Application of RALGDS truncated protein in lung cancer screening

Abstract

The present invention relates to the use of RALGDS truncated protein in lung cancer screening. Specifically, the present invention provides the use of an Anti-RALGDS detection reagent, wherein the detection reagent is a RALGDS truncated protein. The truncated protein of the present invention has a high affinity for Anti-RALGDS, and has high sensitivity and high specificity in lung cancer detection, thereby facilitating a more accurate and earlier diagnosis of lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and in particular to the application of RALGDS truncated protein in lung cancer screening. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world. Its morbidity and mortality rates are increasing year by year. Currently, its morbidity ranks first in the world, posing a serious threat to human health and life.

[0003] Lung cancer is a disease that is good at hiding itself. It often shows clinical symptoms only in the late stage of the disease. 70-80% of lung cancer patients are already in the middle or late stage when they are diagnosed with lung cancer symptoms. Cancer cells have spread and the best time for cure has been missed. The five-year survival rate is low. For patients with early lung cancer, timely treatment can greatly improve the patient's survival rate and quality of life for 5 years or more. Therefore, early diagnosis and effective screening of lung cancer are crucial.

[0004] Therefore, there is an urgent need in this field to develop new biomarkers, detection reagents and detection technologies that can be used for early diagnosis or screening of lung cancer. Summary of the invention

[0005] The purpose of the present invention is to provide new biomarkers, detection reagents and detection technologies for early diagnosis or screening of lung cancer.

[0006] In a first aspect of the present invention, there is provided a use of an Anti-RALGDS detection reagent for preparing a detection agent / kit for detecting lung cancer; wherein the detection reagent is a truncated protein of RALGDS.

[0007] In another preferred embodiment, the truncated protein is a non-full-length protein.

[0008] In another preferred embodiment, the ratio of the length L1 of the RALGDS truncated protein to the length L0 of the RALGDS full-length protein is ≦1 / 2, preferably ≦1 / 3, and more preferably ≦1 / 4.

[0009] In another preferred embodiment, the detection reagent is RALGDS truncated protein.

[0010] In another preferred embodiment, the detection reagent includes one or more RALGDS truncated proteins.

[0011] In another preferred embodiment, the RALGDS truncated protein contains the following fragments in SEQ ID No: 2: positions 1-90, positions 1-211, positions 1-390, positions 210-390, positions 621-951, positions 611-914, positions 621-765, or positions 851-951.

[0012] In another preferred embodiment, the RALGDS truncated protein contains the following fragments in SEQ ID No: 2: positions 851-951, positions 1-90, positions 621-951, or positions 611-914.

[0013] In another preferred embodiment, the RALGDS truncated protein is selected from the following group:

[0014] (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein;

[0015] (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein;

[0016] (Y3) truncated protein 5, whose amino acid sequence is amino acids 621-951 of the RALGDS protein;

[0017] (Y4) truncated protein 6, whose amino acid sequence is amino acids 611-914 of the RALGDS protein;

[0018] (Y5) any combination of Y1 to Y4 above;

[0019] Among them, the amino acid sequence of RALGDS protein is shown in SEQ ID No:2.

[0020] In another preferred embodiment, the RALGDS truncated protein comprises:

[0021] (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein;

[0022] (Y4) truncated protein 6, whose amino acid sequence is amino acids 611-914 of RALGDS protein; and

[0023] (Ya) any truncated protein selected from the group consisting of:

[0024] (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein;

[0025] (Y3) Truncated protein 5, whose amino acid sequence is amino acids 621-951 of RALGDS protein.

[0026] In another preferred embodiment, the RALGDS truncated proteins include: (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein; (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein; and (Y4) truncated protein 6, whose amino acid sequence is amino acids 611-914 of the RALGDS protein.

[0027] In another preferred embodiment, the detection of lung cancer is to detect the level of Anti-RALGDS in the sample to be tested.

[0028] In another preferred embodiment, the samples to be tested include: blood samples, plasma samples and serum samples.

[0029] In another preferred embodiment, the lung cancer patient is an Anti-RALGDS antibody positive patient.

[0030] In another preferred embodiment, the lung cancer patient is a male patient or a female patient.

[0031] In another preferred embodiment, the detection kit contains an instruction manual, which describes the detection method.

[0032] In a second aspect of the present invention, an isolated human RALGDS truncated protein is provided, wherein the truncated protein is selected from the group consisting of:

[0033] (A1) a polypeptide of amino acids 851 to 951 in SEQ ID NO: 2;

[0034] (A2) a polypeptide comprising amino acids 1 to 90 of SEQ ID NO: 2;

[0035] (A3) a polypeptide comprising amino acids 621 to 951 of SEQ ID NO: 2;

[0036] (A4) A polypeptide comprising amino acids 611 to 914 of SEQ ID NO:2.

[0037] In another preferred embodiment, the truncated protein is a polypeptide having amino acids 851 to 951 of the amino acid sequence of SEQ ID NO: 1.

[0038] In the third aspect of the present invention, an isolated polynucleotide is provided, characterized in that it comprises a nucleotide sequence selected from the group consisting of:

[0039] (a) a polynucleotide encoding a truncated protein as described in the second aspect of the present invention;

[0040] (b) A polynucleotide complementary to the polynucleotide (a).

[0041] In another preferred embodiment, it is characterized in that the polynucleotide encodes a polypeptide selected from the following group:

[0042] (A1) a polypeptide of amino acids 851 to 951 in SEQ ID NO: 2;

[0043] (A2) a polypeptide comprising amino acids 1 to 90 of SEQ ID NO: 2;

[0044] (A3) a polypeptide comprising amino acids 621 to 951 of SEQ ID NO: 2;

[0045] (A4) A polypeptide comprising amino acids 611 to 914 of SEQ ID NO:2.

[0046] In another preferred example, the sequence of the polynucleotide is the corresponding coding sequence in SEQ ID No: 1.

[0047] In the fourth aspect of the present invention, a vector is provided, characterized in that it contains the polynucleotide described in the third aspect of the present invention.

[0048] In the fifth aspect of the present invention, a genetically engineered host cell is provided, characterized in that it contains the vector described in the fourth aspect of the present invention, or the polynucleotide described in the third aspect of the present invention is integrated into its genome.

[0049] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0050] In another preferred embodiment, the host cell is Escherichia coli or yeast.

[0051] In a sixth aspect of the present invention, a method for preparing a RALGDS truncated protein is provided, characterized in that the method comprises:

[0052] (a) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for expression;

[0053] (b) isolating the RALGDS truncated protein from the culture.

[0054] In a seventh aspect of the present invention, a kit is provided, the kit comprising:

[0055] (a) a first container, and an Anti-RALGDS detection reagent in the first container, wherein the detection reagent is a RALGDS truncated protein;

[0056] (b) an instruction manual, wherein the instruction manual describes the detection method.

[0057] In another preferred embodiment, the detection method is to detect the level of Anti-RALGDS antibodies in the sample to be tested, and is used for early screening or auxiliary diagnosis of lung cancer.

[0058] In another preferred embodiment, the samples to be tested include: blood samples, plasma samples and serum samples.

[0059] In another preferred embodiment, the RALGDS truncated protein is selected from the following group:

[0060] (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein;

[0061] (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein;

[0062] (Y4) truncated protein 6, whose amino acid sequence is amino acids 611-914 of the RALGDS protein;

[0063] or a combination thereof.

[0064] In an eighth aspect of the present invention, a detection reagent for lung cancer detection is provided, wherein the detection reagent is a RALGDS truncated protein, and the RALGDS truncated protein comprises:

[0065] (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein;

[0066] (Y4) truncated protein 6, whose amino acid sequence is amino acids 611-914 of RALGDS protein; and

[0067] (Ya) any truncated protein selected from the group consisting of:

[0068] (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein;

[0069] (Y3) Truncated protein 5, whose amino acid sequence is amino acids 621-951 of RALGDS protein.

[0070] In another preferred embodiment, the RALGDS truncated proteins include: (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein; (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein; and (Y4) truncated protein 6, whose amino acid sequence is amino acids 611-914 of the RALGDS protein.

[0071] In the ninth aspect of the present invention, there is provided a use of the kit described in the seventh aspect of the present invention or the detection reagent described in the eighth aspect of the present invention, characterized in that it is used to prepare a kit for early screening or auxiliary diagnosis of lung cancer by detecting the level of Anti-RALGDS in a sample to be tested.

[0072] In another preferred embodiment, the samples to be tested include: blood samples, plasma samples and serum samples.

[0073] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The truncation sites for the RALGDS autoantibodies are shown.

[0075] Figure 2 The scatter plots are shown for each RALGDS truncated protein after serum reactions from healthy, benign, and lung cancer patients.

[0076] Figure 3 The receiver operating curves (ROC) of four RALGDS fragments (1-90, 621-951, 611-914, and 851-951) that were significantly elevated in lung cancer sera and had strong antigenic epitope activity were shown. DETAILED DESCRIPTION

[0077] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly developed a RALGDS truncated protein that can bind to the autoantibodies (Anti-RALGDS) against RALGDS of lung cancer patients with high specificity. The truncated protein of the present invention not only has a shorter amino acid sequence and a smaller molecular weight, but also has a high specific affinity with the Anti-RALGDS antibody in the serum, and has no or substantially no cross-reaction with other antibodies in the blood or serum, thus significantly improving the sensitivity and specificity of lung cancer diagnosis and screening. The present invention was completed on this basis.

[0078] Experiments show that the various RALGDS truncated proteins of the present invention (such as RALGDS truncated proteins 1-90, 621-951, 611-914 and 851-951) can more effectively and sensitively detect Anti-RALGDS in serum samples at an early stage, thereby facilitating early screening and auxiliary diagnosis of lung cancer.

[0079] the term

[0080] As used herein, the terms "comprising" and "including" may be open, closed, or semi-closed. Therefore, the terms may also include "consisting of" or "consisting essentially of".

[0081] Autoantibodies

[0082] Autoantibodies are antibodies produced by the body against its own organs, cells or cell components.

[0083] At present, autoantibodies to certain proteins have become markers for lung cancer, such as autoantibodies to the following proteins: p53, NY-ESO-1, CYFRA, etc.

[0084] As used herein, the terms "Anti-RALGDS", "autoantibodies to RALGDS" are used interchangeably to refer to autoantibodies against RALGDS produced in a subject (such as a human). It should be understood that Anti-RALGDS generally includes a plurality of different Anti-RALGDS, and these different Anti-RALGDS often target different binding sites.

[0085] The present invention's research shows that in the blood of some lung cancer patients, there are autoantibodies against RALGDS (Anti-RALGDS antibodies), and these autoantibodies can be used as biomarkers for detecting lung cancer. Furthermore, the inventors unexpectedly discovered that certain truncated proteins of RALGDS can bind to these Anti-RALGDS in blood samples with high specificity and high affinity, and are therefore particularly suitable for early screening and auxiliary diagnosis of lung cancer.

[0086] RALDGS

[0087] As used herein, the terms "RALGDS protein" and "RALGDS polypeptide" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of human breast cancer susceptibility protein RALGDS (SEQ ID NO: 2).

[0088] As used herein, the terms "RALGDS truncated protein", or "RALGDS truncated polypeptide", "RALGDS truncation", or "RALGDS truncation", or "RALGDS truncation mutant" are used interchangeably and refer to a polypeptide formed by truncating the amino acid sequence of the breast cancer susceptibility protein RALGDS.

[0089] RALDGS acts as a guanine nucleotide exchange factor (GEF) to activate RalA or RalB GTPases and plays an important role in intracellular trafficking. It interacts with R-Ras, H-Ras, K-Ras, and Rap and acts as an effector molecule. During bacterial clearance, it recognizes 'Lys-33'-linked polyubiquitinated TRAF3 and subsequently mediates the assembly of an extracellular complex.

[0090] The nucleotide sequence and amino acid sequence of human RALDGS are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0091] Truncated proteins of the present invention

[0092] As used herein, "truncated protein of the present invention", "truncated polypeptide of the present invention" and "polypeptide of the present invention" are used interchangeably and refer to polypeptides formed by truncating the amino acid sequence of RALGDS. The polypeptides can undergo antigen-antibody reaction with Anti-RALGDS in the blood of lung cancer patients, thereby being used for qualitative or quantitative detection of Anti-RALGDS in the sample to be tested.

[0093] As used herein, "truncated protein of the present invention" does not include full-length protein. And the ratio of the length L1 of the truncated protein to the length L0 of the full-length protein is ≤ 1 / 2, preferably ≤ 1 / 3, and more preferably ≤ 1 / 4.

[0094] As used herein, the term "RALGDS truncated protein 1-90" refers to a truncated protein formed by the amino acid sequence of positions 1 to 90 of the RALGDS protein after the amino acid sequence of the RALGDS protein is truncated. "RALGDS truncated protein 621-951", "RALGDS truncated protein 611-914" and "RALGDS truncated protein 851-951" have similar meanings.

[0095] In the present invention, suitable samples to be tested are blood samples or any samples derived from blood and containing antibody components in blood. Representative samples to be tested include (but are not limited to): blood samples, plasma samples and serum samples.

[0096] As used herein, "isolated" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.

[0097] As used herein, "isolated polypeptide of the present invention" means that the RALGDS truncated protein of the present invention is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the polypeptide of the present invention using standard protein purification techniques. Substantially pure polypeptides can produce a single major band on a non-reducing polyacrylamide gel.

[0098] The polypeptide of the present invention can be a recombinant polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide of the present invention can be a product of chemical synthesis (e.g., prepared using peptide synthesis technology), or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects and mammalian cells). Depending on the host used in the recombinant production scheme, the polypeptide of the present invention can be glycosylated, or can be non-glycosylated. The polypeptide of the present invention may also include or not include an initial methionine residue.

[0099] The present invention also includes fragments, derivatives and analogs of the RALGDS truncated protein of the present invention. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same anti-RALGDS binding activity as the RALGDS truncated protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify the polypeptide). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0100] In the present invention, "RALGDS truncated protein" also includes variant forms having the same Anti-RALGDS binding function as the RALGDS truncated protein of the present invention. These variant forms include (but are not limited to): one or more (usually 1-30, preferably 1-25, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, when a small amount of amino acids with similar or similar properties are substituted, the binding function of the RALGDS truncated protein of the present invention with Anti-RALGDS is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the binding activity of the RALGDS truncated protein of the present invention with Anti-RALGDS.

[0101] The present invention also provides analogs of RALGDS truncated proteins. The difference between these analogs and the RALGDS truncated proteins of the present invention may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0102] Modifications (usually without altering the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during polypeptide synthesis and processing or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase).

[0103] In the present invention, "conservative variant polypeptides of RALGDS truncated proteins" refer to polypeptides formed by replacing at most 8, preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the corresponding RALGDS truncated protein (taking the sequence of positions 1-90 or 621-951 in SEQ ID NO:2 as an example, and other RALGDS truncated proteins are similar). These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0104] Table 1

[0105]

[0106]

[0107] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. As used herein, "degenerate variants" in the present invention refer to nucleic acid sequences that encode RALGDS truncated proteins but differ from the coding region sequence of wild-type human RALGDS.

[0108] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0109] The present invention also relates to nucleic acid fragments that hybridize with the above sequences. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR) to obtain and / or isolate a polynucleotide encoding the RALGDS truncated protein of the present invention.

[0110] The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

[0111] The nucleotide sequence of the RALGDS truncated protein of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA samples prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences.

[0112] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0113] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0114] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0115] The method of using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The DNA / RNA fragments amplified can be separated and purified by conventional methods such as by gel electrophoresis.

[0116] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the RALGDS truncated protein coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology.

[0117] The polynucleotide sequence of the present invention can be used to express or produce recombinant RALGDS truncated proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0118] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human RALGDS truncated protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0119] (2) Host cells cultured in a suitable culture medium;

[0120] (3) Isolate and purify proteins from culture medium or cells.

[0121] In the present invention, the polynucleotide sequence encoding the RALGDS truncated protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988) and vectors derived from baculovirus expressed in insect cells. In short, as long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0122] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the RALGDS truncated protein and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0123] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0124] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0125] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells, etc.

[0126] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0127] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0128] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0129] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0130] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0131] Diagnostic Applications

[0132] The RALGDS truncated protein or polypeptide of the present invention has many uses, including (but not limited to): detecting the presence of Anti-RALGDS in a blood sample, thereby providing useful information for early diagnosis or auxiliary diagnosis of lung cancer.

[0133] The present invention also relates to diagnostic tests for quantitatively or qualitatively detecting Anti-RALGDS levels. These tests are well known in the art.

[0134] A method for detecting the presence of Anti-RALGDS in a sample is to detect the specific binding of RALGDS truncated protein with Anti-RALGDS. Typically, the method comprises: contacting a sample (such as a plasma or serum sample) with the RALGDS truncated protein; observing whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of Anti-RALGDS in the sample.

[0135] Tests and kits for diagnostics

[0136] The present invention also provides a detection agent and a kit for early or auxiliary diagnosis of lung cancer, which comprises one or more RALGDS truncated proteins of the present invention as a detection agent.

[0137] The main advantages of the present invention include:

[0138] (a) The truncated protein of the present invention has a high affinity to Anti-RALGDS in blood samples of lung cancer patients.

[0139] (b) The truncated protein of the present invention has almost no binding with other unrelated antibodies in the blood sample, which can reduce nonspecific cross-reactions and improve the signal-to-noise ratio, specificity and accuracy of detection.

[0140] (c) The truncated protein of the present invention can detect Anti-RALGDS in blood samples with high sensitivity.

[0141] (d) The truncated protein of the present invention has a short sequence and can be prepared on a large scale by recombination or polypeptide synthesis.

[0142] (e) The diagnosis or detection of lung cancer by the truncated protein of the present invention only requires serum or plasma samples, without the need for tissue samples for detection, thus avoiding patients' resistance to such detection, helping to detect early-stage lung cancer patients, prolonging patients' survival cycle and improving their quality of life, and having a high prospect for clinical application.

[0143] (f) The truncated proteins of the RALGDS protein described in the present invention can induce a more obvious antigen-antibody reaction in the serum of lung cancer patients than the RALGDS protein, especially the truncated proteins located in the middle and carboxyl end of the full-length RALGDS molecule have a higher screening ability for lung cancer serum.

[0144] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0145] Example 1

[0146] Candidate expression segments of RALGDS

[0147] In this embodiment, based on the sequence of human RALGDS protein, the present invention designs a series of truncated antigen proteins for detecting lung cancer RALGDS autoantibodies, and the candidate truncation sites are as follows: Figure 1 .

[0148] The nucleic acid sequence of human RALGDS is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. Some representative secondary structures are shown in Table 1.

[0149] Table 1

[0150] Location Secondary structure 791-793 Beta strand 795-797 Beta strand 799-807 Beta strand 809-820 Beta strand 825-835 Helix 836-839 Turn 843-845 Helix 846-853 Beta strand 856-859 Beta strand 862-864 Beta strand 866-869 Helix 877-882 Beta strand

[0151] In this example, the candidate truncated proteins of each RALGDS are represented by the number of digits of their starting and ending amino acids, as shown in Table 2.

[0152] Table 2 RALGDS truncated proteins

[0153] serial number Based on the sequence information in SEQ ID No: 2 Truncation protein 1 Contains amino acids 1-90 of RALGDS protein Truncation protein 2 Contains amino acids 1-211 of RALGDS protein Truncation protein 3 Contains amino acids 1-390 of RALGDS protein Truncation protein 4 Contains amino acids 210-390 of RALGDS protein Truncation protein 5 Contains amino acids 621-951 of RALGDS protein Truncated protein 6 Contains amino acids 611-914 of RALGDS protein Truncation protein 7 Contains amino acids 621-765 of RALGDS protein Truncation protein 8 Contains amino acids 851-951 of RALGDS protein

[0154] These truncated protein fragments cover all the antigenic epitopes of RALGDS (http: / / www.iedb.org / ). At the same time, the truncated expressed molecules are easier to obtain higher yields in prokaryotic expression systems, and are further used for the selection and large-scale production of protein fragments with the best antigenic activity and better specificity.

[0155] Example 2 Preparation method of RALGDS truncated antigen protein

[0156] In this example, based on the structures of the truncated antigen proteins in Example 1, each RALGDS truncated protein was prepared by a recombinant method. The preparation method comprises the following steps:

[0157] (1) Construction of recombinant plasmid expressing RALGDS truncated protein

[0158] The nucleotide sequence of the lung cancer antigen RALGDS truncated protein was prepared by PCR. A series of RALGDS truncated protein gene sequences corresponded to truncations of RALGDS protein at amino acids 1-90, 1-211, 1-390, 210-390, 621-951, 611-914, 621-765 or 851-951.

[0159] The plasmid vector containing 6XHis-labeled PET28(a) was extracted, double-digested with Ncol and Ndel, and the double-digested plasmid fragment was recovered by gel electrophoresis; the RALGDS truncated protein gene fragment was double-digested with Ncol and Ndel, and the double-digested gene fragment was recovered by electrophoresis and placed at -20°C for use; the double-digested plasmid fragment and the double-digested gene fragment were connected at a ratio of 1:3-10 using T4 ligase at 16°C overnight, and the recombinant plasmid after connection was PET28(a)-RALGDS(1-9 0), PET28(a)-RALGDS(1-211), PET28(a)-RALGDS(1-390), PET28(a)-RALGDS(210-390), PET28(a)-RALG DS(621-951), PET28(a)-RALGDS(611-914), PET28(a)-RALGDS(621-765), PET28(a)-RALGDS(851-951).

[0160] (2) Screening and identification of recombinant plasmids

[0161] The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), coated on LB plates containing ampicillin (60ug / ml), and incubated overnight at 37°C; the next day, recombinant plasmid-transformed colonies and control colonies (empty PGEM-5zf plasmid-transformed bacteria) were randomly picked, and the plasmids were extracted respectively, and double-digested with Ndel and Ncol. After enzyme digestion, electrophoresis was performed, and the corresponding target fragments and vectors were visible, indicating that the expression vector was successfully constructed, which was a positive expression bacterium;

[0162] (3) High-efficiency expression of recombinant protein engineering bacteria

[0163] The positive expression bacteria were inoculated into a test tube containing 2 ml LB medium (60 ug / ml ampicillin), shaken at 37°C for 4 h, and IPTG was added at a final concentration of 0.5 mmol / l. The induction was continued at 37°C for 6 h, and the precipitated bacteria were collected by centrifugation. The precipitated bacteria were broken, and the supernatant and precipitate were respectively subjected to SDS-PAGE detection. The expressed target protein was obtained in the supernatant;

[0164] (4) Purification of expressed protein

[0165] Select a single colony of highly expressed recombinant protein engineering bacteria, inoculate it into a triangular flask containing 100ml LB liquid medium, add ampicillin to a final concentration of 60ug / ml, and place it in a 37°C shaker overnight. The next day, inoculate the bacterial solution and LB medium into a triangular flask containing 1000ml LB (ampicillin 60ug / ml) culture medium at a ratio of 1:10, culture for 4h, add IPTG (final concentration of 0.5mmol / l), and continue to induce for 6h at 37°C. Centrifuge the 1000ml engineering bacteria induced to express fusion protein, centrifuge at high speed (10000rpm) and low temperature (4°C) for 10min, resuspend the precipitated bacteria in 1 / 10 lysis buffer (50mM Tris-Hcl, 10mMEDTA, 15mM NaCL, 10mM DTT) of the original centrifugation volume, sonicate the bacteria in an ice bath, centrifuge at high speed (12000rpm) and low temperature (4°C) for 30min, and collect the bacterial supernatant.

[0166] After washing the equilibrated nickel ion affinity chromatography column with equilibration solution (20mM PB PH7.4), the supernatant of the bacterial solution collected by ultrasound was directly loaded onto the column at a loading flow rate of 1.5ml / min. After loading, the column was washed with equilibration solution, and the protein was eluted with elution solution containing 20, 50, 100, 200, and 500mM imidazole in sequence. The proteins at each elution peak were collected and detected by SDS-PAGE electrophoresis.

[0167] The results showed that the following recombinant proteins were prepared with high purity: truncated protein 1, truncated protein 2, truncated protein 3, truncated protein 4, truncated protein 5, truncated protein 6, truncated protein 7 and truncated protein 8, respectively.

[0168] Example 3

[0169] RALGDS truncations 1-90, 621-951, 611-914, and 851-951 have good specificity for lung cancer detection

[0170] The purified recombinant proteins prepared in Example 2 were diluted with carbonate buffer (50 mM, pH 9.6) and coated on the ELISA plate at 100 ul / well and 100 ng / well of protein at 4°C overnight. After washing, 200 ul / well of 10% calf serum in PBS was added and blocked at 37°C for 2 h. After washing, the plate was kept at 4°C for later use.

[0171] The indirect ELISA test population included 46 lung cancer patients, 30 patients with benign lung diseases (including chronic obstructive pulmonary disease, tuberculosis, pneumonia, pneumoconiosis, etc.), and 42 healthy controls matched with the age of lung cancer cases. The basic information of lung cancer patients is shown in Table 3.

[0172] Table 3

[0173] Lung cancer patients (n=46) Age Range 26～79 Mean age 58 Gender Male 18 female 28

[0174] A study published in the journal Oncology Reports found that RALGDS is one of the upregulated genes in lung squamous cell carcinoma (LUSC), and overexpression of RALGDS in LUSC is associated with poor prognosis (Kamalakaran, S., Varadan, V., Gierman, HJ, & Huang, RS (2015). Blood genomics of non-small cell lung cancer: a pilot study to examine circulating tumor cells. Oncology reports, 33 (1), 2-16.).

[0175] The reactivity of RALGDS autoantibodies in the sera of patients with lung cancer (46 cases), patients with benign lung diseases (30 cases, including chronic obstructive pulmonary disease, tuberculosis, pneumonia, pneumoconiosis, etc.) and healthy people (42 cases) was detected by indirect enzyme-linked immunosorbent assay.

[0176] Serum or plasma samples were diluted 1:110 with phosphate buffer and added to the microwells for reaction (50 ml / well). After washing the unbound serum or plasma components with washing solution, horseradish peroxidase (HRP)-coupled anti-human IgG was added to each well for reaction. Then the reaction substrate TMB (3,3',5,5'-tetramethylbenzidine) was added for color development. Stop solution (1NHCl) was added and the absorbance was read at 450nm single spectrum on an enzyme reader (OD).

[0177] The results are as follows Figure 2 As shown in Table 3. After the truncated proteins of each RALGDS were reacted with the sera of healthy, benign, and lung cancer patients, the above scatter plots were obtained. These truncated proteins generally induced more obvious antigen-antibody reactions in the sera of lung cancer patients, that is, the OD value showed an increasing trend in the lung cancer group.

[0178] Table 4

[0179]

[0180]

[0181] The Kruskal-Wallis test showed that the OD signal increase results of RALGDS truncations 1-90 (P=0.0145), 621-951 (P=0.0373), 611-914 (P=0.0449), and 851-951 (P=0.0018) in the serum of lung cancer patients were statistically significant (P<0.05), which proved that the level of RALGDS autoantibodies in the serum of lung cancer patients was significantly higher than that in healthy people and patients with benign lung diseases.

[0182] The above results show that the above truncated proteins of the present invention have good reactivity with lung cancer serum, but weak reactivity with healthy and benign people, which proves that they have good specificity and have the potential to be used for lung cancer screening.

[0183] Example 4. The screening ability of RALGDS truncation 851-951 for lung cancer serum is significantly better than other protein fragments

[0184] The present inventors used receiver operating characteristic curve (ROC) analysis to analyze four RALGDS fragments significantly increased in lung cancer serum: 1-90, 621-951, 611-914 and 851-951.

[0185] The analysis results are as follows Figure 3 shown.

[0186] Using healthy subjects as controls, when the Youden index of the ROC curve was the largest, the detection performance of each fragment of AKAP4 was shown in Table 5.

[0187] Table 5

[0188] RALGDS fragment Sensitivity (%) Specificity (%) Area under the curve (AUC) 1-90 30.43 92.50 0.6709 621-951 34.78 87.18 0.6207 611-914 28.26 89.19 0.6337 851-951 39.02 93.94 0.7180

[0189] Taking benign patients as controls, when the Youden index of the ROC curve was the largest, the detection performance of each fragment of AKAP4 was shown in Table 6.

[0190] Table 6

[0191]

[0192]

[0193] The above results indicate that four preferred RALGDS truncated proteins (or RALGDS fragments): 1-90, 621-951, 611-914 and 851-951 can be used as detection reagents for early diagnosis or auxiliary diagnosis of lung cancer, and can be used for quantitative or qualitative detection of Anti-RALGDS autoantibodies in patient blood samples.

[0194] In addition, according to Figure 3The results of the areas under the curves, combined with the sensitivity and specificity analysis, show that: whether using healthy people or benign patients as controls, the screening ability of the 851-951 amino acids of RALGDS for lung cancer serum is significantly better than other protein fragments.

[0195] discuss

[0196] There are literature reports that RALGDS promotes the survival of cancer cells in skin cancer research models by activating the JNK / SAPK pathway. This study identified RalGDS as a key component of the Ras-dependent cancer occurrence signaling pathway (González-García A, Pritchard CA, Paterson HF, Mavria G, Stamp G, Marshall CJ. RalGDS is required for tumor formation in a model of skin carcinogenesis. Cancer Cell. 2005 Mar; 7(3): 219-26. doi: 10.1016 / j.ccr.2005.01.029. PMID: 15766660.).

[0197] In breast cancer studies, RalA GTPase activated by RALGDS makes cancer cells more invasive, and this effect can be eliminated by overexpressing the N segment of the RILP molecule (Wang Z, Zhou Y, Hu X, Chen W, Lin X, Sun L, Xu X, Hong W, Wang T. RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS. Cell Death Dis. 2015 Oct 15; 6(10): e1923. doi: 10.1038 / cddis.2015.266. PMID: 26469971; PMCID: PMC4632296.).

[0198] RALGDS is an important effector molecule of the KRAS signaling pathway. As a driver gene of lung cancer, KRAS plays a vital role in the progression of lung cancer, and the level of RALGDS is the embodiment of the function of the KRAS gene.

[0199] Prior to the present invention, there has been no report on RALGDS protein autoantibodies, nor any prior art related to lung cancer.

[0200] The inventors' research unexpectedly found that RALGDS is a lung cancer-related autoantigen and that Anti-RALGDS autoantibodies exist in the blood of lung cancer patients.

[0201] Due to the large molecular weight of RALGDS (the sequence number in the UniProtKB database is Q12967-2), the yield of prokaryotic cell expression in its full-length state is low; and many antigenic epitopes in the full-length state are difficult to be fully exposed in the products of the prokaryotic cell expression system, so the activity of such protein products for lung cancer screening is bound to be low; however, the prokaryotic expression system is still the most economical and efficient bioreactor at present; secondly, the human immune system is exposed to a large number of antigen molecules in daily life, which inevitably have active epitopes similar to lung cancer-related antigens, which will cause cross-reactions of autoantibodies to lung cancer antigens and other antigens, reducing the specificity of lung cancer screening. Truncating the lung cancer antigen to reduce the complexity of its antigenic epitope can greatly reduce the probability of cross-reaction.

[0202] The inventors truncated the RALGDS protein and screened it, and unexpectedly found that some of the specific truncated proteins formed had high affinity and high specificity binding to Anti-RALGDS autoantibodies. The RALGDS truncated protein of the present invention has a highly immunogenic active epitope, which can significantly improve the sensitivity of lung cancer screening, while reducing nonspecific cross-reactions, and improving the signal-to-noise ratio, specificity and accuracy of detection.

[0203] The truncated protein fragments of the present invention are easy to be recombinantly expressed or synthesized into polypeptides, and can obtain high yields in particular in a prokaryotic expression system, facilitating large-scale production.

[0204] Early screening for lung cancer is helpful. The RALGDS truncated protein of the present invention can be used for screening of lung cancer (especially early screening), and is particularly suitable for routine screening of people who have no symptoms related to lung cancer. Lung cancer can be detected as early as possible before symptoms appear, thereby prompting clinicians to take relevant follow-up diagnosis or take corresponding treatment measures or decisions for patients earlier.

[0205] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of an Anti-RALGDS detection reagent in preparing a kit, characterized in that: The kit is used for screening or auxiliary diagnosis of lung cancer; wherein the detection reagent is RALGDS truncated protein; Wherein, the truncated protein is selected from the following group: (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein; (Y2) truncated protein 1, whose amino acid sequence is amino acids 1-90 of the RALGDS protein; (Y5) a combination of the above Y1 to Y2; Among them, the amino acid sequence of RALGDS protein is shown in SEQ ID No:

2.

2. The use according to claim 1, characterized in that The RALGDS truncated protein is a recombinant protein.

3. The use according to claim 1, characterized in that The detection reagent also includes one or more RALGDS truncated proteins selected from the following group: (Y3) truncated protein 5, whose amino acid sequence is amino acids 621-951 of the RALGDS protein; (Y4) Truncated protein 6, whose amino acid sequence is amino acids 611-914 of RALGDS protein.

4. The use according to claim 1, characterized in that The RALGDS truncated protein is a combination of the following Y1 to Y2: (Y1) truncated protein 8, whose amino acid sequence is amino acids 851-951 of the RALGDS protein; (Y2) Truncated protein 1, whose amino acid sequence is amino acids 1-90 of RALGDS protein.

5. The use according to claim 1, characterized in that The detection reagent is used to detect the level of Anti-RALGDS in the sample to be tested.

6. The use according to claim 5, characterized in that The samples to be tested include: blood samples, plasma samples and serum samples.

7. The use according to claim 1, characterized in that The truncated protein is produced from a prokaryotic or eukaryotic host cell using recombinant technology, and the host cell is Escherichia coli.

8. The use according to claim 7, characterized in that The host cell is a genetically engineered host cell, which contains a vector containing a polynucleotide encoding the RALGDS truncated protein, or a polynucleotide encoding the RALGDS truncated protein is integrated into the genome.

9. The use according to claim 1, characterized in that The kit contains: (a) a first container, and an Anti-RALGDS detection reagent in the first container, wherein the detection reagent is a RALGDS truncated protein; (b) an instruction manual, wherein the instruction manual describes the detection method.

10. The use according to claim 9, characterized in that The detection method is to detect the level of Anti-RALGDS in the sample to be tested.

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