Hemoglobin camelid nanobody and preparation method and application thereof

Abstract

The application discloses a hemoglobin camel-derived nanobody and a preparation method and application thereof. The hemoglobin camel-derived nanobody is an amino acid sequence shown in SEQ ID NO: 7, is a hemoglobin camel-derived nanobody specific to hemoglobin derived from a bactrian camel, has small molecules, can easily penetrate, has high specificity, good stability, is easy to produce and transform, has wide application prospects, and can be applied to the fields of biomedical research, clinical diagnosis and treatment and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a hemoglobin nanobody and a preparation method and application thereof. BACKGROUND

[0002] Hemoglobin (Hb) is a crucial component in red blood cells, which is an iron-containing oxygen transport metalloprotein and plays an important role in the metabolism of organisms and the innate immune system. The content of Hb is a key physiological indicator for evaluating whether the human body is anemic and other blood diseases, and is also one of the important standards for measuring the health status of the human body. At present, the determination methods of hemoglobin concentration mainly include electrophoresis, liquid chromatography, absorbance spectrophotometry, electrochemical method, spectrophotometry and immunization method. However, the traditional methods have certain errors, which are not conducive to general survey and rapid diagnosis due to the complicated sample processing, the influence of hemoglobin family proteins, many interference factors, high maintenance cost, long experimental period, missed detection or misjudgment.

[0003] As a highly specific biological recognition molecule, anti-human hemoglobin antibody ensures that the antibody can distinguish and only bind to human hemoglobin, and does not produce cross-reaction with structurally similar but different source or function proteins. This specificity effectively avoids the detection result deviation caused by non-specific binding, such as false negative and false positive phenomena. It shows very high stability in the detection process, which can resist the influence of various potential interference factors, including non-specific proteins, lipids, sugars and other small molecule compounds and possible chemical or physical treatment condition changes. This anti-interference ability ensures the accuracy and reliability of the detection result, which is crucial for clinical diagnosis and treatment plan.

[0004] Among them, nanobody has the same antigen binding capacity and structural stability as conventional monoclonal antibody due to the extremely small size of protein molecules. Compared with traditional antibodies, nanobody has the advantages of strong antigen binding capacity, high stability, low production cost, easy production, low immunogenicity and unique biological distribution and pharmacokinetic characteristics, and has broad prospects in biomedical research and clinical application. It has shown potential as a biosensor in many fields such as medicine, environment and food analysis, and has brought revolutionary changes to the diagnosis, treatment and basic scientific research of diseases.

[0005] In practical application, if the detection antibody and the sample are derived from the same species, cross-reaction occurs between the detection antibody and the endogenous immunoglobulin in the sample, causing non-specific recognition, i.e. light chain heavy chain interference, resulting in misjudgment. SUMMARY

[0006] The present application aims to provide a hemoglobin camel-derived nanobody with high sensitivity and strong specificity, and a preparation method thereof, which expands the antibody selection range of users in practical applications, such as applications in research on the structure and function of hemoglobin, construction of disease models, blood disease detection, blood transfusion compatibility detection, immunotherapy, and drug development and delivery, so as to avoid errors caused by non-specific recognition of antibodies derived from the same species in practical applications.

[0007] To achieve the above-mentioned purpose, the present application provides a hemoglobin camel-derived nanobody, and the amino acid sequence of the hemoglobin camel-derived nanobody is shown in SEQ ID NO: 7.

[0008] The present application provides a nucleic acid molecule, which is used for encoding the above-mentioned hemoglobin camel-derived nanobody, and the nucleic acid sequence of the nucleic acid molecule is shown in SEQ ID NO: 6.

[0009] The present application provides a preparation method of a hemoglobin camel-derived nanobody, which is used for preparing the above-mentioned hemoglobin camel-derived nanobody, and the preparation method comprises the following steps:

[0010] S1, a hemoglobin antigen is separated from human blood cells to prepare a hemoglobin solution;

[0011] S2, the bactrian camel is immunized four times by using the hemoglobin solution, and the booster immunization is performed every 20, 60, and 40 days after the initial immunization;

[0012] S3, the lymphocytes with immunity are obtained 15 days after the four immunizations, the total RNA of the lymphocytes is extracted to construct a cDNA library, and the cDNA library is used as a template to perform specific amplification to obtain a nanobody nucleic acid sequence, and the nanobody nucleic acid sequence is used to construct a phage antibody library;

[0013] S4, the VHH-12 sequence with hemoglobin specificity is screened from the phage antibody library in step S3;

[0014] S5, the VHH-12 sequence in step S4 is subjected to recombinant expression to obtain the hemoglobin camel-derived nanobody.

[0015] The present application provides a phage antibody library, and the phage antibody library is obtained by the above-mentioned preparation method, and the phage antibody library comprises the above-mentioned nucleic acid sequence of SEQ ID NO: 6.

[0016] The present application provides a detection kit, which comprises the above-mentioned hemoglobin camel-derived nanobody or the hemoglobin camel-derived nanobody obtained by the above-mentioned preparation method.

[0017] The application discloses a hemoglobin camel-derived nanobody specific to hemoglobin from a bactrian camel, which is small in molecule, easy to penetrate, high in specificity, good in stability, easy to produce and transform, and can be applied to the fields of biomedical research, clinical diagnosis and treatment, and has wide application prospects.

[0018] In the preparation method, the bactrian camel is immunized with hemoglobin as an antigen, total RNA in lymphocytes of jugular vein blood of the bactrian camel is then extracted, and the total RNA is converted into cDNA through reverse transcription technology. On this basis, the VHH (nanobody) fragment is amplified, and high-efficiency connection is carried out with a phagemid vector, so that a phage antibody library rich in diversity is constructed. Through a three-round enrichment screening process, the nanobody sequence with high specificity to hemoglobin is successfully separated from the library, and the camel-derived nanobody targeting hemoglobin is obtained through recombinant expression technology. In the preparation method, only four immunizations are needed for the bactrian camel to obtain the antibody against hemoglobin.

[0019] The hemoglobin camel-derived nanobody is not only small in molecular weight, but also has excellent stability and strong tissue penetration, and can accurately recognize and combine hidden antigen epitopes which are usually difficult to access, so that the hemoglobin camel-derived nanobody has great potential in the fields of biomedical research, diagnosis and treatment of clinical diseases and the like. Meanwhile, the hemoglobin camel-derived nanobody has the characteristics of high solubility and strong stability, can be obtained in large quantities through genetic modification and humanization, and has realized intracellular delivery of intracellular antibody drugs in clinical practice.

[0020] Through experiments, the EC50 value of the hemoglobin camel-derived nanobody in the application and hemoglobin is 10.63nM, and the affinity constant KD value is 2.94x10 -7 M, which proves that the hemoglobin camel-derived nanobody obtained in the application has good effect and high affinity, and has high sensitivity for hemoglobin recognition.

[0021] Compared with a traditional glycated hemoglobin or hemoglobin detection kit, the hemoglobin camel-derived nanobody in the kit of the application has a significant improvement in stability. Meanwhile, the detection sensitivity and specificity of the kit of the application are improved, early detection and continuous monitoring of diseases are provided with strong support, and the shortcomings of a traditional human monoclonal antibody, such as large molecular weight, low stability, poor tissue penetration, high production cost and batch difference, are optimized. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Figure 1 is a schematic diagram of the immunization process of the bactrian camel in Example 1 of the present application.

[0023] Figure 2 Figure 2 is a data graph of the P / N values of each well plate in Example 1 of the present application.

[0024] Figure 3 Figure 3 is a schematic diagram of the nucleotide sequence of VHH-12 in Example 1 of the present application.

[0025] Figure 4 Figure 4 is a schematic diagram of the purification results of VHH-12-Fc in Example 1 of the present application.

[0026] Figure 5 Figure 5 is a schematic diagram of the experimental results of the non-competitive ELISA in Example 2 of the present application.

[0027] Figure 6 Figure 6 is a schematic diagram of the experimental results of BLI in Example 2 of the present application.

[0028] Figure 7 Figure 7 is a schematic diagram of the experimental results of the specific binding of VHH-12-Fc in Example 2 of the present application. DETAILED DESCRIPTION

[0029] Embodiments of the present application are described in detail below with reference to the attached drawings, which show by way of example, embodiments in which like numerals indicate like elements or elements having the same or similar function throughout the several views. The embodiments described below are examples intended to explain the present application, and are not to be understood as limiting the present application. Unless otherwise noted, technical or conditions not specified in the embodiments are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained commercially. In the following examples, unless otherwise specified, "%" refers to the percentage by weight.

[0030] 1. The Freund's complete adjuvant and the Freund's incomplete adjuvant used in the present application are commercially available products, purchased from Sigma-Aldrich.

[0031] 2. The formulation of the antigen diluent solution used in the present application is 2.43 g of Tris and 14.61 g of NaCl dissolved in ultrapure water, adjusted to pH 8.0, made up to 1 L, autoclaved at 121°C for 20 min, and 500 mM EDTA was added to a final concentration of 2 mM.

[0032] 3. The formulation of the solid culture medium used in the present application is 10 g of proteose peptone, 5 g of yeast extract, 10 g of NaCl, 100 mL of 20% (w / v) glucose solution, and ultrapure water to make up to 1 L, autoclaved at 121°C for 20 min.

[0033] The preparation method of the hemoglobin camel-derived nanobody in the present application is as follows:

[0034] S1, hemoglobin antigen is separated from human blood cells to prepare a hemoglobin solution;

[0035] S2, the bactrian camel is immunized four times by the hemoglobin solution, and the booster immunization is performed every 20, 60, and 40 days after the initial immunization;

[0036] S3, the lymphocytes with immunity are obtained 15 days after the four immunizations, the total RNA of the lymphocytes is extracted to construct a cDNA library, and the cDNA library is used as a template for specific amplification to obtain a nanobody nucleic acid sequence, and the nanobody nucleic acid sequence is used to construct a phage antibody library;

[0037] S4, the VHH-12 sequence with hemoglobin specificity is screened from the phage antibody library in step S3;

[0038] S5, the VHH-12 sequence in step S4 is recombinantly expressed to obtain a hemoglobin camel-derived nanobody.

[0039] Example 1: Preparation method of hemoglobin camel-derived nanobody

[0040] 1. Preparation of hemoglobin solution:

[0041] Hemoglobin is separated and purified from human blood cells, and then the hemoglobin antigen is dissolved in a phosphate buffer to obtain a hemoglobin solution.

[0042] 2. Immunization of bactrian camel:

[0043] The hemoglobin solution prepared in step 1 is mixed with Freund's complete adjuvant at a volume ratio of 1:1 (500 μg of hemoglobin per bactrian camel), and then the bactrian camel is subcutaneously injected after complete emulsification, and this injection is taken as the primary immunization. Then, the bactrian camel is boosted every 20, 60, and 40 days. The booster immunization refers to mixing the hemoglobin solution with Freund's incomplete adjuvant at a volume ratio of 1:1, and then muscle injecting the bactrian camel after complete emulsification, with a single immunization dose of 500 μg of hemoglobin per bactrian camel. The immunization of the bactrian camel is completed after four immunizations, and the immunization process is as shown in Figure 1 .

[0044] 3. Construction of phage antibody library: 15 days after the end of the last immunization, the bactrian camel's jugular vein blood is collected:

[0045] (1) The extracted double-humped camel jugular vein blood was slowly added to an equal volume of Ficoll separating solution and centrifuged (400g, 25°C, 20min), and the middle lymphocyte layer was taken. The lymphocytes were centrifuged in a centrifuge tube filled with DMEM (500g, 10min), and the cell precipitate was collected. The total RNA in the cell precipitate was extracted using an RNA extraction kit and reverse transcribed into cDNA.

[0046] (2) The cDNA obtained in step (1) was used as a template to amplify the nano sequence by specific primers. The nucleic acid sequence of the forward primer is shown in SEQ ID NO: 1, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO: 2. The DNA polymerase used for amplification is high-fidelity enzyme PrimeSTAR Max DNA Polymerase, and the amplification program is: 98°C, 10s, 57°C, 15s, 72°C, 25s, 30 cycles. The amplified VHH sequence was recovered using a kit.

[0047] (3) The pR2 phagemid was used as a template to amplify the pR2 phagemid sequence by specific primers. The nucleic acid sequence of the forward primer for amplifying the pR2 phagemid is shown in SEQ ID NO: 3, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO: 4. The DNA polymerase used for amplification is high-fidelity enzyme PrimeSTAR GXL DNA Polymerase, and the amplification program is: 98°C, 10s, 53°C, 15s, 68°C, 4min 15s, 30 cycles. Dpn I enzyme was used to specifically remove the template pR2 phagemid DNA in the amplification product, and then the amplified pR2 phagemid sequence was recovered using a kit.

[0048] (4) The nano antibody sequence recovered in step (2) and the pR2 phagemid sequence recovered in step (3) were precisely connected in a 4:1 molar ratio using seamless cloning to obtain a connection product, and the connection product was purified with the help of a kit. Sterile water was used during the elution process to ensure a sterile environment.

[0049] The obtained connection product was efficiently transformed by electroporation technology, and the transformed TG1 competent bacteria were cultured in a 37°C, 200rpm incubator for 1h. 0.2μL and 0.02μL of bacterial liquid were uniformly coated on 90mm solid medium, respectively, and the number of colonies was counted after 13 hours of culture. The total capacity of the constructed antibody library was estimated accordingly. The remaining bacterial liquid was coated on 5 pieces of 150mm solid medium after centrifugation (37°C, 13h), and then the bacterial lawn was scraped and frozen in liquid nitrogen. The phage antibody library was stored at -80°C.

[0050] 4. Screening of specific hemoglobin camel-derived nanobodies:

[0051] (1) Activate the phage antibody library bacteria and add KM13 helper phage, incubate and centrifuge to collect the bacteria, then shake the bacteria overnight. Use PEG precipitation method to enrich the phage, measure the titer of the enriched phage, and when the titer of the phage is 1 x 10 11 pfu, obtain the amplified phage.

[0052] The wells without coating hemoglobin are used as the negative control group (the negative control group is coated with hemoglobin antigen diluent solution), and the wells coated with hemoglobin solution are used as the hemoglobin group, and the hemoglobin group is coated with hemoglobin solution with a concentration of 0.1 mg / mL into the 96-well immunoplate. Add 1 x 10 11 pfu of amplified phage to the hemoglobin group and the negative control group, respectively, and incubate at room temperature for 1 h. The phage specifically combined with hemoglobin is eluted by trypsin solution, and these phage are used to infect TG1 bacteria. Take the bacterial liquid of the hemoglobin group and the negative control group after being infected by the phage, dilute and plate, and take 50 μL and 5 μL of the supernatant of the diluted hemoglobin group and the negative control group and plate on the solid culture medium, and make the number of phage plaques in the hemoglobin group greater than that in the negative control group.

[0053] (2) Randomly pick 95 single phage plaques from the solid culture medium in step (1) for overnight activation culture. Add KM13 helper phage to each single phage plaque to promote the release of phage, centrifuge to collect the supernatant after lysis, and obtain monoclonal phage, respectively.

[0054] The hemoglobin group is coated with hemoglobin at a concentration of 1 μg / mL into the immunoplate, and the above-mentioned 95 kinds of monoclonal phage are added, respectively, and incubated at room temperature for 1 h. Use HRP-anti M13 antibody to capture the phage combined with hemoglobin, add TMB developing solution in each well for color development for 5-10 min, and then add 1 M H2SO4 solution to terminate the reaction. Use the enzyme label instrument to accurately measure and record the absorbance ratio of each hemoglobin group (P value) and the negative control group (N value), and the results are shown in Figure 2 Take the phage plaques with P / N > 10 as positive phage plaques to obtain the antibody sequence specific to hemoglobin.

[0055] (3) Pick the phage plaques with P / N value greater than 10 for sequencing analysis, and the nucleic acid sequence of the sequencing primer is shown in SEQ ID NO: 5.

[0056] (4) After comparing and analyzing the measured antibody sequences and excluding the repeated clones, a specific nanobody sequence VHH-12 is obtained, and the nucleotide sequence of VHH-12 is shown in Figure 3 SEQ ID NO: 6.

[0057] 5. Recombinant expression of the Nanobody sequence:

[0058] The VHH-12 sequence obtained in step 4 was integrated into a mammalian expression vector pTT5 containing a signal peptide, and was expressed with a human IgG1-Fc fragment fused at the C-terminus. A TEV enzyme cleavage site was introduced so that the Nanobody monomer portion can be separated by precise cleavage with TEV enzyme when needed. Subsequently, the recombinant plasmid was successfully transfected into HEK-293F cells, and the cells were cultured and the supernatant was collected. The Nanobody Fc fusion protein (VHH-12-Fc) in the supernatant was efficiently purified by rProtein A affinity chromatography technology, and the VHH-12-Fc is the hemoglobin camel-derived Nanobody in the present application, and the amino acid sequence is shown in SEQ ID NO: 7. The purified VHH-12-Fc sample was subjected to SDS-PAGE experiment to verify the purity of the obtained antibody, and the electrophoresis result showed that the purity of VHH-12-Fc in the obtained sample was high, as shown in FIG. 2. Figure 4

[0059] The nucleic acid sequence of the forward primer of the specific primer for amplifying the VHH sequence in the present application is shown in SEQ ID NO: 1, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO: 2:

[0060] 5'-GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG-3'; SEQ ID NO: 1.

[0061] 5'-GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC-3'; SEQ ID NO: 2.

[0062] The nucleic acid sequence of the forward primer of the specific primer for amplifying pR2 in the present application is shown in SEQ ID NO: 3, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO: 4:

[0063] 5'-AGCAGCCGAACAAAAACTCATCTCAGAAGAG-3'; SEQ ID NO: 3.

[0064] 5'-CCATAGCAGGCTGTGCAGCATAGAAAGGTACCACTAAAGGAATTGC-3'; SEQ ID NO: 4.

[0065] The nucleic acid sequence of the sequencing primer in the present application is shown in SEQ ID NO: 5: ​

[0066] 5'-CAGGAAACAGCTATGAC-3'; SEQ ID NO: 5.

[0067] The nucleotide sequence of the hemoglobin camel-derived nanobody of the present application is shown as SEQ ID NO: 6, and the encoded amino acid sequence is shown as SEQ ID NO: 7.

[0068] CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCGCCGTGCAGGCAGGGGGGCATCTGAGACTCTCCTGTACAGCCTCTGGATTGGCTTTTGATAATCGTGCCATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAACCGGTCTCATGTATTAGTACGAGTGGTCGTAGCACATACTATAAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAAAGCACGCTGTATCTGCAAATGAACAGCCTGAAACCAGAAGACACTGCCGTGTATTACTGTGCGGCCCAGGCCGGAGGTCTGCTGGGTTGGGTACCGAGGTCCTGTCCGCGCGGTCGCTCGTATCTTGGTGCGAAGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA; SEQ ID NO: 6.

[0069] QLQLVESGGGAVQAGGHLRLSCTASGLAFDNRAMGWFRQAPGKEREPVSCISTSGRSTYYKDSVKGRFTISRDNA KSTLYLQMNSLKPEDTAVYYCAAQAGGLLGWVPRSCPRGRSYLGAKWGQGTQVTVSS; SEQ ID NO: 7.

[0070] Example 2: Validation experiment of hemoglobin camel-derived nanobody

[0071] 1. To evaluate the binding property of VHH-12-Fc to hemoglobin, non-competitive ELISA was used: hemoglobin at a concentration of 5 pg / mL was coated in the immunoplate, and 1:5 gradient dilution of VHH-12-Fc was added, incubated at room temperature for 1 h. Then, HRP anti-IgGl Fc antibody was added to detect the bound VHH-12-Fc. As shown in Figure 5 , the EC50 value of VHH-12-Fc to hemoglobin was 10.63 nM.

[0072] 2、In order to further quantify the affinity between the hemoglobin camel-derived nanobody (VHH-12-Fc) and hemoglobin in the present application, BLI experiment was used to characterize the affinity of VHH-12-Fc. First, biotin was used to label hemoglobin to obtain biotinylated hemoglobin (biotin-Hb). Then, it was immobilized on the SA biosensor, and different concentration gradients of VHH-12-Fc were set to detect the affinity of different concentration gradients of VHH-12-Fc to hemoglobin. The results are shown in Figure 6 The affinity constant KD value of VHH-12-Fc to hemoglobin is 2.94 x 10 -7 M.

[0073] Generally, the affinity constant KD value of antibody is considered to have strong affinity with the target in the range of 10 -12 to 10 -6 M. The affinity constant KD value of nanobody is in the sub-nanomolar to low nanomolar level. This range indicates that the nanobody has very strong binding ability to specific antigens and has high affinity. The affinity constant KD value of VHH-12-Fc to hemoglobin in the present application is 2.94 x 10 -7 M, which is within the above-mentioned affinity constant range, indicating that VHH-12-Fc in the present application has high affinity to hemoglobin. This high affinity characteristic makes it have great application potential in the field of drug research and development.

[0074] 3、In order to verify the specificity of VHH-12-Fc, non-competitive ELISA was used to characterize the binding of VHH-12-Fc to hemoglobin and other eight antigens: hemoglobin (Hb), SARS-CoV-2 receptor binding domain (SARS-CoV-2-RBD), SARS receptor binding domain (SARS-RBD), Vibrio parahaemolyticus thermostable direct hemolysin (TDH), ovalbumin (OVA), bovine serum albumin (BSA), green fluorescent protein (GFP), matrix metalloproteinase-1 (collagenase) (MMP-1) and polyphenol oxidase (PPO) were coated in the immunoplate at a concentration of 5 μg / mL. Add 1:5 gradient dilution of VHH-12-Fc, incubate at room temperature for 1 h, then add HRP anti-IgG1 Fc antibody to detect the bound VHH-Fc. The results are shown in Figure 7 The experimental results show that the hemoglobin camel-derived nanobody only specifically binds to hemoglobin and does not show any cross-reactivity with the other eight antigens, thereby confirming its high antigen specificity.

[0075] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the spirit and scope of the present application.

Claims

1. A hemoglobin camel-derived nanobody, characterized in that, The amino acid sequence of the hemoglobin camel-derived nanobody is shown in SEQ ID NO:

7.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is used to encode the hemoglobin camel-derived nanobody of claim 1, and the nucleic acid sequence of the nucleic acid molecule is shown in SEQ ID NO:

6.

3. A test kit, characterized in that, Includes the hemoglobin camel-derived nanobody as described in claim 1.

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