Oral cancer marker linear epitope fusion peptide and its application

Abstract

The present invention discloses an oral cancer marker linear epitope fusion peptide and an application thereof. The oral cancer marker linear epitope fusion peptide comprises a fusion peptide 1 and a fusion peptide 2; the nucleotide sequence of the fusion peptide 1 is: SKDQIKKLTSLKNKLERRQNRSQNPVQPIGPQTPKACDSK; the nucleotide sequence of the fusion peptide 2 is: SKDQIKKLTSLKNKLERRQNEDERWTNNFREYNL; the oral cancer marker linear epitope fusion peptide can be fused with a T cell epitope to promote the occurrence of an immune response. The generated antiserum has high affinity and specificity, laying a good foundation for the further preparation of high-affinity monoclonal antibodies and the development of detection kits. The fusion peptide 1 and the fusion peptide 2 can replace the full-length MMP-1 as a calibrator of the detection kit through chemical coupling treatment.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, in particular to an oral cancer marker linear epitope fusion peptide and application thereof. Background Art

[0002] Oral cancer is the sixth most common cancer worldwide, with approximately 657,000 new cases reported annually, according to the World Health Organization. The five-year survival rate for oral cancer patients is approximately 50%. However, statistics from 2012 to 2016 found that the survival rates for stages I, II, III, and IV were 79.9%, 71.0%, 56.5%, and 35.6%, respectively. These data indicate that early detection and treatment of oral cancer can significantly improve survival rates.

[0003] Oral mucosal testing is currently a crucial tool in the clinical evaluation of oral cancer. Tissue biopsy of the lesion remains the gold standard for oral cancer diagnosis. However, due to the heterogeneity of oral cancer, accurately selecting the biopsy site presents a significant challenge. According to the World Health Organization, oral mucosal screening has a sensitivity and specificity of 0.5-0.99 and 0.64-0.99, respectively. It is also costly, time-consuming, and requires high levels of clinical expertise. Furthermore, it is invasive. Therefore, oral cancer detection indicators with high sensitivity, good specificity, and ease of use are crucial for early detection and treatment of oral cancer.

[0004] Among the many oral cancer markers, matrix metalloproteinase-1 (MMP-1) is considered the most promising. MMP-1 levels in the saliva of most oral cancer patients are approximately 83 times higher than in controls, demonstrating high discrimination, with a sensitivity and specificity of 69.5% and 95%, respectively. Because the sample tested is saliva, the test is essentially non-invasive, simple, and convenient for at-home testing, making MMP-1 an ideal marker.

[0005] Comprehensive research has shown that MMP-1 is the earliest identified member of the MMP family. This family gene primarily encodes centrocyte collagenase, which participates in extracellular matrix remodeling and is closely associated with migration, agitation, and inflammation during tumorigenesis. MMP-1 expression is also elevated in some other cancer tissues. The human body contains 24 genes encoding proteins in this family, which share a high degree of homology, posing significant challenges to the development of MMP-1-specific antibodies and subsequent detection kits. Summary of the Invention

[0006] The present invention aims to provide an oral cancer marker linear epitope fusion peptide and its application. The oral cancer marker linear epitope fusion peptide can be fused with a T cell epitope to promote the occurrence of an immune response. The antiserum produced has high affinity and specificity, laying a good foundation for the further preparation of high-affinity monoclonal antibodies and the development of detection kits. Fusion peptide 1 and fusion peptide 2 can replace the full-length MMP-1 as a calibrator for the detection kit through chemical coupling treatment.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] An oral cancer marker linear epitope fusion peptide, comprising fusion peptide 1 and fusion peptide 2;

[0009] The nucleotide sequence of the fusion peptide 1 is:

[0010] SKDQIKKLTSLKNKLERRQNRSQNPVQPIGPQTPKACDSK;

[0011] The nucleotide sequence of the fusion peptide 2 is:

[0012] SKDQIKKLTSLKNKLERRQNEDERWTNNFREYNL.

[0013] Preferably, the fusion peptide 1 and the fusion peptide 2 are extracted from matrix metalloproteinase-1 or obtained by chemical synthesis.

[0014] An application of an oral cancer marker linear epitope fusion peptide, wherein fusion peptide 1 and fusion peptide 2 are coupled to a carrier protein or fused with a T cell epitope, and used to immunize animals to obtain MMP-1-specific immune serum or further prepare polyclonal antibodies and monoclonal antibodies.

[0015] Preferably, the carrier protein is bovine serum albumin (BSA) or hemocyanin (KLH); the T cell epitope is a small molecule peptide derived from malaria, and the nucleotide sequence of the small molecule peptide is:

[0016] SKDQIKKLTSLKNKLERRQN.

[0017] The invention discloses an application of a linear epitope fusion peptide as an oral cancer marker, wherein the fusion peptide 1 and the fusion peptide 2 are used for detecting antigens or antibodies of MMP-1.

[0018] The invention discloses an application of a linear epitope fusion peptide of an oral cancer marker, wherein the fusion peptide 1 and the fusion peptide 2 are chemically coupled to replace the full-length MMP-1 as a calibrator of a detection kit.

[0019] After adopting the above technical solution, the present invention has the following advantages compared with the background technology: the fusion peptide 1 and fusion peptide 2 of the present invention are suitable for being obtained by chemical synthesis due to their short peptide segments, which are simple, convenient and low-cost to obtain; the oral cancer marker linear epitope fusion peptide can be fused with the T cell epitope to promote the occurrence of an immune response, and the resulting antiserum has high affinity and specificity, laying a good foundation for the further preparation of high-affinity monoclonal antibodies and the development of detection kits. At the same time, the synthesis of the complete antigen of the linear epitope, including the T cell epitope and the linear epitope, is conducive to the production of high-titer serum, and due to the single epitope, the production of unnecessary antibodies is reduced, which is conducive to the production of specific antibodies; in addition, the fusion peptide 1 and fusion peptide 2 can replace the full-length MMP-1 as a calibrator for the detection kit through chemical coupling treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Homology analysis of MMP-1 and MMP-8, where the boxes are two linear epitopes;

[0021] Figure 2 This is a diagram showing the positions of the fusion peptide 1 and fusion peptide 2 of the present invention in the structure of MMP-1;

[0022] Figure 3 This is a graph showing the immune titer of the fusion peptide 1 immune serum against MMP-1 and MMP-8;

[0023] Figure 4 The graph shows the immune titer of the fusion peptide 2 immune serum against MMP-1 and MMP-8. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] See also Figures 1 to 4 , an oral cancer marker linear epitope fusion peptide, comprising fusion peptide 1 and fusion peptide 2;

[0026] The nucleotide sequence of the fusion peptide 1 is:

[0027] SKDQIKKLTSLKNKLERRQNRSQNPVQPIGPQTPKACDSK;

[0028] The nucleotide sequence of the fusion peptide 2 is:

[0029] SKDQIKKLTSLKNKLERRQNEDERWTNNFREYNL.

[0030] The fusion peptide 1 and the fusion peptide 2 are extracted from matrix metalloproteinase-1 or obtained through a chemical synthesis method.

[0031] An application of an oral cancer marker linear epitope fusion peptide, wherein fusion peptide 1 and fusion peptide 2 are coupled to a carrier protein or fused with a T cell epitope, and used to immunize animals to obtain MMP-1-specific immune serum or further prepare polyclonal antibodies and monoclonal antibodies.

[0032] The carrier protein is bovine serum albumin (BSA) or hemocyanin (KLH); the T cell epitope is a small molecule peptide derived from malaria, and the nucleotide sequence of the small molecule peptide is:

[0033] SKDQIKKLTSLKNKLERRQN.

[0034] The invention discloses an application of a linear epitope fusion peptide as an oral cancer marker, wherein the fusion peptide 1 and the fusion peptide 2 are used for detecting antigens or antibodies of MMP-1.

[0035] The invention discloses an application of a linear epitope fusion peptide of an oral cancer marker, wherein the fusion peptide 1 and the fusion peptide 2 are chemically coupled to replace the full-length MMP-1 as a calibrator of a detection kit.

[0036] 1. Mouse Immunity Test

[0037] For the first immunization of 6-8 week old Balb / C mice, fusion peptide 1 and fusion peptide 2 were diluted to 1 mg / mL (in 0.01 mol / L PBS buffer) as immunogens, mixed with equal volumes of Freund's complete adjuvant, and fully emulsified. The emulsified antigens were inoculated subcutaneously in the back of the neck at multiple points into three mice at a dose of 50 μg / mouse (0.1 ml per mouse). Fourteen days later, a second immunization was performed using an equal volume of Freund's incomplete adjuvant emulsified with the immunogens at the same dose as the first immunization, with four booster immunizations.

[0038] 2. Identification of immune titer

[0039] One week after the fifth immunization, 200 μL of blood was collected from the tip of the tail and centrifuged to collect serum. The serum titer was measured by enzyme-linked immunosorbent assay (ELISA).

[0040] 1) Determination of antiserum titer, the steps are as follows:

[0041] (1) Coating: MMP-1 and MMP-8 proteins were diluted to 10 μg / mL with 0.05 mol / L carbonate buffer (pH 9.6), and 100 μL / well was added to the ELISA plate. The plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded, and the plate was washed three times with PBST buffer (pH 7.2). The washing solution was then spun dry.

[0042] (2) Blocking: Add 150 μL of blocking solution (5% BSA) to each well and block at 37°C for 1 h. Shake dry the liquid in the wells, wash the plate three times with PBST buffer (pH 7.2), and pat dry the washing solution.

[0043] (3) Add antiserum: Add 100 μL of antiserum diluted in blocking buffer (5% BSA) to each well. Dilute the antibody starting at 1:1000 in a 0.01 M PBS skim milk solution in a 2-step dilution, for a total of 8 dilution steps. Add 100 μL per well. Incubate at 37°C for 60 min. Wash three times with PBST buffer (pH 7.2) and pat dry. Serum from unimmunized mice was also included as a negative control.

[0044] (4) Add enzyme-labeled secondary antibody: add 100 μL HRP-goat anti-mouse IgG (5000-fold diluted in PBS) to each well, incubate at 37°C for 30 min, wash three times with PBST buffer (pH 7.2), and pat dry.

[0045] (5) Color development: Mix the horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine solution and 30% hydrogen peroxide at a volume ratio of 1:1, add 100 μL to each well, and incubate at 37°C for 15 min. Then, add 50 μL of stop solution (2 mol / L H2SO4) to each well.

[0046] (6) Reading determination: Read the absorbance (OD) with a microplate reader at a wavelength of 450nm. The dilution corresponding to 2.5 times the OD of the negative control is the antiserum titer. The test results are as follows: Figure 3 and Figure 4 As shown in the figure, Bx represents fusion epitope x, -x- represents mouse number, and MMP-x represents MMP family protein member.

[0047] The results showed that the fusion peptides 1 and 2 of the present invention can promote the occurrence of immune response, and the antiserum produced has high affinity and specificity, laying a good foundation for the further preparation of high-affinity monoclonal antibodies and the development of detection kits. Fusion peptides 1 and 2 can replace the full-length MMP-1 as calibrators for detection kits through chemical coupling treatment.

[0048] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An oral cancer marker linear epitope fusion peptide, characterized in that: The oral cancer marker linear epitope fusion peptide is fusion peptide 1 or fusion peptide 2; The amino acid sequence of the fusion peptide 1 is: SKDQIKKLTSLKNKLERRQNRSQNPVQPIGPQTPKACDSK; The amino acid sequence of the fusion peptide 2 is: SKDQIKKLTSLKNKLERRQNEDERWTNNFREYNL.

2. The oral cancer marker linear epitope fusion peptide according to claim 1, characterized in that: The fusion peptide 1 or fusion peptide 2 is obtained by chemical synthesis.

3. A use of the oral cancer marker linear epitope fusion peptide according to any one of claims 1 to 2, characterized in that: The fusion peptide 1 or fusion peptide 2 is coupled to a carrier protein and used to immunize animals to obtain MMP-1-specific immune serum or further prepare monoclonal antibodies.

4. The use of an oral cancer marker linear epitope fusion peptide according to claim 3, characterized in that: The carrier protein is bovine serum albumin BSA or hemocyanin KLH.

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