LRG1 nano antibody and application thereof

Abstract

The embodiment of the invention discloses an LRG1 nano antibody and application thereof. The LRG1 nano antibody provided by the invention has the advantages of simple structure, low cost, high affinity, good thermal stability, high tissue penetrability and epitope accessibility, low immunogenicity and the like, can be used for preparing diagnostic reagents and therapeutic drugs for castration resistant prostate cancer (CRPC), provides new technology and drug support for accurate diagnosis and treatment of CRPC, and has a relatively good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an LRG1 nanobody and its applications. Background Technology

[0002] Prostate cancer (PCa) is the second most common malignant tumor in men. In recent years, the incidence of prostate cancer has been rising annually, with an increasing rate of increase. Most newly diagnosed prostate cancer patients are diagnosed at an advanced stage, and many already have distant metastases at initial diagnosis. Patients with extensively metastatic prostate cancer cannot receive local radical treatment. However, newly diagnosed metastatic prostate cancer patients who undergo endocrine therapy are unlikely to develop castration-resistant prostate cancer (CRPC). Most localized prostate cancers that progress to CRPC are also prone to distant metastasis, transforming into metastatic CRPC. Once patients enter the CRPC stage, the prognosis is generally very poor, with the five-year relative survival rate dropping from 97.5% to 30.6%, and the median survival usually less than two years, making it a challenging aspect of clinical treatment.

[0003] In recent years, nanobodies have demonstrated significant advantages and enormous application potential in various biotechnology fields, including diagnostics and therapy, due to their unique characteristics. Compared to the complex VH-VL antigen recognition structure of traditional antibodies, nanobodies can bind to antigen protein epitopes that monoclonal antibodies cannot recognize through longer CH3 binding, achieving affinity in the nanomolar or even picomolar range. They can be easily genetically engineered to form new structures, enabling multispecific and multifunctional chimeric antibodies. They possess superior physicochemical properties, with higher conformational and thermal stability. They exhibit deep and rapid tissue penetration, which is crucial for penetrating solid tumor tissues. Furthermore, they are non-immunogenic or have low immunogenicity, allowing for rapid selection in display libraries for high-throughput screening, thus significantly reducing production costs and shortening development time. Currently, nanobodies are widely used in clinical research for various diseases.

[0004] Leucine-rich α-2 glycoprotein 1 (LRG1), encoded by the lrg1 gene located at 19p13.3 in humans, was first isolated from human serum in 1977. Current research has confirmed that LRG1 protein can serve as a novel molecular target for the precise diagnosis and targeted therapy of CRPC. Therefore, developing specific nanobodies targeting LRG1 provides new ideas and directions for the precision diagnosis and treatment of CRPC, possessing significant research value and promising application prospects. Summary of the Invention

[0005] Therefore, this invention provides an LRG1 nanobody and its application.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides an LRG1 nanobody comprising aLR-P1F3, aLR-P1H2, aLR-P1B5, aLR-P1H5, aLR-P1G7, aLR-P1H4, aLR-P3N9, aLR-P1C10, aLR-P3N5, and aLR-P3N8, wherein,

[0008] aLR-P1F3 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:1~3;

[0009] aLR-P1H2 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:4~6;

[0010] aLR-P1B5 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:7~9;

[0011] aLR-P1H5 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:10~12;

[0012] aLR-P1G7 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:13~15;

[0013] aLR-P1H4 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:16~18;

[0014] aLR-P3N9 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:19~21;

[0015] aLR-P1C10 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:22~24;

[0016] aLR-P3N5 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:25~27;

[0017] aLR-P3N8 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:28~30.

[0018] Furthermore, the full-length sequences of the heavy chain variable regions of aLR-P1F3, aLR-P1H2, aLR-P1B5, aLR-P1H5, aLR-P1G7, aLR-P1H4, aLR-P3N9, aLR-P1C10, aLR-P3N5, and aLR-P3N8 are shown in SEQ ID NO:31~40, respectively.

[0019] According to a second aspect of the present invention, the present invention provides a nucleotide encoding the LRG1 nanobody as described above.

[0020] According to a third aspect of the present invention, the present invention provides a carrier containing the nucleotides described above.

[0021] According to a fourth aspect of the present invention, the present invention provides a cell containing the carrier described above.

[0022] According to a fifth aspect of the present invention, the present invention provides the use of the LRG1 nanobody as described above, the nucleotide as described above, the carrier as described above, or the cell as described above in the preparation of a diagnostic reagent for castration-resistant prostate cancer.

[0023] According to a sixth aspect of the present invention, the present invention provides the use of the LRG1 nanobody as described above, the nucleotide as described above, the carrier as described above, or the cell as described above in the preparation of a therapeutic agent for castration-resistant prostate cancer.

[0024] The embodiments of the present invention have the following advantages:

[0025] 1. Using nanobodies (VHH) is simple in structure, low in cost, only 12-15 kDa, does not require heavy and light chain pairing, has no glycosylation requirements, and can be expressed in high yield in prokaryotic or yeast systems, significantly reducing costs and improving batch-to-batch consistency.

[0026] 2. Achieving higher affinity through phage display and affinity maturation: The nanobodies of this invention can achieve nM affinity through screening / mutation. Stronger binding ability can lead to higher detection sensitivity, stronger pathway blocking ability, and lower dosage requirements.

[0027] 3. Stability mutation significantly improves Tm (or freeze-thaw resistance): Through frame region stability optimization, Tm or residual activity under accelerated conditions is significantly improved, making it easier to store, transport and use for a long time, which is superior to existing control antibodies that lack stability verification.

[0028] 4. Small molecular structure improves tissue penetration and epitope accessibility: Nanobodies are small in size and can penetrate deep into solid tumors or inflamed tissues, with more sufficient binding sites and better therapeutic or imaging effects.

[0029] 5. Humanization reduces immunogenicity and is more suitable for clinical translation: Sequence optimization reduces the risk of ADA and its safety is superior to that of non-humanized antibodies or polyclonal antibodies. Attached Figure Description

[0030] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0031] Figure 1 This is a map of the circular double-stranded DNA plasmid vector for expressing the recombinant protein of this invention (circular).

[0032] Figure 2 This is a linear map of the circular double-stranded DNA plasmid vector for expressing the recombinant protein of this invention.

[0033] Figure 3 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P1F3 of this invention are shown below.

[0034] Figure 4 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P1B5 of the present invention are shown below.

[0035] Figure 5 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P1H2 in this invention are shown below.

[0036] Figure 6 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P1G7 of this invention are shown below.

[0037] Figure 7 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P1H4 in this invention are shown below.

[0038] Figure 8 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P3N9 of this invention are shown below.

[0039] Figure 9 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P1C10 of this invention are shown below.

[0040] Figure 10 The SDS-PAGE electrophoresis diagram, chromatogram, and quantitative analysis results of P3N5 of this invention are shown below.

[0041] Figure 11 The affinity test results of P1B5 provided by this invention;

[0042] Figure 12 The affinity test results for P1H2 provided by this invention;

[0043] Figure 13 The affinity test results for P1H4 provided by this invention;

[0044] Figure 14 The affinity test results of P3N5 provided by this invention;

[0045] Figure 15 The affinity test results of P3N9 provided by this invention. Detailed Implementation

[0046] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0047] Example 1: Screening and preparation of LRG1 nanobodies

[0048] 1. Screening process

[0049] 1.1 First round of screening

[0050] 1.1.1 Remove the screening antigen from the -80℃ freezer and thaw it on ice;

[0051] 1.1.2 Coat the screening antigen onto the immunotubes (50µg / tube, coating solution: CBS, pH 9.6, 2ml / tube), and incubate overnight at 4°C with slow rotation. Simultaneously, coat the tubes with 50µg of 3% BSA as a control.

[0052] 1.1.3 Discard the liquid in the overnight coated immunotubes, add 2 ml of PBS buffer and wash the immunotubes 3 times at room temperature, rotating for 5 min each time;

[0053] 1.1.4 Add 2 ml of blocking buffer (3% PBSTB) and incubate at room temperature for 2 hours by rotation.

[0054] 1.1.5 Discard the liquid in the sealed immunosorbent tube and add 2 ml of PBS buffer to wash the immunosorbent tube 3 times at room temperature, rotating for 5 min each time;

[0055] 1.1.6 Discard the washing solution in the immunotherapy tube, add 2 ml of PBS buffer, calculate and add 50 μl of the prepared phage library according to the following formula as the first round of screening input phage library, and incubate at room temperature by rotation for 1 h:

[0056]

[0057] Where V is the volume of added phage (µl), and T library Phage titer;

[0058] 1.1.7 Discard the liquid in the immunoassay tube, add 2 ml of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer and wash the immunoassay tube 20 times at room temperature, rotating for 5 min each time;

[0059] 1.1.8 Discard the liquid in the immunosorbent assay tube, remove as much residual liquid as possible, add 1 ml of 0.25 mg / ml Trypsin solution, and elute by rotating at room temperature for 30 min;

[0060] 1.1.9 Add 10 μl of 10% AEBSF to stop elution, and transfer the solution in the immunoassay tube to a new 1.5 ml centrifuge tube, which is the first round of phage screening elution solution.

[0061] 1.2 First-round phage eluent titer detection

[0062] 1.2.1 The SS320 strain stored at -80℃ was streaked onto 2×YT solid medium (Tet) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium (Tet) and incubated overnight at 37℃.

[0063] 1.2.2 Transfer 250 μl of overnight culture to 5 ml of 2×YT liquid medium (Tet), incubate at 37°C and 250 rpm for approximately 45-60 min until OD is reached. 600 It is 0.5-0.55;

[0064] 1.2.3 Take 10 μl of the first round of phage elution buffer and serially dilute it 10-fold in a 1.5 ml centrifuge tube. Repeat this process 12 times. Specifically, take 10 μl of the first round of phage elution buffer and dilute it to 100 μl. Then take another 10 μl of the first round of phage elution buffer and dilute it to 100 μl. Repeat this process 12 times to a total of 10⁻¹². Shake well to mix.

[0065] 1.2.4 Add 90 μl of SS320 bacterial culture to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min;

[0066] 1.2.5 Take 5 μl from each dilution centrifuge tube and add it to 2×YT solid medium (Amp), and incubate overnight at 37°C upside down;

[0067] 1.2.6 The statistical panel clearly distinguishes the number of single colonies at different dilutions. The number of phage particles per milliliter of phage solution, i.e., the phage library titer, is calculated using the following formula:

[0068]

[0069] Where T is the phage titer (unit: pfu / ml), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0070] 1.3 Amplification of the first round of phage elution buffer

[0071] 1.3.1 The SS320 strain, which was stored at -80℃, was streaked onto 2×YT solid medium (Tet) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium (Tet) and incubated overnight at 37℃.

[0072] 1.3.2 Transfer 250 μl of overnight culture to 5 ml of 2×YT liquid medium (Tet), incubate at 37°C and 250 rpm for approximately 45-60 min until OD is reached. 600 The value is 0.5-0.55;

[0073] 1.3.3 Add 500 μl of the phage elution buffer obtained after the first round of screening to OD. 600 The solution is in a bacterial culture of 0.5-0.55 (the remaining eluent is stored at 4°C).

[0074] 1.3.4 Continue culturing at 37℃ and 250 rpm for 30 min;

[0075] 1.3.5 Spread all bacterial culture evenly onto a 150 mm circular agar plate containing 100 μg / ml Amp and 2% agarose, and incubate overnight at 37°C;

[0076] 1.3.6 Take the overnight cultured circular plate, add 6 ml of 2×YT liquid medium to the surface of the plate, gently scrape off the colonies with a spreader, and collect the bacterial solution into a 15 ml centrifuge tube. This is the amplified bacterial sublime library. At the same time, use a spectrophotometer to measure the OD of the bacterial solution. 600 The value is the OD value of the bacterial library in the elution buffer. 600 The value is then adjusted by adding glycerol to a final concentration of 20%, which constitutes the first round of bacterial library.

[0077] 1.3.7 Calculate the corresponding bacterial volume of the elution buffer bacterial library according to the following formula, and transfer it to 100 ml of 2×YT liquid medium (containing 100 μg / ml Amp and Tet) to achieve initial OD. 600 =0.1:

[0078]

[0079] Where V is the volume of the transferred bacterial culture (in μl), and OD600 is the OD600 of the constructed elution bacterial library. 600 ;

[0080] 1.3.8 Incubate at 37℃ and 250rpm until the bacterial OD600 reaches 0.5-0.55;

[0081] 1.3.9 Calculate and add helper phage M13K07 according to the following formula to make the ratio of bacteria to phages = 1:20:

[0082]

[0083] Where V is the volume (in ml) of helper phage added. For the auxiliary phage titer used, OD 600 OD of bacterial culture 600 value;

[0084] 1.3.10 Continue culturing at 37℃ and 250 rpm for 30 min;

[0085] 1.3.11 Add 50 μg / ml Kana and 0.2 mM IPTG to the final concentration, and incubate overnight at 30°C and 250 rpm.

[0086] 1.4 First round of phage purification

[0087] 1.4.1 Transfer the overnight culture to a new 50ml centrifuge tube and centrifuge at 4000 rpm for 10 min at 4°C;

[0088] 1.4.2 Transfer the supernatant after centrifugation to a new 50ml centrifuge tube, add 1 / 4 volume of 4℃ pre-cooled 20% PEG / 2.5M NaCl, mix thoroughly, and place on ice for 30min;

[0089] 1.4.3 Centrifuge at 4000 rpm and 4℃ for 20 min, discard the supernatant, and remove as much residual liquid as possible;

[0090] 1.4.4 Add 1 ml of PBS to resuspend the precipitate, transfer the resuspended solution to a new 1.5 ml centrifuge tube, and centrifuge at 13000 rpm and 4°C for 20 min;

[0091] 1.4.5 Transfer the supernatant after centrifugation to a new 1.5ml centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl solution, mix well and place on ice for 10min;

[0092] 1.4.6 Centrifuge at 13000 rpm and 4℃ for 10 min, discard the supernatant, and resuspend the precipitate in 1 ml PBS;

[0093] 1.4.7 Centrifuge at 13000 rpm, 4℃ for 2 min. Transfer the supernatant to a new 1.5 ml centrifuge tube. This is the first round of phage selection library. Aliquot 100 μl / tube. Store at -80℃ for long-term storage, or at -20℃ for short-term storage (1-2 weeks).

[0094] 1.4.8 First round of screening phage sub-library titer detection, the method is the same as 1.2.

[0095] 1.5 Second Round of Screening

[0096] The screening method is the same as in 1.1. The input phage is 1 ml of the phage sub-library obtained from the first round of screening, which is used as the input phage library for the second round of screening to obtain the phage elution buffer for the second round of screening.

[0097] 1.6 Second round of phage eluent titer detection

[0098] The method is the same as 1.2.

[0099] 1.7 Amplification and purification of the second round of elution buffer

[0100] The method is the same as 1.3-1.4, to obtain the second round of phage sub-libraries.

[0101] 1.8 Second-round screening of phage sub-library titer detection

[0102] The method is the same as 1.2.

[0103] 1.9 Monoclonal ELISA Detection

[0104] 1.9.1 The SS320 strain, which was stored at -80℃, was streaked onto 2×YT solid medium (Tet) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 ml of 2×YT medium (Tet) and incubated overnight at 37℃.

[0105] 1.9.2 Transfer 250 μl of overnight culture to 5 ml of 2×YT liquid medium (Tet), incubate at 37°C and 250 rpm for approximately 45-60 minutes until OD is reached. 600 The value is 0.5-0.55;

[0106] 1.9.3 Take 10 μl of phage elution buffer after the third round of screening and serially dilute it 10-fold in a 1.5 ml centrifuge tube. Repeat this process 12 times. Specifically, take 10 μl of the phage library and dilute it to 100 μl, then take another 10 μl of the phage library and dilute it to 100 μl, and so on, for a total of 12 serial dilutions. Shake well to mix.

[0107] 1.9.4 Add 90 μl OD to each dilution centrifuge tube 600 Mix the bacterial solution with a pH of 0.5-0.55 thoroughly.

[0108] 1.9.5 Continue incubation at 37℃ and 250 rpm for 30 min;

[0109] 1.9.6 Spread the bacterial culture evenly onto a solid culture medium plate containing 100 μg / ml Amp and incubate overnight at 37°C;

[0110] 1.9.7 Randomly pick single colonies from the culture medium plates after overnight culture and place them into sterile 96-well cell culture plates (P1-P2). Add 200 µl of 2×YT medium (containing 100 µg / ml Amp and Tet) to each well and incubate at 37°C overnight.

[0111] 1.9.8 Take 2µl of the overnight culture and transfer it to a new 96-well cell culture plate containing 200µl of 2×YT liquid medium (containing 100µg / ml Amp and Tet) per well. Incubate at 37°C for 5 hours. Store the overnight culture before transfer at 4°C.

[0112] 1.9.9 Calculate and add helper phage M13K07 to each well according to the following formula to achieve a bacterial count: phage count = 1:20:

[0113]

[0114] Where V is the volume (in ml) of helper phage added. The titer of the helper phage used;

[0115] 1.9.10 Incubate at 37℃ for 30 min, add Kana to a final concentration of 50 µg / ml and 0.2 mM IPTG, and incubate at 30℃ overnight.

[0116] 1.9.11 After overnight incubation, the 96-well culture plate was centrifuged at 4000 rpm for 10 min at 4°C and stored at 4°C for later use;

[0117] 1.9.12 Coat the ELISA plate with the screening antigen (1 ng / μl, coating solution is CBS pH 9.6, 100 μl / well), and simultaneously coat with BSA as a control, and incubate overnight at 4°C;

[0118] 1.9.13 Discard the liquid in the overnight coated microplate, add 200µL of PBS buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time;

[0119] 1.9.14 Add 200 μl of blocking buffer (3% BSA) to each well to block the microplate and incubate at room temperature for 1 h;

[0120] 1.9.15 Discard the blocking solution, add 200µL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time;

[0121] 1.9.16 Add 120 µl of 3% BSA to each well and (add 0.02% Tween 20 followed by 80 µl of the supernatant from centrifugation in step 2.9.11), and incubate at room temperature for 2 h;

[0122] 1.9.17 Discard the liquid in the microplate, add 200µl of PBST buffer to each well and wash 3 times, 10min each time;

[0123] 1.9.18 Add M13 Bacteriophage Antibody (HRP) and Mouse Mab, diluted 1:40000 in blocking buffer, 100 μl / well, and incubate at room temperature for 1 h;

[0124] 1.9.19 Discard the liquid in the ELISA plate, add 200µl of PBST buffer to each well and wash 3 times, 10 min each time;

[0125] 1.9.20 Add 100 µl of TMB single-component chromogenic solution to each well, incubate in the dark for 2-3 min, then add 100 µl of 1MHcl to each well to stop the reaction. Read the OD value using a microplate reader. 450 Values, record and save.

[0126] 1.10 Positive clone ELISA secondary validation

[0127] To rule out false positive results, clones initially identified as positive were subjected to a second ELISA verification, using the same method as in 1.9.

[0128] 1.11 Sequencing of positive clones

[0129] Positive monoclonal antibodies were selected based on ELISA test data and secondary validation data.

[0130] Take 5 µl of positive clone culture from the monoclonal ELISA plate (1.9.7) and inoculate it into 1 ml of 2×YT medium (containing 100 µg / ml Amp and Tet). Incubate at 37°C and 250 rpm until OD. 600Bring the solution to 0.8-1.0 (approximately 6-8 hours), take 0.5 ml of bacterial culture for sequencing, and store the remaining bacterial culture at 4°C.

[0131] 1.12 Sequence Analysis

[0132] The sequenced sequences were analyzed using GENtle software for sequence alignment, and the antibody sequences were translated into amino acids using GENtle software.

[0133] 2.1 The results of the three rounds of screening are shown in Table 1 below.

[0134] Table 1

[0135]

[0136] The results showed that after three rounds of screening, ELISA monoclonal validation was performed.

[0137] 2.2 Monoclonal ELISA Detection Results

[0138] After screening with immunoassay tubes, 192 monoclonal antibodies (P1 (A1-H12) and P2 (A1-H12) were randomly selected for ELISA detection. The yellow-background clones represent the OD of the target antigen. 450 Value greater than BSA control OD 450 The value is 3 times the original value and the reading is greater than 0.5. The results are shown in Tables 2-1 to 2-4 below.

[0139] Table 2-1

[0140]

[0141] Table 2-2

[0142]

[0143] Table 2-3

[0144]

[0145] Table 2-4

[0146]

[0147] 2.3 Sequence Diversity Results

[0148] After screening, a total of 142 clones were selected for sequencing. 135 sequences were normal, and 7 failed. The nanobody sequences were obtained from the sequencing results based on the pre- and post-sequence sequences. The 135 sequences were translated into amino acids, sorted, and subjected to multiple sequence alignment. Finally, 10 final nanobody sequences were selected. The full-length sequence information of the heavy chain variable region is as follows:

[0149] aLR-P1F3:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQPGGSLRLSCAASTSIFSINATAWYRRAPGKQRELVALITKGGYINYAESVKGRFAISRDTAKNTLYLQMNSLKPEDTGVYYCNTVIGGVMGYWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:31);

[0150] aLR-P1H2:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQLELVVVISSDGSINSTESVKGRFSISRDNNKNTVYLLMNSLKPEDTAIYYCNTHLKKYSWSIRSFEYWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:32);

[0151] aLR-P1B5:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQAGGSLRLSCVGSGSIFSVNTMGWYRQAPGKQRELVATITSDDNTTYADSVKGRFTISRDNAKNTVYLQMNSLKFEDTAVYYCYARRRMWLGRQYDYWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:33);

[0152] aLR-P1H5:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQPGGSLRLSCAASGYGFGIYVMGWYRQSPGKQREPVAVIHSGSITNYTDSVKGRFTVSRDSVKNTVYLQMSRLNPEDTAVYYCYVVIRIGYNNTYRGVWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:34);

[0153] aLR-P1G7:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQPGGSLRLSCGISGRIFRASVMGWYRQAPGKERELVATITGDGVTKYDGLVKGRFTISRDDAKNTVYLQMDSLRPEDTAVYYCNAHFTILFTTRDYWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:35);

[0154] aLR-P1H4:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQAGGSLRLSCEASGRTFSSYAMGWFRQAPGKEREFVAAISWSGDSTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEETAVYYCNARRVGSPGSWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:36);

[0155] aLR-P3N9:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQPGGSLRLSCAASRRIASIVVMGWYRQAPGMQRELVATITNGGTTNYVDSVKGRFTISRDNAENSVYLQMNSLKPEDTAVYYCNGKMRFWSVGFWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:37);

[0156] aLR-P1C10:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQAGGSLRLSCTPSRRTFRWTSMGWFRQAPGKEREFVAAITSSGGYTYYADSVKGRFTISRDNAKNTVYLQMNNLQRDDTAVYYCNTAFLPRDWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:38);

[0157] aLR-P3N5:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQSGGSLTLSCGASGRIFRNSIMGWYRQAPGQERELVATITGDGVTKYDGSVKGRFTISRDNAKNTAFLQMNSLKPEDTGVYLCNVHFTILFTTRDYWGQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC(SEQ ID NO:39);

[0158] aLR-P3N8:CCATGGDYKDDDDKIGIMAVQLVESGGGLVQSGGSLRLSCTASGRISSFNVMGWYRQAPGKQRELVARITLGGSTNYVDSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCVRGYSRLFAEWSQGTQVTVSSGSGSNQLHHHHHH*GCGGCCGC (SEQ ID NO: 40).

[0159] The three complementarity-determining regions (CDR1, CDR2, and CDR3) of the nanobody were obtained using GENtle software, and the results are shown in Table 3 below.

[0160] Table 3

[0161]

[0162] 3.1 PCR amplification of the entire genome (MIX) using primers

[0163] Forward primers: phiS3 5'-CACAGGAAACAGCTATGACCATGATTA; phiS2 5'-ATGAAATACCTATTGCCTACGG;

[0164] Reverse primers: psiR2 5'-CGTTAGTAAATGAATTTTCTGTATGAGG; psiR3 5'-GCGTAACGATCTAAAGTTTTGTCG.

[0165] 3.1.1 Gene fragment amplification: Prepare 30 μl or 50 μl systems as shown in Table 4 below in a 200 μl PCR reaction tube (using nanobody sequences as templates).

[0166] Table 4

[0167]

[0168] 3.1.2 Place the 200μl PCR reaction tube with the sample added on a vortex mixer and shake for 3-4 seconds to mix the reaction components evenly. Then place the reaction tube in a small centrifuge and centrifuge for 4-5 seconds.

[0169] 3.1.3 Place the centrifuged reaction tubes into the PCR instrument in sequence. The reaction program is as follows: pre-denaturation 96℃, 3min; 96℃, 30s, 57℃, 30s, 72℃, 65s, 23-30 cycles, final extension 72℃, 3min; incubation 72℃, 10min.

[0170] 3.1.4 Full-length gene amplification: Prepare the reaction system as shown in Table 5 below in a 200 μl PCR reaction tube (50 μl, 1 tube).

[0171] Table 5

[0172]

[0173] 3.1.5 Place the 200μl PCR reaction tube with the sample added on a vortex mixer and shake for 3-4 seconds to mix the reaction components evenly. Then place the reaction tube in a small centrifuge and centrifuge for 4-5 seconds.

[0174] 3.1.6 Place the centrifuged reaction tubes into the PCR instrument in sequence and refer to the PCR program settings in step 3.1.3.

[0175] 3.2 Site-directed mutagenesis primer amplification PCR

[0176] 3.2.1 Full-length gene amplification: Prepare the reaction system as shown in Table 6 below in a 200µl PCR reaction tube.

[0177] Table 6

[0178]

[0179] 3.2.2 Place the 200μl PCR reaction tube with the sample added on a vortex mixer and shake for 5-6 seconds to mix the reaction components evenly. Then place the reaction tube in a small centrifuge and centrifuge for 4-5 seconds.

[0180] 3.2.3 Place the centrifuged reaction tubes into the PCR instrument in sequence, and refer to the PCR program settings in step 3.1.3.

[0181] 4. Connection Conversion Coating Screening

[0182] 4.1 Recombination and Linkage Reaction System

[0183] The reaction systems shown in Table 7 were prepared in a 200µl 96-well PCR plate.

[0184] Table 7

[0185]

[0186] After adding the 200µl PCR reaction plate, centrifuge it for 3 seconds in a microplate centrifuge, and finally place it in a PCR instrument and react at 50℃ for 20 minutes.

[0187] 4.2 Conversion Operation

[0188] 4.2.1 Remove competent cells from the -80℃ freezer and thaw them in the refrigerator compartment;

[0189] 4.2.2 Add all the ligation product to the corresponding competent cells and place them in a 4°C freezer for 8 minutes.

[0190] 4.2.3 After the ice bath, place the competent cells on the PCR instrument heating plate at 42°C for 90 seconds. After the heat shock, remove the competent cells and turn off the PCR instrument.

[0191] 4.2.4 Then place the competent cells in a 4°C freezer for 4 minutes;

[0192] 4.2.5 Take all the competent cell sap, spread it on a solid culture medium, and then place the solid culture dish in an incubator for 8 hours;

[0193] 4.2.6 Select 8 single colonies on a solid culture dish and perform PCR clone screening on the colonies. The PCR reaction system for bacterial detection is shown in Table 8 below.

[0194] Table 8

[0195]

[0196] Set up the bacterial PCR instrument reaction according to the following program: pre-denaturation 96℃, 3min; 96℃, 30s, 57℃, 30s, 72℃, 65s, 23-30 cycles; final extension 72℃, 3min; incubation 72℃, 10min.

[0197] 4.2.7 Select positive clones based on the bacterial detection chart for testing and verification.

[0198] After the sequencing verification was confirmed to be correct, plasmid extraction was performed on it.

[0199] 5. Plasmid extraction

[0200] The plasmid used in this invention is a circular double-stranded DNA expression vector, approximately 5871 bp in length. This plasmid contains the bacterial origin of replication (ori), enabling stable replication in host cells, and includes an antibiotic resistance gene for screening and maintaining positive clones after transformation. The plasmid contains a promoter, transcription terminator, and inducible regulatory elements for exogenous gene expression, allowing for controlled expression of the target gene. A secretion signal peptide coding sequence is located upstream of the target gene insertion region to guide the expression product to a specific cellular compartment, and a tag sequence can be fused for subsequent detection and purification. The plasmid also contains a multiple cloning site, providing various restriction endonuclease recognition sites, facilitating flexible cloning and construction of different target genes. This plasmid is compact, functionally complete, and suitable for the expression and preparation of recombinant proteins.

[0201] 5.1 Preparation of Materials and Reagents

[0202] Materials: packed column, microporous filter column, centrifuge tube, EP tube, filter screen.

[0203] Reagents: Buffer P1 (P1 with added RNase A, store at 4℃), RNase A (store at -20℃), Buffer P2 (if SDS precipitates, preheat in a 37-42℃ water bath), Buffer P3, ER Buffer (store at 4℃), Elution Buffer, QBT, QC, isopropanol, 75% ethanol, deionized water.

[0204] Reagent preparation

[0205] 75% ethanol: Prepare anhydrous ethanol and deionized water in a 3:1 ratio, mix well and store at room temperature.

[0206] Buffer P1 with added Rnase A: Rnase A and P1 solutions were prepared at a ratio of 6:1000, mixed well and stored at 4°C.

[0207] 5.2 Collect 100 ml of overnight (12-16 h) cultured bacterial solution into a pre-labeled 50 ml centrifuge tube. Collect approximately 45 ml of bacterial solution at a time (verify that the labels match). Centrifuge at 8000 rpm for 4 min using an angle rotor centrifuge or at 4500 rpm for 15 min using a horizontal rotor centrifuge. Discard the supernatant and retain the bacterial cells. Repeat the above steps once more, and mark the first centrifuge tube cap in each row of a 36-well acrylic rack with a checkmark to indicate that the collection is complete. Add 10 ml of Buffer P1 containing RNase A to the centrifuge tube and vortex for 6 min until no obvious clumps of bacterial cells are visible.

[0208] 5.3 Add 10 ml of Buffer P2 to the centrifuge tube (if SDS precipitation occurs, preheat in a 37-42℃ water bath until no reagent precipitate remains), then immediately and gently rotate manually 3-9 times or mix on a mixer at 45000 rpm for 60 seconds. At this point, the bacterial solution will change from turbid to a viscous liquid.

[0209] 5.4 After adding 10 ml of Buffer P3 to the centrifuge tube, immediately invert it manually 5-15 times or mix it at 70,000 rpm for 90 seconds. Once a white flocculent precipitate appears, centrifuge at 11,000 rpm for 4 minutes in an angle rotor centrifuge or at 4,500 rpm for 4 minutes in a horizontal rotor centrifuge.

[0210] 5.5 Add 10 ml of ER Buffer to the centrifuged 50 ml centrifuge tube, manually invert 3-5 times to mix thoroughly, or place on a mixer and mix at 45000-70000 rpm for 10 seconds.

[0211] 5.6 Before the filtration and loading step, add 25 ml of QBT reagent to the chromatography column, which is placed on a centrifuge rack and filled with packing material, to equilibrate the packing material.

[0212] 5.7 After filtering the sample treated with ER Buffer to remove the white precipitate, add the remaining liquid to a clean, labeled chromatography column.

[0213] 5.8 After the liquid has finished dripping, add 60 ml of QC gravity column chromatography solution to the chromatography column.

[0214] 5.9 After the QC titration is complete, place the chromatography column into a clean, labeled 50mL centrifuge tube and verify that the tube numbers match. Add 10mL of Elution Buffer and allow it to elute by gravity.

[0215] 5.10 After elution, gently press the chromatography column against the centrifuge tube wall and then quickly remove it. Add 7 mL of isopropanol to the collected filtrate, invert and mix 3-5 times, and centrifuge at 11000 rpm for 15 min in an angle rotor centrifuge or at 4500 rpm for 15 min in a horizontal centrifuge.

[0216] 5.11 Add 75% ethanol: After centrifugation, gently pour off the supernatant, place the centrifuge tubes on the rack in order, and add 5 ml of 75% ethanol to each tube to rinse the precipitate thoroughly. After adding the sample, centrifuge at 11000 rpm for 10 min in an angle rotor centrifuge or at 3700 rpm for 10 min in a horizontal centrifuge.

[0217] 5.12 After centrifugation, gently discard the supernatant and invert the centrifuge tubes onto a piece of paper in ascending order of size. Add 50-500 μl of deionized water to the location of the plasmid and agitate 5-25 times to fully dissolve the plasmid in the water. When the liquid no longer adheres to the tube wall, the plasmid has been completely eluted. Use a pipette to transfer the dissolved liquid into the corresponding microporous filter column according to the centrifuge tube number. Centrifuge at 14000 rpm for 15-30 minutes. After centrifugation, transfer the plasmid into the corresponding EP tube according to the microporous filter column number.

[0218] 5.13 Place 1.5ml EP tubes on a 96-well aliquot plate for plasmid aliquoting, and use the sample sent for testing for various assays.

[0219] 5.14 Use an ELISA reader to determine and record the concentration, and record the corresponding concentration on the side wall of the EP tube.

[0220] 6. Protein expression and purification

[0221] 6.1 Transform the successfully constructed plasmid into BL21(DE3), incubate overnight at 37°C, and pick single clones and shake overnight.

[0222] 6.2 Small-scale induction to explore optimal induction conditions: Inoculate the overnight culture at a 1:1000 ratio into 3 ml LB, and incubate at 37°C for 4-5 hours until the bacterial growth rate reaches OD. 600 =0.6-0.8, add different concentrations of IPTG (0.1-1mM), induce at different temperatures. As the temperature decreases, the induction time is prolonged, such as 4-5h at 37℃, 6-8h at 30℃, and 16-20h at 16℃. Take the bacterial suspension before and after induction under different induction conditions, run gel, stain, and observe the induction results.

[0223] 6.3 After finding the optimal induction conditions, inoculate the bacterial culture at a ratio of 1:1000 into 2L of LB medium and shake at 37°C until the bacterial culture reaches its OD value. 600 =0.6-0.8, aspirate 20 μl of bacterial solution for gel running, and then induce protein expression under the appropriate induction conditions obtained in the preliminary experiment, aspirate 20 μl of bacterial solution for gel running.

[0224] 6.4 Take out the induced bacterial culture, 4000g, and centrifuge at 4℃ for 15min.

[0225] 6.5 Discard the supernatant, weigh the contents, add 10 ml of Lysis buffer (1:100 with protease inhibitor) per gram of bacteria, resuspend, and place on ice for about 30 minutes.

[0226] 6.6 Pressure Breakdown: Drain the alcohol from the high-pressure homogenizer, rinse twice with water, equilibrate once with Lysis buffer, add bacterial solution, pressurize, and run the bacterial solution three to five times until it is transparent and non-viscous.

[0227] 6.7 Collect the broken bacterial culture, 12000g, centrifuge at 4℃ for 20min, separate the supernatant and precipitate, and keep 20μl of each sample for gel running.

[0228] 6.8 Add 2 mL of Ni-NTA to the purification column, filter with ethanol, rinse with water, and add Lysis buffer to equilibrate the column.

[0229] 6.9 Resuspend Ni-NTA in Lysis buffer, add it to the supernatant, mix well, and incubate on a shaker at 4°C for 2 hours.

[0230] 6.10 Pass the supernatant through a column at 4°C and collect 20 μL of the filtrate.

[0231] 6.11 Wash the column three times with 5 ml of wash buffer and collect 20 μl of filtrate sample.

[0232] 6.12 Add 1 ml of Elution buffer, incubate for 5 min, collect the eluent, repeat 5 times, collect 5 ml of eluent in the same tube, and keep 20 μl of sample.

[0233] 6.13 Run the protein samples obtained during the experiment into a gel, stain with Coomassie Brilliant Blue for 1 hour, and decolorize until the background blue is light and clear protein bands are visible.

[0234] 6.14 Analyze the protein bands of each sample and perform subsequent operations based on the results: If there are no impurities or few and light impurities in the eluted protein sample, dialysis and concentration can be performed; if there are many impurities, purification is required before dialysis and concentration.

[0235] 6.15 Dialysis: Add the sample into the dialysis membrane, clamp both ends, and dialyze overnight at 4°C.

[0236] 6.16 Concentration: Select a suitable concentration tube according to the molecular weight of the sample and concentrate at low speed at 4℃. After concentration, measure the protein concentration, aliquot and label, quick-freeze in liquid nitrogen, and then store in a freezer at -80℃.

[0237] The results of protein expression and purification are shown in Table 9 below.

[0238] Table 9

[0239]

[0240] Example 2: Affinity Detection of LRG1 Nanobody

[0241] To accurately assess the binding affinity of candidate nanobodies to the LRG1 antigen, surface plasmon resonance (SPR) technology was used for systematic analysis. The LRG1 antigen was immobilized using a His-tagged chip, and the nanobodies were used as the mobile phase analyte.

[0242] In preliminary experiments, the buffer system underwent extensive screening and optimization. Ultimately, 1×PBS, 0.01% Tween 20, 0.1% BSA, 100 mM NaCl, and 50 μM EDTA were selected as the running buffer, which effectively ensured signal stability and the specificity of the binding reaction. Based on these optimized conditions, three rounds of repeated assays were performed on six nanobodies: P1B5, P1H2, P1H4, P3N5, P3N9, and P1C0. The results showed that, except for P1C0, which showed no specific binding, the other five nanobodies could effectively bind to LRG1, and good kinetic curves were obtained. Figures 11-15 Compared to the pre-optimization version, the baseline of the sensor chromatograms remained stable within the first 100 seconds of analysis, with no significant drift or noise interference. All antibody binding signals exhibited typical concentration dependence, meaning the signal response intensity increased with increasing analyte concentration, consistent with the expected kinetic characteristics.

[0243] SPR experimental results show that the equilibrium dissociation constants (K0) of P1B5, P1H2, P1H4, P3N5, and P3N9 are... D The values ​​were 0.987, 0.963, 0.987, 1.073, and 0.943 nM, respectively, indicating that the above antibodies have a high affinity interaction with LRG1.

[0244] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An LRG1 nanobody, characterized in that, Including aLR-P1F3, aLR-P1H2, aLR-P1B5, aLR-P1H5, aLR-P1G7, aLR-P1H4, aLR-P3N9, aLR-P1C10, aLR-P3N5, and aLR-P3N8, among which, aLR-P1F3 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:1~3; aLR-P1H2 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:4~6; aLR-P1B5 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:7~9; aLR-P1H5 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:10~12; aLR-P1G7 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:13~15; aLR-P1H4 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:16~18; aLR-P3N9 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:19~21; aLR-P1C10 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:22~24; aLR-P3N5 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:25~27; aLR-P3N8 has complementary determinant regions CDR1, CDR2 and CDR3 of SEQ ID NO:28~30.

2. The LRG1 nanobody according to claim 1, characterized in that, The full-length sequences of the heavy chain variable regions of aLR-P1F3, aLR-P1H2, aLR-P1B5, aLR-P1H5, aLR-P1G7, aLR-P1H4, aLR-P3N9, aLR-P1C10, aLR-P3N5, and aLR-P3N8 are shown in SEQ ID NO:31~40.

3. A nucleotide, characterized in that, Encodes the LRG1 nanobody as described in claim 1.

4. A carrier, characterized in that, It contains the nucleotides as described in claim 3.

5. A cell, characterized in that, It contains the carrier as described in claim 4.

6. The use of the LRG1 nanobody of claim 1, the nucleotide of claim 3, the carrier of claim 4, or the cell of claim 5 in the preparation of a diagnostic reagent for castration-resistant prostate cancer.

7. The use of the LRG1 nanobody of claim 1, the nucleotide of claim 3, the carrier of claim 4, or the cell of claim 5 in the preparation of a therapeutic agent for castration-resistant prostate cancer.

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