Preparation method and application of GST (glutathione S-transferase) nano antibody

Abstract

The invention relates to the technical field of biology, and discloses a preparation method and application of a GST nano antibody, and the preparation method comprises the steps of animal immunization, lymphocyte separation, nano antibody gene library construction, screening and purification. According to the method, the nano antibody specifically bound with the GST protein can be accurately screened out, the problem of poor specificity of a polyclonal antibody is effectively avoided, compared with a complicated preparation process of a monoclonal antibody, alpaca is used as an immune animal, and a conventional molecular biological technology is combined, so that the preparation process is relatively simple and convenient, the cost is remarkably reduced, and the method is suitable for industrial production. The prepared GST nano antibody is small in molecular weight, has good tissue penetrating ability, high stability and strong affinity, and can play a better role in application scenes such as protein detection and purification.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing GST nanobodies and their applications. Background Art

[0002] GST is a class of enzymes with multiple biological functions, participating in various biological processes in cells, such as detoxification, anti-oxidation and signal transduction. In the field of biotechnology, GST is often used as a fusion tag to promote the expression, purification and detection of recombinant proteins. Traditional antibodies against GST are mainly polyclonal antibodies and monoclonal antibodies. Although polyclonal antibodies are relatively simple to prepare, they have poor specificity and large batch-to-batch variability. Monoclonal antibodies have better specificity and uniformity, but the preparation process is complex, the cost is high, and the molecular weight is large, resulting in poor penetration in some application scenarios. Nanobodies are single-domain antibodies derived from the variable region of heavy chain antibodies from camels or sharks. They possess advantages such as small molecular weight, high stability, strong affinity, low immunogenicity, and ease of expression and modification. Nanobodies can rapidly penetrate tissues and bind to target antigens, showing great application potential in protein research, disease diagnosis, and treatment. However, the preparation methods and related applications of GST-targeted nanobodies have not yet been fully developed. Therefore, there is an urgent need to develop an efficient method for preparing GST nanobodies. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing GST nanobodies and their applications, in order to solve the problems mentioned in the background art, such as poor specificity, complex preparation, high cost, and insufficient penetration ability of existing GST antibodies.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing GST nanobodies, comprising the following steps: Step 1: Immunize animals: Select healthy alpacas, mix purified GST protein with Freund's complete adjuvant, emulsify thoroughly, and immunize alpacas by subcutaneous injection at multiple points. After the initial immunization, booster immunizations are performed every 2 weeks by mixing purified GST protein with Freund's incomplete adjuvant and emulsifying. Step 2, lymphocyte isolation: After the alpaca's last immunization, peripheral blood of the alpaca is collected, and lymphocytes are isolated by centrifugation to obtain peripheral blood mononuclear cells; Step 3: Constructing a nanobody gene library: Total RNA was extracted from peripheral blood mononuclear cells, and the variable region gene of the nanobody was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified variable region gene of the nanobody was ligated with the expression vector pET-28a and transformed into E. coli competent cells to construct a nanobody gene library. Step 4: Screening: The nanobody gene library was inoculated onto LB solid medium containing kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated onto LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Then, the expression of nanobodies was induced by IPTG for 4 hours. Positive clones that could specifically bind to GST protein were screened by ELISA (enzyme-linked immunosorbent assay). Step 5, Purification: The selected positive clones were subjected to DNA sequencing to determine the amino acid sequence of the nanobody. The positive clones were inoculated into LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Expression was induced by IPTG. The expressed nanobody was purified by Ni-NTA affinity chromatography column. The elution peak was collected and impurities were removed by dialysis to obtain the purified GST nanobody.

[0005] As a preferred embodiment of the present invention, the purified GST protein in step one is mixed with Freund's complete adjuvant at a volume ratio of 1:1.

[0006] As a preferred embodiment of the present invention, in step one, the number of booster immunizations is 4 to 5, and the booster immunization dose is the same as the initial immunization dose, with each alpaca receiving 500 to 800 μg per injection.

[0007] As a preferred embodiment of the present invention, in step two, peripheral blood of alpacas is collected 7-10 days after the last immunization of the alpaca.

[0008] As a preferred technical solution of the present invention, in step two, peripheral blood is slowly added to the lymphocyte separation medium, centrifuged at 2000-2500 rpm for 20-30 minutes at room temperature, and the cells in the middle white membrane layer are collected, which are peripheral blood mononuclear cells.

[0009] As a preferred embodiment of the present invention, the reverse transcription reaction system in step three comprises 4 μL of reverse transcription buffer, 1 μL of dNTPs (10 mM), 1 μL of random primers (10 μM), 0.5 μL of RNase inhibitor, 1 μL of reverse transcriptase, 2 μg of RNA template, and 20 μL of RNase-free water. The reaction conditions are 42–70 °C for 15–60 minutes. The PCR reaction system comprises 5 μL of PCR buffer, 4 μL of dNTPs (2.5 mM), 1 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, 0.5 μL of Taq DNA polymerase, and 50 μL of sterile water. The reaction conditions are 94 °C pre-denaturation for 5 minutes, 94 °C denaturation for 30 seconds, 55–60 °C annealing for 30 seconds, 72 °C extension for 1 minute, for a total of 30 cycles, and a final extension at 72 °C for 10 minutes.

[0010] As a preferred embodiment of the present invention, the concentration of kanamycin in step four is 50 μg / mL.

[0011] As a preferred embodiment of the present invention, the ELISA (enzyme-linked immunosorbent assay) method in step four specifically involves coating GST protein onto an ELISA plate, 100 μL per well (concentration 1 μg / mL), and incubating overnight at 4°C; the next day, discard the liquid in the wells, wash three times with PBS containing 0.05% Tween-20 (PBST), add 200 μL of 5% skim milk to each well, and block at 37°C for 1 hour; discard the blocking solution, and add the supernatant of the induced expression nanoantibody. Add 100 μL of HRP-labeled goat anti-His tag antibody (1:5000 dilution) to each well and incubate at 37°C for 1 hour. Wash three times with PBST, add 100 μL of HRP-labeled goat anti-His tag antibody (1:5000 dilution) to each well, and incubate at 37°C for 1 hour. After washing five times with PBST, add 100 μL of TMB substrate solution to each well and react at room temperature in the dark for 15 minutes. Finally, add 50 μL of 2MH2SO4 to stop the reaction. Measure the absorbance at 450 nm and select clones with absorbance significantly higher than the negative control as positive clones.

[0012] The GST nanobody described in this invention is used in protein detection and protein purification.

[0013] Compared with existing technologies, the beneficial effects of this invention are as follows: The preparation method of this GST nanobody, through multiple immunizations of alpacas and screening using enzyme-linked immunosorbent assay (ELISA), can accurately screen for nanobodies that specifically bind to GST proteins, effectively avoiding the problem of poor specificity of polyclonal antibodies. Compared with the complex preparation process of monoclonal antibodies, using alpacas as immunization animals and combining conventional molecular biology techniques, the preparation process is relatively simple and the cost is significantly reduced. The prepared GST nanobodies have small molecular weights, good tissue penetration ability, high stability, and strong affinity, and can play a better role in applications such as protein detection and purification. Attached Figure Description

[0014] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0015] 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1

[0017] Please see Figure 1 The technical solution of this invention: a method for preparing GST nanobodies, comprising the following steps: Step 1: Immunize animals: Select healthy alpacas, mix purified GST protein with Freund's complete adjuvant, emulsify thoroughly, and immunize alpacas by subcutaneous injection at multiple points. After the initial immunization, booster immunizations are performed every 2 weeks by mixing purified GST protein with Freund's incomplete adjuvant and emulsifying. Step 2, lymphocyte isolation: After the alpaca's last immunization, peripheral blood of the alpaca is collected, and lymphocytes are isolated by centrifugation to obtain peripheral blood mononuclear cells; Step 3: Constructing a nanobody gene library: Total RNA was extracted from peripheral blood mononuclear cells, and the variable region gene of the nanobody was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified variable region gene of the nanobody was ligated with the expression vector pET-28a and transformed into E. coli competent cells to construct a nanobody gene library. Step 4: Screening: The nanobody gene library was inoculated onto LB solid medium containing kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated onto LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Then, the expression of nanobodies was induced by IPTG for 4 hours. Positive clones that could specifically bind to GST protein were screened by ELISA (enzyme-linked immunosorbent assay). Step 5, Purification: The selected positive clones were subjected to DNA sequencing to determine the amino acid sequence of the nanobody. The positive clones were inoculated into LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Expression was induced by IPTG. The expressed nanobody was purified by Ni-NTA affinity chromatography column. The elution peak was collected and impurities were removed by dialysis to obtain the purified GST nanobody.

[0018] In step one, the purified GST protein is mixed with Freund's complete adjuvant at a volume ratio of 1:1.

[0019] In step one, the booster immunization is performed four times, with the booster immunization dose being the same as the initial immunization dose, which is 800 μg per alpaca per injection.

[0020] In step two, peripheral blood of alpacas is collected 7 days after their last immunization.

[0021] In step two, peripheral blood is slowly added to the lymphocyte separation medium and centrifuged at 2000 rpm for 30 minutes at room temperature. The cells in the middle white membrane layer are collected, which are peripheral blood mononuclear cells.

[0022] In step three, the reverse transcription reaction system includes 4 μL of reverse transcription buffer, 1 μL of dNTPs (10 mM), 1 μL of random primers (10 μM), 0.5 μL of RNase inhibitor, 1 μL of reverse transcriptase, 2 μg of RNA template, and RNase-free water to a final volume of 20 μL. The reaction conditions are 42°C for 60 minutes. The PCR reaction system includes 5 μL of PCR buffer, 4 μL of dNTPs (2.5 mM), 1 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, 0.5 μL of Taq DNA polymerase, and sterile water to a final volume of 50 μL. The reaction conditions are 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 1 minute, for a total of 30 cycles, and a final extension at 72°C for 10 minutes.

[0023] In step four, the concentration of kanamycin is 50 μg / mL.

[0024] In step four, the ELISA (enzyme-linked immunosorbent assay) method specifically involves coating GST protein onto an ELISA plate, 100 μL per well (concentration 1 μg / mL), and incubating overnight at 4°C. The next day, the liquid in the wells is discarded, and the plate is washed three times with PBS containing 0.05% Tween-20 (PBST). 200 μL of 5% skim milk is added to each well, and the plate is blocked at 37°C for 1 hour. The blocking solution is discarded, and 100 μL of the supernatant of the induced expression nanobody is added to each well, and the plate is incubated at 37°C for 1 hour. The plate is washed three times again with PBST, and 100 μL of HRP-labeled goat anti-His tag antibody (1:5000 dilution) is added to each well, and the plate is incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of TMB substrate solution is added to each well, and the plate is incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of 2MH2SO4 is added to terminate the reaction, and the absorbance is measured at 450 nm. Clones with absorbance significantly higher than the negative control are selected as positive clones.

[0025] Example 2

[0026] Please see Figure 1 The technical solution of this invention: a method for preparing GST nanobodies, comprising the following steps: Step 1: Immunize animals: Select healthy alpacas, mix purified GST protein with Freund's complete adjuvant, emulsify thoroughly, and immunize alpacas by subcutaneous injection at multiple points. After the initial immunization, booster immunizations are performed every 2 weeks by mixing purified GST protein with Freund's incomplete adjuvant and emulsifying. Step 2, lymphocyte isolation: After the alpaca's last immunization, peripheral blood of the alpaca is collected, and lymphocytes are isolated by centrifugation to obtain peripheral blood mononuclear cells; Step 3: Constructing a nanobody gene library: Total RNA was extracted from peripheral blood mononuclear cells, and the variable region gene of the nanobody was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified variable region gene of the nanobody was ligated with the expression vector pET-28a and transformed into E. coli competent cells to construct a nanobody gene library. Step 4: Screening: The nanobody gene library was inoculated onto LB solid medium containing kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated onto LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Then, the expression of nanobodies was induced by IPTG for 4 hours. Positive clones that could specifically bind to GST protein were screened by ELISA (enzyme-linked immunosorbent assay). Step 5, Purification: The selected positive clones were subjected to DNA sequencing to determine the amino acid sequence of the nanobody. The positive clones were inoculated into LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Expression was induced by IPTG. The expressed nanobody was purified by Ni-NTA affinity chromatography column. The elution peak was collected and impurities were removed by dialysis to obtain the purified GST nanobody.

[0027] In step one, the purified GST protein is mixed with Freund's complete adjuvant at a volume ratio of 1:1.

[0028] In step one, the booster immunization is performed 5 times, and the booster immunization dose is the same as the initial immunization dose, which is 650μg per alpaca per injection.

[0029] In step two, peripheral blood of alpacas is collected 8 days after their last immunization.

[0030] In step two, peripheral blood is slowly added to the lymphocyte separation medium and centrifuged at 2200 rpm for 25 minutes at room temperature. The cells in the middle white membrane layer are collected, which are peripheral blood mononuclear cells.

[0031] In step three, the reverse transcription reaction system includes 4 μL of reverse transcription buffer, 1 μL of dNTPs (10 mM), 1 μL of random primers (10 μM), 0.5 μL of RNase inhibitor, 1 μL of reverse transcriptase, 2 μg of RNA template, and RNase-free water to a final volume of 20 μL. The reaction conditions are 55°C for 40 minutes. The PCR reaction system includes 5 μL of PCR buffer, 4 μL of dNTPs (2.5 mM), 1 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, 0.5 μL of Taq DNA polymerase, and sterile water to a final volume of 50 μL. The reaction conditions are 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, for a total of 30 cycles, and a final extension at 72°C for 10 minutes.

[0032] In step four, the concentration of kanamycin is 50 μg / mL.

[0033] In step four, the ELISA (enzyme-linked immunosorbent assay) method specifically involves coating GST protein onto an ELISA plate, 100 μL per well (concentration 1 μg / mL), and incubating overnight at 4°C. The next day, the liquid in the wells is discarded, and the plate is washed three times with PBS containing 0.05% Tween-20 (PBST). 200 μL of 5% skim milk is added to each well, and the plate is blocked at 37°C for 1 hour. The blocking solution is discarded, and 100 μL of the supernatant of the induced expression nanobody is added to each well, and the plate is incubated at 37°C for 1 hour. The plate is washed three times again with PBST, and 100 μL of HRP-labeled goat anti-His tag antibody (1:5000 dilution) is added to each well, and the plate is incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of TMB substrate solution is added to each well, and the plate is incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of 2MH2SO4 is added to terminate the reaction, and the absorbance is measured at 450 nm. Clones with absorbance significantly higher than the negative control are selected as positive clones.

[0034] Example 3

[0035] Please see Figure 1 The technical solution of this invention: a method for preparing GST nanobodies, comprising the following steps: Step 1: Immunize animals: Select healthy alpacas, mix purified GST protein with Freund's complete adjuvant, emulsify thoroughly, and immunize alpacas by subcutaneous injection at multiple points. After the initial immunization, booster immunizations are performed every 2 weeks by mixing purified GST protein with Freund's incomplete adjuvant and emulsifying. Step 2, lymphocyte isolation: After the alpaca's last immunization, peripheral blood of the alpaca is collected, and lymphocytes are isolated by centrifugation to obtain peripheral blood mononuclear cells; Step 3: Constructing a nanobody gene library: Total RNA was extracted from peripheral blood mononuclear cells, and the variable region gene of the nanobody was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified variable region gene of the nanobody was ligated with the expression vector pET-28a and transformed into E. coli competent cells to construct a nanobody gene library. Step 4: Screening: The nanobody gene library was inoculated onto LB solid medium containing kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated onto LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Then, the expression of nanobodies was induced by IPTG for 4 hours. Positive clones that could specifically bind to GST protein were screened by ELISA (enzyme-linked immunosorbent assay). Step 5, Purification: The selected positive clones were subjected to DNA sequencing to determine the amino acid sequence of the nanobody. The positive clones were inoculated into LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Expression was induced by IPTG. The expressed nanobody was purified by Ni-NTA affinity chromatography column. The elution peak was collected and impurities were removed by dialysis to obtain the purified GST nanobody.

[0036] In step one, the purified GST protein is mixed with Freund's complete adjuvant at a volume ratio of 1:1.

[0037] In step one, the booster immunization is performed 5 times, and the booster immunization dose is the same as the initial immunization dose, which is 500μg per alpaca per injection.

[0038] In step two, peripheral blood of alpacas is collected 10 days after their last immunization.

[0039] In step two, peripheral blood is slowly added to the lymphocyte separation medium and centrifuged at 2500 rpm for 20 minutes at room temperature. The cells in the middle white membrane layer are collected, which are peripheral blood mononuclear cells.

[0040] In step three, the reverse transcription reaction system includes 4 μL of reverse transcription buffer, 1 μL of dNTPs (10 mM), 1 μL of random primers (10 μM), 0.5 μL of RNase inhibitor, 1 μL of reverse transcriptase, 2 μg of RNA template, and RNase-free water to a final volume of 20 μL. The reaction conditions are 70°C for 15 minutes. The PCR reaction system includes 5 μL of PCR buffer, 4 μL of dNTPs (2.5 mM), 1 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, 0.5 μL of Taq DNA polymerase, and sterile water to a final volume of 50 μL. The reaction conditions are 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 1 minute, for a total of 30 cycles, and a final extension at 72°C for 10 minutes.

[0041] In step four, the concentration of kanamycin is 50 μg / mL.

[0042] In step four, the ELISA (enzyme-linked immunosorbent assay) method specifically involves coating GST protein onto an ELISA plate, 100 μL per well (concentration 1 μg / mL), and incubating overnight at 4°C. The next day, the liquid in the wells is discarded, and the plate is washed three times with PBS containing 0.05% Tween-20 (PBST). 200 μL of 5% skim milk is added to each well, and the plate is blocked at 37°C for 1 hour. The blocking solution is discarded, and 100 μL of the supernatant of the induced expression nanobody is added to each well, and the plate is incubated at 37°C for 1 hour. The plate is washed three times again with PBST, and 100 μL of HRP-labeled goat anti-His tag antibody (1:5000 dilution) is added to each well, and the plate is incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of TMB substrate solution is added to each well, and the plate is incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of 2MH2SO4 is added to terminate the reaction, and the absorbance is measured at 450 nm. Clones with absorbance significantly higher than the negative control are selected as positive clones.

[0043] GST Nanobody Performance Testing

[0044] Purity testing

[0045] The purity of the GST nanobodies prepared in the above examples was determined by high performance liquid chromatography, and the results are shown in the table below:

[0046] As can be clearly seen from the charts, the GST nanobodies prepared in the three examples all have high purity and the purity values ​​are quite similar, indicating that the preparation method has good stability and reproducibility and can stably prepare high-purity GST nanobodies.

[0047] Affinity testing

[0048] The affinity between the GST nanobodies prepared in the examples and the GST protein was detected using Biacore technology. The dissociation constants (KD values) obtained are shown in the table below:

[0049] As can be seen from the charts, the GST nanobodies prepared in the three examples all have strong affinity for GST protein, and the affinity levels are similar, further demonstrating that the nanobodies obtained by this preparation method have stable and excellent performance in binding GST protein.

[0050] Stability testing

[0051] The GST nanobodies prepared in the examples were stored at different temperatures (4℃, 25℃, and 37℃), and their binding activity with GST protein was detected by ELISA at different time points (day 1, day 7, day 14, day 21, and day 28). The stability was assessed by the retention rate of binding activity. The data are shown in the table below:

[0052] As can be clearly seen from the chart, at lower temperatures (4°C), the binding activity retention rate of GST nanobodies decreases relatively slowly, indicating good stability. As the temperature increases, the rate of decrease in binding activity retention rate accelerates, but the stability change trends of the nanobodies in the three examples are similar at different temperatures, further demonstrating that the nanobodies obtained by this preparation method have consistency and reliability in terms of stability.

[0053] Application Verification

[0054] Protein detection application: Using the GST nanobodies prepared in the above examples, Western blot detection was performed on samples containing GST fusion proteins. The detection results were compared with those of traditional polyclonal antibodies and monoclonal antibodies. The band intensity was quantified by the gray value of the bands, and the signal-to-noise ratio (band intensity / background intensity) was calculated to evaluate the detection effect. The data are as follows:

[0055] The results show that the signal-to-noise ratio of the GST nanobody prepared in this invention is significantly higher than that of traditional polyclonal antibodies and also better than that of traditional monoclonal antibodies in different embodiments. This indicates that the nanobody can more clearly identify and bind to GST fusion proteins, with clearer detection bands and lower background interference, proving that it has high accuracy and sensitivity in protein detection.

[0056] Protein purification application: Using the prepared GST nanobody, the GST fusion protein was purified by affinity chromatography. The purity of the GST fusion protein before and after purification was determined by high-performance liquid chromatography (HPLC), and compared with traditional purification methods. The data are as follows:

[0057] The results showed that the purity of GST fusion proteins in different samples was significantly improved after purification using the GST nanobody of the present invention, and was significantly higher than that after purification by traditional methods. This fully demonstrates that the nanobody can effectively remove impurities and greatly improve the purity of target proteins in the field of protein purification, indicating that the nanobody has practical application value in the field of protein purification.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing GST nanobodies, characterized in that, The following steps are involved: Step 1: Immunize animals: Select healthy alpacas, mix purified GST protein with Freund's complete adjuvant, emulsify thoroughly, and immunize alpacas by subcutaneous injection at multiple points. After the initial immunization, booster immunizations are performed every 2 weeks by mixing purified GST protein with Freund's incomplete adjuvant and emulsifying. Step 2, lymphocyte isolation: After the alpaca's last immunization, peripheral blood of the alpaca is collected, and lymphocytes are isolated by centrifugation to obtain peripheral blood mononuclear cells; Step 3: Constructing a nanobody gene library: Total RNA was extracted from peripheral blood mononuclear cells, and the variable region gene of the nanobody was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified variable region gene of the nanobody was ligated with the expression vector pET-28a and transformed into E. coli competent cells to construct a nanobody gene library. Step 4: Screening: The nanobody gene library was inoculated onto LB solid medium containing kanamycin and incubated overnight at 37°C. Single colonies were picked and inoculated onto LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Then, the expression of nanobodies was induced by IPTG for 4 hours. Positive clones that could specifically bind to GST protein were screened by ELISA (enzyme-linked immunosorbent assay). Step 5, Purification: The selected positive clones were subjected to DNA sequencing to determine the amino acid sequence of the nanobody. The positive clones were inoculated into LB liquid medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Expression was induced by IPTG. The expressed nanobody was purified by Ni-NTA affinity chromatography column. The elution peak was collected and impurities were removed by dialysis to obtain the purified GST nanobody.

2. The method for preparing GST nanobodies according to claim 1, characterized in that, The purified GST protein in step one is mixed with Freund's complete adjuvant at a volume ratio of 1:

1.

3. The method for preparing GST nanobodies according to claim 1, characterized in that, In step one, the number of booster immunizations is 4 to 5, and the booster immunization dose is the same as the initial immunization dose, which is 500 to 800 μg per alpaca per injection.

4. The method for preparing GST nanobodies according to claim 1, characterized in that, In step two, peripheral blood of alpacas is collected 7-10 days after the last immunization.

5. The method for preparing GST nanobodies according to claim 1, characterized in that, In step two, peripheral blood is slowly added to the lymphocyte separation medium and centrifuged at 2000-2500 rpm for 20-30 minutes at room temperature. The cells in the middle white membrane layer are collected, which are peripheral blood mononuclear cells.

6. The method for preparing GST nanobodies according to claim 1, characterized in that, The reverse transcription reaction system in step three includes 4 μL reverse transcription buffer, 1 μL dNTPs (10 mM), 1 μL random primers (10 μM), 0.5 μL RNase inhibitor, 1 μL reverse transcriptase, 2 μg RNA template, and RNase-free water to a final volume of 20 μL. The reaction conditions are 42–70 °C for 15–60 minutes. The PCR reaction system includes 5 μL PCR buffer, 4 μL dNTPs (2.5 mM), 1 μL each of forward and reverse primers (10 μM), 2 μL cDNA template, 0.5 μL Taq DNA polymerase, and sterile water to a final volume of 50 μL. The reaction conditions are 94 °C pre-denaturation for 5 minutes, 94 °C denaturation for 30 seconds, 55–60 °C annealing for 30 seconds, 72 °C extension for 1 minute, for a total of 30 cycles, and a final extension at 72 °C for 10 minutes.

7. The method for preparing GST nanobodies according to claim 1, characterized in that, In step four, the concentration of kanamycin is 50 μg / mL.

8. The method for preparing GST nanobodies according to claim 1, characterized in that, The ELISA (enzyme-linked immunosorbent assay) method in step four is as follows: 100 μL of GST protein (concentration 1 μg / mL) is coated onto an ELISA plate and incubated overnight at 4°C. The next day, the liquid in the wells is discarded, and the plate is washed three times with PBS containing 0.05% Tween-20 (PBST). 200 μL of 5% skim milk is added to each well, and the plate is blocked at 37°C for 1 hour. The blocking solution is discarded, and 100 μL of the supernatant of the induced expression nanobody is added to each well. The plate is incubated at 37°C for 1 hour. The plate is washed three times again with PBST, and 100 μL of HRP-labeled goat anti-His tag antibody (1:5000 dilution) is added to each well. The plate is incubated at 37°C for 1 hour. After washing five times with PBST, 100 μL of TMB substrate solution is added to each well, and the plate is incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of 2MH2SO4 is added to terminate the reaction. The absorbance is measured at 450 nm, and clones with absorbance significantly higher than the negative control are selected as positive clones.

9. The application of a GST nanobody according to any one of claims 1-8, characterized in that, The GST nanobodies are used in protein detection and protein purification.

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