Monoclonal nanoantibody for recognizing Epstein-Barr virus gp42 protein and its application and monoclonal nanoantibody library
The camel nanoantibody library was constructed through phage display technology, and monoclonal nano-antibody that specifically recognized the EB virus gp42 protein was screened, which solved the problem of lack of effective antibodies against the EB virus gp42 protein in the prior art, achieved high affinity and specific antibody development, and had broad clinical application value.
Patent Information
- Application Number
- CN202510075055.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art has failed to effectively develop specific monoclonal nano-antibody against the EB virus gp42 protein, resulting in a lack of effective prevention, diagnosis and treatment methods.
Through phage display technology, total RNA extracted from alpaca lymphocytes was reverse transcription and PCR amplification, and the gene fragment of the variable region of the camel nanoantibodies was obtained, and the camel nanoantibodies were ligated through molecular cloning to build the camel nanoantibodies library. Finally, a single-clonal nanoantibodies specifically recognized the EB virus gp42 protein were obtained through high-throughput ELISA screening.
Three monoclonal nano-antibody with high affinity and specific recognition of EB virus gp42 protein were obtained, which can be widely used to detect the expression of EB virus gp42 protein, diagnose the occurrence and development of EB virus pathogenic tumors, evaluate the efficacy of drugs for treating EB virus pathogenic tumors, and treat EB virus pathogenic tumors.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of phage display of nano antibodies, in particular to a monoclonal nano antibody for recognizing Epstein-Barr virus gp42 protein, an application thereof and a monoclonal nano antibody library. Background Art
[0002] Epstein-Barr virus (Epstein-Barr virus) is a member of the genus Lymphotropic virus of the family Herpesviridae. It is one of the earliest identified oncogenic viruses and also the virus with the widest range of infection. More than 90% of adults worldwide have been infected with Epstein-Barr virus, and most people will become virus secretors for life after being infected with the virus without any clinical symptoms. However, Epstein-Barr virus may be activated under certain circumstances, leading to more than 1%-1.5% of malignant cancers worldwide, including various lymphomas (Hodgkin lymphoma, Burkitt lymphoma) and epithelial cell cancers (nasopharyngeal carcinoma and 10% of gastric cancer). Therefore, research on this virus is of great significance, but there is still no effective vaccine or targeted treatment for Epstein-Barr virus.
[0003] Epstein-Barr virus is an enveloped virus that infects host cells by membrane fusion of surface membrane proteins and host cells. Studies have found that the surface glycoprotein gp42 of Epstein-Barr virus plays an indispensable role in the process of Epstein-Barr virus infecting B cells. Epstein-Barr virus has the characteristics of being tropistic for B lymphocytes, and can establish latent infection in B lymphocytes, stimulating cell proliferation and transformation. During the infection process, gp42 participates in key links such as the binding of the virus to B cells and membrane fusion. When Epstein-Barr virus approaches B cells, gp42 interacts with specific molecules on the surface of B cells (such as HLA class II molecules) to initiate the fusion program between the virus and the cell, allowing the genetic material of the virus to enter the interior of the B cell, thereby establishing an infection state. gp42 also works synergistically with other Epstein-Barr virus proteins. This synergistic effect helps the virus interact with host cells more effectively and improves the success rate of infection.
[0004] Existing studies have also found that monoclonal antibodies that bind to different epitopes on gp42 can prevent EB virus from fusing with host cells and can be used to treat diseases caused by EB virus infection. At the same time, antibodies against gp42 protein can also be used to detect the presence or level of gp42 protein in serum and tissues, thereby detecting EB virus and diagnosing diseases caused by EB virus infection, and can also prevent EB virus infection.
[0005] Phage display technology refers to the technology of fusing exogenous proteins or peptides with specific phage capsid proteins and displaying them on the surface of phages. This technology can simulate the process of natural selection, realize high-throughput screening of target molecule-specific receptors, and perfectly combine the genotype and phenotype of the screened protein molecules. It is often used in the screening of monoclonal antibodies against different target proteins. Nanobodies refer to a class of heavy chain single-domain antibodies. Compared with other antibodies, the light chain is naturally missing and the size is only one-tenth of the traditional IgG antibody. Therefore, it has the advantages of high penetration, good stability, and simple humanization. It is widely used in the research and development of antibody drugs or the clinical diagnosis of diseases. Common nanobodies are mainly derived from camels and certain cartilaginous fish.
[0006] Therefore, the development and screening of specific monoclonal nanoantibodies against gp42 protein has important clinical significance for the prevention, diagnosis and treatment of related diseases caused by EB virus infection. Summary of the invention
[0007] In view of the deficiencies of the above prior art, the present invention provides a monoclonal nano antibody that recognizes Epstein-Barr virus gp42 protein, its application, and a monoclonal nano antibody library. The present invention first extracts the total RNA of alpaca lymphocytes (PBMCs) immunized with Epstein-Barr virus gp42 protein, and synthesizes cDNA by a reverse transcription kit; then PCR amplification is performed using the cDNA as a template to obtain the variable region gene fragment of the camel-derived nano antibody; the variable region gene fragment of the camel-derived nano antibody is connected to the phage display vector by molecular cloning, and transformed into the host bacteria competent state, and a phage display camel-derived nano antibody library is successfully constructed; using the camel-derived nano antibody library, with Epstein-Barr virus gp42 protein as the target protein, through in vitro phage display antibody enrichment and panning and high-throughput ELISA screening, finally 3 camel-derived monoclonal nano antibody sequences with high affinity and specific recognition of Epstein-Barr virus gp42 protein are obtained. It is specifically achieved by the following technologies.
[0008] In a first aspect of the present invention, a monoclonal nanobody that recognizes Epstein-Barr virus gp42 protein is provided, which is monoclonal nanobody EBV-gp42-1023, EBV-gp42-1024 or EBV-gp42-1025;
[0009] The amino acid sequence of the heavy chain complementary determining region CDR1 of the monoclonal nanobody EBV-gp42-1023 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.3;
[0010] The amino acid sequence of the heavy chain complementary determining region CDR1 of the monoclonal nanobody EBV-gp42-1024 is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.7;
[0011] The amino acid sequence of the heavy chain complementary determining region CDR1 of the monoclonal nanobody EBV-gp42-1025 is shown in SEQ ID NO.9, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.10, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.11.
[0012] Furthermore, according to claim 1, the monoclonal nanoantibody that recognizes the Epstein-Barr virus gp42 protein is characterized in that the amino acid sequence of the monoclonal nanoantibody EBV-gp42-1023 is as shown in SEQ ID NO.4; the amino acid sequence of the monoclonal nanoantibody EBV-gp42-1024 is as shown in SEQ ID NO.8; the amino acid sequence of the monoclonal nanoantibody EBV-gp42-1025 is as shown in SEQ ID NO.12.
[0013] Furthermore, the gene sequence encoding the nanobody EBV-gp42-1023 is shown as SEQ ID NO.13, the gene sequence encoding the nanobody EBV-gp42-1024 is shown as SEQ ID NO.14, and the gene sequence encoding the nanobody EBV-gp42-1025 is shown as SEQ ID NO.15.
[0014] The present invention has carried out recombinant expression and related detection on the above three monoclonal nanobodies provided. Through ELISA detection, the three monoclonal nanobodies we obtained can specifically recognize human Epstein-Barr virus gp42 protein.
[0015] The second aspect of the present invention provides a biomaterial, wherein the biomaterial comprises any one of the following:
[0016] (1) A nucleic acid molecule encoding the monoclonal Nanobody described in any one of the above;
[0017] (2) A recombinant vector comprising a gene fragment encoding any one of the above-mentioned monoclonal nanobodies, or comprising the above-mentioned nucleic acid molecule;
[0018] (3) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express the monoclonal nanobody described in any one of the above;
[0019] (4) An antibody derivative, comprising any one of the monoclonal nanobodies described above.
[0020] The third aspect of the present invention provides a product, which is a pharmaceutical composition or a detection product;
[0021] The pharmaceutical composition contains any one of the above-mentioned monoclonal nanoantibodies or derivatives of monoclonal nanoantibodies;
[0022] The detection product comprises any one of the above-mentioned monoclonal nanoantibodies or the above-mentioned biological materials.
[0023] Furthermore, the detection product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.
[0024] The fourth aspect of the present invention provides an application of the monoclonal nanobody for recognizing Epstein-Barr virus gp42 protein as described in any one of the above, wherein the application includes any one of the following aspects:
[0025] (1) Used for detecting the expression of EB virus gp42 protein for purposes other than disease diagnosis and treatment, or for detecting the occurrence and development of EB virus-induced tumors, or for evaluating the efficacy of drugs for treating EB virus-induced tumors;
[0026] (2) Products used to detect the expression of EB virus gp42 protein, or to diagnose EB virus-induced tumors, or to evaluate the efficacy of drugs for treating EB virus-induced tumors;
[0027] (3) Used for the preparation of drugs for treating Epstein-Barr virus-induced tumors.
[0028] In a fifth aspect of the present invention, a nanoantibody library is provided for screening the monoclonal nanoantibodies described in any one of the above items; the nanoantibody library is obtained by extracting total RNA from natural camel-derived PBMCs cells, performing reverse transcription and PCR amplification to obtain the gene sequence of the antibody variable region VHH; the gene sequence of the antibody variable region VHH is connected to a phage expression vector and transformed into competent cells.
[0029] Compared with the prior art, the advantages of the present invention are as follows: the present invention utilizes phage display technology to provide a monoclonal nanoantibody (3 in total) that specifically recognizes Epstein-Barr virus gp42. These nanoantibodies have very high affinity and specificity for Epstein-Barr virus gp42 protein and can be widely used to detect the expression of Epstein-Barr virus gp42 protein, or to detect the occurrence and development process of Epstein-Barr virus-pathogenic tumors, or to evaluate the efficacy of drugs for treating Epstein-Barr virus-pathogenic tumors, or to treat Epstein-Barr virus-pathogenic tumors. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The electrophoresis results of total RNA extracted from PBMCs cells after animal immunization in Example 1.
[0031] Figure 2 The agarose gel electrophoresis result of the first round of PCR products in Example 1 is shown.
[0032] Figure 3 The agarose gel electrophoresis result of the second round of PCR products in Example 1 is shown.
[0033] Figure 4 The following are the results of Protein A resin purification of three nanobodies. DETAILED DESCRIPTION
[0034] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0035] Example 1: Construction of a camel-derived specific nanoantibody library recognizing Epstein-Barr virus gp42, in vitro enrichment and screening of nanoantibodies
[0036] 1. Construction of camel-derived phage nanoantibody library
[0037] (1) Animal immunization
[0038] A 2-year-old female alpaca was purchased (provided by Qingdao Kangda Aibo Biotechnology Co., Ltd.) and immunized with Epstein-Barr virus gp42 protein (AntibodySystem). 1000 μg of Epstein-Barr virus gp42 was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points in the alpaca. Immunization was performed every 2 weeks, and venous blood was collected from the alpaca 7 days after each immunization, and the serum titer was determined by indirect ELISA.
[0039] After 4 rounds of immunization, 80 ml of venous blood was extracted and PBMCs were isolated from camel blood using Tianjin Haoyang's camel peripheral blood mononuclear cell isolation kit (Cat. No. LDS1078). The cells were added to 1 ml of trizol solution (Invitvogen, Cat. No. 15596018) and lysed using Thermo's PureLink TMThe total RNA of the cells was extracted using RNA extraction kit (Cat. No. 12183020) and detected by 1% agarose gel electrophoresis. Figure 1 shown.
[0040] (2) Total RNA from natural alpaca PBMCs cells was selected as a template and reverse transcribed using Vazyme's HiScript®III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Cat. No.: R312-02) to obtain cDNA;
[0041] Using cDNA as a template and Vazyme's Phanta® Max Super-Fidelity DNA Polymerase (Cat. No.: P505-d3), the gene sequence of the nanobody heavy chain variable region VHH was obtained through two rounds of PCR amplification.
[0042] The two-round PCR reaction systems are shown in Tables 1 and 2 below, and the PCR reaction procedures are shown in Table 3 below.
[0043] Table 1 First round PCR reaction system
[0044] Table 2 Second round PCR reaction system
[0045] Table 3 PCR reaction procedure
[0046] The results of 1% agarose gel electrophoresis of the first round of PCR products are as follows Figure 2 As shown in Figure 2, DNA fragments of about 740 bp to 630 bp were recovered. The results of 1% agarose gel electrophoresis of the second round of PCR products are shown in Figure 2. Figure 3 As shown, a DNA fragment of about 450 bp was recovered.
[0047] (3) The target fragment obtained by PCR and the vector pCANTAB5E (Amersham biosciences, phage display system) were digested with SfiI double site restriction enzyme (digestion conditions: 50℃ for 5-6 h), and then ligated with T4 ligase (NEB) at 16℃ overnight (ligation conditions: 16℃ for 15 h; 65℃ for 10 min inactivation) to the vector pCANTAB 5E. The restriction enzyme digestion system of the vector and DNA target fragment is shown in Table 4 below, and the ligation system is shown in Table 5 below.
[0048] Table 4 Enzyme digestion system of vector and target DNA fragment
[0049] Table 5 Connection system
[0050] (4) The ligation product was transformed into TG1 electrocompetent cells (Lucigen) by electroporation, plated on 2YT-Amp-Glucose solid culture medium (same recipe as below), and cultured at 37°C overnight.
[0051] The formula of the 2YT solid culture medium is: 1.6% (W / V) Tryptone, 1% (W / V) Yeast Extract, 0.5% (W / V) NaCl, dissolved in 1 L of distilled water.
[0052] The formula of the 2YT-Amp-Glucose culture medium is: 100 μg / ml of ampicillin and 2% glucose are added to the 2YT solid culture medium.
[0053] (5) Scrape the colonies on the culture medium, mix them evenly, add glycerol to a final concentration of 15%, and store them at -80°C; inoculate the bacterial solution into 2YT-Amp-Glucose medium and culture at 37°C for 1-2 h until the OD reaches 0. 600 =0.4-0.6.
[0054] Add helper phage and culture at 37°C for 45 min-1 h; centrifuge the bacterial solution at 3000-5000 rpm, discard the supernatant, resuspend the bacteria with an equal volume of 2YT-Amp-Kan medium, and culture overnight at 30°C; the next day, transfer the supernatant into a new centrifuge tube by centrifugation; add 1 / 4 volume of 5×PEG / NaCl solution, mix thoroughly, place on ice or in a 4°C environment, and let stand for 1-2h; then centrifuge and discard the supernatant, resuspend the precipitate with about PBS, and finally obtain a monoclonal nanoantibody library, which is stored at 4°C or -20°C.
[0055] The formula of the 2YT-Amp-Kan culture medium is as follows: 100 μg / ml of ampicillin and 50 μg / ml of kanamycin are added to the 2YT culture medium.
[0056] Example 2: In vitro enrichment and high-throughput monoclonal screening of camel-derived nanoantibodies (VHH) sequences specific for Epstein-Barr virus gp42 protein
[0057] 1. Experimental reagents and materials
[0058] The experimental reagents and materials are shown in Table 6 below.
[0059] Table 6
[0060] 2. Immunotube solid phase panning
[0061] (1) Coating: Coat the immunotubes with 50 μg / ml antigen Epstein-Barr virus gp42 protein, 1 ml per tube, overnight at 4°C; wash the immunotubes 3 times with 5 ml PBST.
[0062] (2) Blocking: 5 ml 5% skim milk / PBST, block at 30℃ for 1 h. Wash twice with 5 ml PBS.
[0063] (3) Incubation: Add 1 ml to the immunotube, with a total volume of 1-2×10 12 pfu of nanolibrary phages. Incubate at 30°C for 1.5h.
[0064] (4) Washing: Wash 6-8 times with 5 ml PBST.
[0065] (5) Elution: Add 1 ml of Gly-HCl (pH=2.2) to the immunotube to elute the phage, and incubate at room temperature with shaking for about 6-8 minutes. Add Tris-HCl (pH=9.6) to neutralize the solution to pH=7.0-8.0.
[0066] 3. Determine the titer of phage after elution
[0067] (1) Cultivate E. coli TG1 until OD 600 = 0.4-0.6. Mix 10 μL of diluted eluted phage and 190 μL of E. coli TG1.
[0068] (2) After the mixture was incubated at 37°C for 30 min, it was poured onto 2×YT-A (Amp 100 μg / ml) solid culture medium and cultured at 37°C overnight.
[0069] 4. Amplification of eluted phage
[0070] (1) Directly aspirate 800 μl of the eluted phage solution and add it to 10 ml of the logarithmic phase TG1 bacterial culture. Incubate at 37°C for 30 min and incubate at 220 rpm for 30 min.
[0071] (2) Add the bacterial solution to 30 ml of 2YT-Amp-Glucose medium and culture at 37°C and 220 rpm until the OD is about 0.4-0.6. Add 3×10 11 pfu helper phage, incubate at 37°C for 30 min, and then culture at 220 rpm for 45 min-1 h.
[0072] (3) Centrifuge the bacterial solution at 3000-5000 rpm and discard the supernatant. Resuspend the bacteria in an equal volume of 2YT-Amp-Kan medium. Incubate overnight at 30℃ and 220 rpm.
[0073] (4) The next day, centrifuge the bacterial solution at 8000 rpm, 4°C, for 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5×PEG / NaCl solution. After thorough mixing, place on ice or at 4°C for 1-2 h. Centrifuge and discard the supernatant, and resuspend the precipitate in about 1 ml of PBS. Centrifuge to precipitate impurities and transfer the supernatant to a new centrifuge tube.
[0074] 5. Test the titer of amplified phage
[0075] The test method is the same as step 2. Repeat steps 1 to 4 to perform the second to fourth rounds of enrichment panning. The specific panning method is shown in Table 7 below.
[0076] Table 7
[0077] The results of in vitro enrichment and panning are shown in Table 8 below.
[0078] Table 8
[0079] 6. Polyclonal phage ELISA detection
[0080] (1) Coating: Coat the immunoplate with 4 μg / ml Epstein-Barr virus gp42 protein, 100 μl, at 4°C overnight. Coat the control wells with 100 μl PBS; wash three times with 300 μl PBST.
[0081] (2) Blocking: Block with 300 μl 5% skim milk / PBST at 30°C for 1 h; wash 2-3 times with 300 μl PBST.
[0082] (3) Incubation: Dilute the phage after each round of amplification with PBS, and increase the dilution factor by three times. The initial concentration is 10 12 pfu / ml. Add 100 μl of diluted amplification phage to each well and incubate at 30℃ for 1 h.
[0083] (4) Washing: Wash three times with 300 μl PBST.
[0084] (5) Secondary antibody: Add 100 μl of secondary antibody diluent (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.
[0085] (6) Washing: Wash three times with 300 μl PBST.
[0086] (7) Color development: Add 100 μl of TMB color development solution and color for 3-8 min in the dark, then add 100 μl of 2M HCl to terminate the reaction. Read the result on a microplate reader (450 nm-620 nm). The results of the polyclonal phage ELISA test are shown in Table 9 below.
[0087] Table 9
[0088] 7. Monoclonal phage ELISA screening
[0089] (1) Select an appropriate round number (the fourth round), dilute the eluted phage to an appropriate concentration, infect TG1 in the logarithmic phase, and plate it.
[0090] (2) The next day, 192 single clones were picked from the plate and inoculated into two 96-deep-well plates (600 μl of 2YT-Amp-Glucose medium was added to each well) and cultured at 37°C and 250 rpm for 2 h until the OD value of the bacterial solution was 0.4-0.6.
[0091] Pipette 100 μl of bacterial solution into each well of the cell culture plate, add sterile glycerol (final concentration 20%-25%) to each well, mix well and store at -20°C.
[0092] (3) Add helper phage to the remaining 96-well plate culture medium. The amount of helper phage added = (0.6 × liquid volume × 5 × 10 8 ×30) pfu. Incubate at 37°C for 30 min and shake at 250 rpm for 45 min-1 h at 37°C.
[0093] (4) Centrifuge the 96-well plate at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial solution in each well with 600 μl of 2YT-Amp-Kan medium and culture overnight at 30°C and 250 rpm with shaking.
[0094] (5) The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 min and take the supernatant for ELISA experiment.
[0095] (6) Coating: Coat the immunoplate with 4 μg / ml Epstein-Barr virus gp42 protein, 100 μl, overnight at 4°C. Coat the control wells with 100 μl PBS. Wash three times with 300 μl PBST.
[0096] (7) Blocking: Block with 300 μl 5% skim milk / PBST at 30°C for 1 h. Wash 2-3 times with 300 μl PBST
[0097] (8) Incubation: Add 100 μl of supernatant phage to each well and incubate at 30°C for 1 h.
[0098] (9) Washing: Wash three times with 300 μl PBST.
[0099] (10) Secondary antibody: Add 100 μl of secondary antibody diluent (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.
[0100] (11) Washing: Wash three times with 300 μl PBST.
[0101] (12) Color development: Add 100 μl of TMB colorimetric solution and color for 3-8 min in the dark. Add 100 μl of 2M HCl to terminate the reaction. Read the result with an ELISA reader (450 nm-620 nm).
[0102] The results of the ELISA test of monoclonal phage No. 1-192 are shown in Tables 10-13 below.
[0103] Table 10 ELISA test results of monoclonal phage No. 1-48
[0104] Table 11 ELISA test results of monoclonal phage No. 49-96
[0105] Table 12 ELISA test results of monoclonal phage No. 97-144
[0106] Table 13 ELISA test results of monoclonal phage No. 145-192
[0107] According to the above table results, OD 600 (Antigen Group)-OD 600 (Control group) those greater than 1 were defined as positive clones, and these positive clones were sequenced to exclude erroneous antibody sequences and repeated antibody sequences. Finally, three high-affinity antibody sequences were obtained, namely high-affinity nanoantibodies that specifically recognize Epstein-Barr virus gp42 protein, named EBV-gp42-1023, EBV-gp42-1024, and EBV-gp42-1025, and sequenced.
[0108] The amino acid sequence of the heavy chain complementary determining region CDR1 of the nanobody EBV-gp42-1023 is GFTFSSYW, as shown in SEQ ID NO.1; the amino acid sequence of the heavy chain complementary determining region CDR2 is IHPGDGRT, as shown in SEQ ID NO.2; the amino acid sequence of the heavy chain complementary determining region CDR3 is NAPALDS, as shown in SEQ ID NO.3.
[0109] The amino acid sequence of the nanobody EBV-gp42-1023 is:
[0110] QVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYW MSWVRQAPGKGLEWVSF IHPGDGRT TYADSVKGRFTISTDNARNTLYLQMNNLKPEDTAVYYC NAPALDS WGQGTQVTVSS, as shown in SEQ ID NO.4.
[0111] The amino acid sequence of the heavy chain complementary determining region CDR1 of the nanobody EBV-gp42-1024 is GRAFSSGG, as shown in SEQ ID NO.5; the amino acid sequence of the heavy chain complementary determining region CDR2 is ISWNGGRT, as shown in SEQ ID NO.6; the amino acid sequence of the heavy chain complementary determining region CDR3 is HAFGIKPF, as shown in SEQ ID NO.7.
[0112] The amino acid sequence of the nanobody EBV-gp42-1024 is:
[0113] QVQLVESGGGLVQAGGSLRLSCTAS GRAFSSGG MAWFRQAAGKEREFLAD ISWNGGRT YYADSVKGRFTISRDITKNTFYLQMNSLQSEDTAVYYC HAFGIKPF WGQGIQVTVSS, as shown in SEQ ID NO.8.
[0114] The amino acid sequence of the heavy chain complementary determining region CDR1 of the nanobody EBV-gp42-1025 is GFSLDYYA, as shown in SEQ ID NO.9; the amino acid sequence of the heavy chain complementary determining region CDR2 is INSGSSST, as shown in SEQ ID NO.10; the amino acid sequence of the heavy chain complementary determining region CDR3 is HALRMGA, as shown in SEQ ID NO.11.
[0115] The amino acid sequence of the nanobody EBV-gp42-1025 is:
[0116] QVQLVESGGGLVQPGGSLRLSCTAS GFSLDYYA ISWFRQAPGKGLEWVSE INSGSSST HYADSVKGRFTISRDNAKNTVDLQMDNLKPDDTAVYYC HALRMGA WGQGTQVTVSS, as shown in SEQ ID NO.12.
[0117] The gene sequence encoding the nanobody EBV-gp42-1023 is:
[0118] caggtgcagctcgtggagtcagggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcaccttcagtagctactggatgtcttgggtccgtcaggctccagggaagggactcgagtgggtctcatttattcatcctggtgacggtaggac aacttatgcagactccgtgaagggccggttcaccatctccacagacaacgccaggaacacactgtatctgcaaatgaacaacctgaaacctgaggacacggccgtgtattactgtaacgcgcccgcgctggattcctggggccaggggacccaggtcaccgtctcctca, as SEQ Shown as ID NO.13.
[0119] The gene sequence encoding the nanobody EBV-gp42-1024 is:
[0120] caggtgcagctcgtggagtcggggggagggttggtgcaggctgggggctctctgagactctcctgtacagcctctggacgcgctttcagtagcggtggcatggcgtggttccgccaggctgcagggaaggagcgtgagtttctagcagatattagctggaatggcggtagaacatattatgcagactccgtgaagggccgattcaccatttcgagagacattaccaagaacaccttctatttgcaaatgaacagtctgcagtccgaagacacggccgtttattattgtcacgcgtttgggataaaacccttctggggccaggggatccaggtcaccgtctcctca, as shown in SEQ ID NO.14.
[0121] The gene sequence encoding the nanobody EBV-gp42-1025 is:
[0122] caggtgcagctcgtggagtcagggggaggcttggtgcagcctggggggtctctgagactctcctgtacagcctctggattcagtttggattattatgccataagctggttccgccaggccccagggaaggggctcgagtgggtctcagaaattaatagcggtagtagcagcacacactatgcagactccgtgaagggccgatttaccatctccagagacaacgccaagaacacggtcgatcttcaaatggacaacctgaaacctgacgacacggccgtctattactgtcatgcattgcgaatgggtgcctggggccaggggacccaggtcaccgtctcctcg, as shown in SEQ ID NO.15.
[0123] Example 3: Recombinant expression of EBV-gp42-1023, EBV-gp42-1024 and EBV-gp42-1025 of monoclonal nanobodies
[0124] The gene sequences of the three nanobodies obtained by screening were subcloned into the pATX2 vector (ATAGENIX), with restriction sites EcoR1 / Not1 and human IgG Fc added to the C-terminus. Xten CHO cells were transfected. After 72 h of transfection, the cells were removed by centrifugation, and the culture supernatant was purified using Protein A resin (12% Non-reduced SDS-PAGE), as shown in Figure 4 shown.
[0125] Example 4: Identification of monoclonal nanobodies EBV-gp42-1023, EBV-gp42-1024 and EBV-gp42-1025
[0126] 1. ELISA detection of monoclonal nanoantibodies EBV-gp42-1023, EBV-gp42-1024 and EBV-gp42-1025
[0127] (1) Coating: Coat the immunoplate with 5 μg / ml Epstein-Barr virus gp42 protein, 100 μl, overnight at 4°C. Coat the control wells with 100 μl PBS; wash three times with 300 μl PBST.
[0128] (2) Blocking: Block with 300 μl 3% BSA / PBS at 37°C for 1.5 h; wash 2-3 times with 300 μl PBST.
[0129] (3) Incubation: Dilute the monoclonal nanoantibody with PBS in 5-fold increments. The initial concentration is 10 μg / ml. Add 100 μl of the diluted nanoantibody to each well. Incubate at 37°C for 1 h.
[0130] (4) Washing: Wash three times with 300 μl PBST.
[0131] (5) Secondary antibody: Add 100 μl of secondary antibody diluent (Goat anti Human (H+L)-HRP, 1:5000) (AntibodySystem, Catalog No. PHB96431) and incubate at 37°C for 30 min.
[0132] (6) Washing: Wash three times with 300 μl PBST.
[0133] (7) Color development: Add 100 μl of TMB color development solution and color for 8 min in the dark, then add 100 μl of 2 M HCl to terminate the reaction. Read on a microplate reader (450 nm-620 nm)
[0134] The ELISA test results are shown in Table 14 below.
[0135] Table 14
[0136] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.
Claims
1. A monoclonal nanobody that recognizes Epstein-Barr virus gp42 protein, characterized in that: It is a monoclonal nanobody EBV-gp42-1023, EBV-gp42-1024 or EBV-gp42-1025; The amino acid sequence of the heavy chain complementary determining region CDR1 of the monoclonal nanobody EBV-gp42-1023 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.3; The amino acid sequence of the heavy chain complementary determining region CDR1 of the monoclonal nanobody EBV-gp42-1024 is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.7; The amino acid sequence of the heavy chain complementary determining region CDR1 of the monoclonal nanobody EBV-gp42-1025 is shown in SEQ ID NO.9, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.10, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.
11.
2. The monoclonal nanobody that recognizes Epstein-Barr virus gp42 protein according to claim 1, characterized in that: The amino acid sequence of the monoclonal nanobody EBV-gp42-1023 is shown in SEQ ID NO.4; the amino acid sequence of the monoclonal nanobody EBV-gp42-1024 is shown in SEQ ID NO.8; the amino acid sequence of the monoclonal nanobody EBV-gp42-1025 is shown in SEQ ID NO.
12.
3. The monoclonal nanobody that recognizes Epstein-Barr virus gp42 protein according to claim 1, characterized in that: The gene sequence encoding the nanobody EBV-gp42-1023 is shown in SEQ ID NO.13, the gene sequence encoding the nanobody EBV-gp42-1024 is shown in SEQ ID NO.14, and the gene sequence encoding the nanobody EBV-gp42-1025 is shown in SEQ ID NO.
15.
4. A biomaterial, characterized in that: The biological material includes any one of the following: (1) A nucleic acid molecule encoding the monoclonal nanobody according to any one of claims 1 to 3; (2) A recombinant vector comprising a gene fragment encoding the monoclonal nanobody according to any one of claims 1 to 3, or comprising the nucleic acid molecule; (3) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express the monoclonal nanobody according to any one of claims 1 to 3; (4) An antibody derivative, comprising the monoclonal nanobody according to any one of claims 1 to 3.
5. A product, characterized in that: The product is a pharmaceutical composition or a detection product; The pharmaceutical composition contains the monoclonal nanobody or a derivative of the monoclonal nanobody according to any one of claims 1 to 3; The detection product comprises the monoclonal nanobody according to any one of claims 1 to 3, or the biological material according to claim 4.
6. The product according to claim 5, characterized in that The detection product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.
7. Use of a monoclonal nanobody for recognizing Epstein-Barr virus gp42 protein according to any one of claims 1 to 3, characterized in that: The application includes any one of the following aspects: (1) Used for detecting the expression of EB virus gp42 protein for purposes other than disease diagnosis and treatment, or for detecting the occurrence and development of EB virus-induced tumors, or for evaluating the efficacy of drugs for treating EB virus-induced tumors; (2) Products used to detect the expression of EB virus gp42 protein, or to diagnose EB virus-induced tumors, or to evaluate the efficacy of drugs for treating EB virus-induced tumors; (3) Used for the preparation of drugs for treating Epstein-Barr virus-induced tumors.
8. A nanobody library, characterized in that Used to screen the monoclonal nanobody according to any one of claims 1 to 3; the nanobody library is obtained by extracting total RNA from natural camel-derived PBMCs cells, performing reverse transcription and PCR amplification to obtain the gene sequence of the antibody variable region VHH; the gene sequence of the antibody variable region VHH is connected to a phage expression vector and transformed into competent cells.