Preparation method and application of EGFR single-chain antibody and recombinant IgG antibody thereof

Abstract

This invention relates to the field of genetic engineering, and in particular to a method for preparing and using an EGFR single-chain antibody and its recombinant IgG antibody. This application discloses an EGFR single-chain antibody and its amino acid sequence, the amino acid sequence of which is shown in SEQ ID NO:1, and a screening method for the EGFR single-chain antibody. Based on this, a recombinant EGFR-IgG full-length antibody containing the nucleotide sequence encoding the EGFR single-chain antibody is further disclosed. This antibody can be used in the early diagnosis, molecular subtyping, and preparation of personalized treatment drugs or diagnostic reagents for tumors. Furthermore, this antibody provides an important research tool for exploring the molecular mechanisms of EGFR in autoimmune diseases and tumor development, and provides strong support for the diagnosis, treatment decisions, and prognostic assessment of various solid tumors.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a method for preparing and applying an EGFR single-chain antibody and its recombinant IgG antibody. Background Technology

[0002] Epidermal growth factor receptor (EGFR), or ErbB-1, is a core member of the ErbB transmembrane receptor tyrosine kinase family. As a transmembrane glycoprotein, EGFR plays a crucial driving role in the occurrence, development, and metastasis of various malignant tumors, and is an important target for tumor molecular diagnosis and treatment. EGFR is frequently highly expressed in various solid tumors, including non-small cell lung cancer, breast cancer, colorectal cancer, and head and neck cancer, and its expression level is closely related to tumor invasiveness, clinical stage, and patient prognosis. Accurate detection of EGFR has significant clinical value for early tumor diagnosis, personalized treatment, and prognostic assessment.

[0003] In the development of EGFR detection technology, immunohistochemistry remains the mainstream method for clinical pathological diagnosis due to its advantages such as ease of operation, moderate cost, and ability to directly locate in situ expression in tissues. High-specificity antibodies are the core diagnostic tool of this method. Currently, the EGFR immunohistochemistry (IHC) market is still dominated by imported products: Ventana's CONFIRM anti-EGFR (5B7) and Dako's 2-18C9 have received FDA registration approval, while Novocastra's H11 antibody and Zytomed Systems' EP2 antibody are also widely used for IHC analysis of clinical samples. Although some domestic biotech companies have developed EGFR polyclonal or monoclonal antibodies, these are mostly limited to research applications and lag behind international standards in terms of sensitivity, stability, and clinical validation, thus failing to achieve large-scale industrial application of in vitro diagnostic (IVD) reagents.

[0004] While traditional monoclonal antibody technology has made significant progress, it has inherent limitations: the complete immunoglobulin molecule has a complex structure and a large molecular weight (about 150 kDa), which limits its tissue penetration ability and makes it difficult to effectively penetrate the dense matrix of solid tumors; in addition, high production costs, long preparation cycles and limited yields also restrict its widespread application in the diagnostic field.

[0005] Single-chain antibodies (scFv) are fusion proteins created through genetic engineering by linking the heavy chain variable region (VH) and light chain variable region (VL) of immunoglobulins using flexible peptide linkers. Compared to traditional antibodies, scFv exhibits significant advantages: smaller molecular weight, stronger tissue penetration, allowing them to penetrate deep into solid tumors; shorter in vivo half-life and faster metabolic rate, effectively reducing drug accumulation toxicity; avoidance of non-specific binding and complement activation; and extremely low immunogenicity. scFv antibodies combine precise targeting capabilities with extremely low immunogenicity, demonstrating significant application value in areas such as targeted tumor therapy and the development of in vitro diagnostic reagents.

[0006] In conclusion, the development of highly specific and high-affinity anti-EGFR single-chain antibodies is of significant scientific importance and clinical application value for improving the accuracy of early tumor diagnosis, optimizing targeted therapy strategies, and dynamically monitoring efficacy. Summary of the Invention

[0007] The purpose of this invention is to provide an EGFR single-chain antibody and a screening method thereof.

[0008] Another object of the present invention is to provide a method for preparing and applying a recombinant IgG antibody based on an EGFR single-chain antibody.

[0009] This application combines genetic engineering technology with phage display technology. By constructing a rabbit-derived anti-EGFR phage single-chain antibody library using phage display technology, high-affinity EGFR single-chain antibodies are obtained through systematic screening. Furthermore, recombinant IgG antibodies of EGFR single-chain antibodies suitable for tumor diagnosis are developed, providing a new technological pathway for developing diagnostic antibodies that meet clinical requirements.

[0010] The technical solution adopted to achieve the purpose of this invention is: In a first aspect, this application discloses an EGFR single-chain antibody, referred to as scFv-03 in this application, the amino acid sequence of which is shown in SEQ ID NO:1.

[0011] The SEQ ID NO:1 is: DVVMTQTPSSVSAAVGGTVTINCQASESISNYLAWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFTLTISGMKAEDAATYYCQSGYYGVGATFGAGTNVEIKKESGSVSSEQLA QFRSLDQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMTWVRQAPGKGLEWIGTIHTGGSAYYATWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGSGWDGFDPWDPGTLVTISS.

[0012] Secondly, this application discloses a method for screening EGFR single-chain antibodies, comprising the following steps: Step (1) Prepare the EGFR-scFv phage antibody library.

[0013] Step (2) Screening for scFvs that specifically target EGFR.

[0014] Step (3) Analysis and identification using the monoclonal phage-ELISA method.

[0015] Further, step (1) of preparing the EGFR-scFv phage antibody library includes the following steps: a. Synthesize recombinant EGFR protein, express and purify EGFR protein using a eukaryotic expression system, immunize New Zealand white rabbits with this antigen protein, isolate lymphocytes from spleen tissue, and obtain cDNA library through RNA extraction and reverse transcription.

[0016] b. The antibody heavy chain variable region (VH) and light chain variable region (VL) gene fragments were obtained by PCR technology, and the recombinant scFv gene sequence was constructed.

[0017] Furthermore, the recombinant scFv gene sequence was obtained by splicing the VH and VL genes using SOE-PCR technology.

[0018] c. After the recombinant scFv gene was directionally inserted into the phage vector, it was introduced into TG1 competent cells by electroporation to complete the construction of the primary antibody library.

[0019] Furthermore, the phage vector is Pcomb3xSS.

[0020] Furthermore, the antibody library has a capacity of 1.2 × 10⁻⁶. 8 pfu / mL, random sampling PCR verification showed that the size of the inserted gene fragment was consistent with that of approximately 96.5%.

[0021] d. Prepare an EGFR-scFv phage antibody library by infecting the primary antibody library with bacteriophage.

[0022] Furthermore, using VCSM13 to assist phage infection of the primary antibody library, an EGFR-scFv phage antibody library (abbreviated as phage antibody library in this application) was successfully prepared and its titer was determined.

[0023] Furthermore, in step (2), in screening scFv that specifically targets EGFR, recombinant EGFR protein is used as a solid-phase antigen, and the phage antibody library is subjected to four rounds of biological panning by combining solid-phase screening in immunotubes with in situ screening in tissue sections.

[0024] Furthermore, the four rounds of biological screening include the following steps: the first round of immunotube screening, the second round of immunotube screening, the third round of immunotube screening, and the fourth round of in situ screening of tissue sections.

[0025] In the first three rounds of immunopanning, EGFR antigen was diluted to 100 μg / mL, 50 μg / mL, and 20 μg / mL using buffer, respectively. The phage recovery titer increased from 4.8 × 10⁻⁶ in the first round. 6 The pfu / mL was increased to 1.12 × 10⁻⁶ in the fourth round. 9 The enrichment efficiency reached 233-fold with pfu / mL, demonstrating that phage clones that specifically bind to EGFR antigen were effectively enriched in the phage antibody library.

[0026] Thirdly, this application relates to a nucleic acid that encodes the EGFR single-chain antibody.

[0027] Fourthly, this application relates to a recombinant antibody expression vector containing the aforementioned nucleic acid.

[0028] Fifthly, this application discloses a recombinant EGFR-IgG full-length antibody, wherein the recombinant EGFR-IgG comprises a nucleotide sequence encoding the EGFR single-chain antibody.

[0029] Furthermore, the IgG monoclonal antibody is of rabbit origin.

[0030] Sixthly, this application discloses the application of the EGFR single-chain antibody, the nucleic acid, the recombinant antibody expression vector, and the recombinant EGFR-IgG full-length antibody in the preparation of reagents for detecting immune diseases and tumors.

[0031] Seventhly, this application discloses the application of the EGFR single-chain antibody, the nucleic acid, the recombinant antibody expression vector, and the recombinant EGFR-IgG full-length antibody in the preparation of immune diseases and anti-tumor drugs.

[0032] Compared with the prior art, the beneficial effects of the present invention are: This application discloses an EGFR single-chain antibody that achieves high affinity and high specificity for EGFR antigens, effectively solving the technical problems of large molecular weight and poor tissue penetration of traditional intact antibodies, and significantly improving the sensitivity and accuracy of EGFR detection in tumor tissues.

[0033] In the screening process of the EGFR single-chain antibodies, a rich and diverse EGFR-scFv phage antibody library was first successfully constructed. The phage antibody library was then subjected to four rounds of biological panning through a combination of immunotube solid-phase screening and in situ screening of tissue sections to gradually enrich and obtain high-affinity EGFR-specific scFv.

[0034] Secondly, a recombinant EGFR-IgG full-length antibody was constructed, expressed, and purified. This antibody molecule not only exhibits high affinity but also possesses good target recognition specificity, enabling precise localization of EGFR protein expressed in situ in tumor tissues. Therefore, it can be applied in the early diagnosis, molecular subtyping, and preparation of personalized treatment drugs or detection reagents for tumors. At the same time, this antibody also provides an important research tool for exploring the molecular mechanisms of EGFR in autoimmune diseases and tumor development, and provides strong support for the diagnosis, treatment decisions, and prognostic assessment of various solid tumors. Attached Figure Description

[0035] Figure 1 This is a technical roadmap for an embodiment of the present invention; Figure 2 This is an SDS-PAGE electrophoresis image of the Ni affinity chromatography purified EGFR recombinant antigen in Example 2 of the present invention. Figure 3 The results of antibody serum titer determination using the indirect ELISA method in this invention; Figure 4 This is an agarose gel electrophoresis image of PCR amplification of the VH and VL genes in this invention; Figure 5 This is an agarose gel electrophoresis image of the VH-Linker-VL gel in this invention; Figure 6 This is an agarose gel electrophoresis image of the enzyme digestion products of pcomb3XSS plasmid (A) and scFv fragment (B) in this invention; Figure 7 This invention identifies the capacity and insertion rate of the primary antibody library; Figure A shows the serially diluted primary antibody library, and Figure B shows the bacterial culture PCR identification of TG1 transformed by Pcomb3xSS-scFv. Figure 8 This invention relates to the determination of EGFR-scFv phage antibody library titers. Figure 9 This invention demonstrates the identification of single clones through four rounds of screening; where: Figures A, B, C, and D are PCR identification diagrams of bacterial cultures after one, two, three, and four rounds of screening, respectively; Figure 10 The results of polyclonal phage-ELISA analysis in this invention show the specific enrichment of phage antibody libraries. Figure 11 The above figures are for screening positive monoclonal phage-ELISA clones in this invention; wherein: Figure A shows the ELISA assay of positive phage clones, Figure B shows the rescreening of positive clones, Figure C shows the ELISA analysis of 18 positive clones against EGFR using phage monoclonal ELISA, and Figure D shows the heatmap of positive clones. Figure 12 This is a sequence of positive clone affinities in this invention. Figure 13 SDS-PAGE was used in this invention to identify the expression of recombinant antibodies. Figure 14 This is an SDS-PAGE image of the recombinant antibody purified by Protein A affinity chromatography in this invention; Figure 15 Western blot was used in this invention to identify the binding ability of the recombinant antibody; Figure 16 This invention provides the specificity of recombinant antibody detection using ELISA. Figure 17 This invention uses ELISA to detect the affinity of recombinant antibodies. Figure 18 This is the immunohistochemical staining result of the EGFR recombinant antibody in breast cancer tissue in this invention. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0038] The main research technologies in this application are as follows: Figure 1 As shown: Recombinant EGFR protein was prepared using a eukaryotic expression system for immunizing New Zealand rabbits. Total RNA was extracted from spleen lymphocytes and reverse transcribed. The variable regions of the antibody light chain (VL) and heavy chain (VH) were amplified using primer design and linked to form the scFv gene fragment. The scFv gene was cloned into the Pcomb3xSS vector and electroporated into TG1 competent cells. An EGFR-scFv phage antibody library was constructed using VCSM13 helper phages. Four rounds of panning were performed using immunotubes as the solid-phase matrix and EGFR as the target antigen. The enrichment of the phage antibody library was detected by polyclonal phage-ELISA. Positive clones were screened from the enriched phages using monoclonal phage-ELISA and sent for analysis. Positive clones were confirmed by Sanger sequencing. Recombinant antibodies were prepared using a eukaryotic expression system, and their excellent specificity and binding ability were confirmed by ELISA, Western blot, and IHC.

[0039] Part 1: Construction of EGFR Single-Chain Antibody Example 1: Induction and purification of recombinant EGFR protein (1) Eukaryotic expression of recombinant EGFR protein Expi293F cells were cultured to a density of 4.5–5.5 × 10⁻⁶. 6 Cells / mL and viability ≥95%, the cell suspension was adjusted to 3×10⁶ cells / mL using preheated culture medium. 6 Cells / mL, gently vortex to mix and transfer to a sterile transfection culture vessel; dilute 80 μg pcDNA3.4-EGFR plasmid (pcDNA3.4-EGFR plasmid synthesized by GenScript Biotech) with OPTI-MEM medium to a final volume of 2 mL, and mix 320 μL PEI40000 transfection reagent with an equal volume of OPTI-MEM. After mixing the two solutions in equal volumes, let stand at room temperature for 15 min; then add the complex dropwise to the cell suspension and place it in a dynamic culture at 37℃, 8% CO2, and 125 r / min to continuously express the target protein; when the cell viability drops to 60%, stop the culture, collect the culture supernatant, centrifuge at 4000g for 30 min at 4℃ to remove cell debris, clarify the supernatant and freeze at -80℃ for later use.

[0040] (2) Isolation and purification of EGFR recombinant protein 10 column volumes of equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) were pumped into the Ni-NTA column at a flow rate of 0.5 mL / min to ensure complete equilibration of the packing material. The protein sample prepared in the above steps was loaded into the column, and after natural flow-through, non-specific binding components were washed again with 10 column volumes of equilibration buffer. Gradient elution was performed sequentially with 50 mM, 100 mM, 150 mM, and 200 mM imidazole elution buffers, and the protein solutions corresponding to each concentration elution peak were collected stepwise and stored at 4°C for later use. After elution, the chromatography column was washed with 10 column volumes of equilibration buffer, and then perfused with preservation solution containing 20% ​​ethanol to more than 2 / 3 of the column volume. The packing material was sealed and stored at 4°C. The purified target protein was resuspended in 250 mM imidazole refolding buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). 8.0), transferred to a dialysis bag; immersed in 4°C pre-cooled PBS dialysis buffer (10mM phosphate, 137mM NaCl, 2.7mM KCl, pH 7.4), dialyzed for 24h with magnetic stirring, changing the dialysis buffer every 2h during the process, and gradually reducing the imidazole concentration to 0mM; collected the protein solution in the dialysis bag, centrifuged at 12000g for 10min to remove the precipitate and obtain the protein solution.

[0041] The expression and purity of the obtained EGFR recombinant protein were detected using SDS-PAGE, with a theoretical molecular weight of 198 kDa. Figure 2 As shown, M represents the protein molecular weight marker, 1 represents the stock solution, 2 represents the flow-through solution, 3 represents the washing solution, 4 represents 50 mM imidazole elution buffer E0, 5 represents 50 mM imidazole elution buffer E0 + β-ME (in this application, β-ME refers to β-mercaptoethanol), 6 represents 100 mM imidazole elution buffer E1, 7 represents 100 mM imidazole elution buffer E1 + β-ME, 8 represents 150 mM imidazole elution buffer E2, and 9 represents 200 mM imidazole elution buffer E3. The purified protein sample showed a clear, single band, indicating that the protein had been successfully expressed and had high purity, and that the elution conditions with 100 mM imidazole elution buffer were optimal. Based on the relative density of the band, the purity of the EGFR protein was calculated to be approximately 90%. Quantitative detection was performed using a BCA protein assay kit, and the concentration of recombinant EGFR protein was calculated to be 1.1 mg / mL according to the standard curve. The high-purity protein solution was aliquoted into EP tubes and stored at -80°C for long-term storage.

[0042] Example 2 Construction of EGFR-scFv phage antibody library (1) Immunization of New Zealand white rabbits with EGFR recombinant protein An immunogen was prepared by mixing an equal volume of EGFR protein solution with Freund's complete adjuvant (CFA) at a concentration of 1 mg / mL. The immunogen was subcutaneously injected into the back of rabbits, with each rabbit receiving 400 μg of recombinant EGFR protein. Subsequently, three booster immunizations were performed every 14 days using incomplete Freund's adjuvant (IFA).

[0043] (2) Harvesting and titer determination of antiserum Three days after the last immunization, whole blood was collected via cardiac biopsy. The blood was allowed to stand at 37°C for 1 hour, then incubated overnight at 4°C to extract serum. The serum was centrifuged at 3000g for 15 minutes at 4°C, and the supernatant was collected and filtered through a 0.22μm filter membrane for sterilization. The serum was aliquoted into sterile EP tubes, and glycerol was added to a final concentration of 15%–45%. After thorough mixing, the tubes were labeled and stored at -80°C for long-term storage, avoiding repeated freeze-thaw cycles.

[0044] Serum titers were determined using an indirect ELISA method: First, antigen coating was performed. The recombinant EGFR antigen was diluted to a working concentration of 50 μg / mL with carbonate coating buffer (50 mM Na2CO3 / NaHCO3, pH 9.6), and 100 μL / well was accurately spotted onto a 96-well ELISA plate and incubated overnight at 4°C. Second, washing was performed. The liquid in the wells was patted dry, and 200 μL of PBST (PBS containing 0.05% Tween-20) was added to each well. The plate was washed three times for 5 min each time. Third, blocking was performed. 200 μL of blocking buffer containing 5% bovine serum albumin was added to each well, and the plate was blocked on a horizontal shaker at 37°C for 2 h. The plate was then washed three times with PBST for 5 min each time. Fourth, incubate the primary antibody. Serially dilute the immunized rabbit polyclonal serum to 1:2040000 using blocking buffer. Add pre-adsorbed healthy rabbit serum to each negative control well. Spot 100 μL of working solution into each well and incubate at 37°C with shaking for 1 hour. Wash three times with PBST for 5 minutes each time. Fifth, incubate the secondary antibody. Dilute HRP-labeled donkey anti-rabbit IgG secondary antibody 1:10000 in blocking buffer. Add 100 μL to each well and react at 37°C in the dark with shaking for 1 hour. Wash three times with PBST for 5 minutes each time. Sixth, perform color development. Accurately add 100 μL of TMB single-component substrate solution to each well. After color development at room temperature in the dark for 10 minutes, add 50 μL of 2M H2SO4 to terminate the reaction. Finally, use a microplate reader to detect the absorbance value at 450 nm. A positive threshold is defined as the ratio of the average absorbance of positive wells (P) to the average absorbance of negative control wells (N) ≥ 2.1. Figure 3 The results showed that when the diluted immunized rabbit serum was 1:2048000, the absorbance value at 450 nm was significantly higher than that of the negative control group. The antibody titer of the immunized rabbit serum was calculated to be 1:512000 using the standard curve, indicating that the immunized rabbit serum had a high antibody concentration, meeting the requirements for subsequent experiments.

[0045] (3) Isolation of rabbit spleen cells and extraction of total RNA After intraperitoneal anesthesia with a hysterosalpingography (HMA), the spleen of experimental animals was aseptically removed and placed in pre-cooled PBS balanced salt solution. After removing the membrane, the spleen parenchyma was cut into tissue blocks and spread evenly on a 100 μm nylon cell strainer. The strainer was suspended above a culture dish containing 10 mL of erythrocyte lysis buffer. The tissue blocks were gently ground, and cell dissociation buffer was added simultaneously. After complete tissue dissociation, the strainer was rinsed with 20 mL of cold PBS. The filtrate was collected and filtered through a 40 μm cell strainer to obtain a single-cell suspension. An equal volume of the cell suspension and lymphocyte separation buffer was added to a 15 mL conical tube. The tube was centrifuged at 600 g for 25 min at 20 °C. The lymphocyte layer was precisely aspirated into a new centrifuge tube, and 10 mL of PBS washing buffer containing EDTA (pH 7.2) was added. The tube was centrifuged at 400 g for 10 min to remove platelets. The washing process was repeated twice (250 g, 8 min). The cell concentration was measured using an automated cell counter and adjusted to 5 × 10⁶ cells / mL. 6 cells / mL available for later use.

[0046] Take 5×10 6 Lymphocytes were lysed after adding 1 mL of Trizol reagent and vortexing for 30 s, followed by ice bath for 5 min. 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 s to form an emulsion. The emulsion was incubated at 4 °C for 5 min and then centrifuged at 12000 g for 15 min. The supernatant was transferred to an RNase-free EP tube, and an equal volume of pre-chilled isopropanol was added. The tube was incubated at 4 °C for 10 min. Centrifugation at 12000 g for 10 min at 4 °C revealed a white precipitate at the bottom of the EP tube. The tube was washed twice with 1 mL of 75% ethanol, centrifuged at 7500 g for 5 min, and then air-dried in a clean bench for 5 min. The RNA precipitate was dissolved in 20 μL of LEPC water and stored at -80 °C for long-term storage.

[0047] (4) Reverse transcription to synthesize cDNA Using PrimeScript TM IV 1st strand cDNA Synthesis Mix Reverse Transcription Kit: Add 4 μL of 5×PrimeScript IV cDNA Synthesis Mix, 2 μL of Random 6 mers, 2 μL of template RNA, and 12 μL of RNase-Free dH2O to an EP tube and mix gently. Place the EP tube in a PCR instrument and react under the following conditions: 42℃ for 15 min, 70℃ for 15 min, and 4℃ forever. After completion, store at -80℃.

[0048] (5) Cloning and splicing of scFv gene Primer Design and Synthesis: Based on rabbit germline gene sequences of antibodies collected in the International Immunogenetic Database (IMGT), this invention employs bioinformatics methods to systematically analyze the conserved backbone region (FR) of the rabbit IgG variable region (Fv). Considering the high diversity of light chain (VL) and heavy chain (VH) genes, following the principles of degenerate primer design, a primer set covering multiple gene families was constructed using PrimerPremier 5.0 software. All primers were synthesized by Cyspor Biotech Co., Ltd., diluted to working concentration (10 μM), and stored at -20℃ protected from light.

[0049] To construct the scFv gene, a flexible linker peptide was inserted between the VL and VH genes using overlap extension PCR (SOE-PCR). The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:22, in order to maintain the flexible spatial conformation of the antibody domain.

[0050] The SEQ ID NO:22 is: KESGSVSSEQLAQFRSLD.

[0051] Amplification of antibody variable regions VH and VL: Using cDNA synthesized by reverse transcription as a template, the antibody heavy chain variable region (VH) and light chain variable region (VL) genes were amplified separately. The PCR reaction system (50 μL) contained: 25 μL Premix Taq (TaKaRa Taq) TM Version 2.0 plus dye), 1 μL of forward and reverse primers, 1 μL of cDNA template, and sterile deionized water were added to the final volume; the thermal cycling program was set as follows: 98℃ pre-denaturation for 1 min; followed by 26 cycles of amplification (98℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 60 s); and finally, 72℃ final extension for 2 min. After amplification, 25 μL of the product was subjected to 1% agarose gel electrophoresis at a constant voltage of 120V for 25 min. Using the DL2000 DNA Marker as a reference, the target bands VH and VL were observed under a gel imaging system at approximately 350 bp. Figure 4 As shown, 1-4 are VL amplification products; 5-8 are VH amplification products.

[0052] Purification and recovery of PCR products: After rapid gel excision, the target band was purified using an agarose gel DNA recovery kit (Omega Bio-tek D2500). The DNA concentration (A260 / A280 = 1.8~2.0) and integrity were determined by Nanodrop. A template-free negative control was set up throughout the experiment to ensure no non-specific amplification contamination.

[0053] scFv gene assembly: Using purified VH and VL gene fragments as templates, a single-chain antibody (scFv) gene was constructed using overlap extension PCR. 50 ng each of the VH and VL gene fragments were added to a 50 μL reaction mixture containing 25 μL PremixTaq, 1 μL VL-F, 1 μL VH-R, and sterile deionized water to the final volume. After amplification, 25 μL of the product was subjected to 1% agarose gel electrophoresis. The target band scFv was observed to be approximately 750 bp. Figure 5 As shown. The target band was recovered by gel extraction, purified using a gel extraction kit, and after the concentration and purity were determined, it was aliquoted and stored at -20℃.

[0054] (6) Constructing the Pcomb3xSS-scFv recombinant plasmid Enzyme digestion of scFv and Pcomb3xSS vectors: Following a double digestion system, the Pcomb3xSS plasmid and scFv fragment were digested separately using enzymes... Sfi I. Enzyme digestion treatment: Add 20 μg Pcomb3xSS / scFv and 15 μL FastDigest enzyme. Sfi Add 15 μL of 10× QuickCut Buffer and sterile deionized water to the final volume, and incubate the enzyme digestion reaction at 50°C for 1 hour. After the reaction, perform agarose gel electrophoresis on 5 μL of the digestion product to verify successful digestion. Figure 6 As shown, Figures A and B are agarose gel electrophoresis images of the digestion products of the pcomb3XSS plasmid and the scFv fragment, respectively. The target band was rapidly digested on the gel, then purified and its concentration determined.

[0055] Construction of scFv and Pcomb3xSS recombinant vectors: The purified and recovered scFv gene fragment was mixed with the linearized Pcomb3xSS vector at a 1:3 molar ratio, 25 μL of T4 DNA ligase was added, and sterile deionized water was added to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 16 °C for 30 min. After the ligation reaction, the ligation product was desalted using a DNA fragment recovery kit. The specific steps included: mixing the reaction solution with Binding Buffer at a 1:1 volume ratio, passing the mixture through a column to adsorb DNA, washing with Wash Buffer, and then eluting the purified product with 15 μL of Elution Buffer.

[0056] The purified ligation product was analyzed for concentration and purity using Nanodrop, then aliquoted and stored at -20°C. A negative control without the insert fragment and a control without ligase were included in the experiment to ensure the specificity of the ligation reaction.

[0057] (7) Construction and capacity identification of phage antibody library Electroporation of Pcomb3xSS-scFv ligation product: 10 μL of purified ligation product was added to 100 μL of TG1 competent cells, gently mixed, and incubated on ice for 10 min. The mixture was then aliquoted into pre-chilled 2 mm electroporation cuvettes and incubated for 10 min. Electroporation was performed using a Bio-Rad Gene Pulser Xcell at 2400 V, 25 μF, 200 Ω, and 5 ms. 1 mL of preheated SOC medium was added to the electroporation cuvette, gently mixed, and transferred to a 5 mL centrifuge tube. The centrifuge tube was incubated at 37 °C and 220 rpm for 1 h, followed by centrifugation at 4000 g for 5 min, and the supernatant was discarded. The cells were resuspended in 2 mL of SOC medium, and 100 μL of the bacterial suspension was used for library volume determination. The remaining bacterial suspension was evenly spread onto 20 150 mm cells. Incubate overnight at 37°C on 2×YT-AG agar plates. The next day, wash the colonies off the plates with 2×YT liquid medium, centrifuge at 4°C and 4000g for 5 min, discard the supernatant, and resuspend the cells in 3 mL of 2×YT-AG liquid medium. Add 60% glycerol to the bacterial culture to a final concentration of 20%-30%, aliquot and store at -80°C to construct a primary antibody library.

[0058] Identification of primary antibody libraries: First, storage capacity and abundance determination: Take 100 μL of the electroporated bacterial culture and dilute it using a 10-fold serial dilution method. Add 100 μL of the bacterial culture to 900 μL of SOC medium and mix well (10... -1 (Dilution), dilute sequentially to 10 -8 ; Take 100 μL of each dilution and spread it on a 2×YT-AG plate, and incubate overnight at 37℃; count the number of colonies on the plate the next day.

[0059] like Figure 7 As shown in (A), the primary antibody library size, calculated by colony counting, is 1.2 × 10⁻⁶. 8 CFU.

[0060] Second, recombination rate identification: 28 single colonies were randomly selected from the library volume measurement plate and inoculated into test tubes containing 5 mL of 2×YT medium. The colonies were cultured overnight at 37°C and 220 rpm. 1 μL of bacterial culture was used as a template for amplification, and PCR amplification was performed using vector-specific primers. The amplification products were analyzed by agarose gel electrophoresis, and the positive recombination rate was calculated.

[0061] like Figure 7 As shown in (B), 27 clones showed a specific band at 1000 bp, with a positive clone rate of 96.4%, indicating that the library recombination efficiency met the requirements for subsequent screening.

[0062] Third, diversity identification: Thirty single colonies were randomly selected from the library abundance assay plate and inoculated into 5 mL of 2×YT medium, and cultured overnight at 37°C and 220 rpm. One mL of the bacterial culture was sent to Genewiz Biotechnology Co., Ltd. for Sanger sequencing. Sequence diversity was analyzed using SnapGene software, which showed that the inserted fragment completely matched the target sequence and presented a unique variable region sequence, indicating that a diverse primary antibody library was successfully constructed.

[0063] Construction of the EGFR-scFv phage antibody library: Bacterial culture from the primary antibody library was inoculated into 50 mL of 2×YT-A liquid medium and cultured at 37℃ with shaking at 220 rpm until the logarithmic growth phase (OD50). 600 =0.6); add helper phage VCSM13 at a phage titer / log phase host concentration (MOI) ratio of 20:1, incubate at 37°C for 30 min, then incubate at 37°C with shaking at 220 rpm for 30 min; centrifuge at 4°C and 8000g for 10 min, discard the supernatant, resuspend the precipitate in 50 mL of 2×YT-AK liquid medium, and incubate at 37°C and 220 rpm for at least 12 h; the next day, centrifuge at 4°C and 8000g for 15 min, carefully aspirate the supernatant, add 1 / 5 volume of PEG / NaCl solution (20% PEG8000, 2.5M NaCl), incubate at 4°C for 1 h to precipitate the phage; centrifuge at 4°C and 12000g for 30 min, discard the supernatant, resuspend the precipitate in sterile PBS, and store at 4°C for later use.

[0064] Phage antibody library titer determination: TG1 bacterial culture was pre-cultured to the logarithmic growth phase (OD2). 600 =0.6); Add 10 μL of phage stock solution to 90 μL of PBS buffer, vortex to mix, and prepare 10 -1 The diluent was serially diluted 10-fold to 10... -12 From 10 -6 Up to 10 -12 Take 10 μL of each diluent and mix it with 200 μL of logarithmic TG1 bacterial suspension. Incubate at 37°C for 30 min. Spread the mixture evenly on a 2×YT-K solid plate and incubate overnight at 37°C upside down. Count the number of plaques the next day. Figure 8 As shown, the calculated titer of the phage antibody library is 6.2 × 10⁻⁶. 12 The pfu / mL titer meets the requirements, indicating that the library has high infectious activity, laying the foundation for subsequent antibody screening.

[0065] Example 3: Biopanning and Enrichment of EGFR-scFv Phage Antibody Library (1) Preparation of helper phage VCSM13 The VCSM13 helper phage stock solution was serially diluted 100-fold with sterile PBS buffer to prepare 10 6 10 8 10 10 Three dilution gradients were used for subsequent infection experiments. Take 100 μL of fresh TG1 bacterial culture (OD0.05). 600 =0.8) was mixed with 10 μL of diluted VCSM13 phage, gently mixed, and incubated at 37°C for 30 min, followed by shaking culture at 37°C and 220 rpm for 30 min to complete phage infection. After infection, 4 mL of supernatant was added to the mixture, mixed thoroughly, and poured onto 2×YT antibiotic-free solid medium. The mixture was then incubated overnight at 37°C to form phage plaques. The next day, a single phage plaque was picked and mixed with 5 mL of fresh TG1 bacterial suspension (OD) 600 =0.8) Mix and incubate at 37℃ and 220rpm for 2h with shaking to allow the phage to fully amplify; pour 5mL of the amplified mixture into 500mL of fresh 2×YT liquid medium, incubate at 37℃ and 220rpm for 1h with shaking, then add kanamycin to a final concentration of 70μg / mL and continue incubation overnight; the next day, centrifuge at 8000g and 4℃ for 15min, take 80% of the supernatant, add 1 / 5 volume of PEG / NaCl solution, and let stand at 4℃ for 1h to precipitate the phage; centrifuge at 12000g and 4℃ for 30min, discard the supernatant, resuspend the precipitate in 8mL of sterile PBS, aliquot into 1.5mL EP tubes, and store at 4℃ for later use.

[0066] (2) First round of immunotube screening Using the same carbonate coating buffer as in Example 2, the EGFR antigen was diluted to 100 μg / mL and added to 1 mL of the solution in an immunotube. The tube was coated overnight at 4°C. The next day, the plate was washed 5 times with PBST solution (0.05% Tween-20) for 5 min each time. 2 mL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 h. After incubation, the plate was washed 5 times with PBST solution for 5 min each time. 1 mL of PBS buffer was added to each well, followed by 1 mL of phage single-chain antibody library. The plate was incubated at 37°C for 1 h. After incubation, the plate was washed 10 times with 0.1% TBST to remove unbound phages. 500 μL of 0.1 M glycine-HCl acidic elution buffer was added to each well, and the plate was shaken on a constant temperature shaker for 10 min. 500 μL of 1 M Tris-HCl neutralization buffer was added immediately, and the elution was collected. This was the phage enrichment product of the first round of screening.

[0067] Take 10 μL of eluent and dilute it to 10 μL using a 10-fold serial dilution method. -8For each dilution, take 100 μL and spread it on a 2×YT-AG agar plate. Incubate overnight at 37°C. Count the colonies the next day and calculate the phage titer using the formula: Titer (pfu / mL) = Colony count × Dilution factor × 10.

[0068] After infecting TG1 bacterial suspension with 500 μL of eluent, spread it onto 2×YT-AG agar plates and incubate overnight at 37°C. The next day, wash the colonies off the plates with 2×YT liquid medium, centrifuge at 4°C and 4000g for 5 min, discard the supernatant, and resuspend the bacterial cells in 1 mL of 2×YT-AG liquid medium. Add 500 μL of the resuspended bacterial cells to 50 mL of 2×YT-A medium and incubate at 37°C with shaking until OD reaches 100°C. 600 =0.4; Add helper phage VCSM13 at a phage titer / log phase host concentration (MOI) ratio of 20:1, incubate at 37°C for 30 min, then incubate at 37°C and 220 rpm with shaking for 30 min. Centrifuge at 8000g for 10 min, discard the supernatant, resuspend the precipitate in 50 mL of 2×YT-AK medium, and incubate overnight at 37°C and 220 rpm. Purify the amplified phage using the PEG / NaCl precipitation method, following the same steps as for helper phage preparation. After determining the secondary library titer, proceed to the next round of screening. Optimize the screening conditions progressively in each round, reducing the antigen concentration and increasing the number of washes to increase the screening pressure.

[0069] (3) Second round of immunotube screening Dilute the EGFR antigen to 50 μg / mL with carbonate coating buffer, add 1 mL to an immunotube, and coat overnight at 4°C. Complete the second round of screening using the same method as the first round.

[0070] (4) Third round of immunotube screening Dilute the EGFR antigen to 20 μg / mL with carbonate coating buffer, add 1 mL to an immunotube, and coat overnight at 4°C. Complete the third round of screening using the same method as the first round.

[0071] (5) Fourth round of in situ screening of tissue sections Select one FFPE tissue slide that is antigen-positive and one FFPE tissue slide that is antigen-negative. Place the breast cancer tissue slides in a 60°C oven for 1 hour, then immerse them sequentially in xylene I and II for 10 minutes each, and 100%, 95%, 80%, and 70% ethanol for 5 minutes each. Immerse the slides in preheated sodium citrate buffer and heat in a 95°C water bath for 15 minutes, then allow to cool naturally to room temperature. Streak the tissue slides with a streak pen and block the negative-selection slides with 2% MPBS for 1 hour. Remove the blocking solution from the tissues, add 100 μL of blocking phage to each negative-selection slide, and incubate for 1 hour. Simultaneously, block the selected tissue slides (positive-selection slides) with 2% MPBS for 1 hour. Begin positive selection by transferring the phage from the negative-selection slides to the positive-selection slides and incubating with a rotating incubator at 80 rpm for 1 hour. Wash the tissue slides with PBST, rotating and washing 40 times for 3 minutes each time. Use a pipette to take 250 μL of the solution. Slowly add 0.1 mgly-HCl to a positive sieve slide and incubate at room temperature for 10 min. Quickly transfer the solution to a 1.5 mL EP tube, add 250 μL of 1 M Tris-HCl (pH 9.6) to neutralize, and gently mix the sample by pipetting.

[0072] As shown in Table 1, after four rounds of "binding-elution-amplification" cycles, the phage recovery titer decreased from 4.8 × 10⁻⁶ in the first round. 6 The pfu / mL was increased to 1.12 × 10⁻⁶ in the fourth round. 9 The enrichment efficiency reached 233-fold with pfu / mL, demonstrating that phage clones that specifically bind to EGFR antigen were effectively enriched in the phage antibody library.

[0073] Table 1

[0074] To verify whether the recombinant phage particles after screening contained the target gene scFv, single colonies were randomly selected after each round of selection for colony PCR analysis. For example... Figure 9 As shown, all clones amplified a characteristic band of about 1000 bp, indicating that after multiple rounds of screening, the phage vectors carrying the target fragment were specifically eluted, and the antibody was effectively enriched.

[0075] Example 4 Identification of EGFR-scFv phage antibody library (1) Polyclonal Phage-ELISA EGFR protein (100 ng / well) and negative control BSA (100 ng / well) were coated onto 96-well microplates and incubated overnight at 4°C to complete antigen immobilization. The coating solution was discarded, and 250 μL of PBS buffer containing 0.1% Tween-20 was added to each well. The plates were washed twice at room temperature with shaking for 5 min each time. 100 μL of PBS blocking buffer containing 5% BSA was added to each well, and the plates were incubated at 37°C for 2 hours. h, block non-specific binding sites; discard blocking solution, wash 3 times with PBST for 5 min each time; add 100 μL of the original library, first-round screening secondary library, second-round screening secondary library, third-round screening secondary library solution, and fourth-round screening output library to each well, and incubate at 37℃ for 2 h to allow the antibody to specifically bind to the antigen; wash 3 times with PBST for 5 min each time to thoroughly remove unbound phage particles; dilute the HRP-labeled Anti-M13 secondary antibody 1:5000 with PBS buffer containing 5% BSA, add 100 μL to each well, and incubate at 37℃ for 1 h; wash 3 times with PBST for 5 min each time to remove unbound secondary antibody; add 100 μL of TMB chromogenic substrate to each well, and incubate at 37℃ in the dark for 30 min for HRP-catalyzed colorimetric reaction; add 50 μL of 2M to each well. H2SO4 was used as a stop solution to terminate the colorimetric reaction, and the solution changed from blue to yellow. The absorbance of each well was read at a wavelength of 450 nm using an ELISA reader. Each sample was set up in duplicate wells, and the binding activity was calculated by taking the average value. The positive threshold was defined as the signal-to-noise ratio (P / N) of the absorbance of the experimental well (P) to that of the negative control well (N) ≥ 2.1.

[0076] After each round of biological screening, the proportion of high-affinity clones in the library gradually increases. Polyclonal phage-ELISA compares the OD values ​​of phage populations from different rounds. 450 The value is used to determine whether there is specific enrichment. For example... Figure 10 As shown, the OD of unselected recombinant phages bound to EGFR 450 The initial OD value was 0.45. After the first round of screening, the binding signal significantly increased to 1.6. After the second round of screening, the OD value increased to 2.01, and after the third round, the OD value increased to 2.1. The signal increase slowed down after the fourth round of screening. The polyclonal phage-ELISA data, together with the above phage titer assay results, showed that the four rounds of screening increased the enrichment efficiency of specific scFv phages by nearly 233 times, proving that antigen-specific phages were effectively enriched, allowing for the next step of single-clone screening.

[0077] (2) Monoclonal Phage-ELISA First, obtain the supernatant of the recombinant phage. Pick a single colony of TG1 and inoculate it into 5 mL of 2×YT liquid medium, incubating overnight at 37°C with shaking at 220 rpm. Transfer the overnight culture to 50 mL of 2×YT liquid medium and incubate at 37°C with shaking at 220 rpm until OD (dose elapsed). 600 =0.8; Take 1 mL of bacterial culture, add 100 μL of the positive phage library selected in the fourth round, and incubate at 37℃ and 220 rpm for 1 h with shaking to complete phage infection; dilute and plate on 2×YT-A plates to obtain multiple single colonies. Clone screening: Randomly pick 372 single colonies from the phage library plate selected in the fourth round, numbered A1-A372, and inoculate them into 96-well deep-well plates containing 1 mL of 2×YT-AT medium, and incubate overnight at 37℃ and 220 rpm with shaking. Secondary amplification: Take 5 μL of overnight culture and transfer it to 500 μL of fresh 2×YT-A medium, numbered B1-B372, and incubate at 37℃ and 220 rpm with shaking for 2 h. Add 10 μL of helper phage VCSM13 to each well at an MOI of 20:1, incubate at 37°C for 30 min, then add kanamycin to a final concentration of 50 μg / mL, and incubate overnight at 30°C and 220 rpm with shaking. The next day, centrifuge the 96-well plate at 4°C and 3000 g for 15 min, and carefully aspirate the supernatant to a new plate, avoiding aspiration of bacterial precipitate.

[0078] Then, monoclonal phage-ELISA was performed. 100 ng / well of EGFR protein and 100 ng / well of negative control BSA were coated onto 96-well microplates and incubated overnight at 4°C. After blocking, 100 μL of concentrated monoclonal recombinant phage solution was added to each well, and the plates were incubated at 37°C for 2 h. Subsequent procedures were the same as for polyclonal phage-ELISA. Figure 11 As shown, a positive result was determined by a signal-to-noise ratio (P / N) ≥ 2.1 between the absorbance of the experimental well (P) and the negative control well (N), resulting in 156 positive clones. One hundred clones with higher OD values ​​were collected and repeated for testing, with negative (NC) and blank controls (BL) added. Figure 12 As shown, 18 positive clones were finally obtained through screening.

[0079] (3) Antibody affinity ranking Antigen binding affinity was detected using ELISA, and antibody affinity was ranked. Eighteen positive clones were prepared from phage supernatant and then serially diluted six times. 30 μL of each diluted sample was added to an ELISA plate, and the antibody affinity was measured using OD200. 450 Absorbance is used to assess binding capacity. For example... Figure 12As shown, affinity assays based on ELISA plate coating showed that clones scFv-02, scFv-03, scFv-04, scFv-06, scFv-09, scFv-15, scFv-106, and scFv-107 exhibited significant binding activity to EGFR antigen and were selected as positive monoclonal clones for subsequent analysis.

[0080] (4) Sequencing of specific single-chain antibodies High-affinity EGFR-resistant phage monoclonal antibodies were obtained through multiple rounds of biological panning and Sanger sequencing performed by Genewiz Biotechnology Co., Ltd. Sequencing analysis showed that the gene sequences of scFv-02, scFv-03, and scFv-06 were identical; the gene sequences of scFv-04 and scFv-106 were identical; and the gene sequences of scFv-15 and scFv-107 were identical, indicating they originated from the same clone. scFv-09 was excluded due to the presence of an early stop codon.

[0081] Based on sequence integrity considerations, scFv-03, scFv-106, and scFv-107 were selected for subsequent functional studies. Both the VH and VL genes possess complete open reading frames, and no premature stop codons were found, indicating that the structurally complete single-chain antibody variable region gene sequences were successfully obtained. This complete gene framework provides a molecular basis for subsequent antibody preparation.

[0082] in: The scFv-03 sequence is (SEQ ID NO:2): DVVMTQTPSSVSAAVGGTVTINCQASESISNYLAWYQQKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFTLTISGMKAEDAATYYCQSGYYGVGATFGAGTNVEIKKESGSVSSEQLA QFRSLDQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMTWVRQAPGKGLEWIGTIHTGGSAYYATWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCARGSGWDGFDPWDPGTLVTISS; The scFv-106 sequence is (SEQ ID NO:3): AQVLTQTPSPVSAAVGGTVTISCQASQSVYNNNYLSWYQLKPGQPPKLLIYLASTLASGVPSRFKGSGSGTEFTLTISDVQCDDAATYYCLGGYSNGGDNGFGGGTELEILKESGSVSSEQ LAQFRSLDQSLEESGGRLVTPGTPLTLTCTVSGFSLSSFAMSWVRQTPGKGLEYIGFVDIDGYTGYASWAKGRFTISKTSTTVDLKIASPTTEDTATYFCVRNIWSQDLWGPGTLVTVSS; The scFv-107 sequence is (SEQ ID NO:4): ELVMTQTPSSVSAAVGGTVTINCQASESINNYLSWYQHKPGQPPKLLIYGASNLESGVPSRFRGSGSGTEFTLTISGMKAEYAATYYCQSGYYGVGATFGAGTKVEIKKESGSVSSEQLA QFLTLDQSVDESGGRLVTPGTPLTLTCTVSGFSLSSYTMGWFRQAPGEGLEYIGTISTGGSASYASWAKGRFTFSRTPTTVDLKITSPTSEDTATYFCARGSGVDGFDPWGQGTLVTISS.

[0083] Part Two: Construction and Detection of Rabbit-Derived Recombinant EGFR-IgG Full-Length Antibody Example 5 Expression and purification of rabbit-derived recombinant full-length EGFR-IgG (1) Construction of recombinant antibody expression vector To construct the pcDNA3.4-scFv recombinant expression vector, the VL and VH gene fragments were amplified by PCR using scFv-03, scFv-106, and scFv-107 plasmids as templates, with specific primers shown in Tables 2 and 3 below. Using pcDNA3.4-Rabbit-Heavy chain and pcDNA3.4-Rabbit-Light chain plasmids as templates, the CH region of the heavy chain was amplified by PCR. 1-3 And the light chain constant region CL. Nucleic acid electrophoresis analysis showed that the amplified products of the light chain variable region (VL) and the heavy chain variable region (VH) exhibited characteristic bands of approximately 350 bp, while the constant region CH... 1-3The VH and CL fragments were located at 1000 bp and 300 bp, respectively. The molecular weight of the amplified products matched the theoretical predictions, and no non-specific amplification bands were observed. After recovering the target fragments using a gel purification kit, homologous recombination technology was used to combine VH and CH. 1-3 VL and CL were directionally ligated. The intact heavy chain gene (VH-CH) was confirmed by 1% agarose gel electrophoresis. 1-3 The light chain gene (VL-CL) and the light chain gene (VL-CL) showed specific bands of 1400 bp and 750 bp, respectively, which were consistent with the expected full-length sequence calculation results, confirming that the genome was correctly assembled.

[0084] Table 2. Specific primers for amplifying light chain variable region genes.

[0085] Table 3. Specific primers for amplifying the variable region of the heavy chain.

[0086] use Hind III and EcoR The eukaryotic expression vector pcDNA3.4 was digested by double restriction endonuclease I, and the recovered genes were identified by gel electrophoresis. The recovered target genes and the linearized vector were ligated by homologous recombination, and the three genes were successfully cloned into the pcDNA3.4 vector.

[0087] (2) Extraction of recombinant plasmids 100 μL of the correctly sequenced bacterial culture was inoculated into 100 mL of LB-A medium and cultured continuously at 37°C with shaking for 16 h. Plasmid purification was performed using the OMEGA EZNA® Endo-free Plasmid DNA Mini Kit, and the experimental procedure is as follows. First, centrifuge 100 mL of bacterial culture at 12000 g for 10 min at room temperature to completely remove the supernatant. Add 4 mL of Solution I containing RNase A to the precipitated bacterial cells and vortex to fully suspend them. Add 4 mL of Solution II dropwise, gently invert and mix 4-6 times, then let stand at room temperature for 3 min. Add 2 mL of pre-chilled N3 Buffer and gently invert the centrifuge tube 8-10 times to mix. Centrifuge the mixture at 12000 g for 10 min, transfer the supernatant to a new centrifuge tube, add 10% volume of endotoxin removal buffer, gently mix, and incubate on ice for 10 min. Place the sample in a 42℃ water bath for 5 min, then centrifuge at 4000 g for 3 min at room temperature. Add 50% volume of pre-chilled anhydrous ethanol to the supernatant, gently mix, and let stand for 2 min. Transfer the mixture to a DNA purification column, centrifuge at 4000 g for 3 min to collect nucleic acids. Discard the waste liquid, add 3 mL of HBC binding buffer, and centrifuge at 4000 g for 3 min. Repeat the washing steps, adding 3 mL of HBC binding buffer. After centrifugation with DNA elution buffer, leave empty for 2 min to remove residual ethanol; transfer the purification column to a sterile centrifuge tube, add 350 μL of preheated elution buffer, let stand for 2 min, centrifuge at 4000 g for 10 min to collect high-purity plasmid DNA, aliquot and store at -20℃ for long-term storage.

[0088] (3) Expression of recombinant antibodies The density of the cultured Expi293F cells reached 4.5~5.5×10⁻⁶. 6 When the cell count is 3 × 10⁶ cells / mL and the viability is ≥ 95%, adjust the cell suspension to 3 × 10⁶ cells / mL using preheated culture medium. 6Cells / mL were gently vortexed and transferred to a sterile transfection culture vessel. 80 μg of purified plasmid DNA (light to heavy chain mass ratio 2:1) was dissolved in 2 mL of OPTI-MEM medium. Separately, 320 μL of PEI40000 transfection reagent was mixed with an equal volume of OPTI-MEM. The two solutions were then mixed in equal volumes and incubated at room temperature for 15 min to form a stable DNA-cationic polymer complex. The complex was added dropwise to the cell suspension while gently shaking at 60 rpm to ensure uniform distribution. The transfection system was incubated at 37°C, 8% CO2, and 125 r / min for continuous expression of the target protein. Cell status was dynamically monitored using a fluorescence microscope, and culture was terminated when cell viability decreased to 60%. The culture supernatant was collected and centrifuged at 4000 g for 30 min at 4℃ to remove cell debris. The clarified supernatant was stored at -80℃ for later use. On day 5 post-transfection, 20 μL of the supernatant sample was taken and added to 5× Loading bufer buffer containing 2% β-mercaptoethanol. The sample was then denatured in a metal bath at 100℃ for 10 min. SDS-PAGE analysis was performed using a Tris-Glycine system with 12% separating gel / 5% stacking gel. Electrophoresis was performed at a constant voltage of 120 V for 90 min. After Coomassie brilliant blue staining, the expression efficiency of the target bands was evaluated using a gel imaging system.

[0089] like Figure 13 As shown, 1-3 represent the original solution, concentrated solution, and concentrated solution + β-ME of pcDNA3.4-scFv-03; 4-6 represent the original solution, concentrated solution, and concentrated solution + β-ME of pcDNA3.4-scFv-106; and 7-9 represent the original solution, concentrated solution, and concentrated solution + β-ME of pcDNA3.4-scFv-107. SDS-PAGE analysis showed that an intact antibody band of approximately 140 kDa was visible under non-reducing conditions. After reduction with 2% β-mercaptoethanol, it dissociated into a 50 kDa heavy chain and a 25 kDa light chain subunit, consistent with the theoretical molecular weight of IgG. This indicates that the recombinant antibody was successfully expressed in mammalian cells, and that the antibody was expressed secretoriously.

[0090] (4) Affinity purification and concentration of recombinant antibodies Antibody proteins were purified from cell supernatants after transfection with recombinant plasmids using Protein A affinity chromatography medium. A gravity flow chromatography column system was constructed using Protein A affinity chromatography medium (1 mL column bed volume). The system was washed with 10 column volumes of sterile deionized water to remove stored ethanol, followed by equilibration with 5 column volumes of binding buffer (20 mM PBS, pH 7.4) until baseline stability. Cell culture supernatant, clarified through a 0.22 μm filter, was loaded onto the column at a constant flow rate (1 mL / min). Incubation at room temperature for 60 min promoted specific binding of the Fc fragment to the ligand. The flow-through was collected by opening the bottom valve, and non-specifically adsorbed proteins were washed with 5 column volumes of binding buffer. Three column volumes of low-pH elution buffer (0.1 M glycine-HCl, pH 2.7) were added stepwise to dissociate the antibody-resin complex. The elution buffer was immediately adjusted to physiological pH with neutralization buffer (1 M Tris-HCl, pH 8.5) to prevent protein denaturation. The purified product was transferred to a 10 kDa molecular weight cutoff ultrafiltration centrifuge tube and centrifuged at 4000 mL / min at 4°C. Gradient centrifugation was performed to concentrate the sample to the target volume. The concentration of the sample was determined using the BCA protein quantification kit. A standard curve was constructed. Three technical replicates were set for each sample. The absorbance value at 562 nm was measured using an ELISA reader and the protein content was calculated. 2 μg of purified sample was taken for denaturing electrophoresis analysis. Coomassie brilliant blue staining was used to assess the purification purity.

[0091] like Figure 14 As shown, 1-9 represent the stock solution, flow-through solution, washing solution, elution buffer E0, elution buffer E0+β-ME, elution buffer E1, elution buffer E1+β-ME, elution buffer E2, and elution buffer E3, respectively. After purification, the protein was concentrated by ultrafiltration and analyzed by SDS-PAGE. The target band was around 140 kDa. Quantitative detection was performed using a BCA protein detection kit, and the concentrations of recombinant antibodies against pcDNA3.4-scFv-03, pcDNA3.4-scFv-106, and pcDNA3.4-scFv-107 were calculated to be 2.38 mg / mL, 1.15 mg / mL, and 1.28 mg / mL, respectively.

[0092] Example 6 Identification and functional analysis of rabbit-derived recombinant full-length EGFR-IgG (1) Western blot detection of recombinant antibody binding To detect the binding specificity of recombinant antibodies to EGFR, three recombinant antibodies were used as primary antibodies, EGFR protein was used as the positive group, and VEGF and ESA proteins were used as control groups. The specificity was verified by Western blot, and the specific steps are as follows. A Tris-Glycine system with 12% separating gel / 5% stacking gel was used, with 10 μL of sample loaded into each well. Initially, electrophoresis was performed at a constant voltage of 60 V for 40 min to allow the sample to pass through the stacking gel. This was then increased to 120 V for 80 min until the bromophenol blue front migrated to 1 cm from the bottom of the gel. PVDF membranes were cut according to the pre-stained protein molecular weight standard, activated with methanol for 5 min, and then pre-equilibrated in transfer buffer for 15 min. Under ice bath conditions, the transfer clamp was assembled in the following order: cathode plate-sponge pad-3 layers of filter paper-separating gel-PVDF membrane-3 layers of filter paper-sponge pad-anode plate. Air bubbles were removed from the layers using a roller. The transfer tank was filled with pre-cooled buffer, and low-temperature transfer was performed at a constant current of 340 mA for 90 min. After transfer, the PVDF membrane was washed 3 times with TBST buffer and blocked with 5% BSA blocking solution on a horizontal shaker (60 rpm) at room temperature for 60 min. It was then incubated with purified antibody at 4°C with shaking overnight. Finally, it was washed 5 times with TBST buffer, 5 times each time. min; incubate with HRP-labeled goat anti-rabbit IgG secondary antibody at room temperature for 2 h; after thorough washing, evenly cover with ECL chemiluminescent substrate, acquire signals using a chemiluminescent imaging system under dark conditions, and dynamically adjust the exposure time according to the signal intensity.

[0093] like Figure 15 As shown, 1-4 represent EGFR recombinant protein, VEGF recombinant protein, ESA recombinant protein, and BSA, respectively. scFv-03, scFv-106, and scFv-107 show clear bands at approximately 198 kDa, consistent with the expected molecular weight of EGFR recombinant protein; however, they do not bind to VEGF recombinant protein or ESA recombinant protein. These results indicate that the three recombinant antibodies have good binding ability.

[0094] (2) ELISA detection of recombinant antibody affinity To determine the specificity of the recombinant antibody and verify the existence of antibody cross-reactivity, EGFR, VEGF, and BSA were coated onto 96-well plates. EGFR protein served as the positive group, VEGF protein as the control group, and BSA as the negative control. The purified recombinant antibody was used as the primary antibody, and HRP-labeled donkey anti-rabbit IgG was used as the secondary antibody. Antibody cross-reactivity was assessed by ELISA to verify antibody specificity. Figure 16 The results showed that the recombinant antibody had a significantly higher affinity for the EGFR recombinant protein than for other proteins, demonstrating that the expressed antibody had good specificity.

[0095] To detect the antibody affinity of the recombinant antibody, an ELISA method was used. 0.5 μg and 2 μg EGFR antigen were coated onto the plate, and the antigen concentration was fixed at 10⁻⁶ sqFv. 6 10 5 10 4 10 3 10 2 10, 1, 10 -1 10 -2 10 -3 10 -4 The reaction was conducted at a concentration of ng / mL. The results showed that, with the same EGFR coating amount, OD increased with increasing scFv concentration. 450 The signal value also increased. This result indicates that the reaction of scFv with EGFR antigen is concentration-dependent, and that scFv has specific binding activity to EGFR antigen. Furthermore, the antibody affinity constants of scFv-03, scFv-106, and scFv-107 were calculated to be 1.28 × 10⁻⁶. -11 2.69×10 -11 2.63×10 -11 ,like Figure 17 As shown.

[0096] (3) Immunohistochemical detection of recombinant antibody specificity To determine whether the recombinant antibody specifically recognizes the native conformation EGFR antigen on breast cancer tissue sections, this experiment evaluated the staining effect using immunohistochemistry, with rabbit EGFR antibody as a positive control and PBS used as a negative control instead of the primary antibody. Breast cancer pathological slides were dewaxed by baking in a 60℃ constant temperature drying oven for 60 min. They were then dehydrated sequentially with gradient ethanol (100%, 95%, 80%, 70%) for 5 min each, cleared with xylene (10 min each in tanks I and II), and equilibrated with PBS buffer (pH 7.4). The slides were then immersed in preheated 0.01 M sodium citrate buffer (pH 6.0) and heat-induced antigen retrieval at 95℃ for 15 min. After natural cooling to room temperature, the slides were rinsed three times with PBS buffer for 3 min each time. A 3% H2O2 methanol solution was added to cover the tissue area, and the slides were incubated at room temperature in the dark for 10 min to block endogenous peroxidase activity. The slides were then rinsed three times with PBS buffer by shaking for 3 min each time. A PBS blocking solution containing 5% normal goat serum was added evenly, and the slides were blocked on a horizontal shaker (50 rpm) at room temperature for 1 h. After removing residual liquid, the slides were washed three times with PBS buffer. The purified anti-EGFR antibody was diluted to the working concentration using PBS containing 2.5% normal goat serum, and 100 μL of the solution was added to each slide. μL of working solution was incubated overnight in a humidified chamber at 4°C. The cells were then rinsed three times with PBS buffer, 3 min each time. A reaction amplification agent was added, and the cells were incubated at room temperature for 15 min. The cells were then rinsed three times with PBS, 3 min each time. The PBS solution was removed, and a highly sensitive enzyme-labeled anti-mouse / rabbit IgG polymer was added. The reaction was carried out at room temperature for 15 min, and three PBS washes were completed. DAB chromogenic solution was mixed at a 1:50 ratio, and 100 μL of working solution was added to each slide for 5 min of light-protected color development. The intensity of the brownish-red signal was dynamically monitored under a microscope. The reaction was terminated with deionized water. The cells were counterstained with hematoxylin for 10 s, treated with 0.1% hydrochloric acid ethanol differentiation solution for 2 s, and rinsed with running water for 10 min to achieve nucleus bluening. After dehydration with graded ethanol, the cells were cleared with xylene. After mounting with neutral resin, digital images were acquired using an upright optical microscope at 200× field of view.

[0097] like Figure 18 As shown, the staining results indicate that scFv-03 exhibits a clear brown signal in the cell membrane region, with significantly reduced background interference compared to the negative control. Compared to the positive control, the staining intensity of scFv-03 is slightly lower by about 15%, and a small amount of non-specific nuclear staining is present. scFv-106 and scFv-107 show no obvious signal in the cell membrane and cytoplasm regions.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An EGFR single-chain antibody, characterized in that: The amino acid sequence of the EGFR single-chain antibody is shown in SEQ ID NO:

1.

2. The method for screening EGFR single-chain antibodies as described in claim 1, characterized in that: Includes the following steps: Step (1) Prepare the EGFR-scFv phage antibody library; Step (2) Screening for scFvs that specifically target EGFR: Step (3) Analysis and identification using the monoclonal phage-ELISA method.

3. The method for screening EGFR single-chain antibodies as described in claim 2, characterized in that: The preparation of the EGFR-scFv phage antibody library in step (1) includes the following steps: a. Synthesizing recombinant EGFR protein, expressing and purifying EGFR protein using a eukaryotic expression system, immunizing New Zealand white rabbits with this antigen protein, isolating lymphocytes from spleen tissue, and obtaining a cDNA library through RNA extraction and reverse transcription; b. Obtaining antibody heavy chain variable region and light chain variable region gene fragments using PCR technology, and constructing a recombinant scFv gene sequence; c. Directively inserting the recombinant scFv gene into a phage vector, and then introducing it into TG1 competent cells by electroporation to complete the construction of the primary antibody library; d. Using phage to infect the primary antibody library to prepare the EGFR-scFv phage antibody library.

4. The method for screening EGFR single-chain antibodies as described in claim 3, characterized in that: The phage vector in c is Pcomb3xSS.

5. A nucleic acid, characterized in that: The nucleic acid encodes the EGFR single-chain antibody as described in claim 1.

6. A recombinant antibody expression vector, characterized in that: The recombinant antibody expression vector contains the nucleic acid as described in claim 5.

7. A recombinant EGFR-IgG full-length antibody, characterized in that: The recombinant EGFR-IgG full-length antibody includes a nucleotide sequence encoding the EGFR single-chain antibody as described in claim 1.

8. The recombinant EGFR-IgG full-length antibody as described in claim 7, characterized in that: The IgG monoclonal antibody is of rabbit origin.

9. The application of the EGFR single-chain antibody as described in claim 1, the nucleic acid as described in claim 5, the recombinant antibody expression vector as described in claim 6, and the recombinant EGFR-IgG full-length antibody as described in any one of claims 7-8 in the preparation of in vitro detection reagents for immune diseases and tumors.

10. The use of the EGFR single-chain antibody as described in claim 1, the nucleic acid as described in claim 5, the recombinant antibody expression vector as described in claim 6, and the recombinant EGFR-IgG full-length antibody as described in any one of claims 7-8 in the preparation of immune diseases and antitumor drugs.

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