are detected and analyzed using immunoblotting methods on lung cancer patient serum samples and healthy human serum samples.
In one embodiment, the presence of the protein of interest and other proteins that interact with the protein of interest are screened, isolated, purified and assayed using an antibody-agar coupled spin column immunoprecipitation method for lung cancer patient serum samples and healthy human serum samples.
It was found that under physiological conditions, the normally encoded human MutS homologous protein 2 (human MutS homologue, hMSH) and its partner hMSH3, hMSH6 are important members of the family of DNA Mismatch repair (MMR) proteins, normally located in the nucleus, for maintaining genomic stability. Earlier further studies have found that hMSH2, hMSH3, hMSH6 are ectopic expressed on the surface of tumors of epithelial origin and EBV transformed B lymphocytes, and identified that this ectopic expressed membrane hMSH2 (membrane-hMSH 2, mhMSH 2) is an endogenous, stress protein ligand recognized by vγ9δ2t cells.
The invention creatively researches and analyzes lung cancer patients by further exploring the action and significance of hMSH2 and the soluble forms of the interaction proteins thereof in the Vγ9δ2T cell mediated cancer immune escape and the application value of the hMSH2 and the interaction proteins thereof in the early diagnosis and prognosis of clinical cancers, utilizes the technical means such as serum protein extraction, immunoblotting and/or immune co-precipitation and the like, and utilizes the technical means such as serum protein extraction, normal serum and lung cancer cell line whole cell lysate to determine the existence form and expression difference of the soluble hMSH2, hMSH3 and hMSH6 of lung cancer, screens, separates, purifies and identifies the soluble forms of other proteins which interact with the soluble hMSH2 in the normal serum and lung cancer whole cell lysate and further explores the action and significance of the soluble hMSH2 and the interaction proteins thereof in the Vγ9δ2T cell mediated lung cancer natural immune, and provides clues and basis for the application value in the early diagnosis and prognosis of lung cancer.
The invention researches find that the protein can be used as a serum diagnostic marker for diagnosing lung cancer, such as a full-length soluble hMSH3 protein with the molecular weight of 130KD, a full-length soluble hMSH6 protein with the molecular weight of 160KD, a soluble hMSH6 protein fragment with the molecular weight of 45KD-50KD, and the like. The serum diagnostic markers can be used for guiding the diagnosis practice of lung cancer, and are beneficial to enriching the diagnosis and treatment technology of lung cancer.
Drawings
FIG. 1 shows the expression of serum soluble hMSH2 in lung cancer, lung nodules and healthy volunteers; wherein A, B, C, D is the gray scale comparison of the bands of MW 50, 105 and 170KD protein in the WB (western blot) analysis of three groups of human serum soluble hMSH2 respectively.
FIG. 2 shows the expression of serum soluble hMSH3 in lung cancer, lung nodules and healthy volunteers; wherein A, B, C, D is the WB analysis of three groups of human serum soluble hMSH3 and the gray scale comparison of MW 130, 50 and 40KD protein bands.
FIG. 3 shows the expression of serum soluble hMSH6 in lung cancer, lung nodules and healthy volunteers; wherein A, B, C, D is the WB analysis of three groups of human serum soluble hMSH6 and the gray scale comparison of MW 160, 100 and 45KD protein bands.
FIG. 4 is the expression of serum soluble Hsp70 from lung cancer, lung nodules and healthy volunteers; wherein A, B, C is the WB analysis of three groups of human serum soluble Hsp70 and the gray scale comparison of 70 and 45KD protein bands.
FIG. 5 is an IP validation of normal serum soluble hMSH2, hMSH3, hMSH6 chemically modified and truncated forms; wherein A, B, C are WB results of soluble hMSH2 (a), hMSH3 (B) and hMSH6 (C) IP samples in serum of healthy volunteers, respectively.
FIG. 6 shows interactions between normal serum soluble hMSH2, hMSH3, hMSH 6; wherein, A illustrates that hMSH2 (C-terminal) can be co-precipitated by using hMSH3 and hMSH6 as bait proteins; b illustrates that hMSH2 (N-terminal) can be co-precipitated by using hMSH3 and hMSH6 as bait proteins; c illustrates that hMSH3 can be co-precipitated by using hMSH2 and hMSH6 as bait proteins; d illustrates that hMSH6 can be co-precipitated using hMSH2 and hMSH3 as bait proteins.
FIG. 7 is the interaction of normal serum soluble hMSH2, hMSH3, hMSH6 with other co-localized proteins; wherein A illustrates that hMSH2, hMSH3 or hMSH6 is taken as a bait protein and fails to coprecipitate Exo-1; b illustrates the failure of co-precipitation of PMS2 with hMSH2, hMSH3 or hMSH6 as bait proteins; c shows that hMSH2 and hMSH3 can be used as bait proteins to weakly coprecipitate Hsp27.
FIG. 8 shows interactions of NCI-H520 whole cell lysate soluble hMSH2, hMSH3, hMSH 6; wherein, A illustrates that hMSH2 (C-terminal) (MW 105, 50 KD) can be co-precipitated by using hMSH3 or hMSH6 as bait protein; b illustrates that hMSH2 (N-terminus) (MW 105, 50 KD) can be co-precipitated using hMSH2 as a decoy protein; c, hMSH2 and hMSH3 are taken as bait proteins, so that hMSH3 (MW 130 KD) can be weakly co-precipitated; d, using hMSH2 and hMSH6 as bait proteins to co-precipitate hMSH6 (35 KD band is the clearest; 50KD and 65KD have weak bands); e illustrates the failure of co-precipitation of Exo-1 with hMSH2, hMSH3 or hMSH6 as bait proteins; f illustrates failure of co-precipitation of PMS2 with hMSH2, hMSH3 or hMSH6 as bait proteins; g illustrates the failure of co-precipitating Hsp27 with hMSH2, hMSH3 or hMSH6 as decoy proteins.
FIG. 9 shows WB analysis of NCI-H520 cell culture supernatant soluble hMSH2, hMSH3, hMSH6, exo-1, PMS2, hsp 27; wherein A-G represents soluble forms of hMSH2 (C-terminal), hMSH2 (N-terminal), hMSH3, hMSH6, exo-1, PMS2 and Hsp27 which are not detected in culture supernatant stock solutions of NCI-H520 cells with different densities at different times; h represents soluble hMSH2 (105,60KD) (C-terminal) in the concentrated culture supernatant; i represents soluble hMSH2 (60 KD) (N-terminal) in the concentrated culture supernatant; j represents soluble hMSH3 (80 KD) in the concentrated culture supernatant; k represents hMSH6 (55,60KD) in the concentrated culture supernatant; l represents Exo-1 (40,55,70,85KD) in the concentrated culture supernatant; m represents PMS2 (60,70KD) in the concentrated culture supernatant; n represents Hsp27 (27 KD) in the concentrated culture supernatant.
FIG. 10 is a mass spectrum sequencing result of NCI-H520 whole cell lysate 45-55KD strips; wherein, the bold font in A shows hMSH2 protein sequence (coverage rate 35%) matched with NCI-H520 cell lysate 45-55KD peptide fragment sequencing; the bold font in B shows hMSH6 protein sequence (coverage 12%) matched by NCI-H520 cell lysate 45-55KD peptide sequencing.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention provides a lung cancer serum diagnostic marker, which comprises at least one of a full-length soluble hMSH3 protein with the molecular weight of 130KD, a full-length soluble hMSH6 protein with the molecular weight of 160KD and a soluble hMSH6 protein fragment with the molecular weight of 45KD-50KD, which are regulated up relative to the expression of serum of a healthy person.
Preferably, the lung cancer serum diagnostic marker further comprises a full length soluble Hsp70 protein having a molecular weight of 70KD which is down-regulated relative to the expression of healthy human serum.
The lung cancer serum diagnosis marker has different expression levels in the serum of the lung cancer patient and the serum of the healthy person, and the expression is obviously up-regulated or the expression is obviously down-regulated, so that whether the patient suffers from lung cancer or not can be assisted to diagnose by detecting the expression level of the corresponding diagnosis marker, and further technical support is provided for the definitive diagnosis of the lung cancer.
The lung cancer serum diagnosis marker can be applied to preparation of lung cancer diagnosis reagents, diagnosis chips, diagnosis kits or detection equipment.
For example, a specific example provides a lung cancer serum diagnosis chip, on which an antibody corresponding to the above lung cancer serum diagnosis marker is immobilized, and the antibody can specifically bind to the corresponding marker. Preferably, the antibody is a monoclonal antibody. The antibody can be combined with the target protein marker by fixing on a lung cancer serum diagnosis chip, the expression level of the target protein marker in a serum sample can be detected by utilizing the luminescence of the luminescent marker by further adopting, but not limited to, the double-antibody sandwich method detection principle, and the diagnosis of whether a patient from which the serum sample is derived has lung cancer can be assisted.
The invention further provides a separation and identification method of soluble protein related to lung cancer, which comprises the following steps:
extracting serum proteins from a serum sample of a lung cancer patient;
the related antibodies of hMSH2 protein, hMSH3 protein and hMSH6 protein are used for separating and identifying the extracted serum proteins by using an immunoblotting method and/or a co-immunoprecipitation method.
In a specific example, the separation and identification method further comprises the step of removing albumin and immunoglobulin in serum proteins after extracting the serum proteins in the serum sample of the lung cancer patient, and the subsequent separation and identification is to perform separation and identification on the serum proteins from which the albumin and the immunoglobulin are removed.
Further, the isolation and identification method further comprises a step of detecting the concentration of serum proteins from which albumin and immunoglobulin are removed using a BCA protein concentration detection method.
Specifically, the invention uses an immunoblotting method to detect and analyze the existence form and the expression difference of the target protein of the serum sample of the lung cancer patient and the serum sample of the healthy person.
Furthermore, the invention uses an antibody-agar coupled spin column immunoprecipitation method to screen, separate, purify and identify the existence form of the target protein and other proteins interacted with the target protein for the serum sample of the lung cancer patient and the serum sample of the healthy person.
The invention creatively researches and analyzes lung cancer patients by further exploring the action and significance of hMSH2 and the soluble forms of the interaction proteins thereof in the Vγ9δ2T cell mediated cancer immune escape and the application value of the hMSH2 and the interaction proteins thereof in the early diagnosis and prognosis of clinical cancers, utilizes the technical means such as serum protein extraction, immunoblotting and/or immune co-precipitation and the like, and utilizes the technical means such as serum protein extraction, normal serum and lung cancer cell line whole cell lysate to determine the existence form and expression difference of the soluble hMSH2, hMSH3 and hMSH6 of lung cancer, screens, separates, purifies and identifies the soluble forms of other proteins which interact with the soluble hMSH2 in the normal serum and lung cancer whole cell lysate and further explores the action and significance of the soluble hMSH2 and the interaction proteins thereof in the Vγ9δ2T cell mediated lung cancer natural immune, and provides clues and basis for the application value in the early diagnosis and prognosis of lung cancer. The invention researches find that the protein can be used as a serum diagnostic marker for diagnosing lung cancer, such as a full-length soluble hMSH3 protein with the molecular weight of 130KD, a full-length soluble hMSH6 protein with the molecular weight of 160KD, a soluble hMSH6 protein fragment with the molecular weight of 45KD-50KD, and the like. The serum diagnostic markers can be used for guiding the diagnosis practice of lung cancer, and are beneficial to enriching the diagnosis and treatment technology of lung cancer.
The lung cancer serum diagnosis marker and application thereof, and the method for separating and identifying soluble protein related to lung cancer are described in further detail by combining specific examples.
The main reagents and detection equipment used in the following examples are described below:
reagent: anti-MSH2 (ab 70270), anti-MSH2 (ab 92473), anti-MSH3 (ab 154521), anti-MSH6 (ab 208940), anti-Hsp70 (ab 5442), anti-PMS2 (ab 110638); anti-Exonuclease1 (ab 155553) was purchased from Abcam corporation;
anti-MSH2 (15520-1-AP), anti-MSH3 (22393-1-AP), anti-MSH6 (18120-1-AP), anti-HSP27 (18284-1-AP) are all purchased from Proteintech company;
goat anti-rabbit IgG200 (ZB 5301), goat anti-mouse IgG200 (ZB 5305) were all purchased from zhongshan gold bridge corporation;
mouse anti-rabit IgG (Conformation Specific) (L27A 9) mAb (HRP Conjugate) (5127S), normal Rabbit IgG (2729S) were all purchased from CST corporation;
Pierce TM ECL Western Blotting Substrate(Pierce，32109)、Pierce Albumin/IgG Removal Kit(Thermo Scientific TM 89875)、Thermo Scientific TM Pierce TM Co-Immunoprecipitation Kit(Thermo Scientific TM 26149 The molecular weight standards (10-170 kD), protein A+G Agarose (Fast Flow, inlet split) were all purchased from Biyunsan Corp.
Experimental instrument: electrophoresis apparatus (BIO-RED); a transfer film instrument (BIO-RED); pipettes (gilsen); a mass spectrometer (Thermo Scientific Q Exactive); liquid chromatography (Thermo Dionex Ultimate 3000 RSLCnano).
Cell lines: the lung cancer cell line NCI-H520 is presented by the teaching subject group of the basic medical institute He Wei of the national academy of medical sciences.
The lung cancer patient serum, lung nodule patient serum and healthy human (volunteer) serum used in the examples were derived from the university center for tumor control. All serum sources were informed consent of the patient or volunteer and approved by the medical ethics committee of the middle mountain university tumor control center, guangzhou female child medical center.
This example first investigated the soluble forms of hMSH2 and its partner hMSH3, hMSH6 in lung cancer, lung nodule patient serum, normal serum (healthy human serum) and lung cancer cell line whole cell lysate supernatant, and other proteins that interact with soluble hMSH2 in normal serum and lung cancer whole cell lysate supernatant.
1. Experimental method
1. Immunoblotting (Western blot, WB)
(1) Conventionally preparing 10% SDS-PAGE separating gel and concentrating gel;
(2) mixing protein sample and molecular weight standard with 5×loading buffer solution in equal volume, boiling for 10min;
(3) loading electrophoresis: firstly, keeping a constant pressure of 80V, and keeping a constant pressure of 120V after a sample is put into a separation gel until bromophenol blue swims to the bottom of the gel;
(4) soaking SDS-PAGE gel in a transfer buffer solution;
(5) cutting filter paper and PVDF membrane into gel size, and soaking in membrane transferring liquid and methanol for 30sec and 5min respectively;
(6) placing filter paper, gel, PVDF film and filter paper on a cathode electric rotating plate in sequence from bottom to top, wherein no bubbles are generated between each layer, and rotating for 2 hours at a constant current of 100V;
(7) immersing the PVDF film in the ponceau dye solution, shaking for 3-5min or longer until clear strips appear, and photographing and recording; rinsing with distilled water for 2-3 times, each for 3-5min, removing ponceau, and performing subsequent WB detection;
(8) immersing the membrane into 5% skimmed milk powder, and sealing for 1h at room temperature;
(9) primary antibody was diluted 1:1000 and incubated overnight at 4 ℃; washing the membrane with 0.1% PBST for 7 min/time for three times;
wherein horseradish peroxidase (HRP) labeled secondary antibody is diluted 1:2000 and incubated for 1h at room temperature; washing the membrane with 0.1% PBST for 7 min/time for three times; mixing luminescent substrate 1:1, dripping onto PVDF film, chemiluminescence, developing color, and preserving the result.
2. Co-immunoprecipitation (Co-immunopotentiation, co-IP)
2.1 immobilized antibodies
(1) The amino connecting arm is coupled with agar and reagent to be balanced to room temperature;
(2) diluting 20 Xcoupling buffer (contained in 26149 coprecipitation kit, the same applies below) to 1X with ultrapure water, 200. Mu.L of 1 Xcoupling buffer is required for each Co-IP reaction;
(3) slowly rotating an amino connecting arm at the bottom of the bottle to couple agar so as to enable the agar to be uniformly suspended; adding 50 mu L of agar to the spin column with the tip cut off; putting into a centrifuge tube, centrifuging for 1min at 1000g, and discarding waste liquid;
(4) the agar was washed with 200. Mu.L of 1 Xcoupling buffer, and the waste liquid was centrifuged and discarded, and repeated once;
(5) gently sucking the bottom of the pipe column onto filter paper, discarding excessive liquid, and inserting a bottom plug; 10. Mu.g of antibody (hMSH 2/hMSH3/hMSH 6), 20 Xcoupling buffer and ultrapure water were added to an agar-containing spin column to give a total volume of 200. Mu.L;
(6) sodium cyanoborohydride (3. Mu.L/200. Mu.L reaction system) was added to the fume hood; capping, and performing rotary incubation at room temperature for 90-120min;
(7) removing the bottom plug, reserving, and removing the cap; placing the rotary column in a collecting pipe, centrifuging to collect filtrate and detecting the coupling condition of the antibody;
(8) 200. Mu.L of 1×coupling buffer was added, and the filtrate was centrifuged off and repeated once; 200 mu L Quenching Buffer was added and the filtrate was centrifuged off;
(9) gently sucking the bottom of the pipe column onto filter paper, removing excessive liquid, and inserting a bottom plug; adding 200 mu L Quenching Buffer, adding sodium cyanoborohydride into a fume hood, capping, and incubating for 15min with gentle shaking;
loosening the plug and the cap, placing the rotary column in a collecting pipe, centrifuging and discarding the filtrate; the agar was washed twice with 200. Mu.L of 1 Xcoupling buffer, and centrifuged to prepare Co-IP.
2.2 Pre-clear Using control agarose resin
(1) Control agarose resin was added to the spin column at a ratio of 1mg sample to 80. Mu.L control agarose resin (40. Mu.L agarose resin);
(2) centrifuging and discarding the storage buffer;
(3) adding 200 μl of 1×coupling buffer, centrifuging, and discarding the filtrate;
(4) adding a sample, and incubating at 4 ℃ for 30-60min;
(5) 1000g was centrifuged for 1min, spin columns coupled to the resin were discarded, and the filtrate was retained for Co-IP.
2.3Co-IP
(1) Diluting the sample with IP Lysis/Washing Buffer, and recommending a reaction volume of 100-500 mu L;
(2) washing the antibody-agar coupling spin column twice with IP Lysis/Washing Buffer; 200. Mu.L/time, centrifuge;
(3) gently sucking the bottom of the pipe column onto filter paper, discarding residual liquid, and inserting a bottom plug;
(4) adding the sample into a corresponding rotary column, and incubating overnight at 4 ℃;
(5) centrifuging, placing a spin column into a new collecting tube, adding 200 mu L of IP Lysis/Washing Buffer, and centrifuging; repeating for 3 times;
(6) putting the spin column into a new collecting tube, adding 10 mu L of an absorption Buffer, and centrifuging;
(7) adding 50 mu L of an absorption Buffer, incubating for 5min at room temperature, centrifuging, and collecting a target protein filtrate;
(8) mass spectrometry sequencing: and cutting gel of a target protein sample obtained from Co-IP after SDS-PAGE electrophoresis, and identifying unknown proteins by using a biological mass spectrum.
3. Serum protein extraction
200 mu L of protein extract (1 mu L of PMSF,1 mu L Proteinase inhibitor cocktail and 2 mu L Phosphatase inhibitor are added to 100 mu L of serum) is added to each 100 mu L of serum, and the mixture is fully mixed and kept at 4 ℃ for 5min; centrifuging at 12000rpm at 4deg.C for 10min to obtain supernatant.
4. Immunoglobulin removal such as serum albumin/IgG
(1) 30. Mu.L of the supernatant was diluted to 75. Mu.L with Binding/Washing Buffer (89875 kit components);
(2) adding the protein diluent into a rubber bed, capping, slightly shaking and suspending, and placing the column into a centrifuge tube; incubating the rotary shaking table at room temperature for 10min;
(3) removing plugs and caps, putting the column into a centrifuge tube, and centrifuging 10000g for 1min;
(4) adding the filtrate into the gel bed again, and covering with a plug and a cap; rotating the shaking table chamber for incubation for 10min;
(5) removing plugs and caps, placing the column into a centrifuge tube, centrifuging 10000g for 1min, and collecting filtrate;
(6) adding 75 mu L of Binding/Washing Buffer into a gum bed, centrifuging 10000g for 1min, and collecting the filtrate in the previous step into the same centrifuge tube to obtain the target protein solution for removing albumin and immunoglobulin.
5. BCA protein quantification
(1) Preparation of protein standards
(1) 1.2mL of protein standard preparation solution is taken and added with protein standard substance (30 mg BSA), and 25mg/mL of protein standard solution is prepared after full dissolution, namely, the protein standard solution is used or stored for a long time at minus 20 ℃;
(2) and (3) taking a proper amount of 25mg/mL protein standard, diluting to a final concentration of 0.5mg/mL, and then using or storing for a long time at-20 ℃.
(2) BCA working solution preparation
Adding 1 volume BCA reagent B (50:1) into 50 volumes of BCA reagent A according to the number of samples to prepare a proper amount of BCA working solution, and fully and uniformly mixing; the BCA working solution is stable within 24 hours at room temperature.
(3) Protein concentration detection
(1) Adding 0, 1, 2, 4, 8, 12, 16 and 20 μl of standard substances into a 96-well plate, and supplementing the standard substance dilution to 20 μl, wherein the corresponding concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL respectively;
(2) adding a proper volume of sample to a 96-well plate, and adding standard diluent to complement if the sample is less than 20 mu l; note the recorded sample volume;
(3) 200 μl BCA working solution is added to each well, and the wells are left at 37 ℃ for 20-30min;
(4) measurement of A with an enzyme-labeled instrument 562 Or a wavelength A value of 540-595 nm;
(5) the protein concentration of the sample was calculated from the standard curve and the sample volume used.
2. Detection results and analysis
1. Expression of soluble hMSH2 in serum of lung cancer, lung nodule patients and healthy volunteers and its form
The experiment found that the sera of lung cancer, lung nodule patients and normal healthy volunteers all had different levels of soluble hMSH2 protein expression at Molecular Weights (MW) of 105KD, 170KD, 50KD, whereas the normally encoded nuclear hMSH2 and ectopic expression of epithelial-derived tumor cells of membrane hMSH2 were all full-length proteins with MW of about 105KD (fig. 1 a). Western blot gray-scale analysis of sera from lung cancer (n=26), lung nodules (n=13) and healthy volunteers (n=26) showed no significant statistical difference in expression of soluble hMSH2 (105 KD, 170KD, 50 KD) in disease and control groups (B-D in fig. 1). Experiments report the existence of soluble hMSH2 in lung cancer, lung nodule and healthy volunteer serum, and the use of target antibodies to hybridize out obvious bands at MW 105KD, 170KD, 50KD, suggesting that the existence of variants in serum due to chemical modification or gene deletion, in addition to normally encoded full-length proteins.
2. Expression and form of serum soluble hMSH3 in lung cancer, lung nodule patients and healthy volunteers
hMSH3 is a partner protein of hMSH2, and both can form mutsβ heterodimer to participate in recognition and repair of long-chain damaged DNA. The experimental results of WB analysis of soluble hMSH3 from three serum samples showed clearer protein bands at MW 130KD (full length protein), 50KD and 40KD (fig. 2 a). Wherein, the expression of full-length soluble hMSH3 with MW 130KD in serum of lung cancer patients was significantly increased compared to healthy volunteers (< 0.05) whereas the expression of soluble hMSH3 with 50KD, 40KD in each group was not significantly statistically different (B-D in fig. 2). The experimental result shows that the full-length soluble hMSH3 with MW 130KD is expected to be used as a serum diagnosis marker of lung cancer.
3. Expression and form of serum soluble hMSH6 in lung cancer, lung nodule patients and healthy volunteers
hMSH6 is also a partner of hMSH2, both of which can form mutsα heterodimers, and are involved in prognosis of endometrial cancer and osteosarcoma development and survival. The soluble hMSH6 in the three serum samples was found to have clearer protein bands at 160KD, 130KD, and 50KD (fig. 3 a). Wherein, the expression of soluble hMSH6 at MW 160KD, 45-50KD in serum of lung cancer patients is significantly reduced compared to healthy volunteers (< 0.05 for P); there was no significant statistical difference in the expression at 100KD for each group (B-D in FIG. 3).
4. Expression and form of serum soluble Hsp70 in lung cancer, lung nodule patients and healthy volunteers
Heat shock protein 70 (Hsp 70) is another protein ligand recognized by γδ T cells. Experiments found that the expression of serum Hsp70 from lung cancer patients was significantly reduced at 70KD compared to healthy volunteers (P < 0.005); there was also a distinct band at 45KD, but there was no significant statistical difference in protein expression between groups (a-C in fig. 4).
5. IP validation of normal serum soluble hMSH2, hMSH3, hMSH6 chemically modified or truncated forms
As the WB results show that the positions of the soluble hMSH2, hMSH3 and hMSH6 protein strips of the lung cancer, lung nodule patients and healthy volunteers are basically the same, the soluble target protein is further purified and verified in the healthy volunteer serum with higher expression level of the soluble hMSH2, hMSH3 and hMSH6 by adopting the immune coprecipitation and WB technology in experiments. Experiments further demonstrated that there are three forms of soluble hMSH2 in serum of healthy volunteers, 105, 170, 50KD, to which normal rabbit IgG also binds non-specifically (fig. 5 a); hMSH3 appears more in the band, suggesting that the target protein content or antibody concentration in the sample is not appropriate (B in fig. 5); the distinct bands of hMSH6 were hybridized at all MW 130, 105, 85, 65 and 43KD, suggesting that post-IP sample concentration increased sensitivity to detection of soluble protein of interest, and that normally encoded full length hMSH6 with MW of about 160KD may form fragmented truncated forms in serum due to degradation (C in fig. 5).
6. Interaction of normal serum soluble hMSH2, hMSH3, hMSH6
The experiments further examined the interaction of serum-soluble hMSH2 with its partner hMSH3, hMSH6 in normal healthy volunteers. The co-immunoprecipitation results with three soluble hMSH as decoy proteins showed that hMSH2, hMSH3, hMSH6IP groups all showed clear bands at 55KD, suggesting interactions of soluble hMSH2, hMSH3, hMSH6 in serum (fig. 6 a-D). The experimental results also suggest that, unlike the homo-or heterodimeric forms formed by the interaction of full-length hMSH2, hMSH3, hMSH6 in the nucleus, soluble hMSH2 in serum interacting with the partner protein may be a truncated form of full-length hMSH2 that retains the normal N, C end.
7. Normal serum soluble hMSH2, hMSH3, hMSH6 and Exo-1, PMS2 and Hsp27 interaction verification
In view of the normal interaction of hMSH2 in the nucleus with Exo-1C-terminal sites to regulate the process of DNA damage repair and co-localization with Hsp 27; PMS2 forms heterodimer mutlα with MLH1, which is involved in DNA damage repair and tumorigenesis. In the experiment, soluble hMSH2, hMSH3 and hMSH6 are taken as bait proteins, and interaction between the bait proteins and soluble Exo-1, PMS2 and Hsp27 possibly existing in serum is analyzed by adopting co-immunoprecipitation and Western blotting. Experiments have found that no specific protein bands appear for each IP packet, suggesting that they may not have a direct effect in serum or that they have too weak binding capacity to each other (a-C in fig. 7).
8. NCI-H520 whole cell lysate soluble hMSH2, hMSH3, hMSH6 and Exo-1, PMS2, hsp27 interaction verification
The interaction among the soluble hMSH2, hMSH3 and hMSH6 of NCI-H520 whole cell lysate and the interaction between the soluble hMSH2, hMSH3 and hMSH6 and the soluble Exo-1, PMS2 and Hsp27 of the whole cell lysate are detected by adopting the same experimental conditions. Experiments have found that there is an interaction between soluble hMSH2 and hMSH3, hMSH6 in NCI-H520 whole cell lysates (a-D in fig. 8). Using soluble hMSH2, hMSH3, hMSH6 as decoy proteins, their interactions with Exo-1, PMS2, hsp27, which may be present in whole cell lysates, were analyzed by co-immunoprecipitation and WB, and found that each IP packet had no specific protein bands, consistent with the results obtained for serum samples, suggesting that they may not have a direct effect in serum or that they have too weak binding capacity to each other (E-G in FIG. 8).
9. Expression and form of soluble hMSH2, hMSH3, hMSH6 and Exo-1, PMS2, hsp27 in NCI-H520 cell culture supernatant
The experiment simultaneously detects the existence of soluble hMSH2, hMSH3 and hMSH6, exo-1, PMS2 and Hsp27 in culture supernatants of NCI-H520 cells which are cultured and starved for different times and planted with different densities. Experimental results show that the different planting densities (5 x 10 4 cells/well, 10 x 10 4 cells/well, 20 x 10 4 cells/well) NCI-H520 cells were incubated in 24-well plates for 24H, and no presence of soluble hMSH2, hMSH3, hMSH6, exo-1, PMS2, hsp27 was detected in NCI-H520 cell culture supernatants collected after varying periods of starvation by changing fresh complete culture broth (fig. 9 a-G). The cell culture supernatants after ultrafiltration concentration detected traces of soluble hMSH2, hMSH3, hMSH6, exo-1, PMS2, hsp27 proteins (H-N in FIG. 9). Experimental results indicate that culture supernatants of NCI-H520 cells are present in soluble forms of the above proteinsThe formula, however, is extremely low in initial content.
10. Mass spectrum identification of soluble hMSH2 new variants in normal serum and NCI-H520 whole cell lysate
After the normal serum is dyed by an IP sample line R250, MW 170 and 50KD protein bands are cut for mass spectrum identification, and the enzymolysis peptide segments of the two bands are found to be not matched with the protein sequences of hMSH2, while the bands at 45-55KD of NCI-H520 whole cell lysate are respectively matched with the protein sequences of hMSH2 and hMSH6, which indicates that hMSH2 proteins at the MW 170 and 50KD of the serum are possibly covered by other high-abundance proteins with the same MW due to too low abundance. The matching rate of the peptide fragment sequence obtained by the identification of NCI-H520 whole cell lysate MW 45-55KD protein band mass spectrum with the hMSH2, hMSH6 protein sequence (SEQ ID NO.1 and SEQ ID NO.2 respectively) is shown as A and B in FIG. 10.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Guangzhou urban women and children medical center
<120> lung cancer serum diagnosis marker and application thereof, and separation and identification method of lung cancer related soluble protein
<160> 2
<170> SIPOSequenceListing 1.0
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<213> Natural Sequence (Natural Sequence)
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Met Ala Val Gln Pro Lys Glu Thr Leu Gln Leu Glu Ser Ala Ala Glu
1 5 10 15
Val Gly Phe Val Arg Phe Phe Gln Gly Met Pro Glu Lys Pro Thr Thr
20 25 30
Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu
35 40 45
Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly Val Ile
50 55 60
Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln Ser Val Val Leu
65 70 75 80
Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp Leu Leu Leu Val Arg
85 90 95
Gln Tyr Arg Val Glu Val Tyr Lys Asn Arg Ala Gly Asn Lys Ala Ser
100 105 110
Lys Glu Asn Asp Trp Tyr Leu Ala Tyr Lys Ala Ser Pro Gly Asn Leu
115 120 125
Ser Gln Phe Glu Asp Ile Leu Phe Gly Asn Asn Asp Met Ser Ala Ser
130 135 140
Ile Gly Val Val Gly Val Lys Met Ser Ala Val Asp Gly Gln Arg Gln
145 150 155 160
Val Gly Val Gly Tyr Val Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys
165 170 175
Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu Ile
180 185 190
Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu Thr Ala Gly
195 200 205
Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Arg Gly Gly Ile Leu Ile
210 215 220
Thr Glu Arg Lys Lys Ala Asp Phe Ser Thr Lys Asp Ile Tyr Gln Asp
225 230 235 240
Leu Asn Arg Leu Leu Lys Gly Lys Lys Gly Glu Gln Met Asn Ser Ala
245 250 255
Val Leu Pro Glu Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala
260 265 270
Val Ile Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln
275 280 285
Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile
290 295 300
Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu Asp Thr
305 310 315 320
Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys Cys Lys Thr Pro
325 330 335
Gln Gly Gln Arg Leu Val Asn Gln Trp Ile Lys Gln Pro Leu Met Asp
340 345 350
Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu Val Glu Ala Phe Val Glu
355 360 365
Asp Ala Glu Leu Arg Gln Thr Leu Gln Glu Asp Leu Leu Arg Arg Phe
370 375 380
Pro Asp Leu Asn Arg Leu Ala Lys Lys Phe Gln Arg Gln Ala Ala Asn
385 390 395 400
Leu Gln Asp Cys Tyr Arg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn
405 410 415
Val Ile Gln Ala Leu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu
420 425 430
Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser
435 440 445
Lys Phe Gln Glu Met Ile Glu Thr Thr Leu Asp Met Asp Gln Val Glu
450 455 460
Asn His Glu Phe Leu Val Lys Pro Ser Phe Asp Pro Asn Leu Ser Glu
465 470 475 480
Leu Arg Glu Ile Met Asn Asp Leu Glu Lys Lys Met Gln Ser Thr Leu
485 490 495
Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys
500 505 510
Leu Asp Ser Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys
515 520 525
Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile
530 535 540
Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser Leu Asn
545 550 555 560
Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala Gln Asp Ala
565 570 575
Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly Tyr Val Glu Pro Met
580 585 590
Gln Thr Leu Asn Asp Val Leu Ala Gln Leu Asp Ala Val Val Ser Phe
595 600 605
Ala His Val Ser Asn Gly Ala Pro Val Pro Tyr Val Arg Pro Ala Ile
610 615 620
Leu Glu Lys Gly Gln Gly Arg Ile Ile Leu Lys Ala Ser Arg His Ala
625 630 635 640
Cys Val Glu Val Gln Asp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr
645 650 655
Phe Glu Lys Asp Lys Gln Met Phe His Ile Ile Thr Gly Pro Asn Met
660 665 670
Gly Gly Lys Ser Thr Tyr Ile Arg Gln Thr Gly Val Ile Val Leu Met
675 680 685
Ala Gln Ile Gly Cys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile
690 695 700
Val Asp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys
705 710 715 720
Gly Val Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu
725 730 735
Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg
740 745 750
Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Ser Glu
755 760 765
Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala Thr His Phe
770 775 780
His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pro Thr Val Asn Asn Leu
785 790 795 800
His Val Thr Ala Leu Thr Thr Glu Glu Thr Leu Thr Met Leu Tyr Gln
805 810 815
Val Lys Lys Gly Val Cys Asp Gln Ser Phe Gly Ile His Val Ala Glu
820 825 830
Leu Ala Asn Phe Pro Lys His Val Ile Glu Cys Ala Lys Gln Lys Ala
835 840 845
Leu Glu Leu Glu Glu Phe Gln Tyr Ile Gly Glu Ser Gln Gly Tyr Asp
850 855 860
Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Leu Glu Arg Glu Gln Gly
865 870 875 880
Glu Lys Ile Ile Gln Glu Phe Leu Ser Lys Val Lys Gln Met Pro Phe
885 890 895
Thr Glu Met Ser Glu Glu Asn Ile Thr Ile Lys Leu Lys Gln Leu Lys
900 905 910
Ala Glu Val Ile Ala Lys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser
915 920 925
Arg Ile Lys Val Thr Thr
930
<210> 2
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<212> PRT
<213> Natural Sequence (Natural Sequence)
<400> 2
Met Ser Arg Gln Ser Thr Leu Tyr Ser Phe Phe Pro Lys Ser Pro Ala
1 5 10 15
Leu Ser Asp Ala Asn Lys Ala Ser Ala Arg Ala Ser Arg Glu Gly Gly
20 25 30
Arg Ala Ala Ala Ala Pro Gly Ala Ser Pro Ser Pro Gly Gly Asp Ala
35 40 45
Ala Trp Ser Glu Ala Gly Pro Gly Pro Arg Pro Leu Ala Arg Ser Ala
50 55 60
Ser Pro Pro Lys Ala Lys Asn Leu Asn Gly Gly Leu Arg Arg Ser Val
65 70 75 80
Ala Pro Ala Ala Pro Thr Ser Cys Asp Phe Ser Pro Gly Asp Leu Val
85 90 95
Trp Ala Lys Met Glu Gly Tyr Pro Trp Trp Pro Cys Leu Val Tyr Asn
100 105 110
His Pro Phe Asp Gly Thr Phe Ile Arg Glu Lys Gly Lys Ser Val Arg
115 120 125
Val His Val Gln Phe Phe Asp Asp Ser Pro Thr Arg Gly Trp Val Ser
130 135 140
Lys Arg Leu Leu Lys Pro Tyr Thr Gly Ser Lys Ser Lys Glu Ala Gln
145 150 155 160
Lys Gly Gly His Phe Tyr Ser Ala Lys Pro Glu Ile Leu Arg Ala Met
165 170 175
Gln Arg Ala Asp Glu Ala Leu Asn Lys Asp Lys Ile Lys Arg Leu Glu
180 185 190
Leu Ala Val Cys Asp Glu Pro Ser Glu Pro Glu Glu Glu Glu Glu Met
195 200 205
Glu Val Gly Thr Thr Tyr Val Thr Asp Lys Ser Glu Glu Asp Asn Glu
210 215 220
Ile Glu Ser Glu Glu Glu Val Gln Pro Lys Thr Gln Gly Ser Arg Arg
225 230 235 240
Ser Ser Arg Gln Ile Lys Lys Arg Arg Val Ile Ser Asp Ser Glu Ser
245 250 255
Asp Ile Gly Gly Ser Asp Val Glu Phe Lys Pro Asp Thr Lys Glu Glu
260 265 270
Gly Ser Ser Asp Glu Ile Ser Ser Gly Val Gly Asp Ser Glu Ser Glu
275 280 285
Gly Leu Asn Ser Pro Val Lys Val Ala Arg Lys Arg Lys Arg Met Val
290 295 300
Thr Gly Asn Gly Ser Leu Lys Arg Lys Ser Ser Arg Lys Glu Thr Pro
305 310 315 320
Ser Ala Thr Lys Gln Ala Thr Ser Ile Ser Ser Glu Thr Lys Asn Thr
325 330 335
Leu Arg Ala Phe Ser Ala Pro Gln Asn Ser Glu Ser Gln Ala His Val
340 345 350
Ser Gly Gly Gly Asp Asp Ser Ser Arg Pro Thr Val Trp Tyr His Glu
355 360 365
Thr Leu Glu Trp Leu Lys Glu Glu Lys Arg Arg Asp Glu His Arg Arg
370 375 380
Arg Pro Asp His Pro Asp Phe Asp Ala Ser Thr Leu Tyr Val Pro Glu
385 390 395 400
Asp Phe Leu Asn Ser Cys Thr Pro Gly Met Arg Lys Trp Trp Gln Ile
405 410 415
Lys Ser Gln Asn Phe Asp Leu Val Ile Cys Tyr Lys Val Gly Lys Phe
420 425 430
Tyr Glu Leu Tyr His Met Asp Ala Leu Ile Gly Val Ser Glu Leu Gly
435 440 445
Leu Val Phe Met Lys Gly Asn Trp Ala His Ser Gly Phe Pro Glu Ile
450 455 460
Ala Phe Gly Arg Tyr Ser Asp Ser Leu Val Gln Lys Gly Tyr Lys Val
465 470 475 480
Ala Arg Val Glu Gln Thr Glu Thr Pro Glu Met Met Glu Ala Arg Cys
485 490 495
Arg Lys Met Ala His Ile Ser Lys Tyr Asp Arg Val Val Arg Arg Glu
500 505 510
Ile Cys Arg Ile Ile Thr Lys Gly Thr Gln Thr Tyr Ser Val Leu Glu
515 520 525
Gly Asp Pro Ser Glu Asn Tyr Ser Lys Tyr Leu Leu Ser Leu Lys Glu
530 535 540
Lys Glu Glu Asp Ser Ser Gly His Thr Arg Ala Tyr Gly Val Cys Phe
545 550 555 560
Val Asp Thr Ser Leu Gly Lys Phe Phe Ile Gly Gln Phe Ser Asp Asp
565 570 575
Arg His Cys Ser Arg Phe Arg Thr Leu Val Ala His Tyr Pro Pro Val
580 585 590
Gln Val Leu Phe Glu Lys Gly Asn Leu Ser Lys Glu Thr Lys Thr Ile
595 600 605
Leu Lys Ser Ser Leu Ser Cys Ser Leu Gln Glu Gly Leu Ile Pro Gly
610 615 620
Ser Gln Phe Trp Asp Ala Ser Lys Thr Leu Arg Thr Leu Leu Glu Glu
625 630 635 640
Glu Tyr Phe Arg Glu Lys Leu Ser Asp Gly Ile Gly Val Met Leu Pro
645 650 655
Gln Val Leu Lys Gly Met Thr Ser Glu Ser Asp Ser Ile Gly Leu Thr
660 665 670
Pro Gly Glu Lys Ser Glu Leu Ala Leu Ser Ala Leu Gly Gly Cys Val
675 680 685
Phe Tyr Leu Lys Lys Cys Leu Ile Asp Gln Glu Leu Leu Ser Met Ala
690 695 700
Asn Phe Glu Glu Tyr Ile Pro Leu Asp Ser Asp Thr Val Ser Thr Thr
705 710 715 720
Arg Ser Gly Ala Ile Phe Thr Lys Ala Tyr Gln Arg Met Val Leu Asp
725 730 735
Ala Val Thr Leu Asn Asn Leu Glu Ile Phe Leu Asn Gly Thr Asn Gly
740 745 750
Ser Thr Glu Gly Thr Leu Leu Glu Arg Val Asp Thr Cys His Thr Pro
755 760 765
Phe Gly Lys Arg Leu Leu Lys Gln Trp Leu Cys Ala Pro Leu Cys Asn
770 775 780
His Tyr Ala Ile Asn Asp Arg Leu Asp Ala Ile Glu Asp Leu Met Val
785 790 795 800
Val Pro Asp Lys Ile Ser Glu Val Val Glu Leu Leu Lys Lys Leu Pro
805 810 815
Asp Leu Glu Arg Leu Leu Ser Lys Ile His Asn Val Gly Ser Pro Leu
820 825 830
Lys Ser Gln Asn His Pro Asp Ser Arg Ala Ile Met Tyr Glu Glu Thr
835 840 845
Thr Tyr Ser Lys Lys Lys Ile Ile Asp Phe Leu Ser Ala Leu Glu Gly
850 855 860
Phe Lys Val Met Cys Lys Ile Ile Gly Ile Met Glu Glu Val Ala Asp
865 870 875 880
Gly Phe Lys Ser Lys Ile Leu Lys Gln Val Ile Ser Leu Gln Thr Lys
885 890 895
Asn Pro Glu Gly Arg Phe Pro Asp Leu Thr Val Glu Leu Asn Arg Trp
900 905 910
Asp Thr Ala Phe Asp His Glu Lys Ala Arg Lys Thr Gly Leu Ile Thr
915 920 925
Pro Lys Ala Gly Phe Asp Ser Asp Tyr Asp Gln Ala Leu Ala Asp Ile
930 935 940
Arg Glu Asn Glu Gln Ser Leu Leu Glu Tyr Leu Glu Lys Gln Arg Asn
945 950 955 960
Arg Ile Gly Cys Arg Thr Ile Val Tyr Trp Gly Ile Gly Arg Asn Arg
965 970 975
Tyr Gln Leu Glu Ile Pro Glu Asn Phe Thr Thr Arg Asn Leu Pro Glu
980 985 990
Glu Tyr Glu Leu Lys Ser Thr Lys Lys Gly Cys Lys Arg Tyr Trp Thr
995 1000 1005
Lys Thr Ile Glu Lys Lys Leu Ala Asn Leu Ile Asn Ala Glu Glu Arg
1010 1015 1020
Arg Asp Val Ser Leu Lys Asp Cys Met Arg Arg Leu Phe Tyr Asn Phe
1025 1030 1035 1040
Asp Lys Asn Tyr Lys Asp Trp Gln Ser Ala Val Glu Cys Ile Ala Val
1045 1050 1055
Leu Asp Val Leu Leu Cys Leu Ala Asn Tyr Ser Arg Gly Gly Asp Gly
1060 1065 1070
Pro Met Cys Arg Pro Val Ile Leu Leu Pro Glu Asp Thr Pro Pro Phe
1075 1080 1085
Leu Glu Leu Lys Gly Ser Arg His Pro Cys Ile Thr Lys Thr Phe Phe
1090 1095 1100
Gly Asp Asp Phe Ile Pro Asn Asp Ile Leu Ile Gly Cys Glu Glu Glu
1105 1110 1115 1120
Glu Gln Glu Asn Gly Lys Ala Tyr Cys Val Leu Val Thr Gly Pro Asn
1125 1130 1135
Met Gly Gly Lys Ser Thr Leu Met Arg Gln Ala Gly Leu Leu Ala Val
1140 1145 1150
Met Ala Gln Met Gly Cys Tyr Val Pro Ala Glu Val Cys Arg Leu Thr
1155 1160 1165
Pro Ile Asp Arg Val Phe Thr Arg Leu Gly Ala Ser Asp Arg Ile Met
1170 1175 1180
Ser Gly Glu Ser Thr Phe Phe Val Glu Leu Ser Glu Thr Ala Ser Ile
1185 1190 1195 1200
Leu Met His Ala Thr Ala His Ser Leu Val Leu Val Asp Glu Leu Gly
1205 1210 1215
Arg Gly Thr Ala Thr Phe Asp Gly Thr Ala Ile Ala Asn Ala Val Val
1220 1225 1230
Lys Glu Leu Ala Glu Thr Ile Lys Cys Arg Thr Leu Phe Ser Thr His
1235 1240 1245
Tyr His Ser Leu Val Glu Asp Tyr Ser Gln Asn Val Ala Val Arg Leu
1250 1255 1260
Gly His Met Ala Cys Met Val Glu Asn Glu Cys Glu Asp Pro Ser Gln
1265 1270 1275 1280
Glu Thr Ile Thr Phe Leu Tyr Lys Phe Ile Lys Gly Ala Cys Pro Lys
1285 1290 1295
Ser Tyr Gly Phe Asn Ala Ala Arg Leu Ala Asn Leu Pro Glu Glu Val
1300 1305 1310
Ile Gln Lys Gly His Arg Lys Ala Arg Glu Phe Glu Lys Met Asn Gln
1315 1320 1325
Ser Leu Arg Leu Phe Arg Glu Val Cys Leu Ala Ser Glu Arg Ser Thr
1330 1335 1340
Val Asp Ala Glu Ala Val His Lys Leu Leu Thr Leu Ile Lys Glu Leu
1345 1350 1355 1360

Claims (6)

1. The application of a detection agent of a lung cancer serum diagnostic marker in preparing a lung cancer diagnostic reagent or detection equipment, wherein the detection agent of the lung cancer serum diagnostic marker comprises at least one of a serum protein extraction reagent, a serum albumin and immunoglobulin removal reagent and an immunoblotting reagent, the serum protein extraction reagent is used for extracting a serum protein sample, the serum albumin and immunoglobulin removal reagent are used for removing albumin and immunoglobulin in the serum protein sample, the immunoblotting reagent is used for detecting the content of the lung cancer serum diagnostic marker in the serum protein sample by performing an immunoblotting method, the lung cancer serum diagnostic marker comprises at least one of a full-length soluble hMSH6 protein with a molecular weight of 160KD and a soluble hMSH6 protein fragment with a molecular weight of 45KD-50KD, and the content of the lung cancer serum diagnostic marker of a lung cancer patient is significantly reduced relative to the content of the lung cancer serum diagnostic marker of a healthy person;
the extraction of the serum protein sample comprises the following steps:
mixing a serum sample to be tested with a serine protease inhibitor, a protease inhibitor cookie and a phosphatase inhibitor, standing and centrifuging to obtain a supernatant to obtain the serum protein sample, wherein the centrifuging speed is 12000rpm;
the immunoblotting reagent is used for:
separating serum protein samples from which albumin and immunoglobulin are removed by SDS-PAGE gel electrophoresis according to the molecular weight, transferring the serum protein samples to a hybridization membrane, carrying out specific detection on lung cancer serum diagnosis markers by using hMSH6 protein antibodies and labeled secondary antibodies, carrying out chromogenic reaction by using the labeled secondary antibodies, and detecting the content of the lung cancer serum diagnosis markers by using a BCA protein quantitative method.
2. The use according to claim 1, wherein the detection agent for the lung cancer serum diagnostic marker further comprises at least one of an Hsp70 protein antibody and a hMSH3 protein antibody;
the lung cancer serum diagnostic marker further comprises at least one of a full-length soluble hMSH3 protein with a molecular weight of 130KD and a full-length soluble Hsp70 protein with a molecular weight of 70KD, wherein the content of the hMSH3 protein in a lung cancer patient is obviously up-regulated relative to the content of the hMSH3 protein in a healthy person, and the content of the Hsp70 protein in the lung cancer patient is obviously down-regulated relative to the content of the Hsp70 protein in the healthy person.
3. A method for separating and identifying lung cancer related soluble proteins for non-diagnostic purposes, which is characterized by comprising the following steps:
extracting a serum protein sample;
removing albumin and immunoglobulin from the serum protein sample;
separating and identifying serum protein samples from which albumin and immunoglobulin are removed by using a relevant antibody of a target protein by using an immunoblotting method, wherein the relevant antibody of the target protein comprises an hMSH6 antibody;
carrying out protein quantitative analysis on the full-length soluble hMSH6 protein with the molecular weight of 160KD and/or the soluble hMSH6 protein fragment with the molecular weight of 45KD-50KD obtained by separation identification by adopting a BCA protein quantitative method;
the serum protein sample extraction comprises the following steps:
mixing a serum sample to be tested with a serine protease inhibitor, a protease inhibitor cookie and a phosphatase inhibitor, standing and centrifuging to obtain a supernatant to obtain the serum protein sample, wherein the centrifuging speed is 12000rpm;
the isolation and identification of serum protein samples from which albumin and immunoglobulin are removed by immunoblotting method comprises:
separating serum protein samples from which albumin and immunoglobulin are removed by SDS-PAGE gel electrophoresis according to the molecular weight, transferring the serum protein samples to a hybridization membrane, carrying out specific detection on lung cancer serum diagnosis markers by using hMSH6 protein antibodies and labeled secondary antibodies, and carrying out chromogenic reaction by using the labeled secondary antibodies.
4. The method for isolating and identifying lung cancer-associated soluble proteins of non-diagnostic interest according to claim 3, wherein the antibodies associated with the protein of interest further comprise hMSH3 antibodies and Hsp70 antibodies, and wherein the method further comprises, after isolating and identifying the serum protein sample from which albumin and immunoglobulins are removed: quantitatively analyzing the full-length soluble hMSH3 protein with the molecular weight of 130 KD;
and/or
The full-length soluble Hsp70 protein with the molecular weight of 70KD is quantitatively analyzed.
5. The method for isolating and identifying lung cancer-associated soluble proteins of non-diagnostic interest according to claim 3 or 4, wherein the presence and expression differences of the target proteins are detected and analyzed by immunoblotting of the serum sample to be tested and the serum sample of a healthy person.
6. The method for isolating and identifying soluble proteins associated with lung cancer, which is not diagnostic according to claim 3 or 4, wherein the presence of the target protein and other proteins interacting with the target protein is screened, isolated, purified and identified by using an antibody-agar coupled spin column immunoprecipitation method on the serum sample to be tested.
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