CN111454916A

CN111454916A - Novel application of glyceraldehyde-3-phosphate dehydrogenase protein or immune fragment thereof

Info

Publication number: CN111454916A
Application number: CN201910048610.1A
Authority: CN
Inventors: 王泽宋
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2020-07-28

Abstract

The invention provides a new application of glyceraldehyde-3-phosphate dehydrogenase protein or an immune fragment thereof, provides an application of glyceraldehyde-3-phosphate dehydrogenase protein or an immune fragment thereof in blood as a target in developing, screening and/or preparing a medicament for preventing and/or inhibiting tumor-related diseases, and also provides an application of an inhibitor or antagonist aiming at the glyceraldehyde-3-phosphate dehydrogenase protein or the immune fragment thereof in preparing a medicament for preventing and/or inhibiting tumor-related diseases.

Description

Novel application of glyceraldehyde-3-phosphate dehydrogenase protein or immune fragment thereof

Technical Field

The invention relates to a new application of glyceraldehyde-3-phosphate dehydrogenase protein or an immune fragment thereof, in particular to a new application of glyceraldehyde-3-phosphate dehydrogenase in blood as a tumor-related disease drug target and an application thereof, and particularly relates to a new application of glyceraldehyde-3-phosphate dehydrogenase in blood or an immune fragment thereof as a drug screening target and an inhibitor and/or antagonist thereof in treating cancer.

Background

Malignant tumor is a common disease and frequently encountered disease in the whole world, and is one of the major diseases harming human health. Malignant tumors are a type of systemic disease involving abnormal proliferation, invasion, and metastasis of cells to other parts of the body. Common symptoms include lumps, abnormal bleeding, chronic cough, weight loss and abnormal intestinal motility. The most common types of cancer in men include lung, prostate, colorectal, and gastric cancer; the most common types of cancer in women include breast, colorectal, lung, and cervical cancer; among children, acute lymphocytic leukemia and brain cancer are the most prevalent types of cancer with the highest incidence, except for the non-hodgkin's lymphoma, continental. About 16.5 ten thousand children under 15 years of age were diagnosed with cancer in 2012. Cancer incidence increases dramatically with age, and a variety of cancer types are also common in developed countries. With the change of life style of people, the incidence of cancer in developing countries is increasing. About 429.2 ten thousand newly-increased cancer cases are newly increased in 2015 of China, and 1.2 ten thousand newly increased every day on average; at the same time, about 281.4 people die from cancer, with an average of 7500 deaths per day. Among them lung and bronchial, gastric, liver, esophageal and colorectal cancers account for three-quarters of all Cancer deaths (CA Cancer J Clin.2016; 66: 115-. The incidence of malignant tumors in China is in a continuous high-incidence trend in recent years, and becomes the leading cause of death of domestic residents.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in the glycolysis process. Has a molecular weight of 37kDa, and catalyzes the reaction of glyceraldehyde 3-phosphate (glyceraldehyde 3-phosphate) to glyceric acid 1, 3-diphosphate (D-glycerate 1, 3-biphosphate). In addition to this well-known metabolic regulation function, GAPDH has been shown to be involved in many non-metabolic regulation processes in recent years, including transcriptional activation (oncogene.2007; 26(18): 2606-2620), etc.

There are studies reporting significant increases in mRNA levels of GAPDH in melanoma (Anticancer research.2013; 35(1): 439-444.) and non-small cell lung cancer tissues (P L OS one.2013; 8(4): e61262), and expression levels are related to tumor malignancy because the important role of GAPDH in glycolysis process and its anti-apoptotic function are also important for tumor cell proliferation and protection, e.g. GAPDH can protect telomere shortening due to the action of chemotherapeutic drugs, but if conditions such as oxidative stress destroy GAPDH function, cells will age or die (Clinical and experimental Pharmacology & physiology.2012; 39: 674-679), the absence of GAPDH will also lead to tumor cell aging (Biochemical Research & physiology & gt; 39: 674-679; the absence of GAPDH is also found in Clinical and Biochemical Research & diagnosis and Clinical diagnosis & gt; 1: 2011-674-679; the observed positive expression levels of GAPDH are also reported in patients (Clinical and Clinical diagnosis & diagnosis.

Through inspection, no report about the correlation between GAPDH and tumorigenesis and development and the application of GAPDH as a tumor and related disease drug screening target is found.

Disclosure of Invention

The main content of the invention relates to the correlation of GAPDH and tumorigenesis and development and the application of GAPDH as a tumor and related disease drug screening target.

The inventor finds in research that glyceraldehyde-3-phosphate dehydrogenase secreted by Hela of human cervical cancer cells is obviously higher than that of HEK293 of human normal cells, and the content of glyceraldehyde-3-phosphate dehydrogenase in blood of cancer patients is obviously higher than that of healthy people. The glyceraldehyde-3-phosphate dehydrogenase can promote the proliferation and migration of cancer cells; the monoclonal antibody resisting glyceraldehyde-3-phosphate dehydrogenase and the monoclonal antibody resisting the immune fragment of glyceraldehyde-3-phosphate dehydrogenase can inhibit tumor growth and melanoma metastasis, and prove that the glyceraldehyde-3-phosphate dehydrogenase or the immune fragment thereof can be used as a target spot for tumor prevention and treatment for drug screening.

Thus, the present invention provides a novel use of glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof in blood.

Specifically, in one aspect, the invention provides application of glyceraldehyde-3-phosphate dehydrogenase protein or immune fragments thereof in blood as targets in development, screening and/or preparation of drugs for preventing and/or inhibiting tumor-related diseases.

According to a specific embodiment of the present invention, in the novel use of the glyceraldehyde-3-phosphate dehydrogenase protein or the immune fragment thereof of the present invention, the glyceraldehyde-3-phosphate dehydrogenase protein or the immune fragment thereof comprises a protein or the immune fragment thereof consisting of the amino acid sequences shown in the following a, b and c:

a. a protein consisting of an amino acid sequence shown in SEQ ID NO.2 or an immune fragment thereof;

b. a protein or an immune fragment thereof with the same or similar function as a obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by a;

c. and (b) the derived protein or the immune fragment thereof, which has more than 75% of homology with the amino acid sequence shown in a and has the same or similar functions with a.

According to a specific embodiment of the invention, the protein or the immune fragment thereof with the same or similar function as a obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by a refers to the derived protein or the immune fragment thereof obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence defined by a, and the derived protein or the immune fragment thereof has the same or similar biological function as the protein consisting of the amino acid sequence shown by SEQ ID NO. 2.

According to a specific embodiment of the present invention, the derived protein or immunogenic fragment thereof having 75% or more homology with the amino acid sequence shown in a and having the same or similar function as a preferably has 80% or more homology with the amino acid sequence shown in a, more preferably more than 85%, more preferably more than 90%, still more preferably more than 95%, particularly preferably more than 98%, still more particularly preferably more than 99% sequence homology. The derived proteins or the immune fragments thereof have the same or similar biological functions with the protein consisting of the amino acid sequence shown in SEQ ID NO. 2.

According to a specific embodiment of the present invention, the glyceraldehyde-3-phosphate dehydrogenase protein or immune fragment thereof of the present invention is used in a novel application for tumor-related diseases including, but not limited to, liver cancer, lung cancer, breast cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma or thyroid tumor.

On the other hand, the invention also provides the application of the glyceraldehyde-3-phosphate dehydrogenase protein or the immune fragment thereof in preparing preparations for promoting the proliferation of tumor cells and/or promoting the migration of tumor cells.

According to a specific embodiment of the present invention, preferably, the tumor cell includes, but is not limited to, a liver cancer cell, a lung cancer cell, a breast cancer cell, a stomach cancer cell, an esophageal cancer cell, a colorectal cancer cell, a pancreatic cancer cell, a cervical cancer cell, a lymphoma cell, or a thyroid tumor cell.

In another aspect, the invention also provides the use of an inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof for the preparation of a medicament for the prevention and/or treatment and/or inhibition of a tumor-related disease.

In another aspect, the invention also provides the use of an inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof for the preparation of a formulation for inhibiting tumor cell proliferation and/or migratory invasion.

According to a particular embodiment of the invention, in the novel use of the glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof according to the invention, the inhibitor or antagonist against the glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof is an antibody, a competing polypeptide or a compound. Preferably, the inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof is an antibody against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof; more preferably a monoclonal antibody. In some embodiments of the invention, the inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof is an inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof in blood.

On the other hand, the invention also provides application of the glyceraldehyde-3-phosphate dehydrogenase protein or the immune fragment thereof, or the polynucleotide for coding the glyceraldehyde-3-phosphate dehydrogenase protein or the immune fragment thereof in preparing a vaccine for preventing and treating tumors.

According to a specific embodiment of the present invention, the glyceraldehyde-3-phosphate dehydrogenase protein or immune fragment thereof of the present invention is used in a novel application, wherein the tumor includes, but is not limited to, liver cancer, lung cancer, breast cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma, and thyroid tumor.

According to some embodiments of the invention, the immunogenic fragment of glyceraldehyde-3-phosphate dehydrogenase comprises a polypeptide fragment consisting of the amino acid sequence shown in SEQ ID No. 5.

According to some embodiments of the invention, the polynucleotide encoding the glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID No.2 or SEQ ID No. 5. In some more specific embodiments, the polynucleotide encoding the glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof comprises the polynucleotide sequence shown in SEQ ID No.1 and its codon-optimized polynucleotide sequence SEQ ID No.3 or SEQ ID No. 4.

In some embodiments of the invention, it is demonstrated that cancer cells secrete glyceraldehyde-3-phosphate dehydrogenase to the cell culture medium in large amounts. According to other specific embodiments of the invention, the content of glyceraldehyde-3-phosphate dehydrogenase in the serum of patients with liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma and thyroid tumor is remarkably higher than that of healthy people. The glyceraldehyde-3-phosphate dehydrogenase is added into a culture medium of a lung cancer cell A549, a liver cancer cell HepG2, a breast cancer cell MDA-MB-231, a stomach cancer cell SGC7901, a pancreatic cancer cell PANC-1, a colorectal cancer cell SW620, an esophageal cancer cell CaES-17, a cervical cancer cell Hela and a lymphoma cell U937, so that the proliferation and migration capacity of the cancer cells are remarkably promoted. The extracellular glyceraldehyde-3-phosphate dehydrogenase was demonstrated to have the ability to promote proliferation and migration of tumor cells. Thus, the glyceraldehyde-3-phosphate dehydrogenase in the blood can be used as the target of the tumor-related disease drugs. Blocking its function can significantly inhibit the growth and metastasis of tumors.

In some embodiments of the invention, the glyceraldehyde-3-phosphate dehydrogenase and the immune fragment thereof (the sequence is shown in SEQ ID NO. 5) are prepared and purified by comprehensively using methods such as plasmid construction, prokaryotic expression, affinity chromatography and the like, and are verified by using analytical methods such as SDS-PAGE, Western blot and MA L DI-TOF-MS and the like, so that the prepared protein is confirmed to be the glyceraldehyde-3-phosphate dehydrogenase or the immune fragment thereof.

In some embodiments of the present invention, the prepared glyceraldehyde-3-phosphate dehydrogenase and immune fragments thereof are used to prepare monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase or immune fragments thereof by combining immune mice, cell fusion, affinity chromatography, and the like, and the monoclonal antibodies are used for inhibiting tumor growth and metastasis.

Specifically, human lung cancer cells A549, liver cancer cells HepG2, breast cancer cells MDA-MB-231, gastric cancer cells SGC7901, pancreatic cancer cells PANC-1, colorectal cancer cells SW620, esophageal cancer cells CaES-17, cervical cancer cells Hela and lymphoma cells U937 are respectively inoculated under the right hind limb subcutaneous tissues of BA L B/c-null mice, after the tumors grow to a certain volume, the glyceraldehyde-3-phosphate dehydrogenase or the immune fragment monoclonal antibodies thereof are regularly injected, meanwhile, the weight and the tumor volume of the mice are regularly monitored, the experiment lasts for 6-7 weeks, the tumor growth difference of a control IgG group and the glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody group is compared after the experiment is ended, and the glyceraldehyde-3-phosphate dehydrogenase or the immune fragment thereof has the effect of inhibiting the growth of the glyceraldehyde-3-phosphate dehydrogenase and can be obviously used as a tumor growth inhibiting effect or a tumor growth inhibiting effect of the glyceraldehyde-3-phosphate dehydrogenase.

In some embodiments of the invention, the invention also successfully inhibits tumor metastasis using an anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody using a mouse model. Specifically, the mouse melanoma B16-F10 cell tail vein is injected into a C57 mouse to form lung metastasis, and meanwhile, the monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase is injected regularly. The experiment lasted 18 days, and after the experiment was completed, the metastasis of melanoma in lung tissue was compared between the control IgG group and the anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody group. The invention discovers that the monoclonal antibody for resisting glyceraldehyde-3-phosphate dehydrogenase inhibits the metastasis of tumor. The result shows that the glyceraldehyde-3-phosphate dehydrogenase has the function of promoting the tumor metastasis, and the tumor metastasis can be obviously inhibited by inhibiting the glyceraldehyde-3-phosphate dehydrogenase. And the glyceraldehyde-3-phosphate dehydrogenase can be used as a target spot for preventing and treating tumors for drug screening.

The amino acid and nucleotide sequences of the invention are as follows:

SEQ ID NO.1：

ATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGGGCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTCAACTACATGGTTTACATGTTCCAATATGATTCCACCCATGGCAAATTCCATGGCACCGTCAAGGCTGAGAACGGGAAGCTTGTCATCAATGGAAATCCCATCACCATCTTCCAGGAGCGAGATCCCTCCAAAATCAAGTGGGGCGATGCTGGCGCTGAGTACGTCGTGGAGTCCACTGGCGTCTTCACCACCATGGAGAAGGCTGGGGCTCATTTGCAGGGGGGAGCCAAAAGGGTCATCATCTCTGCCCCCTCTGCTGATGCCCCCATGTTCGTCATGGGTGTGAACCATGAGAAGTATGACAACAGCCTCAAGATCATCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGACAACGAATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAA

SEQ ID NO.2：

MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFHGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

SEQ ID NO.3：

ATGGGCAAAGTTAAAGTTGGGGTTAATGGGTTTGGGCGGATTGGTCGGTTAGTTACGCGTGCGGCCTTTAATAGCGGGAAAGTTGACATTGTTGCGATTAATGACCCGTTTATTGACTTAAATTACATGGTGTATATGTTCCAGTACGATAGTACCCACGGTAAATTTCATGGTACCGTTAAAGCCGAGAATGGGAAACTGGTGATTAATGGTAATCCGATTACCATCTTTCAAGAGCGTGATCCGAGTAAAATTAAGTGGGGTGATGCAGGTGCAGAATACGTTGTTGAGAGCACCGGAGTTTTTACCACCATGGAAAAAGCAGGCGCACACCTTCAGGGCGGCGCGAAAAGAGTAATTATTTCAGCGCCGAGCGCAGATGCACCAATGTTTGTTATGGGGGTTAATCATGAAAAATACGATAATAGCCTGAAGATCATTAGTAATGCAAGCTGTACAACAAATTGTTTAGCACCGTTAGCCAAAGTTATTCATGATAATTTCGGGATTGTGGAAGGTCTGATGACCACAGTTCACGCAATCACCGCCACCCAGAAGACCGTTGATGGTCCTAGCGGAAAACTGTGGCGTGATGGGAGAGGTGCACTGCAGAATATCATTCCGGCCAGTACAGGTGCCGCGAAAGCAGTTGGTAAAGTTATCCCAGAATTAAATGGCAAACTGACAGGTATGGCATTCAGAGTGCCGACCGCAAACGTTAGCGTCGTTGACCTGACCTGTCGTTTAGAAAAACCGGCAAAATATGATGATATCAAAAAGGTTGTGAAGCAGGCGAGCGAAGGACCGCTGAAAGGCATACTGGGTTATACCGAACATCAAGTTGTTTCTAGCGACTTTAATAGCGATACCCACAGTAGCACCTTTGATGCAGGAGCGGGTATTGCGTTAAATGATCATTTTGTTAAGCTGATTAGCTGGTATGATAACGAATTCGGTTATAGTAATCGGGTTGTTGACCTGATGGCACACATGGCAAGCAAGGAATAA

SEQ ID NO.4：

ATGGTGGAAGGCTTAATGACGACGGTTCATGCGATTACGGCCACGCAGAAAACCGTTGACGGGCCGTCGGGGAAACTGTGGCGGGACGGTCGGGGTGCACTGCAGAACATTATTCCGGCGAGTACCGGTGCAGCCAAAGCAGTTGGTAAAGTTATTCCGGAATTAAACGGAAAACTGACAGGTATGGCATTCCGTGTTCCTACCGCAAATGTGAGCGTTGTTGATCTGACCTGTCGTTTAGAAAAGCCGGCAAAATATGATGATATCAAAAAGGTTGTGAAGCAGGCAAGCGAAGGCCCGCTGAAAGGTATTCTGGGTTATACCGAGCATCAGGTTGTTTCATCAGACTTCAATAGCGACACCCATAGCAGCACCTTTGATGCAGGAGCAGGTATTGCATTAAATGATCATTTTGTTAAGCTGATCAGCTGGTATGATAATGAATTCGGTTATAGTAACCGTGTTGTTGATTTAATGGCACACATGGCAAGCAAGGAGTAA

SEQ ID NO.5：

MVEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNGKLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE

in conclusion, the invention provides the application of glyceraldehyde-3-phosphate dehydrogenase or immune fragments thereof in blood as a drug screening target, and also provides the application of inhibitors and/or antagonists of glyceraldehyde-3-phosphate dehydrogenase or immune fragments thereof in treating cancers.

Drawings

FIG. 1: and (3) testing results of glyceraldehyde-3-phosphate dehydrogenase secreted by human normal cells HEK293 and human cervical carcinoma cells Hela.

FIG. 2: the result of detecting the content of glyceraldehyde-3-phosphate dehydrogenase in serum of cancer patients.

FIG. 3: the recombinant glyceraldehyde-3-phosphate dehydrogenase is expressed. Wherein each lane: m, protein molecular weight standard; 1, no induction; 2, after induction; 3, inducing the supernatant after fragmentation; and 4, inducing precipitation after fragmentation.

FIG. 4: and (3) expressing the recombinant glyceraldehyde-3-phosphate dehydrogenase immune fragment. Wherein each lane: m, protein molecular weight standard; 1, no induction; 2, after induction; 3, inducing the supernatant after fragmentation; and 4, inducing precipitation after fragmentation.

FIG. 5: the result of purification of the recombinant glyceraldehyde-3-phosphate dehydrogenase. Wherein each lane: m, protein molecular weight standard; 1, treating a sample after crushing; 2, flowing out; and 3, eluting.

FIG. 6: and (3) purifying the immune fragment of the recombinant glyceraldehyde-3-phosphate dehydrogenase. Wherein each lane: m, protein molecular weight standard; 1, treating a sample after crushing; 2, flowing out; and 3, eluting.

FIG. 7: western Blot identifies the experimental result of the recombinant glyceraldehyde-3-phosphate dehydrogenase. Wherein each lane: m, protein molecular weight standard; 1, sample after purification.

FIG. 8: western Blot identifies the immune fragment experiment result of the recombinant glyceraldehyde-3-phosphate dehydrogenase. Wherein each lane: m, protein molecular weight standard; 1, sample after purification.

FIG. 9: results of experiments on the promotion of cancer cell proliferation by glyceraldehyde-3-phosphate dehydrogenase.

FIG. 10: results of experiments on the promotion of cancer cell migration by glyceraldehyde-3-phosphate dehydrogenase.

FIG. 11: western Blot for detecting the monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase. Wherein each lane: m, protein molecular weight standard; 1, GAPDH native protein (0.5 μ g); 2, GAPDH native protein (1. mu.g).

FIG. 12: western Blot for detecting the monoclonal antibody of the anti-glyceraldehyde-3-phosphate dehydrogenase immune fragment. Wherein each lane: m, protein molecular weight standard; 1, GAPDH native protein (0.5 μ g); 2, GAPDH native protein (1. mu.g).

FIG. 13: the result of the experiment that the monoclonal antibody of the anti-glyceraldehyde-3-phosphate dehydrogenase can inhibit the growth of the tumor.

FIG. 14: the result of the experiment that the monoclonal antibody of the immune fragment of the anti-glyceraldehyde-3-phosphate dehydrogenase can inhibit the growth of the tumor.

FIG. 15: the result of an experiment that the anti-glyceraldehyde-3-phosphate dehydrogenase antibody inhibits the lung metastasis of the mouse melanoma.

Detailed Description

For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. In the examples, the experimental methods without specifying the specific conditions were conventional methods and conventional conditions well known in the art, or were operated according to the conditions suggested by the instrument manufacturer.

Example 1: the human normal cell HEK293 and the human cervical carcinoma cell Hela secrete glyceraldehyde-3-phosphate dehydrogenase, and the secretion amount of the latter is obviously higher than that of the former.

Inoculating human normal cell HEK293 and human cervical cancer cell Hela with good growth condition into a 6-hole cell culture plate, and culturing for 36 hours by using a DMEM and 10% FBS culture medium until the confluence degree reaches 80% and the cell activity reaches more than 95%. At the moment, the cell culture medium is replaced by fresh DMEM, the cell culture medium is continuously cultured for 12 hours, then the cell culture medium is recovered for Western blot detection, and the sample loading amount is adjusted according to the cell number of the two cells. As shown in FIG. 1, Hela secreted a large amount of glyceraldehyde-3-phosphate dehydrogenase into the culture medium in the same number of human cervical cancer cells as compared with the human normal cells HEK 293.

Example 2: the content of glyceraldehyde-3-phosphate dehydrogenase in serum of cancer patients is obviously higher than that of healthy people.

Collecting serum of healthy people and patients with liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma and thyroid tumor, and detecting the concentration of GAPDH in the serum by Enzyme linked immunosorbent assay (Elisa), wherein the specific experimental method is as follows:

1. sample selection

A healthy person: people who have not been diagnosed by biochemical, imaging or pathological methods and are considered to be non-cancerous for a while. Non-cancer patients here include healthy persons and cancer-related disease patients.

Cancer patients: patients diagnosed with cancer by pathological diagnosis include different types and stages of cancer.

2. Sample collection

Blood was collected from healthy persons and cancer patients, respectively, and allowed to stand at room temperature for 20 minutes, serum was separated by centrifugation (800-.

3. Sample detection

The concentration of glyceraldehyde-3-phosphate dehydrogenase in the collected serum samples was measured by the Elisa method.

4. Results of the study

As shown in FIG. 2, the content of glyceraldehyde-3-phosphate dehydrogenase in the serum of patients with liver cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma, and thyroma is significantly higher than that of healthy persons.

Example 3: in vitro prokaryotic expression, purification and identification of glyceraldehyde-3-phosphate dehydrogenase and immune fragments thereof.

In the embodiment, the glyceraldehyde-3-phosphate dehydrogenase and the immune fragment thereof are prepared and purified by comprehensively using methods such as plasmid construction, prokaryotic expression, affinity chromatography and the like, and are verified by using analytical methods such as SDS-PAGE, Western blot and MA L DI-TOF-MS and the like, so that the prepared protein is confirmed to be the glyceraldehyde-3-phosphate dehydrogenase or the immune fragment thereof.

1. Experimental Material

The main experimental materials included pCzn1 plasmid, TOP10 strain, Protein Marker (Thermo Co.), IPTG, Acr, Bis, Tris (Sigma Co.), SDS (Amresco Co.), TEMED (BIO-RAD Co.), restriction enzyme (TaKaRa), Pfu DNA polymerase (Zoonbio, cat. No. PC12), Tyrptone, Yeast Extract (OXOID Co.), Agarose (Shanghai Gene Co.), DNA gel purification kit, plasmid miniprep kit (AXYGEN Co.), consumables (Fisher Co.), 0.22 μm sterile filter and dialysis bag (Millipore Co.), Ni-IDA affinity chromatography gel (Novagen Co.).

2. Instrumentation and equipment

The main instruments used included Allegra 21R desktop high speed refrigerated centrifuge (BECKMAN corporation), desktop high speed centrifuge (SORVA L corporation), biological L P chromatography system, Mini Protean II vertical plate electrophoresis system, Gel Doc2000 imaging system, horizontal electrophoresis system (BIO-RAD corporation), PTC-200 gene amplification instrument (MJResearch corporation, USA), 320-S pH meter (Mettler Toledo corporation, USA), AR5120 electronic balance (AHOMS corporation), MultiTempIII constant temperature water bath, Hofer Mm V-25 ultraviolet transilluminator (Amersham Pharmacia corporation), ice maker (SANYO corporation, Japan), JY92-2D ultrasonic cell crusher (New Korea research institute, China), ultra clean bench (ROSujing corporation) NANODP 2000(Thermo corporation).

3. Experimental procedures and results

(1) And (5) constructing a plasmid. The full-length protein adopts a PAS (PCR-based Accurate Synthesis) based method to design a full-length splicing primer, and the recombinant plasmid pCzn1-GAPDH is constructed through the steps of PCR, enzyme digestion, connection and the like. Then the obtained recombinant plasmid pCzn1-GAPDH is transferred into TOP10 clone strain, positive clone is picked for sequencing, and the sequencing result is consistent with the polynucleotide sequence shown in SEQ ID NO. 3. The immune fragment adopts a PAS (PCR-based Accurate Synthesis) based method to design a full-length splicing primer, the obtained recombinant plasmid pCzn1-GAPDH-C is transferred into a TOP10 clone strain, a positive clone is selected for sequencing, and the sequencing result is consistent with the polynucleotide sequence shown in SEQ ID NO. 4.

(2) Expression of recombinant protein, adding 1 μ l plasmid pCzn1-GAPDH or pCzn1-GAPDH-C into 100 μ l competent bacteria, placing on ice for 20min, heating at 42 deg.C for 90sec, quickly placing on ice for 5min, adding 600 μ l L B culture solution, shaking at 37 deg.C and 220r/min for 1h, centrifuging, coating on L B plate containing 50 μ g/ml Amp, culturing at 37 deg.C, standing overnight, expressing fusion protein, selecting monoclonal on the conversion plate, inoculating on test tube containing 3ml L B culture solution containing 50 μ g/ml Amp, shaking at 37 deg.C and 220r/min overnight, inoculating on the next day, inoculating on 30ml L B culture solution containing 50 μ g/ml Amp, shaking at 37 deg.C and 220r/min until OD600 is 0.6-0.8 (about 2h), taking out 1ml culture, 10000 r/mM 2min, removing supernatant, adding supernatant, shaking for 10min, adding supernatant, weighing supernatant, adding supernatant, shaking for 10 mM, adding supernatant, taking out supernatant, adding supernatant, taking out.

(3) The purification of recombinant protein was carried out by loading the supernatant solution onto Ni-IDA Binding-Buffer pre-equilibrated Ni-IDA-Sepharose C L-6B affinity column using a low pressure chromatography system at a flow rate of 0.5ml/min, Washing with Ni-IDA Binding-Buffer at a flow rate of 0.5ml/min until the effluent OD280 reached the baseline, Washing with Ni-IDA Washing-Buffer at a flow rate of 1ml/min until the effluent OD280 reached the baseline, eluting the target protein with Ni-IDA Elution-Buffer at a flow rate of 1ml/min, collecting the effluent, adding the collected protein solution into a dialysis bag, dialyzing with 20mM Tris-HCl, 0.15M NaCl, pH8.0 overnight, carrying out 12% SDS-PAGE analysis, and the result is shown in FIGS. 5 and 6. the purified band was identified by MA L DI-TOF-MS method to match the amino acid sequence of SEQ ID NO.2 and SEQ ID NO. 5.

(4) The method comprises the steps of sampling 5 mu l of a sample, loading polyacrylamide gel, running out 90V of the gel layer, increasing the voltage to 200V until electrophoresis is finished, unloading the gel for membrane transfer after the electrophoresis is finished, carrying out membrane transfer at constant voltage of 100V for about 1.5h and keeping constant current of 250mA, unloading the membrane after the electrotransformation is finished, washing the membrane for 4 times by PBST (Poly-N-phenylenebacteria-Sulfonylike acid) 5min each time, placing the membrane in 5% skimmed milk powder sealing solution for sealing at 37 ℃ for 1h, diluting the primary antibody by the sealing solution, placing the membrane in a primary antibody diluting solution for overnight at 4 ℃, washing the membrane for 4 times by PBST 5min each time after the membrane is taken out the next day, diluting a secondary antibody by the sealing solution containing 5% of milk, reacting the membrane for 1h at 37 ℃, taking out the membrane, washing the membrane for 4 times by a clean box for 5min each time after the reaction is finished, developing by exposing L, and carrying out the Western Blot results of Western Blot as shown in a Western Blot 7 and immune fragments as shown in a graph 8.

Example 4: glyceraldehyde-3-phosphate dehydrogenase promotes cancer cell proliferation.

Human lung cancer cells A549, liver cancer cells HepG2, breast cancer cells MDA-MB-231, stomach cancer cells SGC7901, pancreatic cancer cells PANC-1, colorectal cancer cells SW620, esophageal cancer cells CaES-17, cervical cancer cells Hela and lymphoma cells U937 which grow well are respectively inoculated into a 6-well cell culture plate, after being cultured for 24 hours by using a culture medium of DMEM and 10% FBS, the culture medium is replaced by fresh DMEM and 1% FBS and 2 mu g/ml glyceraldehyde-3-phosphate dehydrogenase, and a culture medium of a control group and 2 mu g/ml BSA is continuously cultured for 24 hours, and the cell activity is more than 95%. The cell proliferation of the glyceraldehyde-3-phosphate dehydrogenase treated group and the control group was measured by using the CCK-8 cell activity assay. As shown in FIG. 9, the addition of glyceraldehyde-3-phosphate dehydrogenase significantly promoted the proliferation of human lung cancer cell A549, liver cancer cell HepG2, breast cancer cell MDA-MB-231, stomach cancer cell SGC7901, pancreatic cancer cell PANC-1, colorectal cancer cell SW620, esophageal cancer cell CaES-17, cervical cancer cell Hela, and lymphoma cell U937.

Example 5: glyceraldehyde-3-phosphate dehydrogenase promotes cancer cell migration.

Firstly, adding 500 mu L DMEM culture medium (containing 2% fetal bovine serum) into a 24-well plate, then placing the 24-well plate into a Transwell with 20 mu L Matrigel spread on the bottom, standing the Transwell plate at 37 ℃ for 30min, collecting human lung cancer cell A549, liver cancer cell HepG2, breast cancer cell MDA-MB-231, stomach cancer cell SGC7901, pancreatic cancer cell PANC-1, colorectal cancer cell SW620, esophageal cancer cell CaES-17, cervical cancer cell Hela and lymphoma cell U937, and preparing the cells into 5 × 10 DMEM culture medium⁵The method comprises the following steps of adding 100 mu L cells into each Transwell for suspension, culturing for 36-48 hours at 37 ℃, taking out a Transwell small basket after the culture is finished, sucking upper chamber liquid, fixing for 30 minutes by using 4% paraformaldehyde solution, washing a Transwell membrane once by using PBS (phosphate buffer solution), staining the membrane in a crystal violet solution for 60 minutes, washing the Transwell membrane twice by using PBS (phosphate buffer solution), wiping off Matrigel and cells on the membrane by using a cotton swab, observing and photographing under an inverted phase-contrast microscope, selecting 5 fields of view for each Transwell, and calculating an average value, wherein the result is shown in figure 10, and the addition of glyceraldehyde-3-phosphate dehydrogenase remarkably promotes the migration of human lung cancer cells A549, liver cancer cells HepG2, breast cancer cells MDA-MB-231, stomach cancer cells SGC7901, pancreatic cancer cells PANC-1, colorectal cancer cells SW620, esophageal cancer cells CaES-17, cervical cancer cells Hela and lymphoma cells U937.

Example 6: preparing, purifying and detecting monoclonal antibodies of the glyceraldehyde-3-phosphate dehydrogenase and immune fragments thereof.

1. Experimental Material

Balb/c mice; recombinant GAPDH protein; freund's adjuvant (Sigma Co.); goat anti-mouse-HRP secondary antibody; a ProteinMarker; acr, Bis, Tris, PEG (Sigma), SDS, liquid paraffin (Amresco), TEMED (BIO-RAD), 0.22 μm sterile filter and dialysis bag (Millipore), high-sugar DMEM medium (Gibco), cell counting plate (purchased from Improved Neubauer), 96-well cell culture plate (Costar).

2. Instrumentation and equipment

The main instruments used include Allegra 21R desk-top high-speed refrigerated centrifuge (BECKMAN), desk-top high-speed centrifuge (SORVA L), 320-S pH meter (Mettler Toledo, USA), AR5120 electronic balance (AHOMS), multitempIII constant-temperature water bath (Amersham Pharmacia), ice maker (SANYO, Japan), microplate reader, plate washer, NANODROP2000 (Thermo), ultra clean bench (SUJINGYO, China).

3. Experimental procedures and results

(1) And (3) immunizing animals, namely respectively selecting 8 female Balb/C mice with the age of 6-8 weeks, mixing GAPDH protein with Freund adjuvant for immunization, carrying out immunization according to 100ug per mouse by subcutaneous injection, carrying out boosting immunization once in 2-3 weeks, carrying out blood collection detection, and determining the titer of antiserum against the GAPDH or GAPDH-C protein by an indirect E L ISA method.

(2) And (4) fusing the cells. Myeloma cells and splenocytes were mixed. The cells were placed in a 50ml centrifuge tube, diluted with DMEM basal medium, and then centrifuged at 1000rpm for 5min, and the supernatant was discarded. The tubes were shaken to homogenize the cells. 0.8ml of 50% PEG was slowly added for 90 seconds, then 20-30ml of DMEM medium was added to stop the PEG, and the fused cells were placed in a water bath at 37 ℃ for reaction for 10 minutes. 1000rpm for 5min, the supernatant was discarded and HAT DMEM medium was added. The fused cells were plated in 96-well plates at 100. mu.l per well. The cell culture plate was then placed in CO₂Culturing in an incubator. The cloning rate of the hybridoma cells is more than 50% and the growth state of the cells is good when the hybridoma cells are checked 4 days after fusion.

(3) And (5) purifying the antibody. Loading Protein A agarose gel medium into an affinity purification chromatography column, mixing the ascites of a mouse immunized by GAPDH or GAPDH-C Protein with PBS in equal amount, slowly loading the mixture, eluting the mixture by glycine elution buffer solution after the antibody is combined to obtain the required purified antibody, and immediately dialyzing the mixture in the PBS at 4 ℃ overnight.

(4) The detection of the antibody is carried out by a Western Blot method, namely, sampling 5 mul of recombinant GAPDH protein and natural GAPDH protein, after sampling, running 90V of polyacrylamide gel to finish lamination of the gel, then increasing the voltage to 200V until electrophoresis is finished, after electrophoresis is finished, taking down the gel to carry out membrane conversion, carrying out membrane conversion at constant voltage of 100V for about 1.5h and keeping constant current of 250mA, after electric conversion is finished, taking down the membrane, washing the membrane for 4 times by PBST for 5min each time, sealing the membrane in 5% skimmed milk powder sealing solution for 1h at 37 ℃, diluting the primary antibody with the sealing solution, washing the membrane in a primary antibody diluting solution for 4 ℃ overnight, taking out the membrane on the next day, washing the membrane for 4 times by PBST for 5min each time, diluting the secondary antibody with the sealing solution containing 5% of milk, reacting the membrane for 1h at 37 ℃, after reaction, taking out the membrane, placing the membrane in a clean box, washing the membrane for 4 times, developing the monoclonal antibody fragment of the Western Blot, exposing the Western Blot, wherein the result is shown in a monoclonal antibody 11-based on the Western Blot result.

Example 7: the monoclonal antibody of anti-glyceraldehyde-3-phosphate dehydrogenase can inhibit the growth of tumor.

When human lung cancer cells A549, liver cancer cells HepG2, breast cancer cells MDA-MB-231, stomach cancer cells SGC7901, pancreatic cancer cells PANC-1, colorectal cancer cells SW620, esophageal cancer cells CaES-17, cervical cancer cells Hela and lymphoma cells U937 with good growth state grow to 90% confluency, the cells are collected, the cells are resuspended in DMEM culture medium and counted by using a cell counter, then the cells and Matrigel are mixed according to the proportion of 1:1 and inoculated under the right hind limb of BA L B/c-null mice, each mouse is inoculated with 100 mu L1 × 10 of nude mice⁷And (4) cells. After the tumor grows to a certain volume, the anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody 2.5mg/Kg mouse is periodically injected, and IgG is injected into a control group. Mice body weight and tumor volume were monitored periodically and tumor growth curves were plotted. The experiment was continued for a period of 6-7 weeks, and the difference in tumor growth between the control IgG group and the anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody group was compared after the end of the experiment. The experimental result shown in FIG. 13 shows that the monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase can significantly inhibit the growth of human lung cancer cell A549, liver cancer cell HepG2, breast cancer cell MDA-MB-231, stomach cancer cell SGC7901, pancreatic cancer cell PANC-1, colorectal cancer cell SW620, esophageal cancer cell CaES-17, cervical cancer cell Hela and lymphoma cell U937 tumors. In vivo experiments prove that the glyceraldehyde-3-phosphate dehydrogenase has the function of promoting the growth of tumors,the inhibition of the compound can obviously inhibit the growth of tumors. The glyceraldehyde-3-phosphate dehydrogenase is proved to be capable of being used as a target spot for preventing and treating tumors for drug screening.

Example 8: the monoclonal antibody of the immune fragment of the anti-glyceraldehyde-3-phosphate dehydrogenase can inhibit the growth of tumors.

When human lung cancer cells A549, liver cancer cells HepG2, breast cancer cells MDA-MB-231, stomach cancer cells SGC7901, pancreatic cancer cells PANC-1, colorectal cancer cells SW620, esophageal cancer cells CaES-17, cervical cancer cells Hela and lymphoma cells U937 with good growth state grow to 90% confluency, the cells are collected, the cells are resuspended in DMEM culture medium and counted by using a cell counter, then the cells and Matrigel are mixed according to the proportion of 1:1 and inoculated under the right hind limb of BA L B/c-null mice, each mouse is inoculated with 100 mu L1 × 10 of nude mice⁷And (4) cells. After the tumor grows to a certain volume, the anti-glyceraldehyde-3-phosphate dehydrogenase immune fragment monoclonal antibody is regularly injected into 2.5mg/Kg mice, and IgG is injected into a control group. Mice body weight and tumor volume were monitored periodically and tumor growth curves were plotted. The experiment was continued for a period of 6-7 weeks, and after the end of the experiment, the difference in tumor growth between the control IgG group and the anti-glyceraldehyde-3-phosphate dehydrogenase immune fragment monoclonal antibody group was compared. The experimental result shown in FIG. 14 shows that the anti-glyceraldehyde-3-phosphate dehydrogenase immune fragment monoclonal antibody significantly inhibits the growth of human lung cancer cell A549, liver cancer cell HepG2, breast cancer cell MDA-MB-231, stomach cancer cell SGC7901, pancreatic cancer cell PANC-1, colorectal cancer cell SW620, esophageal cancer cell CaES-17, cervical cancer cell Hela and lymphoma cell U937 tumors. Proves that the immune fragment of glyceraldehyde-3-phosphate dehydrogenase can be used as a target spot for preventing and treating tumors and used for screening medicaments.

Example 9: the monoclonal antibody of the glyceraldehyde-3-phosphate dehydrogenase can inhibit the metastasis of melanoma.

When the melanoma cells B16-F10 in good growth state grow to 90% confluency, the cells are collected, resuspended in DMEM medium, counted by using a cell counter, and injected into C57 mice in tail vein, and each mouse is inoculated with 100 mu L2 × 10⁵And (4) cells, so that lung metastasis is formed. Mice with anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody 2.5mg/Kg injected regularly, control groupIgG was injected. The experiment lasted 18 days, and after the experiment was completed, the metastasis of melanoma in lung tissue was compared between the control IgG group and the anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody group. FIG. 15 shows the results of an experiment showing that the monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase inhibits lung metastasis of melanoma cells. In vivo experiments prove that glyceraldehyde-3-phosphate dehydrogenase has the function of promoting tumor metastasis, and can obviously inhibit tumor metastasis by inhibiting the glyceraldehyde-3-phosphate dehydrogenase. The glyceraldehyde-3-phosphate dehydrogenase is proved to be capable of being used as a target spot for preventing and treating tumors for drug screening.

Sequence listing

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Claims

1. The application of glyceraldehyde-3-phosphate dehydrogenase protein or immune fragments thereof in blood as targets in developing, screening and/or preparing drugs for preventing and/or inhibiting tumor-related diseases.

2. The use according to claim 1, wherein the glyceraldehyde-3-phosphate dehydrogenase or an immune fragment thereof comprises a protein or an immune fragment thereof consisting of the amino acid sequences shown in the following a, b and c:

3. The use of claim 1, wherein the tumor-related disease includes, but is not limited to, liver cancer, lung cancer, breast cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma or thyroid tumor.

4. The application of glyceraldehyde-3-phosphate dehydrogenase protein or immune fragments thereof in preparing preparations for promoting tumor cell proliferation and/or tumor cell migration;

preferably, the tumor cell includes, but is not limited to, a liver cancer cell, a lung cancer cell, a breast cancer cell, a stomach cancer cell, an esophageal cancer cell, a colorectal cancer cell, a pancreatic cancer cell, a cervical cancer cell, a lymphoma cell, or a thyroid tumor cell.

5. Use of an inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof for the manufacture of a medicament for the prevention and/or treatment and/or inhibition of a tumor-related disease.

6. Use of an inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof for the preparation of a formulation for inhibiting tumor cell proliferation and/or migratory invasion.

7. The use according to claim 5 or 6, wherein the inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof is an antibody, a competing polypeptide or compound;

preferably, the inhibitor or antagonist against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof is an antibody against glyceraldehyde-3-phosphate dehydrogenase protein or an immunological fragment thereof; more preferably a monoclonal antibody.

8. Application of glyceraldehyde-3-phosphate dehydrogenase protein or immune fragment thereof, or polynucleotide for encoding glyceraldehyde-3-phosphate dehydrogenase protein or immune fragment thereof in preparing tumor prevention and treatment vaccines.

9. The use according to any one of claims 5 to 8, wherein the glyceraldehyde-3-phosphate dehydrogenase or an immune fragment thereof comprises a protein or an immune fragment thereof consisting of the amino acid sequences shown in the following a, b and c:

10. The use of any one of claims 5 to 9, wherein the tumor includes, but is not limited to, liver cancer, lung cancer, breast cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cervical cancer, lymphoma, thyroid tumor.