CN115184610A - Application of MRPL12 protein in preparation of lung adenocarcinoma detection product - Google Patents
Application of MRPL12 protein in preparation of lung adenocarcinoma detection product Download PDFInfo
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Abstract
The invention discloses an application of MRPL12 protein in the preparation of lung adenocarcinoma detection products, wherein MRPL12 protein and a specific antibody thereof are used as a molecular marker for the first time, and the molecular marker is applied to lung adenocarcinoma detection products such as a kit, test paper, a protein chip and the like, so that the sensitivity and specificity are good, a tool is used for early diagnosis of lung adenocarcinoma, the problems that the existing lung adenocarcinoma molecular marker is not accurate enough and sufficient enough are solved, the timeliness, the sensitivity and the popularity of lung adenocarcinoma diagnosis are improved, the molecular marker has important significance for early diagnosis, early treatment and prognosis evaluation of lung adenocarcinoma, and the application prospect is wide.
Description
Technical Field
The invention relates to the field of medical diagnosis, in particular to application of MRPL12 protein in preparation of lung adenocarcinoma detection products.
Background
Lung cancer is a malignant tumor that originates in the bronchial mucosa or glands of the lung. The incidence and mortality of lung cancer are extremely high on a global scale, and the health of human beings is seriously threatened. The pathogenic factors of lung cancer mainly include smoking, air pollution, ionizing radiation, occupational exposure, heredity and the like. According to the histopathological characteristics, the cancer is divided into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC). Lung adenocarcinoma (LUAD) belongs to NSCLC, and is the most prominent histological type of lung cancer, accounting for 40% -55% of the total diagnosed cases. The lung adenocarcinoma originates from bronchial mucosa epithelium, a few originate from mucus glands of large bronchi, early symptoms are hidden, most patients are diagnosed at an advanced stage or have metastasis, and the five-year survival rate is less than 20%. At present, the treatment methods of lung adenocarcinoma are divided into surgical treatment, chemotherapy, radiotherapy, immunotherapy, targeted therapy, traditional Chinese medicine therapy and the like. Surgical resection is a treatment with a high cure rate for lung adenocarcinoma, but only about 25% of patients are eligible for surgical treatment when they are diagnosed.
A molecular marker is any gene, protein, or the like, whose expression level in a tissue or cell is altered compared to the expression level in a normal or healthy cell or tissue. In recent years, with the development of molecular biology research, tumor molecular markers have become hot spots of clinical tumor treatment, and clinical practice has proved that the tumor molecular markers are helpful for advanced diagnosis, targeted therapy and prognosis monitoring of tumors, but lack specificity and sensitivity. The discovery of the novel tumor molecular marker has important significance for early diagnosis, early treatment and prognosis evaluation of lung adenocarcinoma.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide MRPL12 protein for preparing a lung adenocarcinoma detection product and applying the product to early diagnosis of lung adenocarcinoma.
The purpose of the invention is realized by the following technical scheme: application of MRPL12 protein in preparation of lung adenocarcinoma detection products.
Further, the MRPL12 protein includes all the translation products of MRPL12 gene, i.e., the MRPL12 protein is a single protein; the MRPL12 gene comprises any one of MRP12 wild type, mutant type, splicing type and fragments thereof; the translation product comprises any one of MRP12 protein phosphorylation, acetylation, methylation, ubiquitination, glycosylation modification products and shearing products.
Further, the MRPL12 protein is a specific antibody, and the specific antibody of the MRPL12 protein includes any form of antibody capable of specifically binding to MRPL12, such as an intact antibody molecule, an antibody fragment or a modified antibody.
Further, the specific antibody of MRPL12 protein includes monoclonal antibody, polyclonal antibody; the antibody against MRPL12 protein can be used for immunohistochemical staining, and the MRPL12 protein content in a biopsy sample is detected to be used as a monitoring index for early diagnosis and prognosis of lung adenocarcinoma; also can be used for ELISA, immunoblotting analysis, isotope labeling and fluorescence labeling detection methods.
Furthermore, the detection product is any one of a kit, test paper and a protein chip.
Furthermore, the kit comprises an ELISA detection kit, an immunohistochemical detection kit, an immunoblot detection kit and the like.
Further, the reagent in the kit also comprises a ligand which can indirectly reflect the content or activity of the MRPL12 protein and is combined with the MRPL12 protein; the protein chip comprises a solid phase carrier and an antibody specific to MRPL12 protein fixed on the solid phase carrier, can be used for detecting the expression levels of a plurality of proteins including MRPL12 protein, and can improve the detection precision by detecting the MRPL12 protein and a plurality of markers simultaneously.
The invention has the beneficial effects that: the MRPL12 protein is used for preparing a tool (kit and the like) for diagnosing lung adenocarcinoma, has good sensitivity and specificity, is used for early diagnosis of lung adenocarcinoma, solves the problem that the current lung adenocarcinoma molecular marker is not accurate enough and sufficient enough, improves the timeliness, sensitivity and popularity of lung adenocarcinoma diagnosis, and has important significance for early diagnosis, early treatment and prognosis evaluation of lung adenocarcinoma.
Drawings
FIG. 1 is a Western Blot of MRPL12 protein expression in lung adenocarcinoma cells and normal lung epithelial cells;
FIG. 2 is a bar graph of the ratio of MRPL12 protein expression in lung adenocarcinoma cells and normal lung epithelial cells;
FIG. 3 is a graph showing typical results of immunohistochemical detection of MRPL12 protein expression in lung adenocarcinoma tissue;
FIG. 4 is a graph showing typical results of immunohistochemical detection of MRPL12 protein expression in normal lung cancer tissues;
FIG. 5 is a graph of the statistics of immunohistochemical detection 30 on tissue samples.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.
Examples
Differential expression of MRPL12 protein in lung adenocarcinoma cells and normal lung epithelial cells
1. Cell culture
Culturing human normal lung epithelial cells Beas-2B in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 deg.C with 5% CO 2 Culturing in an incubator. Human LUAD cells (H1395, H1975, PC-9 and HCC 827) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, at 37 ℃ in an incubator containing 5% CO2. Fresh medium was changed for 2-3 days and passaged by trypsinization with 0.25% Ethylene Diamine Tetraacetic Acid (EDTA).
2. Protein sample preparation
Pouring out the culture solution in the culture dish, washing twice by PBS, placing on ice, adding 100ul of lysate, cracking for 10min on ice, and collecting the lysate into a precooled 1.5ml EP tube; adding 5X SDS Loading Buffer into the protein sample according to the proportion; ultrasonic treating in ultrasonic cell pulverizer for 2min, and heating in boiling water for 5min to denature.
3. Immunoblotting
(1) SDS-PAGE gel electrophoresis: preparing SDS-PAGE gel according to the molecular weight of the protein; then slowly adding the protein samples in sequence, 10ul per well; electrophoresis is selected to be constant in pressure, the gel is concentrated to be 70V, the gel is separated to be 130V, and the electrophoresis is stopped when the bromophenol blue indicator reaches the bottom of the gel;
(2) Film transfer: according to the molecular weight of the target protein, and the position of the marker is combined, the protein glue area where the target protein is located is reserved; making a sandwich according to the sequence of sponge-filter paper-glue-PVDF membrane-filter paper-sponge, then placing the sandwich into an electrophoresis tank, performing constant current of 200mA, and transferring the membrane for 2 hours;
(3) And (3) sealing: sealing 5% skimmed milk powder at room temperature for 2h or overnight at 4 deg.C, taking out the membrane, and washing with 1X TBST twice;
(4) Primary antibody incubation: diluting primary antibody with primary antibody diluent according to a proportion; taking out the membrane from the confining liquid, sucking residual liquid by using filter paper, and then incubating at room temperature for 1h or at 4 ℃ overnight in primary antibody; then taking out the membrane, washing for three times by 1X TBST, 5min each time;
(5) And (3) secondary antibody incubation: diluting the secondary antibody and 5% of skimmed milk powder according to a required proportion; the membrane was placed in a secondary antibody, incubated at 37 ℃ for 1h, then removed and washed three times with 1X TBST for 5min each.
(6) Signal detection: ECL chemiluminescence is adopted for color development, and luminous liquid A and B are mixed according to 1:1; after the residual liquid on the membrane is absorbed by filter paper, the membrane is fully contacted with the developing solution, and then the membrane is placed on a developing instrument for detection.
(7) Gray level statistics: protein bands were quantitatively analyzed using ImageJ software.
4. Results of the experiment
FIG. 1 shows protein expression levels of MRPL12 and GAPDH in lung adenocarcinoma cells (H1395, H1975, PC-9 and HCC 827) and normal lung epithelial cells (Beas-2B). As shown in FIG. 2, the expression levels of MRPL12 protein in lung adenocarcinoma cells (H1395, H1975, PC-9 and HCC 827) were 18-fold, 21-fold, 4-fold and 35-fold higher than those of normal lung tissue cells (Beas-2B), respectively, using GAPDH protein as an internal reference.
Application of MRPL12 protein in immunohistochemical kit
1. Tissue sample source
30 lung adenocarcinoma tissue and lung normal tissue chips with array number HLugA060PG02 were purchased from Shanghai core Biotechnology Ltd. 18 male cases and 12 female cases, age 38-83 years, all had tumor primary focus, no metastasis, pathological classification including I-III grade, and lung adenocarcinoma as pathological type. The tissue chips described above have been reviewed by the tissue ethics committee.
2. Experimental methods
S1, baking the slices: placing the tissue chip in a 63 ℃ oven, and baking for 1h;
s2, dewaxing and hydrating: dewaxing is completed in a full-automatic dyeing machine (instrument model: LEICAST 5020), and the dewaxing sequence is sequentially xylene I15 min-xylene II 15min-100% alcohol 7min-90% alcohol 5 min-80% alcohol 5min-70% alcohol 5min;
s3, antigen retrieval: according to the simple operation procedure of PTLink, putting the glass slide into a full-automatic immunohistochemical pretreatment system instrument (instrument model: PT Link) for antigen restoration, and after the antigen restoration is finished, putting the glass slide into distilled water at room temperature to naturally cool the glass slide to the room temperature;
s4 quenching: treating with 3% hydrogen peroxide water solution at room temperature for 10min to inactivate endogenous peroxidase; treating 0.01% ovalbumin solution at room temperature for 20min to eliminate activity of endogenous biotin;
s5, sealing: blocking with PBST containing 5% goat serum, and standing at room temperature for 1h;
s6 primary antibody incubation: taking out the slide, and washing the slide by using PBST buffer solution; adding diluted primary antibody working solution, and storing in a refrigerator at 4 ℃ overnight;
and (2) incubation, blocking and color development of the S7 second antibody: taking out the slide from the refrigerator, re-warming for 45min at room temperature, and then washing with PBST buffer solution; putting the slide into a DAKO full-automatic immunohistochemical staining system instrument (instrument model: autostainer Link 48), and selecting corresponding programs for running blocking, secondary antibody combination and DAB color development;
s8 hematoxylin counterstain: staining with hematoxylin for 1min, soaking in 0.25% hydrochloric acid alcohol (400ml 70% ethanol +1ml concentrated hydrochloric acid) for at least 2s, and washing with tap water for more than 2 min;
s9, mounting and scanning: drying the glass slide at room temperature, and sealing the glass slide by neutral resin; scanning analysis was performed using an Aperio scanner (instrument model: aperio XT).
3. Results of the experiment
The 30 pairs of samples in the tissue chip are pictures taken under the same light microscope and the same exposure intensity. Fig. 3 is a typical lung adenocarcinoma tissue staining picture, and fig. 4 is a typical normal lung adenocarcinoma tissue staining picture. Areas of the same size of different tissue staining pictures were selected, areas of positive staining in the tissues were counted using ImageJ software under the same conditions, and differences between lung adenocarcinoma tissue and normal lung tissue staining were counted, as shown in fig. 5, with a P value of less than 0.0001 (P < 0.05;. P < 0.01;. P < 0.001;. P < 0.0001;. P. 0.0001;. P.;) indicating that the MRPL12 immunohistochemistry kit was used to screen for diagnosis of lung adenocarcinoma cancer, with very high specificity and sensitivity.
The MRPL12 protein sequence table related by the invention is as follows:
the full length of the protein sequence of human MRPL12 is 199aa
MLPAAARPLWGPCLGLRAAAFRLARRQVPCVCAVRHMRSSGHQRCEALAGAPLDNA PKEYPPKIQQLVQDIASLTLLEISDLNELLKKTLKIQDVGLVPMGGVMSGAVPAAAAQEAVE EDIPIAKERTHFTVRLTEAKPVDKVKLIKEIKNYIQGINLVQAKKLVESLPQEIKANVAKAEA EKIKAALEAVGGTVVLE。
The foregoing is illustrative of the preferred embodiments of the present invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and is not to be construed as limited to the exclusion of other embodiments, and that various other combinations, modifications, and environments may be used and modifications may be made within the scope of the concepts described herein, either by the above teachings or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
- Application of MRPL12 protein in preparation of lung adenocarcinoma detection products.
- 2. The use of MRPL12 protein according to claim 1 in the preparation of lung adenocarcinoma assay products, wherein: the MRPL12 protein includes all the translation products of the MRPL12 gene.
- 3. The use of MRPL12 protein of claim 1 in the preparation of a lung adenocarcinoma assay product, wherein: the MRPL12 protein is a specific antibody.
- 4. The use of MRPL12 protein of claim 3 in the preparation of a lung adenocarcinoma assay product, wherein: the specific antibody of the MRPL12 protein is a polyclonal antibody.
- 5. The use of MRPL12 protein of claim 3 in the preparation of a lung adenocarcinoma assay product, wherein: the specific antibody of the MRPL12 protein is a monoclonal antibody.
- 6. The lung adenocarcinoma assay product according to any one of claims 1 to 5, wherein: the lung adenocarcinoma detection product is any one of a kit, test paper and a protein chip.
- 7. The lung adenocarcinoma detection product according to claim 6, wherein: the reagent in the kit also comprises a ligand which can indirectly reflect the content or activity of MRPL12 protein and is combined with MRPL12 protein; the protein chip comprises a solid phase carrier and an antibody specific to MRPL12 protein fixed on the solid phase carrier, and can be used for detecting the expression level of a plurality of proteins including MRPL12 protein.
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| CN202210916155.4A CN115184610A (en) | 2022-08-01 | 2022-08-01 | Application of MRPL12 protein in preparation of lung adenocarcinoma detection product |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070099209A1 (en) * | 2005-06-13 | 2007-05-03 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| US20110064739A1 (en) * | 2008-03-28 | 2011-03-17 | Borlak Juergen | Medicament, compositions, and substances for treating and identifying adenocarcinoma of the lung |
-
2022
- 2022-08-01 CN CN202210916155.4A patent/CN115184610A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070099209A1 (en) * | 2005-06-13 | 2007-05-03 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
| US20110064739A1 (en) * | 2008-03-28 | 2011-03-17 | Borlak Juergen | Medicament, compositions, and substances for treating and identifying adenocarcinoma of the lung |
Non-Patent Citations (2)
| Title |
|---|
| 熊雷 等: "MRPL12 在肺腺癌中的表达和预后分析", 《上海交通大学学报（医学版）》, vol. 41, no. 8, pages 1033 - 1040 * |
| 葛彦和王勤 主编: "《药理学实验操作教程（全视频展示）》", 中国协和医科大学出版社, pages: 212 - 215 * |
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Application publication date: 20221014 |