An antibody of lncrna upk1a-as1 and a preparation method and application thereof

Abstract

The application discloses an antibody of lncRNA UPK1A-AS1 and a preparation method and application thereof, and relates to the technical field of genetic engineering. The immunogen comprises a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 and / or SEQ ID NO. 2. The antibody prepared by immunizing animals with the immunogen can specifically recognize a polypeptide coded by lncRNA UPK1A-AS1, and can also be used for preventing and treating liver cancer.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an antibody against lncRNA UPK1A-AS1, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and its global incidence is increasing year by year. HCC has an insidious onset, with more than 70% of patients already at an advanced stage when they seek medical attention. Sorafenib and lenvatinib targeted therapy are first-line treatments for advanced HCC, but their efficacy is poor. Immune checkpoint inhibitor monotherapy has an overall response rate of only 15%–20% in HCC.

[0003] Therefore, there is a need to provide a new anti-liver cancer drug with good therapeutic effects. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes an immunogen for use in immunizing animals to prepare antibodies that can specifically recognize polypeptides encoded by lncRNA UPK1A-AS1, and can also be used for the prevention and treatment of liver cancer.

[0005] The present invention also provides a biomaterial.

[0006] The present invention also provides a method for preparing polyclonal antibodies.

[0007] The present invention also provides a polyclonal antibody.

[0008] The present invention also provides the use of the above-mentioned polyclonal antibody in the preparation of a drug for the prevention or treatment of liver cancer.

[0009] The present invention also provides a drug for the prevention or treatment of liver cancer.

[0010] The present invention also provides the application of the above-mentioned polyclonal antibody in the preparation of a kit for detecting peptides encoded by lncRNA UPK1A-AS1.

[0011] The present invention also provides a reagent kit.

[0012] An immunogen according to a first aspect of the present invention comprises: a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 and / or a polypeptide with an amino acid sequence as shown in SEQ ID NO.2.

[0013] The immunogen according to embodiments of the present invention has at least the following beneficial effects:

[0014] Previous studies have suggested that lncRNAs lack the ability to encode proteins. This invention is the first to discover that lncRNA UPK1A-AS1 can encode a polypeptide. The polypeptide with the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2 can promote the proliferation of liver cancer cells. Furthermore, when used as an immunogen to immunize animals, it can prepare antibodies that specifically recognize the polypeptide encoded by lncRNA UPK1A-AS1. These antibodies can also be used to treat liver cancer.

[0015] According to some embodiments of the present invention, the immunogen is used to prepare an antibody against the protein encoded by lncRNA UPK1A-AS1. The antibody is selected from polyclonal antibodies or monoclonal antibodies.

[0016] According to some embodiments of the present invention, the immunogen further includes a carrier protein, which is coupled to a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 and / or SEQ ID NO.2. The carrier protein coupled to the polypeptide, acting as an immunogen, is beneficial for stimulating helper T cells and further inducing a B cell immune response.

[0017] According to some embodiments of the present invention, the carrier protein is at least one selected from serum albumin, hemocyanin, ovalbumin, thyroglobulin, cholera toxin B subunit, Escherichia coli unstable toxin B subunit, diphtheria toxoid, and tetanus toxoid. Specifically, the carrier protein may be hemocyanin.

[0018] According to some embodiments of the present invention, the immunogen can be purified by prokaryotic or eukaryotic expression, or it can be prepared by a biotechnology company through chemical synthesis based on its polypeptide sequence.

[0019] A biomaterial according to a second aspect of the present invention comprises at least one of 1) to 4).

[0020] 1) Nucleic acid molecules encoding the immunogen described in the first aspect of the present invention;

[0021] 2) An expression cassette containing the nucleic acid molecules described in 1);

[0022] 3) A recombinant vector comprising the nucleic acid molecule described in 1) or the expression cassette described in 2);

[0023] 4) Recombinant cells comprising the nucleic acid molecule described in 1), the expression cassette described in 2), or the recombinant vector described in 3).

[0024] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the immunogen in host cells. This DNA may include not only a promoter for initiating transcription of the nucleic acid molecule encoding the immunogen, but also a terminator for terminating transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0025] According to some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a nucleic acid molecule encoding the immunogen into the multiple cloning site of the vector.

[0026] According to some embodiments of the present invention, the recombinant biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells. The recombinant biological cells do not contain reproductive material.

[0027] According to some embodiments of the present invention, the recombinant biological cell is a recombinant biological cell obtained by introducing 1) the nucleic acid molecule, 2) the expression cassette or 3) the recombinant vector into a biological cell.

[0028] A method for preparing a polyclonal antibody against lncRNA UPK1A-AS1 according to a third aspect of the present invention includes the following steps:

[0029] Animals were immunized with the immunogen described in the first aspect above to obtain polyclonal antibodies.

[0030] According to some embodiments of the present invention, the animal includes, but is not limited to, rats, cattle, rabbits, horses, sheep, or pigs. Specifically, the animal may be a white rabbit.

[0031] According to some embodiments of the present invention, the sites of immunization include, but are not limited to, subcutaneous tissue on the back, abdomen, armpits, and limbs.

[0032] According to some embodiments of the present invention, in the preparation method, the number of immunizations is 5 to 6.

[0033] According to some embodiments of the present invention, the first immunization is the primary immunization, using Freund's complete adjuvant. All subsequent immunizations are booster immunizations, using Freund's incomplete adjuvant.

[0034] According to some embodiments of the present invention, the interval between the initial immunization and the first booster immunization is 10 to 14 days. Specifically, it can be 12 days.

[0035] According to some embodiments of the present invention, the interval between each booster immunization is 12 to 16 days. Specifically, it can be 14 days.

[0036] According to some embodiments of the present invention, the dose of the immunogen used for primary immunization is 0.6 mg / animal to 0.8 mg / animal. The dose of the immunogen used for booster immunization is 0.25 mg / animal to 0.45 mg / animal.

[0037] According to some embodiments of the present invention, the preparation method further includes affinity purification of collected animal serum to obtain polyclonal antibodies.

[0038] According to some embodiments of the present invention, the polyclonal antibody includes an IgG antibody.

[0039] A polyclonal antibody against lncRNA UPK1A-AS1 according to a fourth aspect of the present invention is prepared by the preparation method described in the second aspect above.

[0040] The polyclonal antibody according to embodiments of the present invention has at least the following beneficial effects:

[0041] The polyclonal antibody described in this embodiment can specifically recognize the polypeptide encoded by lncRNA UPK1A-AS1; it can also be used to prevent and treat liver cancer, significantly inhibiting tumor proliferation, reducing tumor volume and weight, and without the side effects of related drug treatments.

[0042] The use of the above-described polyclonal antibody according to the fifth aspect of the present invention in the preparation of a medicament for the prevention or treatment of liver cancer.

[0043] A medicament for the prevention or treatment of liver cancer according to a sixth aspect of the present invention includes the aforementioned polyclonal antibody. Since the medicament employs all the technical solutions of the polyclonal antibody described above, it possesses at least all the beneficial effects brought about by the technical solutions of the aforementioned embodiments.

[0044] According to some embodiments of the present invention, the drug further includes a pharmaceutically acceptable carrier. The carrier includes at least one selected from diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

[0045] The application of the above-described polyclonal antibody according to the seventh aspect of the present invention in the preparation of a kit for detecting lncRNA UPK1A-AS1 encoded polypeptide.

[0046] According to some embodiments of the present invention, the polypeptide comprises a peptide segment with an amino acid sequence as shown in SEQ ID NO.1 and / or a peptide segment shown in SEQ ID NO.2.

[0047] A kit according to an eighth aspect of the present invention includes the above-described polyclonal antibody. Since the kit employs all the technical solutions of the polyclonal antibody described above, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments. The kit can be used for quantitative and / or qualitative detection of peptides encoded by lncRNA UPK1A-AS1.

[0048] According to some embodiments of the present invention, the application method of the kit is selected from immunospot assay, enzyme-linked immunosorbent assay, Western blotting, immunofluorescence assay, or immunoelectron microscopy.

[0049] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0050] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0051] Figure 1 These are the results of polyribosome profiling analysis of lncRNA UPK1A-AS1;

[0052] Figure 2 The results are from the ORF analysis of lncRNA UPK1A-AS1;

[0053] Figure 3 The results show the expression of six predicted ORFs of lncRNA UPK1A-AS1; where A is a schematic diagram of the ORFs-Flag vector construction, and B and C are the Western blot and immunofluorescence detection results of different ORF expression levels, respectively; the scale bar in C represents 50 μm.

[0054] Figure 4 This is the validation result of the ORF1 and ORF2 encoding ability of lncRNA UPK1A-AS1; where A is the construction mode diagram of ORFs-GFP-Mut vector, and B and C are the Western blot and immunofluorescence detection results of ORF1 and ORF2 encoding ability, respectively; the scale bar in C represents 100μm.

[0055] Figure 5 The results show the effects of ORF1 and ORF2 on the proliferation of liver cancer cells; where A represents the effects of ORF1 and ORF2 on the proliferation of liver cancer cells; B represents the statistical results of the EdU positivity rate; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.01; the scale bar in A represents 50 μm;

[0056] Figure 6 The predicted results for linear B-cell epitopes of LUP1;

[0057] Figure 7 Prediction results of transmembrane domains for LUP1

[0058] Figure 8 The signal peptide prediction results for LUP1;

[0059] Figure 9 The results of the hydrophilicity, immunogenicity, and epitope exposure analysis of LUP1;

[0060] Figure 10 The predicted results for linear B-cell epitopes of LUP2;

[0061] Figure 11 Prediction results for the transmembrane domains of LUP2

[0062] Figure 12 The signal peptide prediction results for LUP2;

[0063] Figure 13 The results of the hydrophilicity, immunogenicity, and epitope exposure analysis of LUP2;

[0064] Figure 14 The results are obtained by ELISA detection of rabbit serum after the fifth immunization with the LUP1 recognition site peptide.

[0065] Figure 15 The results of Western blot analysis of rabbit serum after the fifth immunization with the LUP1 recognition site peptide;

[0066] Figure 16 The results are obtained by ELISA detection of rabbit serum after the fifth immunization with the LUP2 recognition site peptide.

[0067] Figure 17 The results of Western blot analysis of rabbit serum after the fifth immunization with LUP2 recognition site peptides;

[0068] Figure 18 These are the results of the conservation analysis of ORF1 and ORF2;

[0069] Figure 19 This is the result of Western blot detection of the expression of ORF1 and ORF2 in different liver cancer cells by Anti-LUP1 and Anti-LUP2 antibodies;

[0070] Figure 20 The results of Western blot (A) and immunofluorescence (B) detection of ORF1 and ORF2 expression in liver cancer cells with different lncRNA knockouts UPK1A-AS1 using Anti-LUP1 and Anti-LUP2 antibodies are shown; the scale bar in B represents 20 μm.

[0071] Figure 21 The results of silver staining (A) and mass spectrometry (B) detection of the expression of endogenous ORF1 and ORF2 by Anti-LUP1 and Anti-LUP2 antibodies are shown.

[0072] Figure 22 The therapeutic effects of Anti-LUP1 and Anti-LUP2 antibodies on hepatocellular carcinoma mice were investigated. A shows the experimental flowchart and photographs of tumor volume changes in each group of mice; B shows the tumor volume changes in each group of mice; and C shows the tumor weight statistics for each group of mice on day 21. * indicates P < 0.05, ** indicates P < 0.01.

[0073] Figure 23 The results show the effects of Anti-LUP1 and Anti-LUP2 antibodies on the body weight of mice with liver cancer; where A is a gross image of each group of nude mice, B is the change in body weight of each group of nude mice, and C is the change in body weight of each group of nude mice before and after the experiment; ns indicates no significant difference. Detailed Implementation

[0074] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0075] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0076] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0077] Unless otherwise specified, "room temperature" in this invention means an allowable temperature of 25℃±5℃.

[0078] In the following examples, the method for detecting the expression of the target protein using immunofluorescence is as follows:

[0079] ① Cell plating: After digesting and resuspending the cells to be tested, the cells are seeded at a density of 15% in a 35mm confocal dish. The confocal dish is then placed in an incubator and cultured at 37℃ and 5% CO2 for 24 hours. After that, the confocal dish is removed and the cells are observed under a microscope to see if they adhere to the wall (if the cells do not adhere well, poly-L-lysine or gelatin can be used to coat the bottom of the dish first).

[0080] ② Remove the culture medium: Remove the cells from the incubator and add an appropriate amount of ice-cold phosphate buffer to wash for 5 minutes to clean the cells in the confocal dish and remove excess culture medium. Repeat 3 times (if the cells do not adhere firmly, the number of phosphate buffer washes can be reduced).

[0081] ③ Paraformaldehyde fixation: Pre-cool 4% paraformaldehyde solution at 4℃ for 30 min, then add an appropriate amount of pre-cooled paraformaldehyde solution to the cells treated in step ②, fix the cells at room temperature for 30 min, and then wash with phosphate buffer 3 times, each time for 5 min (shake slowly during the washing process to prevent cell detachment).

[0082] ④ Cell permeation: Treat cells treated in step ③ with cell permeation solution (0.2% Triton X-100 solution: prepared by mixing 10 μL of pure Triton X-100 and 5 mL of phosphate buffer) for 10 min (the permeation time should not be too long, otherwise it will easily lead to an increase in cell debris, and the total permeation time should not exceed 15 min).

[0083] ⑤ Blocking: Aspirate the cell permeabilization solution, wash the cells three times with phosphate buffer for 3 minutes each time; after blotting the phosphate buffer with absorbent paper, block with blocking solution (phosphate buffer containing 5% skim milk powder) at room temperature for 2 hours.

[0084] ⑥ Incubate with primary antibody: Blot off the blocking solution with absorbent paper, add an appropriate amount of diluted primary antibody (obtained by dilution with phosphate buffer containing 3% BSA) to the confocal dish, and incubate overnight on a shaker at 4°C.

[0085] ⑦ Incubation with secondary antibody: Wash cells treated in step ⑥ three times with phosphate buffer, 5 min each time. Blot dry any remaining phosphate buffer on the confocal dish with absorbent paper, add an appropriate amount of diluted fluorescently labeled secondary antibody (obtained by dilution with phosphate buffer containing 3% BSA), and incubate at 37°C in the dark.

[0086] ⑧ Counterstaining the nuclei: Wash the cells treated in step ⑦ three times with phosphate buffer, 5 min each time. Add DAPI and stain for 3-5 min to stain the nuclei. Wash three more times with phosphate buffer, 5 min each time. Use absorbent paper to remove any remaining liquid from the confocal dish. Add anti-fluorescence quencher (glycerol) to mount the slide, ensuring the cells are fully immersed. Observe the cells under a laser confocal microscope and acquire images.

[0087] The method for detecting the expression of the target protein using Western blot is as follows:

[0088] ①Total protein extraction: After washing the treated cells with PBS, add protein lysis buffer, scrape off the cells, collect the cell lysis buffer and place it on ice for 30 min to fully lyse, shaking 3 times in between; then centrifuge the cell lysis buffer at 4℃ and 12000rpm for 15 min and collect the supernatant.

[0089] ② Total protein concentration determination and protein denaturation: The total protein concentration in the supernatant obtained in step ① was determined by the BCA method. 1 / 4 volume of 5× loading buffer was added to the supernatant, and the mixture was boiled in boiling water for 10 min to obtain the sample to be tested to denature the protein. After cooling, the sample was stored at -80℃.

[0090] ③ Electrophoresis, transfer and development: Prepare 10% to 12.5% ​​electrophoresis gel, perform electrophoresis on the sample to be tested, cut the gel according to the molecular weight, and transfer the protein on the gel to a PDVF membrane. Incubate with primary antibody at 4°C overnight; after incubation with secondary antibody, develop with ECL system.

[0091] Unless otherwise specified, all liver cancer cells (including Huh7 cells, SK-Hep-1 cells, and MHCC-97H cells) are cultured in DMEM medium containing 10% serum; the culture conditions are a constant temperature incubator containing 5% CO2 at 37°C; routine passage and subsequent experiments are performed when the cell density reaches about 85%, and the medium is changed every 1-2 days.

[0092] Analysis of lncRNA UPK1A-AS1

[0093] (1) Polyribosome profiling analysis showed that lncRNA UPK1A-AS1 was bound to ribosomes, suggesting that it may encode a protein or polypeptide. For example... Figure 1 As shown.

[0094] (2) Analysis of the lncRNA UPK1A-AS1 sequence using the online software ORF1 Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) revealed six open reading frames (ORFs) with ATG as the start codon and encoding amino acid sequences greater than 40 amino acids. These were named ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6. Figure 2 As shown.

[0095] (3) Based on different ORF sequences, design corresponding target fragment primers and add a Flag sequence to the reverse primer so that the 3' end of each ORF has a Flag tag (ORFs-Flag vector construction pattern diagram is shown in the figure). Figure 3(See Figure A). The primer sequences for each ORF are shown in Table 1. Using hepatocellular carcinoma cell line DNA or cDNA as templates, the target fragment was amplified using high-fidelity enzymes. The amplified target fragment and pcDNA3.1(+) plasmid were digested with restriction endonucleases, and then the digested target fragment was ligated with pcDNA3.1(+) plasmid using ligase. The resulting cells were transformed into E. coli, single clones were picked, cultured, sequenced, and the correctly sequenced plasmids were saved and named ORF1-Flag vector, ORF2-Flag vector, ORF3-Flag vector, ORF4-Flag vector, ORF5-Flag vector, and ORF6-Flag vector, respectively. The correctly sequenced plasmids were transfected into cells (recipient cells were SK-Hep-1 cells). The pcDNA3.1(+) plasmid containing only the Flag sequence (denoted as: Flag vector) was used as a control. The expression of ORFs was detected by Western blot and immunofluorescence. The detection results are shown below. Figure 3 As shown in Figures B and C, the results indicate that only ORF1 and ORF2 have encoding capabilities.

[0096] Table 1

[0097]

[0098]

[0099] (4) Design corresponding primers for the target fragments based on different ORF sequences. The primer sequences for each ORF are shown in Table 2. Using hepatocellular carcinoma cell line DNA or cDNA as templates, the target fragments were amplified using high-fidelity enzymes. The amplified target fragments and pEGFP-N1 plasmids were digested with restriction endonucleases, and then the digested target fragments were ligated with ligases to the pEGFP-N1 plasmids. The cells were transformed into E. coli, single clones were picked, cultured, sequenced, and the correctly sequenced plasmids were saved. Using a point mutation kit, the GFP start codon ATG in the vector was mutated to ATT to obtain the ORF1-GFP-Mut vector and the ORF2-GFP-Mut vector, respectively. The GFP start codon ATG in the ORFs and vectors was mutated to ATT to obtain the ORF1-Mut-GFP-Mut vector and the ORF2-Mut-GFP-Mut vector, respectively. The ORFs-GFP-Mut vector construction diagram is shown in Table 2. Figure 4 As shown in Figure A. The above vectors were transfected into cells (recipient cells were MHCC-97H cells). The pEGFP-N1 vector with an unmutated GFP start codon (denoted as GFP-wt vector) and the pEGFP-N1 vector with a mutated GFP start codon (denoted as GFP-Mut vector) without inserted ORF sequences were used as controls. The expression of ORFs was detected by Western blot and immunofluorescence. The detection results are as follows. Figure 4 Figures B and C in the middle show the results. The results indicate that the cells can express ORF1-Mut-GFP and ORF2-Mut-GFP, while the ORF1-Mut-GFP-Mut and ORF2-Mut-GFP-Mut sequences with start codon mutations lose their ability to encode peptides.

[0100] Table 2

[0101]

[0102] In summary, the ORF1 (MDRRQQSAAPTPKLATTKKEKPQKMAI QAPAKTSSLPETPISGYTRYWSAVTHMVSANRARPDSWTERWVRKVCSGPAALASGISTSK FLPHLESPLPSQ) and ORF2 (MRVSRVQERRDSPARQPTPTPQLPVSSGIKGDATWKGWSSGL AQRRCLVHLPSLDGAVIPRGGEHGHGHKNYKWARQGGSHLQSQHCGRPRREDCLSPGV) in lncRNA UPK1A-AS1 encode peptides, which are named LUP1 and LUP2, respectively.

[0103] Effects of LUP1 and LUP2 on the proliferation of liver cancer cells

[0104] MHCC-97H cells transfected with GFP-wt vector, ORF1-GFP-Mut vector, ORF2-GFP-Mut vector, ORF1-Mut-GFP-Mut vector, and ORF2-Mut-GFP-Mut vector, respectively, were used as the treatment subjects, at a ratio of 5 × 10⁻⁶. 3 Cells were seeded per well in a 96-well plate. After appropriate treatment, 100 μL of 50 μM EdU medium (diluted with DMEM medium at a ratio of 1000:1) was added to the 96-well plate and incubated for 2 h. The medium was discarded, and the cells were washed 1-2 times with PBS. The cells were fixed with 100 μL of 4% paraformaldehyde solution for 30 min, and the fixative was discarded. 50 μL of 2 mg / mL glycine solution was added, and the cells were incubated on a shaker for 5 min. The glycine solution was discarded, and the cells were washed once with PBS. 100 μL of permeabilizer was added and incubated for 10 min. The permeabilizer was discarded, and the cells were washed once with PBS. 100 μL of 1×Hoechst staining solution was added to stain the cell nuclei. Five fields of view were taken under a microscope, and the EdU positivity rate was calculated.

[0105] The specific treatments were as follows: 24 hours after cell seeding, cells were subjected to normoxic and hypoxic treatments for 12 hours, respectively. For normoxic treatment, liver cancer cells were placed in a closed cell incubator containing 95% air and 5% CO2, and then cultured in a constant temperature incubator at 37°C. For hypoxic treatment, liver cancer cells were placed in a closed cell incubator containing 1% O2, 5% CO2, and 94% N2, and then cultured in a constant temperature incubator at 37°C.

[0106] Test results as follows Figure 5 As shown, LUP1 and LUP2 can effectively promote the proliferation of liver cancer cells.

[0107] Preparation of polyclonal antibodies against lncRNA UPK1A-AS1

[0108] Comprehensive linear expression, hydrophilicity, immunogenicity, and epitope exposure data (e.g.) Figures 6 to 13 As shown in the diagram, a peptide route was selected for antibody preparation. The recognition site for LUP1 is: NRDRPDSWTERWVRKVC (number: WG-03528, SEQ ID NO.1); the recognition site for LUP2 is: RVSRGQERRDSPARQPT (number: WG-03529, SEQ ID NO.2). Attempts were made to prepare antibodies using the full-length peptides of ORF1 and ORF2, but it was difficult to purify the relevant peptides for subsequent immunization.

[0109] The peptides for the aforementioned recognition sites were synthesized by Wuhan Aiboteke Biotechnology Co., Ltd., and dissolved in PBS buffer. These peptides were then conjugated to hemocyanin (KLH) via thiol groups and used to immunize two experimental-grade white rabbits. After transcross immunization and rabbit sacrifice, affinity-purified antibodies (Anti-LUP1 antibody and Anti-LUP2 antibody) were finally obtained.

[0110] Affinity purification can be performed using the LUP1 recognition site peptide as an antigen to obtain purified Anti-LUP1 antibody, and using the LUP2 recognition site peptide as an antigen to obtain purified Anti-LUP2 antibody. Other affinity purification methods known in the art can also be used to purify polyclonal antibodies.

[0111] The immunization procedure for experimental-grade white rabbits is shown in Table 3.

[0112] Table 3

[0113]

[0114]

[0115] The results of ELISA and Western blot analysis of antiserum obtained after blood collection from immunized animals are as follows: Figures 14 to 17 As shown. Among them, G-03528 is LUP1, and WG-03529 is LUP2.

[0116] The results showed that the antibody concentrations in the serum of rabbits E8135 and E8136 both reached above 1 mg / mL, and the titers were high.

[0117] The binding effects of Anti-LUP1 and Anti-LUP2 antibodies on LUP1 and LUP2

[0118] (1) By analyzing the conservation of peptides LUP1 and LUP2 among primate species (humans, gorillas, and northern white-cheeked gibbons), it was found that LUP1 and LUP2 are well conserved in primates. The results are as follows: Figure 18 As shown.

[0119] (2) Huh7 cells, SK-Hep-1 cells, and MHCC-97H cells transfected with ORF1-Flag vector, ORF2-Flag vector, and Flag vector, respectively, were used as the detection objects. The expression of ORF1 and ORF2 in different liver cancer cells was detected by Western blot (primary antibody was Anti-LUP1 antibody or Anti-LUP2 antibody, secondary antibody was Anti-Rabbit IgG HRP-Linked Antibod); β-actin was used as an internal control. Among them, the unlabeled CoCl2 treatment group was cultured in a constant temperature incubator containing 95% air and 5% CO2 at 37℃ for 24h; the CoCl2 treatment group was treated with 150μM CoCl2 for 24h to simulate the hypoxic microenvironment, and the culture environment was a constant temperature incubator containing 95% air and 5% CO2 at 37℃.

[0120] The results are as follows Figure 19 As shown.

[0121] Anti-LUP1 antibody has a good recognition effect on ORF1; Anti-LUP2 antibody also has a good recognition effect on ORF2; Anti-LUP1 antibody and Anti-LUP2 antibody can be used to detect the expression of ORF1 (LUP1) and ORF2 (LUP2), respectively.

[0122] (3) SK-Hep-1 cells and MHCC-97H cells before and after specifically interfering with UPK1A-AS1 expression (stable transfected with lentiviruses Si-AS1-2 (sequence 5'-GTGAGCAGAGGCCAGGAGAGA-3') and lentiviruses si-AS1-3 (sequence 5'-ATGGCCATAAACATTACAAAT-3'), respectively) were used as detection objects. The expression of ORF1 and ORF2 in different hepatocellular carcinoma cells was detected by Western blot (primary antibody was the Anti-LUP1 antibody or Anti-LUP2 antibody prepared by affinity purification above, and secondary antibody was Anti-Rabbit IgG HRP-Linked Antibody) and immunofluorescence (primary antibody was the Anti-LUP1 antibody or Anti-LUP2 antibody prepared by affinity purification above, and secondary antibody was Anti-Rabbit IgG HRP-Linked Antibody). β-actin was used as an internal control.

[0123] The results are as follows Figure 20 As shown.

[0124] After reducing the expression of endogenous UPK1A-AS1, the expression of LUP1 and LUP2 was also downregulated, which further demonstrates the high specificity of the prepared Anti-LUP1 and Anti-LUP2 antibodies.

[0125] (4) After digesting and resuspending the liver cancer cells, the cells were seeded at 80% cell density in 10cm culture dishes (the cell culture medium was DMEM cell culture medium containing 5% BSA). The cells were harvested the next day, washed twice with PBS, and an appropriate amount of Western lysate and IP cell lysis buffer (Beyotime, P0013) containing protease and phosphatase inhibitors was added. The cells were lysed on ice for 30 min, and the cell lysis buffer was slowly resuspended every 10 min. Then, the cells were centrifuged at 14000g for 5 min, and the supernatant was collected. The corresponding primary antibody (Anti-LUP1 antibody, Anti-LUP2 antibody or IgG) was added, and the cells were incubated overnight on a rotating shaker at 4℃. An appropriate amount of Protein A / GA garose was added, and the cells were incubated for 1 h to 4 h. The proteins that were not bound to the antibody and magnetic beads were washed with pre-cooled PBS, and the proteins that were bound to the antibody and magnetic beads were washed with 2× loading buffer. The cells were then subjected to silver staining. The target band (the band at the recognition position of the corresponding polyclonal antibody) was excised under sterile conditions and sent to the company for mass spectrometry analysis.

[0126] The silver staining results and mass spectrometry detection results are as follows: Figure 21 As shown.

[0127] Liver cancer cells endogenously express ORF1 and ORF2, and Anti-LUP1 and Anti-LUP2 antibodies can recognize the endogenously expressed ORF1 and ORF2, respectively.

[0128] Therapeutic effects of Anti-LUP1 and Anti-LUP2 antibodies on hepatocellular carcinoma in mice

[0129] BALB / c nude mice were provided by the Experimental Animal Center of Southern Medical University and housed in the SPF-grade animal room of Southern Medical University Southern Hospital. The cages and utensils were strictly disinfected, and the mice had free access to food and purified water. The nude mice were observed for clinical symptoms before the experiment.

[0130] MHCC-97H human hepatocellular carcinoma cells were cultured to the logarithmic growth phase, digested with trypsin, centrifuged at 800 rpm for 3 min, washed three times with PBS, and the cell number was adjusted to 1 × 10⁻⁶ cells / year. 8 100 μL of hepatocellular carcinoma cell suspension was injected into the back of BALB / c nude mice (4-6 weeks old, male) at a concentration of 100 cells / mL. Tumors in the tumor-bearing mice grew to approximately 100 mm. 3 ~150mm 3 After measuring the body weight and tumor volume of each mouse and eliminating mice with outliers in terms of weight and tumor volume, the tumor-bearing mice were randomly divided into three groups (n=6 per group): an IgG treatment group (PBS+IgG), an Anti-LUP1 antibody treatment group (Anti-LUP1), and an Anti-LUP2 antibody treatment group (Anti-LUP2). Based on mouse body weight, the IgG treatment group received 100 μg / mouse of IgG antibody via intraperitoneal injection every other day. The Anti-LUP1 and Anti-LUP2 antibody treatment groups received 100 μg / mouse of Anti-LUP1 and Anti-LUP2 antibody via intraperitoneal injection every other day, respectively. The antibody solution was PBS (pH 7.3) containing 50% glycerol. Tumor volume was measured daily using calipers, and mouse body weight was measured. The mice's mental state, skin condition, and food intake were observed and recorded daily. The results were statistically analyzed using a two-way ANOVA method. The specific experimental procedure is as follows: Figure 22 As shown in Figure A.

[0131] Experimental results are as follows Figure 22 and Figure 23 As shown.

[0132] Treatment with Anti-LUP1 and Anti-LUP2 antibodies significantly inhibited tumor proliferation and reduced tumor weight and volume (P<0.5), while having no significant effect on mouse body weight, and no side effects were observed during drug treatment.

[0133] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An antigenic polypeptide produced by translation of lncRNA UPK1A-AS1, characterized in that, The amino acid sequence of the antigenic polypeptide is shown in SEQ ID NO.

2.

2. An immunogen, characterized in that, Includes the conjugate formed by the antigen polypeptide and the carrier protein as described in claim 1; The carrier protein is at least one of serum albumin, hemocyanin, ovalbumin, thyroglobulin, cholera toxin B subunit, Escherichia coli unstable toxin B subunit, diphtheria toxoid, and tetanus toxoid.

3. A biomaterial, characterized in that, Including at least one of 1) to 4), 1) A nucleic acid molecule encoding the antigenic polypeptide of claim 1; 2) An expression cassette containing the nucleic acid molecules described in 1); 3) A recombinant vector comprising the nucleic acid molecule described in 1) or the expression cassette described in 2); 4) A recombinant host cell containing the nucleic acid molecule described in 1), the expression cassette described in 2), or the recombinant vector described in 3).

4. A method for preparing a polyclonal antibody, characterized in that, Includes the following steps: Animals were immunized with the immunogen described in claim 2 to obtain polyclonal antibodies.

5. The preparation method according to claim 4, characterized in that, In the preparation method, the number of immunizations is 5 to 6.

6. A polyclonal antibody, characterized in that, The polyclonal antibody is prepared by the preparation method according to any one of claims 4 or 5.

7. The use of the polyclonal antibody according to claim 6 in the preparation of a medicament for the prevention or treatment of liver cancer.

8. A drug for the prevention or treatment of liver cancer, characterized in that, The drug includes the polyclonal antibody as described in claim 6.

9. The use of the polyclonal antibody of claim 6 in the preparation of a kit for detecting peptides encoded by lncRNA UPK1A-AS1.

10. A reagent kit, characterized in that, The kit includes the polyclonal antibody as described in claim 6.

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