CN114940714B

CN114940714B - Preparation and application of liver cancer marker SOAT1 monoclonal antibody

Info

Publication number: CN114940714B
Application number: CN202210555350.9A
Authority: CN
Inventors: 刘如石; 梁湘辉
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2023-06-16
Anticipated expiration: 2042-05-20
Also published as: CN114940714A

Abstract

The invention obtains SOAT1 gene from liver cancer cell Huh-7, and connects it with expression vector pET-32a to construct recombinant expression vector. After induced expression and protein purification, the purified recombinant SOAT1 protein is used as antigen to immunize BALB/c mice; 5 SOAT1 monoclonal antibody cell strains are obtained by utilizing hybridoma technology and subcloning culture, and finally 4D11 is screened to identify SOAT1 protein in liver cancer cells Huh-7 and clinical liver cancer tissue specimens. The invention successfully prepares the anti-SOAT 1 specific monoclonal antibody by utilizing the recombinant SOAT1 protein, and lays a foundation for the subsequent preparation of liver cancer accompanying diagnosis kit by using the SOAT1 protein, the raw materials and the establishment of a liver cancer diagnosis method based on SOAT1.

Description

Preparation and application of liver cancer marker SOAT1 monoclonal antibody

Technical Field

The invention belongs to the field of immunology, and particularly relates to a liver cancer marker SOAT1 monoclonal antibody and application thereof.

Background

Liver cancer (liver cancer) is a more common malignancy worldwide, with its mortality rate second among all cancers (1). Liver cancer is mainly classified into primary liver cancer and metastatic liver cancer. The focus of primary liver cancer is derived from liver itself, including hepatocellular carcinoma and cholangiocarcinoma, etc., wherein hepatocellular carcinoma accounts for 95% (2) of primary liver cancer; metastatic liver cancer is formed by metastasis of malignant tumors derived from other tissues, such as gastric cancer, colorectal cancer, etc. The liver cancer is hidden, the initial symptoms are not obvious, and the clinical symptoms such as abdominal distension, nausea, emaciation, obstructive jaundice, fever caused by unknown reasons and the like can only appear in the late stage (3). The risk factors of liver cancer are numerous, and there are mainly hepatitis virus (hepatitis B virus, hepatitis C virus) infection, exposure to aflatoxin, alcoholism, obesity, diabetes, etc. (4). At present, the clinical diagnosis of liver cancer is mainly based on pathology, imaging, serum biochemical examination and detection means (5) related to various molecular techniques, and a method for effectively diagnosing early liver cancer is still lacking. Therefore, finding more accurate and early markers and diagnostic methods is a major issue.

Liver cancer is one of the most common malignant tumors, and the high mortality and high invasiveness of the liver cancer bring great medical and economic burden to patients and families. Therefore, early screening and early diagnosis of liver cancer are of great significance in improving prognosis and prolonging survival time of patients. Alpha Fetoprotein (AFP) is a liver cancer serum marker widely used clinically, but has lower sensitivity and specificity, and the AFP level of only one third of liver cancer patients is higher than 100ng/mL; in the absence of malignancy, serum AFP was also elevated in patients with chronic liver disease (9). In recent years, des-gamma-carboxyprothrombin (DCP) has also been used for diagnosing early liver cancer, but the sample size is small, the detection quality is different, and more high-quality researches are required to further prove the feasibility of DCP in diagnosing liver cancer (10). Therefore, it is urgent to develop a novel diagnostic marker for liver cancer with high specificity and sensitivity.

In recent years, the relationship between alterations in lipid metabolism and the development of cancer has been attracting attention of many scholars. It has been shown that adipocytes can drive fatty acid metabolism by increasing CPT1A and electron transport chain complex protein levels, promoting proliferation and migration of breast cancer cells, and thus accelerating cancer progression (11). Furthermore, wang et al (12) found that three lipid metabolism-related genes were highly expressed in hepatocellular carcinoma; and is closely related to the prognosis of cancer, and can be used as an independent prognosis factor of liver cell and liver cancer. Cholesterol is an important member of lipid metabolism and is involved in the process of cancer development and progression. It is pointed out that the addition of cholesterol can reverse the effect of ursolic acid in inhibiting liver cancer growth of liver cells (13). Yet another study on the mechanism of p53 inhibition of liver cancer growth of hepatocytes demonstrated that p53 plays a role in inhibiting proliferation of hepatocytes by inhibiting mevalonate pathway, reducing intracellular cholesterol content (14).

Sterol O-acyltransferase (SOAT), also known as acyl CoA-cholesterol acyltransferase (ACAT), is a protein that localizes to the endoplasmic reticulum membrane, catalyzes the conversion of cholesterol to cholesterol esters, and stores it in cytoplasmic fat droplets (6). Human body has two SOAT genes in total, respectivelyACAT1AndACAT2。from the following componentsACAT1The translated SOAT1 is mainly expressed in adrenal gland, kidney, ovary, liver and the like; and is composed ofACAT2The SOAT2 formed by translation is expressed in intestinal cells (7).In 2019, jiang et al found that SOAT1 levels were significantly up-regulated in early hepatocellular carcinoma associated with hepatitis b virus, and that their high expression was closely related to poor prognosis of liver cancer (8). Therefore, SOAT1 is expected to be a novel marker for diagnosing liver cancer and judging prognosis. SOAT1 is a key enzyme regulating intracellular cholesterol ester storage, and its high expression has been shown to be associated with poor prognosis in various cancers such as glioma, renal cell carcinoma, adrenocortical carcinoma, breast cancer, etc. (15-18). Analysis of the TCGA database by Ren et al found that SOAT1 was most significantly up-regulated in hepatocellular carcinoma and cholangiocarcinoma, and that high expression of SOAT1 was associated with poor prognosis in hepatocellular carcinoma patients (19). Meanwhile, chen et al found that SOAT1 was expressed significantly higher in liver cancer tissue than in cancer tissue by comparing the expression of SOAT1 in liver cancer and in cancer tissue by immunohistochemistry (20). It is speculated that SOAT1 alone or in combination with other serological markers detects or enhances the early liver cancer detection rate. The prokaryotic expression system is used for protein expression, which has the defects of insoluble protein, unstable conformation, low purification rate and the like, and the use of fusion tags can enhance the expression of target proteins and improve the protein solubility (21).

Reference to the literature

1. Chen Yao, yang Xunjun, chen Han, chen Guorong. Expression of SOAT1 in hepatocellular carcinoma tissue and its clinical significance [ J ]. University of medical science, J.J., university of Etsuzhou, 2021;51 (01): 35-9.

2. Kulik L, El-Serag HB. Epidemiology and Management of Hepatocellular Carcinoma. Gastroenterology. 2019;156(2):477-91 e1.

3. Dimitroulis D, Damaskos C, Valsami S, Davakis S, Garmpis N, Spartalis E, et al. From diagnosis to treatment of hepatocellular carcinoma: An epidemic problem for both developed and developing world. World J Gastroenterol. 2017;23(29):5282-94.

4. Cabrera R, Nelson DR. Review article: the management of hepatocellular carcinoma. Aliment Pharmacol Ther. 2010;31(4):461-76.

5. Ronot M, Purcell Y, Vilgrain V. Hepatocellular Carcinoma: Current Imaging Modalities for Diagnosis and Prognosis. Dig Dis Sci. 2019;64(4):934-50.

6. Long T, Sun Y, Hassan A, Qi X, Li X. Structure of nevanimibe-bound tetrameric human ACAT1. Nature. 2020;581(7808):339-43.

7. Sakashita N, Miyazaki A, Takeya M, Horiuchi S, Chang CCY, Chang T-Y, et al. Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues [J] The American Journal of Pathology. 2000;156(1).

8. Jiang Y, Sun A, Zhao Y, Ying W, Sun H, Yang X, et al. Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma. Nature. 2019;567(7747):257-61.

9. Grandhi MS, Kim AK, Ronnekleiv-Kelly SM, Kamel IR, Ghasebeh MA, Pawlik TM. Hepatocellular carcinoma: From diagnosis to treatment. Surg Oncol. 2016;25(2):74-85.

10. Deji, yang Li, wang Yiping. Systematic evaluation of de-gamma-carboxyprothrombin diagnosis of primary liver cancer [ J ]. J.Chinese journal of evidence-based medicine 2020;20 (07): 798-808.

11. Balaban S, Shearer RF, Lee LS, van Geldermalsen M, Schreuder M, Shtein HC, et al. Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration. Cancer Metab. 2017;5:1.

12. Wang W, Zhang C, Yu Q, Zheng X, Yin C, Yan X, et al. Development of a novel lipid metabolism-based risk score model in hepatocellular carcinoma patients. BMC Gastroenterol. 2021;21(1):68.

13. Kim GH, Kan SY, Kang H, Lee S, Ko HM, Kim JH, et al. Ursolic Acid Suppresses Cholesterol Biosynthesis and Exerts Anti-Cancer Effects in Hepatocellular Carcinoma Cells. Int J Mol Sci. 2019;20(19).

14. Moon SH, Huang CH, Houlihan SL, Regunath K, Freed-Pastor WA, Morris JPt, et al. p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression. Cell. 2019;176(3):564-80 e19.

15. Bemlih S, Poirier MD, El Andaloussi A. Acyl-coenzyme A: cholesterol acyltransferase inhibitor Avasimibe affect survival and proliferation of glioma tumor cell lines. Cancer Biol Ther. 2010;9(12):1025-32.

16. Antalis CJ, Arnold T, Rasool T, Lee B, Buhman KK, Siddiqui RA. High ACAT1 expression in estrogen receptor negative basal-like breast cancer cells is associated with LDL-induced proliferation. Breast Cancer Res Treat. 2010;122(3):661-70.

17. Chen L, Peng T, Luo Y, Zhou F, Wang G, Qian K, et al. ACAT1 and Metabolism-Related Pathways Are Essential for the Progression of Clear Cell Renal Cell Carcinoma (ccRCC), as Determined by Co-expression Network Analysis. Front Oncol. 2019;9:957.

18. Lacombe AMF, Soares IC, Mariani BMP, Nishi MY, Bezerra-Neto JE, Charchar HDS, et al. Sterol O-Acyl Transferase 1 as a Prognostic Marker of Adrenocortical Carcinoma. Cancers (Basel). 2020;12(1).

19. Ren M, Xu H, Xia H, Tang Q, Bi F. Simultaneously targeting SOAT1 and CPT1A ameliorates hepatocellular carcinoma by disrupting lipid homeostasis. Cell Death Discov. 2021;7(1):125.

20. Chen Y, Yang X, Chen Y, Chen G, Winkler CA, An P, et al. Impacts of the SOAT1 genetic variants and protein expression on HBV-related hepatocellular carcinoma. BMC Cancer. 2021;21(1):615.

21. Walls D, Loughran ST. Tagging recombinant proteins to enhance solubility and aid purification. Methods Mol Biol. 2011;681:151-75。

Disclosure of Invention

The invention adopts pET-32a expression vector with polyHis tag at two ends, the tag is the most widely used affinity tag at present, the expression and folding of protein are not affected, and the recombinant protein with His tag has high affinity with Ni (II) -nitrosotriacetic acid (Ni2+NTA), which allows the combined recombinant protein to be dissociated by gradient concentration imidazole to achieve the purification effect. The obtained recombinant protein exists in cytoplasm in the form of inclusion bodies and is difficult to dissolve; therefore, the gradient denaturant urea is used for dissolving inclusion bodies, and then the denaturant is removed by a dialysis method, so that the target protein is restored to a natural conformation, and finally the recombinant protein for mouse immunization is obtained. After 4 immunizations, the serum titer of the mice reached 1:3200000, this demonstrates that recombinant SOTA1 has good immunogenicity. Finally 5 anti-SOAT 1 monoclonal antibodies are obtained through cell fusion and subcloning experiments and are respectively named as 1F3, 1G3, 1D6, 2F8 and 4D11. According to the indirect ELISA result, all five antibodies have higher potency affinity. The 4D11 can detect the SOAT1 protein in Western-blot, immunohistochemical and other experiments, which shows that a monoclonal antibody capable of specifically recognizing SOAT1 protein expressed by liver cancer cells is finally obtained, and the invention is finally completed.

The invention firstly provides an anti-SOAT 1 specific monoclonal antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region are respectively CDR1: SFAMS; CDR2: ASISSGGAMYYPDSVQG; CDR3: WTYYGSSYGAMDS; and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody are CDR1: KSSQSLLSSGNQKNYLT; CDR2: WASTRES; CDR3: QNDYTYPLT.

Preferably, the heavy chain amino acid sequence of the monoclonal antibody is MNFGFSLIFLVLVLKGVQCEVKLVESGGGFVKPGGSLKLSCAASGFTFRSFAMSWVRQTPEKRLEWVASISSGGAMYYPDSVQGRFTISRDSAGNILYLEMSSLRSEDTAMYYCARWTYYGSSYGAMDSWGQGTSVTVSS; the light chain amino acid sequences of the monoclonal antibodies were MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLSSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK, respectively.

The invention further provides a nucleotide molecule encoding said monoclonal antibody.

The invention also provides expression vectors comprising said nucleotide molecules.

The invention also provides a SOAT1 monoclonal antibody cell strain which can secrete the anti-SOAT 1 specific monoclonal antibody.

Furthermore, the invention provides application of the monoclonal antibody in preparing a reagent for detecting or diagnosing liver cancer. Also provides a kit for detecting or diagnosing liver cancer by the monoclonal antibody.

At present, the number of monoclonal antibodies for detecting the human SOAT1 antigen is small, but the anti-SOAT 1 monoclonal antibody obtained in the invention has the capability of identifying the natural SOAT1 protein in human liver cancer cells and liver cancer tissues, and the dilution ratio of the antibody of WB and IHC is in a proper range; the titer of the SOAT1 monoclonal antibody can reach 1:204800 has strong binding capacity with SOAT1 antigen and has great application value.

Drawings

The acquisition of the target gene and the construction of the recombinant vector in FIG. 1. Wherein A: electrophoresis pattern of liver cancer cell Huh-7 total RNA; b, a step of preparing a composite material; positive clone colony PCR electrophoresis pattern; c: electrophoresis diagram of recombinant plasmid double enzyme digestion products (Lane M: DNA molecular mass standard; lane 1: before enzyme digestion; lane 2: after enzyme digestion).

FIG. 2 expression and identification of recombinant proteins. Wherein A: SDS-PAGE; b: western blot (Lane M: pre-stained protein relative molecular mass standard; lane 1: before induction; lane 2: after induction; lane 3: after sonication suspension; lane 4: sonication supernatant; lane 5: sonication pellet).

FIG. 3 purification of SOAT1 protein. Wherein A: the dissolution of recombinant proteins in urea of different concentrations (Lane M: pre-stained protein relative molecular mass standard; lane 1-5: urea concentration: 8M, 6M, 4M, 2M, 1M; lane a-c: represents protein suspension, precipitation after suspension centrifugation and supernatant after suspension centrifugation, respectively); b: recombinant protein purity was identified (Lane M: pre-stained protein relative molecular mass standard; lane 1-8: imidazole eluent concentration: 20mM, 30mM, 50mM, 70mM, 90mM, 100mM, 200mM, 300mM; lane 9: hanging column flow-through).

Figure 4 immune mouse serum antibody titer assay.

FIG. 5 purification results of monoclonal antibodies (Lane M: pre-stained protein relative molecular mass standard; lane 1: non-boiled pre-purification ascites; lane 2: boiled pre-purification ascites; lane 3: non-boiled ammonium octoate sulfate precipitation purification ascites; lane 4: boiled ammonium octoate sulfate precipitation purification ascites; lane 5: non-boiled post-purification monoclonal antibody; lane 6: boiled post-purification monoclonal antibody).

FIG. 6 monoclonal antibody subtype identification.

Figure 7 monoclonal antibody titers.

FIG. 8 Western-blot detection of SOAT1 expressed by Huh-7 cells.

FIG. 9 immunohistochemical detection of SOAT1 expressed in liver cancer tissue. Wherein A: normal myocardial tissue in humans; b: normal liver tissue of humans; c: highly differentiated hepatocellular carcinoma (400×).

Detailed Description

According to the invention, the SOAT1 gene is obtained from liver cancer cells Huh-7, an SOAT1 prokaryotic expression vector is constructed, purified SOAT1 protein is used as an immunogen to immunize a mouse, and an anti-SOAT 1 monoclonal antibody is prepared through techniques such as cell fusion, so that a foundation is laid for not only providing raw materials for screening the anti-SOAT 1 antibody, but also further establishing an early liver cancer detection method based on SOAT1.

1. Experimental materials and methods

1. Material

Coli Rosettagamib, plasmid pET-32a, huh-7 hepatoma cells and mouse myeloma cells Sp2/6 are all provided by the laboratory; DMEM medium was purchased from Gibco company, usa; fetal bovine serum was purchased from Shanghai Bei Bo biotechnology limited; SOAT1 upstream and downstream primers were synthesized by Changshaqingke biotechnology Co., ltd; construction of vector-related ExTaq enzyme and restriction endonucleaseEcoR IAndXho IDNA standards were purchased from TaKaRa, japan; agarose gel recovery kits were purchased from Tiangen Biochemical technologies company; protein standards were constructed from BIO-RAD, inc., USA; plasmid miniprep kit, protein G column and mouse source anti-6 XHis-tag monoclonal antibody are purchased from Shanghai Biotechnology Co., ltd; male BALB/c mice (6-8 w) were purchased from Hunan Stokes Levoda laboratory animal Co., ltd; freund's adjuvant, HT, and HAT were all purchased from Sigma company; GAM-IgG-HRP is a benefit taught by the university of mansion Xia Ningshao; goat anti-mouse IgG antibodies were purchased from changsha Ai Bi vitamin technologies limited; the high-sensitivity ECL luminous liquid is purchased from Monaco (Wuhan) biological limited publicA driver; the paraffin sections of the normal cardiac muscle and liver cancer tissues are all given away by the disease department of Xiangya two hospitals; DAB chromogenic kit was purchased from Wuhansai Weibull technologies Co., ltd; the rest reagents are all made or imported analytically pure.

2. Experimental method

1) Acquisition of the Gene of interest

Extracting liver cancer cell Huh-7 total RNA by Trizol method, and obtaining SOTA1 cDNA by reverse transcription. Based on the CDS sequence of SOAT1 in GenBank, the following primers were designed: f:5 '-CGGAATTCATGAAGGAAGTT-GGCAGTC-3'; r:5 '-CCGCTCGAGCTAAAACACGTAACGACAAG-3' -and respectively addingEcoR IAndXho Ithe primer pair is used for PCR amplification of the SOAT1 gene. Reaction conditions: (1) pre-denaturation at 94℃for 2min; (2) denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 30 cycles; (3) final extension at 72℃for 10min. The PCR products were subjected to 1% agarose gel electrophoresis, and the target bands were subjected to gel recovery and the concentration was measured.

2) Recombinant vector construction

The PCR product after the gel recovery and the vector pET-32a are respectively carried outEcoR I/Xho IDouble enzyme cutting, electrophoresis of enzyme cutting product with 1% agarose gel, and recovery of target band cutting gel. And connecting the purified PCR product with a vector pET-32a, converting the PCR product into escherichia coli Rosettagamib, picking positive clones, and sending the positive clones to a company for sequencing after PCR and enzyme digestion identification.

3) SOAT1 recombinant protein induced expression and identification

Positive colonies were picked and transferred to 3mL of ampicillin-containing LB liquid medium, shaking at 37℃and 230rpm overnight. Activated species were grown at 1:100, at 37℃and 230 rpm. When OD is _600nm When the bacterial strain reaches 0.6, 0.1% IPTG is added to induce for 8-9 hours, the centrifugation is carried out for 10 minutes at 12000rpm, bacterial cells are collected, bacteria are broken by an ultrasonic breaking method, bacterial liquid before and after induction, ultrasonic mixture, supernatant after centrifugation and sediment are respectively collected, and the expression condition of target protein is analyzed by SDS-PAGE electrophoresis. Meanwhile, a mouse-derived anti-6 XHis-tag monoclonal antibody is used as a primary antibody, a goat anti-mouse antibody is used as a secondary antibody, and a Western-blot method is used for identifying the target protein.

4) SOAT1 recombinant protein purification

The SOAT1 protein exists mainly in the form of inclusion bodies in the cells. After washing the inclusion bodies of SOAT1 sufficiently, 20 volumes of PBS containing 6M, 5M, 4M, 3M, and 2M Urea was prepared for proteins, and the protein mixture was transferred to dialysis bags and dialyzed from high to low concentration, and replaced every 3 hours. After renaturation, the supernatant is collected by centrifugation, and the protein is purified by adopting a nickel column affinity chromatography mode. The purity of the purified protein was analyzed by SDS-PAGE electrophoresis and the concentration was measured.

5) Mouse immunity and serum antibody titer detection

The purified antibodies were used to immunize 6-8w male BALB/c mice for five rounds. The first four rounds are all injected subcutaneously at multiple points, once every two weeks; the fifth round of immunization was performed by intraperitoneal injection 10 days after the end of the fourth round of immunization. Orbital blood was taken before each immunization, coated with SOAT1 protein, and mouse serum antibody titers were determined by indirect ELISA.

6) Cell fusion

Spleens of five rounds of immunized mice were taken, fully ground, and spleen cells were collected. After cell counting, spleen cells and Sp2/6 cells in logarithmic growth phase were counted at 5:1 to 10:1, 1300rpm, centrifuging for 5min, and discarding the supernatant. The centrifuge tube was placed in warm water at 37℃and PEG solution was slowly added dropwise over 1min. Mixing, and standing for 1min. 5mL of preheated DMEM medium is added within 3min, and the volume is fixed and the centrifugation is carried out. Cells were resuspended in HAT medium containing trophoblast cells and plated according to the number of splenocytes. After 10d of incubation, HT medium was replaced.

7) Hybridoma cell screening and cloning culture

When the fused cells have larger cell clusters, SOAT1 protein is coated, and an indirect ELISA method is adopted to screen positive clones. And (3) performing amplification culture on the screened positive holes, and subcloning by a limiting dilution method after the cells grow to 60%.

8) Large-scale preparation and purification of monoclonal antibodies

9w male BALB/c mice were given intraperitoneal injections of paraffin oil 450-600. Mu.L/mouse. After 10d-15d, hybridoma cells in logarithmic growth phase were collected and their concentration was adjusted to 2X 10 ⁶ The volume of the solution is one per mL,mice were injected in the same manner at a dose of 500. Mu.L/mouse. Ascites fluid was collected after 7d and purified by ammonium octoate sulfate precipitation and Protein G column affinity chromatography.

9) Property analysis of anti-SOAT 1 monoclonal antibody

a. Antibody subtype identification

Positive hybridoma cell line cell supernatants were collected, with anti-IgG 1, igG2a, igG2b, igG3, igM, igGAM antibodies as secondary antibodies, with anti-IgGAM as positive control, and each strain antibody subtype was identified by indirect ELISA.

b. Antibody titer determination

The antibody titers were detected by indirect ELISA, and the purified antibodies were diluted to the same concentration (0.5 mg/mL) followed by gradient dilution to 1:409600, the analytical OD values evaluate antibody titers.

Western blot experiment

Culturing liver cancer cell Huh-7, extracting total protein, performing SDS-PAGE electrophoresis, transferring film, sealing, and washing with TBST. The 5 obtained monoclonal antibodies 1: after 500 dilution, the antibody is used as a primary antibody and incubated overnight at 4 ℃; after the next TBST wash, HRP-labeled goat anti-mouse antibody was added as a secondary antibody for incubation, TBST wash, ECL color development was observed for antibody binding effect.

d. Immunohistochemical identification

After tissue sections were baked, deparaffinized, hydrated, sodium citrate antigen retrieval, 3% hydrogen peroxide to remove endogenous peroxidase, add 1:200 dilution of purified SOAT1 antibody, incubation overnight at 4 ℃; the next day PBST washing, adding secondary antibody, incubating for 1h at room temperature, PBST washing, DAB color development, distilled water termination color development, hematoxylin counterstain cell nucleus, saturated lithium carbonate bluing, gradient alcohol dehydration, xylene transparency, neutral resin sealing, and observing the binding condition of the antibody under a microscope.

e. Antibody variable regions

The 4D11 hybridoma cell RNA was extracted and sent to the golden Biotech Co.Ltd for sequencing.

2. Experimental results

1. Acquisition of target Gene and construction of recombinant vector

Extracting liver cancerThe total RNA of the cell Huh-7, after electrophoresis, was found to appear as three distinct bands on agarose gel, 28S, 18S and 5S, respectively (A in FIG. 1). The bands were clear and bright and can be used in subsequent reverse transcription experiments. The SOTA1 RNA is reversely transcribed into cDNA as a template for PCR, and obvious bands are found near 1500bp through agarose gel electrophoresis, which indicates that the SOTA1 gene can be amplified successfully. After enzyme digestion and connection, the recombinant vector is transformed into competent cells of the escherichia coli, the plates are coated, colonies are randomly picked up for colony PCR, and positive colonies are screened. After electrophoresis, the colony is found to have a specific band at a 1500bp position (B in FIG. 1), and the success of the transformation of the recombinant vector is preliminarily determined. For further identification, plasmids of colony PCR positive bacteria were extracted and subjected toEcoR I/Xho IDouble digestion and electrophoresis showed that the recombinant vector was cut into two parts, the size of the fragment of interest was consistent with the expectations (C in FIG. 1), indicating that the recombinant vector construction was successful. The plasmid is sent to sequencing, the recombinant vector sequence is basically correct, no obvious mutation occurs, and the recombinant vector can be used for subsequent experiments.

2. Expression and identification of SOAT1 recombinant proteins

Recombinant vector with basically correct sequence is induced to express, and SDS-PAGE electrophoresis is carried out after ultrasonic disruption. The results showed that both the post-induction and the sonicated groups showed a distinct protein band at 50KD (a in fig. 2), which initially indicated that recombinant protein expression was successful and was present in the bacterial cells predominantly as inclusion bodies. To further confirm that the band is our desired target protein, western-blot validation was performed using anti-6 XHis-tag as primary antibody. WB results indicate that the anti-His antibodies were able to recognize the expressed proteins and the positions were expected (B in fig. 2). In conclusion, after induction by IPTG, the strain successfully expresses SOAT1 recombinant protein with His tag.

3. SOAT1 recombinant protein purification

Slow renaturation of protein inclusion bodies by urea at gradient concentration, SDS-PAGE gel electrophoresis showed that under 8M, 6M, 4M urea solubilization conditions, a distinct band was visible at 50KD (FIG. 3A), indicating that inclusion bodies were minimally solubilized by 4M urea. In the subsequent nickel column affinity chromatography, the SOAT1 recombinant protein can be eluted in 20mmol/L, 30mmol/L and 50mmol/L imidazole (B in FIG. 3), and the purity is higher, so that the SOAT1 recombinant protein can be used for the next step of mouse immunization.

4. Immune mouse serum antibody titer detection

After immunization of the mice, serum antibody titers were detected with recombinant SOAT1 protein. The preimmune detection value is used as a negative control, and the 2.1 times negative value is used as a positive critical value of the detection result. The results show that after four rounds of immunization, the serum antibody titer of the mice is obviously increased, and the titer can reach 1:3200000 (FIG. 4).

5. Purification and identification of monoclonal antibodies

The results showed that 3 bands appeared in the purified antibody lanes, respectively: a 24kDa light chain, a 55/70kDa heavy chain and a 100kDa heavy chain polymer; in addition, the impurity protein bands appeared in the unpurified ascites lanes, the impurity proteins were significantly reduced in the purified antibodies (including initially purified and refined) lanes, and the target proteins were more apparent in the refined antibodies lanes (FIG. 5). This demonstrates that the purity and concentration of the purified monoclonal antibodies are significantly improved, essentially to the requirements of subsequent experiments. After purification, 5 anti-SOAT 1 monoclonal antibodies, designated 1F3, 1G3, 1D6, 2F8, 4D11, respectively, were finally obtained in total. Monoclonal antibody concentration was determined and calculated using BCA method, as follows: 1F3:0.55mg/mL;1G3:0.55mg/mL;1D6:0.57mg/mL;2F8:0.56mg/mL;4D11:0.75mg/mL.

6. Monoclonal antibody subtype identification

Subtype identification is carried out on the antibody stably secreted by the hybridoma cells obtained by screening by using an indirect ELISA method, and the result shows that: 1F3 is of the IgG2a type and the remaining four monoclonal antibodies are of the IgG1 type (FIG. 6).

7. Monoclonal antibody titer determination

As can be seen from fig. 7, the titers of 1F3, 2F8, and 4D11 all reached 1:204800;1D6 and 1G3 titers reached 1:102400.

8、Western-blot

the results of Western-blot showed that, at 1: at a dilution ratio of 500, 1D6 and 4D11 recognize the SOAT1 protein in Huh-7 cells.

9. Immunohistochemistry

4D11 was selected for immunohistochemical identification. The results showed that brown positive foci (red circle labeling) appeared on liver cancer sections, indicating that 4D11 can recognize the SOAT1 protein in liver cancer tissue sections (fig. 9).

10. Antibody variable regions

Sequencing results were as follows:

heavy chain amino acid full length sequence (140 aa): MNFGFSLIFLVLVLKGVQCEVKLVESGGGFVKPGGSLKLSCAASGFTFRSFAMSWVRQTPEKRLEWVASISSGGAMYYPDSVQGRFTISRDSAGNILYLEMSSLRSEDTAMYYCARWTYYGSSYGAMDSWGQGTSVTVSS.

Reconnection variable region sequence:

CDR1：SFAMS；CDR2：ASISSGGAMYYPDSVQG；CDR3：WTYYGSSYGAMDS。

full length sequence of light chain amino acids (133 aa):

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLSSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK。

light chain variable region sequence:

CDR1：KSSQSLLSSGNQKNYLT；CDR2：WASTRES；CDR3：QNDYTYPLT。

<110> Hunan university of teachers and students

<120> preparation and application of SOAT1 monoclonal antibody serving as liver cancer marker

<160>8

<210>1

<211>140

<212>PRT

<213> SOAT1 monoclonal antibody heavy chain

<400> 1

MNFGFSLIFLVLVLKGVQCEVKLVESGGGFVKPGGSLKLSCAASGFTFRSFAMSWVRQTPEKRLEWVASISSGGAMYYPDSVQGRFTISRDSAGNILYLEMSSLRSEDTAMYYCARWTYYGSSYGAMDSWGQGTSVTVSS 140

<210>2

<211>5

<212>PRT

<213> SOAT1 monoclonal antibody heavy chain CDR1

<400> 2

SFAMS 5

<210>3

<211>5

<212>PRT

<213> SOAT1 monoclonal antibody heavy chain CDR2

<400> 3

ASISSGGAMYYPDSVQG 17

<210>4

<211>13

<212>PRT

<213> SOAT1 monoclonal antibody heavy chain CDR4

<400> 4

WTYYGSSYGAMDS 13

<210>5

<211>133

<212>PRT

<213> SOAT1 monoclonal antibody light chain

<400> 5

MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLSSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK 133

<210>6

<211>17

<212>PRT

<213> SOAT1 monoclonal antibody light chain CDR1

<400> 6

KSSQSLLSSGNQKNYLT 17

<210>7

<211>7

<212>PRT

<213> SOAT1 monoclonal antibody light chain CDR2

<400> 7

WASTRES 7

<210>8

<211>7

<212>PRT

<213> SOAT1 monoclonal antibody light chain CDR3

<400> 8

QNDYTYPLT 9

Claims

1. An anti-SOAT 1 specific monoclonal antibody, characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of said monoclonal antibody are CDR1: SFAMS; CDR2: ASISSGGAMYYPDSVQG; CDR3: WTYYGSSYGAMDS; and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody are CDR1: KSSQSLLSSGNQKNYLT; CDR2: WASTRES; CDR3: QNDYTYPLT.

2. The monoclonal antibody of claim 1, wherein the heavy chain amino acid sequence of the monoclonal antibody is MNFGFSLIFLVLVLKGVQCEVKLVESGGGFVKPGGSLKLSCAASGFTFRSFAMSWVRQTPEKRLEWVASISSGGAMYYPDSVQGRFTISRDSAGNILYLEMSSLRSEDTAMYYCARWTYYGSSYGAMDSWGQGTSVTVSS; the light chain amino acid sequences of the monoclonal antibodies were MESQTQVLMSLLFWVSGTCGDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLSSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYTYPLTFGAGTKLELK, respectively.

3. A nucleotide molecule encoding the monoclonal antibody of any one of claims 1-2.

4. An expression vector comprising the nucleotide molecule of claim 3.

A cell line of an SOAT1 monoclonal antibody, characterized in that it is capable of secreting an anti-SOAT 1 specific monoclonal antibody according to claim 1 or 2.

6. Use of the monoclonal antibody of any one of claims 1-2 in the preparation of a reagent for detecting or diagnosing liver cancer.

7. Kit for detecting or diagnosing liver cancer comprising the monoclonal antibody according to any one of claims 1-2.