A Lamprey Serum Globular Agglutinin, Preparation Method Thereof and Application Therein in Hepatocellular Carcinoma Detection

Abstract

The present invention discloses a lamprey serum globular lectin, a preparation method thereof and its application in the detection of liver cancer, belonging to the technical field of cancer detection. The present invention discovers that glycosylation modification is abnormally highly expressed in liver cancer patients. The lamprey serum globular lectin is isolated and purified from lamprey serum. The lamprey serum globular lectin can specifically bind to glycan structures such as Galβ1-3GlcNAc, GlcNAcβ1-2Man and Manα1-2Man of glycoproteins in liver cancer tissues, and can be used for the diagnosis of liver cancer. The lamprey serum globular lectin of the present invention can diagnose liver cancer efficiently and specifically, is particularly sensitive to the diagnosis of intrahepatic cholangiocarcinoma, has high reference value for the diagnosis of liver cancer, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of cancer detection technology, specifically relating to a lamprey serum spheroid agglutinin, its preparation method, and its application in liver cancer detection. Background Technology

[0002] Primary liver cancer (PLC) is the sixth most common malignant tumor of the digestive system worldwide and the second most common cause of death. Common types of liver cancer include hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), accounting for 75%–85% and 10%–15% of cases, respectively. Currently, histopathological evidence based on specific biomarkers is the "gold standard" for the diagnosis of the vast majority of cancers. By taking samples from suspected cancerous lesions and adjacent tissues, and measuring the expression levels and distribution of biomarkers, it is possible to determine whether cancer is present, which plays a decisive role in the formulation of treatment plans and the assessment of survival prognosis.

[0003] Protein glycosylation is an important post-translational modification, occurring in more than half of all proteins in eukaryotes. Changes in glycosylation, especially alterations in sialylation, are closely related to tumorigenesis, development, and metastasis. Glycan tumor markers are a class of important biomarkers for diagnosing and characterizing human tumor cells. While the vast majority of tumor screening biomarkers are glycoproteins, such as AFP in liver cancer, the sensitivity and specificity of AFP do not fully meet clinical needs. Detecting specific glycoforms modified by biomarker target proteins can improve the sensitivity and specificity of cancer diagnosis.

[0004] Lampreys are the oldest living vertebrates. Their unique evolutionary position and distinctive lifestyle have created unique life characteristics. As a new model animal for developmental and evolutionary biology, the potential application value of lampreys has not yet been effectively developed. To date, there have been no reports on isolating a new spherical lectin from lamprey serum and applying it to the detection of liver cancer. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a lamprey serum spherical lectin, its preparation method, and its application in liver cancer detection. This invention, through glycan microarray technology, has discovered that lamprey serum spherical lectin (LSSL) can specifically bind to the glycoforms of glycoproteins in liver cancer tissues, such as Galβ1-3GlcNAc, GlcNAcβ1-2Man, and Manα1-2Man. By detecting the glycosylation modification status of glycoproteins in liver cancer cells, this invention can efficiently and accurately diagnose liver cancer, thereby improving the sensitivity and / or specificity of liver cancer diagnosis.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A lamprey serum spheroid agglutinin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Another aspect of the present invention provides a method for preparing the above-mentioned lamprey serum spheroid agglutinin, the method comprising obtaining lamprey serum spheroid agglutinin through a heterologous expression system or isolating lamprey serum spheroid agglutinin from lamprey serum.

[0009] Based on the above technical solution, the specific steps for obtaining lamprey serum spheroid lectin through a heterologous expression system are as follows: the coding gene for lamprey serum spheroid lectin is cloned into a recombinant expression vector, introduced into a host cell, and heterologously expressed to obtain lamprey serum spheroid lectin.

[0010] Based on the above technical solution, the host cells further include Escherichia coli host cells, Bacillus subtilis host cells, and yeast host cells.

[0011] Based on the above technical solution, the further steps of separating and obtaining lamprey serum spheroidal agglutinins from lamprey serum mainly include the following steps:

[0012] (1) Dilute lamprey serum with buffer at a volume ratio of 1:2-10, mix with Protein G agarose resin, and incubate with shaking at 1-5℃ for 5-24h.

[0013] (2) Sample loading: Load the sample obtained in step (1) onto the chromatographic column;

[0014] (3) Equilibration: Binding Buffer is added to the column at a rate of 1-2 mL / min;

[0015] (4) Elution: Elution buffer is used to elute the target protein at a rate of 1-2 mL / min. The eluent is collected and the pH of the collected eluent is adjusted to 6-8.

[0016] (5) Based on the electrophoresis results, the eluents of proteins with the same electrophoretic bands are mixed together and dialyzed. The result is obtained after dialysis.

[0017] Based on the above technical solution, the buffer solution mentioned in step (1) further includes PBS buffer and Tris-HCl buffer.

[0018] Based on the above technical solution, further, the Binding Buffer in step (3) consists of: 0.15M NaCl, 20mM Na2HPO4, and pH adjusted to 7.0-7.4.

[0019] Based on the above technical solution, further, the components of the Elution Buffer in step (4) are: 0.1M citric acid, pH adjusted to 2.5-3.0.

[0020] In another aspect, the present invention provides the application of the above-mentioned lamprey serum spheroid agglutinin in the preparation of reagents or kits for detecting liver cancer.

[0021] Based on the above technical solution, the liver cancer further includes hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

[0022] Based on the above technical solution, the detection methods of the reagents or kits further include immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).

[0023] The advantages of this invention over the prior art are as follows:

[0024] 1. This invention is the first to discover that the expression level of glycosylation modification in liver cancer tissue is significantly upregulated compared to that in healthy controls (HC) and patients with liver cirrhosis (LC); lamprey serum spheroidal lectin LSSL can specifically bind to the glycoform structures of glycoproteins such as Galβ1-3GlcNAc, GlcNAcβ1-2Man, and Manα1-2Man in liver cancer tissue, which can be applied to the development of immunohistochemical staining reagents for liver cancer, and thus applied to the diagnosis of clinical liver cancer patients.

[0025] 2. The lamprey serum spheroidal lectin (LSSL) of the present invention has the characteristics of high sensitivity and high specificity for the diagnosis and detection of liver cancer. In the diagnosis and screening of liver cancer, since liver cancer is usually discovered late and is almost incurable once discovered, improving sensitivity is particularly important. Improving sensitivity can increase the diagnosis rate of early liver cancer, screen out subclinical or occult liver cancer from the population, and help improve the cure rate and reduce the mortality rate of liver cancer.

[0026] 3. This invention explores the clinical diagnostic potential and recognition mechanism of lamprey serum spheroidal lectin (LSSL), providing a scientific basis and new ideas for the development of clinical liver cancer diagnosis and screening tools, elucidating the glycosylation changes and patterns of cancer modification, improving the accuracy and sensitivity of clinical disease diagnosis, and better guiding clinical practice. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0028] Figure 1 Microarray diagram of glycan structures such as Galβ1-3GlcNAc, GlcNAcβ1-2Man, and Manα1-2Man for LSSL;

[0029] Figure 2 A statistical chart of grayscale values ​​for GPC3 and LSSL detection of hepatocellular carcinoma;

[0030] Figure 3 ROC curves for GPC3 and LSSL detection of hepatocellular carcinoma;

[0031] Figure 4 Representative images of various types of intrahepatic cholangiocarcinoma detected by GPC3 and LSSL;

[0032] Figure 5 A statistical chart of grayscale values ​​for GPC3 and LSSL detection of intrahepatic cholangiocarcinoma;

[0033] Figure 6 ROC curves for GPC3 and LSSL detection of intrahepatic cholangiocarcinoma. Detailed Implementation

[0034] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Example 1:

[0037] This embodiment provides a method for preparing lamprey serum spheroidal lectin (LSSL), which involves purifying natural LSSL using Protein G agarose resin, and includes the following steps:

[0038] (1) Protein G agarose resin is stored in 20% ethanol and washed twice in PBS before use;

[0039] (2) Lamprey serum was diluted with PBS at a ratio of 1:5, filtered through a 0.22 μm filter membrane, mixed with Protein G agarose resin, and incubated overnight at 4°C by rotating the mixture.

[0040] (3) Wash the machine with 20% ethanol for 30 mL of liquid. Use Binding Buffer to balance the machine. Once the machine reading stabilizes, adjust the reading to “0”.

[0041] (4) Sample loading: Load the sample at a rate of 1 mL / min. After the serum premix in the column has been loaded, equilibrate the machine with Binding Buffer (0.15M NaCl, 20mM Na2HPO4, pH adjusted to 7.0-7.4).

[0042] (5) Equilibration: Add Binding Buffer to the column at a rate of 2 mL / min. Once the reading drops rapidly to a stable level, elution can be performed.

[0043] (6) Elution: Elution buffer (0.1M citric acid, pH adjusted to 2.5-3.0) is used to elute the target protein. When the instrument reading rises, the eluent can be collected, about 500μL / tube.

[0044] (7) During the elution process, stop collecting the sample once the instrument reading is basically stable. If the collected sample is in an acidic liquid environment, a small amount of Tris-HCl needs to be added to neutralize the pH value to about 7.0.

[0045] (8) After elution, wash the column with 20% ethanol for about 5 column volumes. Then store Protein G in 20% ethanol. The column can then be removed and stored for future use.

[0046] (9) Based on the electrophoresis results, proteins with the same electrophoretic bands are mixed together for dialysis. After dialysis, they can be stored at -80℃ for long-term use.

[0047] The amino acid sequence of lamprey serum lectin LSSL, as determined by sequencing, is shown below (SEQ ID NO.1):

[0048]

[0049]

[0050] Example 2:

[0051] The hepatocellular carcinoma and cirrhosis tissue samples used in this embodiment were sourced from the Sixth People's Hospital of Dalian City. Informed consent forms and related contracts were signed, ensuring the legality and compliance of the usage process. The tumor types included moderately differentiated hepatocellular carcinoma, poorly differentiated hepatocellular carcinoma, and clear cell carcinoma. Clinical information included gender, age, and pathological information.

[0052] The specific steps for preparing tissue sections of the above-mentioned liver cirrhosis, moderately differentiated hepatocellular carcinoma, poorly differentiated hepatocellular carcinoma, and clear hepatocellular carcinoma are as follows:

[0053] (1) Fixation: Rinse the collected liver tissue with physiological saline and immediately immerse it in neutral formalin fixative for 30-50 minutes.

[0054] (2) Washing: After the material is fixed, rinse it with running water for several hours or overnight;

[0055] (3) Dehydration: The material was dehydrated in 70%, 80% and 90% ethanol solutions for 30 min each, and then placed in 95% and 100% ethanol solutions twice each for 20 min each time;

[0056] (4) Transparency: Mix equal parts of pure alcohol and xylene for 15 min, then xylene I for 15 min and xylene II for 15 min (until transparent).

[0057] (5) Wax impregnation: Immerse in paraffin I, paraffin II and paraffin III for 50-60 minutes each;

[0058] (6) Embedding: Use tweezers to pick up the paraffin wax mold (metallic) and heat it slightly over an alcohol lamp. Place it on a flat table. Take out the wax cup containing pure paraffin wax from the incubator, pour in a little paraffin wax, heat the tweezers slightly over an alcohol lamp, pick up the material and place it in the wax mold with the cut side down, arrange it neatly, place the embedding box on top, and gently pour in the molten wax.

[0059] (7) Slicing: Cut the wax block into 4-micrometer thick slices and spread them onto a glass slide.

[0060] This invention also includes the purchase of a commercially available tissue microarray for intrahepatic cholangiocarcinoma (Xi'an Zhongke Guanghua Intelligent Biotechnology Co., Ltd.), D109Lv01, with a total of 109 points, including 100 points of intrahepatic cholangiocarcinoma, 4 points of adjacent liver tissue, and 5 points of normal liver tissue. Clinical information includes gender, age, TNM, tumor size, stage, and follow-up information. The diameter of each sample point on the microarray is 1.5 mm, and formalin fixation is used.

[0061] Example 3:

[0062] In this embodiment, phosphatidylinositol proteoglycan-3 (GPC3) was used as a positive control. The efficacy of lamprey serum spheroid lectin LSSL in detecting hepatocellular carcinoma and intrahepatic cholangiocarcinoma was evaluated by immunohistochemical experiments, mainly including the following steps:

[0063] (1) Baking: Bake at 65℃ for about 30 minutes;

[0064] (2) Dewaxing: Xylene I for 15 min, Xylene II for 15 min;

[0065] (3) Hydration: 100% ethanol I for 3 min, 100% ethanol II for 3 min, 95% ethanol I for 3 min, 95% ethanol II for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, 60% ethanol for 3 min, rinsed with PBS 3 times;

[0066] (4) 3% hydrogen peroxide blocking: Add an appropriate amount of hydrogen peroxide, incubate at room temperature for 10 min, and wash with PBS for 3 min × 3 times;

[0067] (5) Blocking: Add CFBS dropwise, block at 37℃ for 1 hour, pour off the blocking solution, do not wash;

[0068] (6) Lectin incubation: Add 3 μg of lectin (LSSL) to the tissue and incubate at 37°C for 2 h. Wash with PBS for 3 min × 3 times.

[0069] (7)1 Anti-incubation: According to the size of the tissue, add an appropriate amount of LSSL antibody or GPC3 antibody, incubate at 37°C for 2 hours or at 4°C overnight, and wash with PBS for 3 minutes × 3 times.

[0070] (8) Add biotin-labeled goat anti-mouse / rabbit IgG polymer, incubate at 37°C for 15 min, and wash with PBS for 3 min × 3 times;

[0071] (9) Add horseradish peroxidase-labeled streptomycin working solution, incubate at 37°C for 15 min, and wash with PBS for 3 min × 3 times;

[0072] (10) Color development: DAB color development solution (1mL clearing solution + 60μL DAB) for 30s, followed by PBS washing for 5min;

[0073] (11) Soak in hematoxylin for 4 minutes, then rinse with water;

[0074] (12) Differentiate with hydrochloric acid and ethanol for 10 seconds, then rinse with water;

[0075] (13) Ammonia solution is used to reverse the blue color, water is used to rinse, and the staining is observed under a microscope (if the staining is light, hematoxylin staining is repeated; if the staining is dark, 75% ethanol is used to decolorize).

[0076] (14) Dehydrate to xylene (the reverse process of hydration), then seal the slide.

[0077] (15) Use a microscope to photograph immunohistochemically stained hepatocellular carcinoma sections and intrahepatic cholangiocarcinoma tissue microarrays, then use Image-Pro Plus software to analyze the gray values ​​of the photographs, and then use Prism software to create bar charts and ROC curves of the gray values ​​of each photograph.

[0078] Experimental results are as follows Figure 1-6 As shown, the results indicate that LSSL can specifically recognize glycoform structures such as Galβ1-3GlcNAc, GlcNAcβ1-2Man, and Manα1-2Man. The existing biomarker GPC3 alone has an AUC of 0.994 for identifying hepatocellular carcinoma (HCC), with a sensitivity and specificity of 0.93 and 1, respectively. GPC3 alone has an AUC of 0.49 for identifying intrahepatic cholangiocarcinoma (ICC), with a sensitivity and specificity of 0.07 and 1, respectively. In contrast, the lectin LSSL of this invention alone has AUCs of 0.65 and 0.91 for identifying hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), respectively. Its sensitivity and specificity for detecting HCC are 0.78 and 0.78, respectively, and its sensitivity and specificity for detecting ICC are 0.88 and 1, respectively. Therefore, it can be seen that the lectin provided in this invention has very high sensitivity and specificity, especially for ICC. The higher the sensitivity, the lower the false negative rate, indicating that the lectin LSSL of this invention can effectively distinguish liver cancer patients from normal healthy people.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lamprey serum spheroid agglutinin, characterized in that, The amino acid sequence of the lamprey serum spheroid lectin is shown in SEQ ID NO.

1.

2. The method for preparing lamprey serum spheroid agglutinin according to claim 1, characterized in that, The preparation method includes obtaining lamprey serum lectins through a heterologous expression system or isolating lamprey serum lectins from lamprey serum.

3. The preparation method according to claim 2, characterized in that, The specific steps for obtaining lamprey serum spheroid agglutinin via a heterologous expression system are as follows: the coding gene for lamprey serum spheroid agglutinin is cloned into a recombinant expression vector, introduced into host cells, and heterologously expressed to obtain lamprey serum spheroid agglutinin.

4. The preparation method according to claim 3, characterized in that, The host cells mentioned include Escherichia coli host cells, Bacillus subtilis host cells, and yeast host cells.

5. The preparation method according to claim 2, characterized in that, The process of isolating lamprey serum spheroid agglutinins from lamprey serum mainly includes the following steps: (1) Dilute lamprey serum with buffer at a volume ratio of 1:2-10, mix with Protein G agarose resin, and incubate with shaking at 1-5℃ for 5-24h; (2) Sample loading: Load the sample obtained in step (1) onto the chromatographic column; (3) Equilibration: Binding Buffer is added to the column at a rate of 1-2 mL / min; (4) Elution: Elution buffer is used to elute the target protein at a rate of 1-2 mL / min. The eluent is collected and the pH of the collected eluent is adjusted to 6-8. (5) Based on the electrophoresis results, the eluents of proteins with the same electrophoretic bands are mixed together and dialyzed. The result is obtained after dialysis.

6. The preparation method according to claim 5, characterized in that, The buffers mentioned in step (1) include PBS buffer and Tris-HCl buffer.

7. The preparation method according to claim 5, characterized in that, The Binding Buffer in step (3) consists of 0.15M NaCl, 20mM Na2HPO4, and pH adjusted to 7.0-7.

4.

8. The preparation method according to claim 5, characterized in that, The components of the elution buffer mentioned in step (4) are: 0.1M citric acid, pH adjusted to 2.5-3.

0.

9. The application of the lamprey serum spheroid agglutinin according to claim 1 in the preparation of reagents or kits for detecting liver cancer; The liver cancer mentioned refers to hepatocellular carcinoma or intrahepatic bile duct carcinoma.

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