VHH chain of anti-GST nano antibody and application of VHH chain

Abstract

The invention discloses a VHH chain of an anti-GST (glutathione S-transferase) nano antibody and application of the VHH chain. The VHH chain of the anti-GST nano antibody comprises amino acid sequences which are arranged as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4; the specific amino acid sequences are as follows: CDR1 is shown as SEQ ID NO: 1, CDR2 is shown as SEQ ID NO: 2, CDR3 is shown as SEQ ID NO: 3, FR1 is shown as SEQ ID NO: 4, FR2 is shown as SEQ ID NO: 5, FR3 is shown as SEQ ID NO: 6, and FR4 is shown as SEQ ID NO: 7. According to the obtained anti-GST nano antibody, it is found through ELISA tests that the affinity of the antibody GSTVHH reaches 54.5 nM, the antibody can be expressed in microorganisms, is good in water solubility and high in stability and has excellent performance, the blank of the prior art can be filled, and development of related fields is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular immunology, and in particular to a VHH chain of an anti-GST nanobody and an application thereof. Background Art

[0002] GST is the abbreviation of glutathione S-transferase, a protein containing 211 amino acids, which exists in most organisms. GST is an important metabolic enzyme in organisms and plays a key role in cell resistance to damage and cancer. GST is an important molecule involved in the metabolism of exogenous and endogenous toxic substances. It can catalyze the reduced glutathione (GSH) to bind to toxic substances, increase the water solubility of toxic compounds so that they can be more easily excreted from the body, thereby achieving the effect of detoxification. Based on this principle, people can make GST into a tag and specifically bind it to an affinity medium with a glutathione ligand to purify the target protein. Then use a specific enzyme to remove GST to obtain the natural protein.

[0003] GST has many advantages, such as ease of use, high efficiency and high affinity. The GST tag can increase the stability and solubility of the fusion protein in cells and increase the expression amount, so that the target protein can be quickly, highly purified and quickly separated by a simple glutathione magnetic bead chromatography method, and GST can be detected under non-denaturing conditions. In addition, GST has a simple structure and the amino acid sequence it carries has almost no immunogenicity to most organisms, so it can be expressed in a variety of biological systems.

[0004] In summary, GST is a very practical fusion protein, which is widely used in the fields of molecular biology and biochemistry. Anti-GST antibodies are indispensable in the detection and imaging of GST, and can be used in experiments such as flow cytometry (FlowCyt), protein blotting (WB), immunohistochemistry of paraffin sections (IHC-P), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA).

[0005] Currently, there are many anti-GST monoclonal or polyclonal antibodies on the market. These antibodies are all traditional antibodies, composed of two heavy chains and two light chains. Such antibodies have poor stability, are intolerant to high temperatures and strong acids and alkalis, and are easily inactivated. Moreover, traditional antibodies are difficult to express, with large batch differences and large differences in activity between different batches of products, making it difficult to ensure the consistency of experimental results. Traditional antibodies are prone to precipitation during later modification, such as labeling with HRP, biotin or fluorescent dyes, which increases production costs. In addition, the use of traditional antibodies in experiments such as IP will bring about the problem of interference between light and heavy chains.

[0006] Therefore, the prior art has defects and needs to be improved. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a VHH chain of an anti-GST nanobody and its application, so as to improve the stability, specificity and affinity of the anti-GST antibody, reduce the production cost and facilitate the promotion and application.

[0008] The technical solution of the present invention is as follows: a VHH chain of an anti-GST nanobody is provided, comprising an amino acid sequence arranged as: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; the specific amino acid sequence is as follows: CDR1 is SEQ ID NO: 1, and the amino acid sequence is: GRTLSSYLMG, CDR2 is SEQ ID NO: 2, and the amino acid sequence is: RVTTGGRITT, CDR3 is SEQ ID NO: 3, and the amino acid sequence is: AAKKPFTDSYDY, FR1 is SEQ ID NO: 4, and the amino acid sequence is: DVQLQESGGGLVQTGGSLRLSCAAS, FR2 is SEQ ID NO: 5, and the amino acid sequence is: WFRQAPGKDREFVA, FR3 is SEQ ID NO: 6, and the amino acid sequence is: YAESVKGRFTISRDNAENTVYLQMNALKPEDTAVYYC, and FR4 is SEQ ID NO: 7, and the amino acid sequence is: WGQGTQVTVSS.

[0009] In one embodiment, the amino acid sequence of the VHH chain of the anti-GST Nanobody is shown in SEQ ID NO: 8, and its amino acid sequence is DVQLQESGGGLVQTGGSLRLSCAASGRTLSSYLMGWFRQAPGKDREFV ARVTTGGRITTYAESVKGRFTISRDNAENTVYLQMNALKPEDTAVYYCAAKKPFTDSYDYWGQGTQVTVSS.

[0010] The present invention also provides a first anti-GST Nanobody, which comprises any one of the amino acid sequences having 95% or more identity with the amino acid sequence shown in SEQ ID NO:8, or having no more than five amino acid mutations compared with the amino acid sequence shown in SEQ ID NO:8.

[0011] The first anti-GST Nanobody, whose CDR1 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 1; and / or, its CDR2 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 2; and / or, its CDR3 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 3.

[0012] The present invention also provides a gene sequence, which encodes the VHH chain of the aforementioned anti-GST nanobody or encodes the aforementioned anti-GST nanobody.

[0013] The method for preparing the nano antibody involves injecting an antigen fused with GST into an alpaca of appropriate age, isolating the alpaca peripheral blood lymphocytes and extracting total RNA, obtaining cDNA by reverse transcription, and then amplifying the nano antibody fragment by two rounds of PCR, and finally cloning it into the pComb3xss vector to construct a phage display library. After two rounds of screening and ELISA verification, the anti-GST nano antibody is expressed in Pichia pastoris. The ELISA test found that the antibody GSTVHH affinity reached 54.5nM, can be expressed in microorganisms, has good water solubility, high stability, and excellent performance, which can fill the gap in the existing technology and promote the development of related fields.

[0014] By adopting the above scheme, the present invention provides a VHH chain of an anti-GST nanobody and its application, uses an antigen containing GST to immunize alpacas, and then constructs an immune library containing a diverse anti-GST nanobody sequence, obtains an amino acid sequence library of anti-GST nanobody by screening through phage display technology, and then successfully obtains an anti-GST nanobody through further artificial transformation and optimization. Compared with the prior art, the technical scheme proposed by the present invention can bring the following beneficial effects:

[0015] (1) Small molecules, suitable for tissue imaging: The two anti-GST nanobodies disclosed in the present invention have small molecular weights and have deeper tissue penetration ability in tissue imaging;

[0016] (2) Easy to prepare: The anti-GST nanobody of the present invention can be efficiently expressed by microbial cells such as Escherichia coli or Pichia pastoris, which is easy to produce and has little batch-to-batch variation;

[0017] (3) High affinity and high specificity: The anti-GST nanobody of the present invention has an affinity of up to 5.45*10 -8 M, that is, equilibrium dissociation constant KD = 5.45*10 -8 M, higher than the affinity of currently published anti-GST nanobodies (such as Figure 1 shown).

[0018] (4) High stability: The anti-GST nanobody of the present invention has extremely high stability. It has been verified that it can withstand a variety of harsh cleaning environments and can remain stable for a long time at room temperature (it can still maintain more than 95% activity after being stored at room temperature for two weeks), which can meet the various experimental needs of researchers. Figure 2 As shown, the anti-GST nanobody was coupled to agarose beads and then immunoprecipitated with the GST-containing GFP protein. In different cleaning solutions (3M sodium chloride, 3M guanidine hydrochloride, 8M urea, 2% TritonX-100, 2% NP40, 0.1% SDS, 1M DTT, 5M DTT), the anti-GST nanobody was able to bind firmly to the target protein. In addition to working in physiological saline, the antibody of the present invention can also tolerate high salt, reducing agents, denaturing agents, detergents and other cleaning environments.

[0019] (5) Has a wide range of applications: The anti-GST nanoantibody of the present invention can be covalently coupled with HRP (horseradish peroxidase) or fluorescent dyes such as FITC, which effectively solves the problem of cumbersome labeling steps in experiments such as ELISA, immunoblotting (WB) and fluorescent labeling, and has broad application prospects in experiments such as protein separation and purification, immunoprecipitation, co-immunoprecipitation, ELISA, and immunoblotting that are fused with GST. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the affinity test between the anti-GST nanobody of the present invention and GST.

[0021] Figure 2 It is a stability comparison chart of the anti-GST Nanobody of the present invention.

[0022] Figure 3 This is a concentration comparison chart of the anti-GST nanobody obtained by the present invention and the antibody obtained by the traditional method.

[0023] Figure 4 This is a comparison chart of the anti-GST nanobody of the present invention labeled with HRP and traditional antibodies in ELISA experiments. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0025] The present invention develops alpaca nanoantibodies with high affinity for GST, which can specifically recognize and capture GST fusion proteins and are used in experiments such as WB, ELISA, IP, CO-IP, and MS. The specific GST development method is as follows: First, GST is used as an antigen for alpaca immunization. During the immunization process, 500 μg of antigen is used for each immunization, and a total of four immunizations are performed, once every two weeks. 5 mL of peripheral blood samples were drawn before each immunization, and 5 mL of peripheral blood samples were drawn again within 5 to 7 days after the fourth immunization, for a total of five peripheral blood samples; first, serum was separated from five tubes of peripheral blood, and serum titer was tested. After the fourth immunization, after the serum was diluted to 1:10^5, the OD450 value was still higher than 0.5, indicating that the serum titer met the requirements. Next, 50 mL of alpaca peripheral blood was drawn, PBMC was extracted by density gradient centrifugation, and washed with PBS and stored in Trizol. Total RNA was extracted from 50mL of peripheral blood PBMC, and the quality of RNA was verified by agarose gel. Subsequently, the extracted RNA was reverse transcribed into cDNA, and two rounds of PCR amplification were performed to obtain a diverse nanobody DNA fragment library. The DNA fragment library was linked to the vector and transformed into TG1 Escherichia coli to obtain a bacterial library. The capacity, clone positive rate and diversity of the bacterial library were measured. Auxiliary phages were used for infection to obtain a phage library, and two rounds of phage screening were performed to enrich phages with specific binding to GST. 192 monoclonal clones were selected for two rounds of Elisa verification, positive clones (OD450>0.5) were screened, and Sanger sequencing was performed on them to obtain positive candidate sequences, which were classified according to the CDR3 region sequence, and these positive sequences were expressed and purified using Pichia pastoris, and the affinity was detected by Elisa. The results showed that the affinity of the VHH sequence to GST reached a high level. Finally, nanobodies against GST were successfully obtained. It can be further applied to the purification of GST fusion proteins, IP, WB, ELISA and other experiments, solving the problems of difficult purification, large batch differences and high production costs of traditional monoclonal antibodies.

[0026] The present invention provides a VHH chain of an anti-GST nanobody, comprising an amino acid sequence arranged as: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; the specific amino acid sequence is as follows: CDR1 is SEQ ID NO: 1, and the amino acid sequence is: GRTLSSYLMG, CDR2 is SEQ ID NO: 2, and the amino acid sequence is: RVTTGGRITT, CDR3 is SEQ ID NO: 3, and the amino acid sequence is: AAKKPFTDSYDY, FR1 is SEQ ID NO: 4, and the amino acid sequence is: DVQLQESGGGLVQTGGSLRLSCAAS, FR2 is SEQ ID NO: 5, and the amino acid sequence is: WFRQAPGKDREFVA, FR3 is SEQ ID NO: 6, and the amino acid sequence is: YAESVKGRFTISRDNAENTVYLQMNALKPEDTAVYYC, and FR4 is SEQ ID NO: 7, and the amino acid sequence is: WGQGTQVTVSS.

[0027] In one embodiment, the amino acid sequence of the VHH chain of the anti-GST Nanobody is shown in SEQ ID NO: 8, and its amino acid sequence is DVQLQESGGGLVQTGGSLRLSCAASGRTLSSYLMGWFRQAPGKDREFV ARVTTGGRITTYAESVKGRFTISRDNAENTVYLQMNALKPEDTAVYYCAAKKPFTDSYDYWGQGTQVTVSS.

[0028] The present invention also provides a first anti-GST Nanobody, which comprises any one of the amino acid sequences having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 8 in claim 2, or having no more than five amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 8 in claim 2.

[0029] The first anti-GST Nanobody, whose CDR1 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 1; and / or, its CDR2 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 2; and / or, its CDR3 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 3, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 3.

[0030] The present invention also provides a gene sequence, which encodes the VHH chain of the aforementioned anti-GST nanobody or encodes the aforementioned anti-GST nanobody.

[0031] Example 1

[0032] Anti-GST Nanobody Expression and Purification

[0033] The gene of the anti-GST nanobody was electrotransferred into Pichia pastoris, and secretory expression was induced. The supernatant was collected and purified using a Ni column and a molecular sieve column, respectively. 10 mg of the anti-GST nanobody was obtained from 200 mL of culture medium.

[0034] Comparative Example 1

[0035] Anti-GST traditional monoclonal antibody (antibody species: Mouse antibody subclass IgG1) expression and purification

[0036] Hek293 cells (200 mL culture medium) were transfected with a secretory plasmid of anti-GST conventional monoclonal antibody. After 72 hours, the cells were collected by centrifugation, lysed with a lysis buffer containing protease inhibitors, and the supernatant was collected. After Protein A affinity chromatography, 1 mg of anti-GST monoclonal antibody was obtained from 200 mL culture medium.

[0037] In comparison, Figure 3 As shown, nanobodies can be expressed and purified by microorganisms, while traditional antibodies need to be expressed and purified by mammalian cells, which has higher culture costs. In addition, for the same culture medium volume, the anti-GST traditional monoclonal antibodies purified by mammalian cells have less mass and lower molar concentration. Figure 3 It can be seen that the anti-GST nanobody of the present invention is easy to prepare and can be expressed in a microbial system such as yeast, with high yield and low cost.

[0038] Example 2

[0039] Application of HRP-labeled anti-GST nanobody in ELISA

[0040] 1. Coating ELISA plate. Dilute GST at a concentration of 4 ng / μL by two times, with a total of 12 concentration gradients. Coat 96-well plates, coat two wells for each concentration, and coat the BSA control wells at the same time. The coating solution is CBS at pH 9.4, the coating volume is 100 μl / well, and the coating is carried out at 4°C overnight.

[0041] 2. Wash the ELISA plate. Pour out the liquid in the wells of the ELISA plate, then add 200 μL PBS to each well to wash the well wall to remove unbound antigens. Wash the ELISA plate 3 times at room temperature, 10 minutes each time.

[0042] 3. Block the ELISA plate. Add 200 μl of blocking solution (PBS containing 3% BSA) to each well to block the ELISA plate and block for 1 hour at room temperature.

[0043] 4. Wash the ELISA plate. Discard the blocking solution, add 200 μl PBST (PBS containing 0.1% Tween20) buffer to each well, and wash the ELISA plate 3 times at room temperature, 10 minutes each time;

[0044] 5. Add antibodies. Dilute the prepared HRP-labeled anti-GST nanoantibody (1 mg / mL) at 1:5000 in PBS containing 3% BSA, add the diluted HRP-labeled anti-GST nanoantibody solution to the ELISA plate, 150 μl per well, and incubate at room temperature for 2 h.

[0045] 6. Wash the ELISA plate. Discard the liquid in the ELISA plate and add 200 μl PBST buffer to each well and wash 6 times, 5 minutes each time;

[0046] 7. Color development and reading. Add 100 μl TMB single-component color development solution to each well, color development for 2-3 min in the dark, add 100 μl 1M HCl to each well to stop color development, read the OD450 value with an enzyme-labeled instrument, record and save.

[0047] Comparative Example 2

[0048] Traditional anti-GST antibody ELISA experiment

[0049] Steps 1 to 4 are the same as the first 4 steps of the experiment in Example 2 (coating - washing - blocking - washing the ELISA plate).

[0050] 5. Incubate with primary antibody. Dilute the anti-GST traditional antibody (1 mg / mL, mouse IgG2a) at 1:5000 in PBS containing 3% BSA. Add the diluted traditional anti-GST antibody solution to the ELISA plate, 150 μl per well, and incubate at room temperature for 2 h.

[0051] 6. Wash the ELISA plate. Discard the liquid in the ELISA plate and add 200 μl PBST buffer to each well and wash 6 times, 5 minutes each time;

[0052] 7. Incubate with secondary antibody. Dilute the anti-mouse conventional secondary antibody (1 mg / mL) at 1:5000 in PBS containing 3% BSA. Add the diluted conventional anti-GST antibody solution to the ELISA plate, 150 μl per well, and incubate at room temperature for 2 h.

[0053] 8. Wash the ELISA plate. Discard the liquid in the ELISA plate and add 200 μl PBST buffer to each well and wash 6 times, 5 minutes each time;

[0054] 9. Color development and reading: Add 100 μl TMB single-component color development solution to each well, color development for 2-3 min in the dark, add 100 μl 1M HCl to each well to stop color development, read the OD450 value with an enzyme-labeled instrument, record and save.

[0055] Comparing the ELISA experimental operation process of nano antibodies (HRP) and traditional antibodies, it can be seen that when performing ELISA experiments with traditional antibodies, it is necessary to first incubate the primary antibody and then the secondary antibody, which takes a lot of time. The anti-GST nano antibody described in the present invention can be efficiently labeled with HRP, and has the characteristics of simple labeling process and high yield. The HRP-labeled anti-GST nano antibody only needs one step of incubation to develop color in the ELISA experiment, which can save at least 2 hours of time, and by Figure 4 It can be seen that when the antigen coating amount is 12.5 ng, the OD450 reading of the HRP-labeled nanobody after color development is still higher than 0.5, while at the same dilution multiple, the traditional antibody is colored after incubation with the primary antibody and the secondary antibody, and the OD450 reading is significantly lower than the nanobody shown in the present invention, indicating that the anti-GST nanobody described in the present invention has higher sensitivity.

[0056] In summary, the present invention provides a VHH chain of an anti-GST nanobody and its application, uses an antigen containing GST to immunize alpacas, and then constructs an immune library containing a diverse anti-GST nanobody sequence, and obtains an amino acid sequence library of anti-GST nanobody by screening through phage display technology, and then successfully obtains an anti-GST nanobody through further artificial transformation and optimization. Compared with the prior art, the technical solution proposed by the present invention can bring the following beneficial effects:

[0057] (1) Small molecules, suitable for tissue imaging: The two anti-GST nanobodies disclosed in the present invention have small molecular weights and have deeper tissue penetration ability in tissue imaging;

[0058] (2) Easy to prepare: The anti-GST nanobody of the present invention can be efficiently expressed by microbial cells such as Escherichia coli or Pichia pastoris, which is easy to produce and has little batch-to-batch variation;

[0059] (3) High affinity and high specificity: The anti-GST nanobody of the present invention has an affinity of up to 5.45*10 -8 M, higher than the affinity of currently published anti-GST nanobodies (such as Figure 1 shown).

[0060] (4) High stability: The anti-GST nanobody of the present invention has extremely high stability. It has been verified that it can withstand a variety of harsh cleaning environments and can remain stable for a long time at room temperature (it can still maintain more than 95% activity after being stored at room temperature for two weeks), which can meet the various experimental needs of researchers. Figure 2 As shown, the anti-GST nanobody was coupled to agarose beads and then subjected to immunoprecipitation experiments on GST-containing GFP protein. In different cleaning solutions (3M sodium chloride, 3M guanidine hydrochloride, 8M urea, 2% TritonX-100, 2% NP40, 0.1% SDS, 1M DTT, 5M DTT), the anti-GST nanobody was able to bind firmly to the target protein; in addition to being able to work in physiological saline, the antibody of the present invention can also tolerate high salt, reducing agents, denaturing agents, detergents and other cleaning environments.

[0061] (5) Has a wide range of applications: The anti-GST nanoantibody of the present invention can be covalently coupled with HRP (horseradish peroxidase) or fluorescent dyes such as FITC, which effectively solves the problem of cumbersome labeling steps in experiments such as ELISA, immunoblotting (WB) and fluorescent labeling, and has broad application prospects in experiments such as protein separation and purification, immunoprecipitation, co-immunoprecipitation, ELISA, and immunoblotting that are fused with GST.

[0062] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A VHH chain of an anti-GST nanobody, characterized in that It includes an amino acid sequence arranged as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; the specific amino acid sequence is as follows: CDR1 is SEQ ID NO: 1, CDR2 is SEQ ID NO: 2, CDR3 is SEQ ID NO: 3, FR1 is SEQ ID NO: 4, FR2 is SEQ ID NO: 5, FR3 is SEQ ID NO: 6, and FR4 is SEQ ID NO:

7.

2. The VHH chain of an anti-GST nanobody according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

8.

3. An anti-GST nanobody, characterized in that: It includes any amino acid sequence that has 95% or more identity with the amino acid sequence shown in SEQ ID NO: 8 in claim 2, or has no more than five amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 8 in claim 2.

4. An anti-GST Nanobody according to claim 3, characterized in that, Its CDR1 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO:1, or having one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO:

1.

5. An anti-GST Nanobody according to claim 3, characterized in that, Its CDR2 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO:2, or having one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO:

2.

6. An anti-GST Nanobody according to claim 3, characterized in that, Its CDR3 includes any one of the amino acid sequences having 90% or more identity with the amino acid sequence shown in SEQ ID NO:3, or has one or two amino acid mutations compared with the amino acid sequence shown in SEQ ID NO:

3.

7. A gene sequence, characterized in that It encodes the VHH chain of the anti-GST Nanobody according to claim 1 or 2 or it encodes the anti-GST Nanobody according to claim 3 or 4 or 5 or 6.

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