Nano antibody of acanthamoeba protozoa and application

Abstract

The invention provides an acanthamoeba protozoa nano antibody, which is characterized in that the acanthamoeba protozoa nano antibody comprises complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is as shown in SEQ ID NO.3. The nano antibody aiming at the acanthamoeba protozoa is obtained by screening on the basis of an alpaca natural antibody library, and the nano antibody is excellent in solubility, stability, resistance to aggregation, refolding performance and expression yield, low in expression cost and capable of being widely applied and popularized to pathogen detection of tap water, swimming pools, contact lens boxes and clinical patient specimens. Meanwhile, a targeted drug developed based on the antibody makes up the blank of treatment of amebic keratitis at present, and has important clinical value and market potential.

Description

Technical Field

[0001] The invention relates to a nanobody against Acanthamoeba and its application, belonging to the field of biotechnology. Background Art

[0002] Acanthamoeba is an amoeba widely present in the natural environment and can be isolated from soil, tap water, and air. Infections caused by Acanthamoeba can lead to Acanthamoeba keratitis and encephalitis, with high rates of blindness and fatality, imposing a heavy burden on society. Research data shows that among the reported cases of Acanthamoeba keratitis since 1980, 85% of the cases are related to wearing contact lenses. Factors such as rinsing the lenses with tap water and swimming after wearing suggest that Acanthamoeba may exist and cause disease in environments such as tap water, swimming pools, and contact lens cases. The clinical diagnosis of Acanthamoeba keratitis is relatively difficult. Moreover, the life cycle of Acanthamoeba has two stages: trophozoites and cysts. When the surrounding environment is not conducive to its growth, it will cause the transformation of amoeba trophozoites into cysts. The double-layer cyst wall structure of cysts has strong resistance to the environment and drugs, and has varying degrees of resistance to most antibiotics, making the infection chronic. The continuous survival of cysts at the infection site is the fundamental reason for the chronicity, intractability, and recurrence of Acanthamoeba infections in clinical practice. Therefore, it is very difficult to clinically cure Acanthamoeba infections.

[0003] Nanobodies are the smallest functional antigen-binding fragments derived from heavy-chain antibodies (HCAbs) in camelids. They have high stability and high affinity for antigen binding, and can interact with protein clefts and enzyme active sites. Therefore, nanobodies can provide new ideas for the design of small molecule enzyme inhibitors from peptide mimetic drugs. Due to having only a heavy chain, the production of nanobodies is easier than that of monoclonal antibodies. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, can be produced in large quantities at low cost. Therefore, nanobodies have great value and development prospects in the treatment and diagnosis of diseases. However, there is currently no nanobody product against Acanthamoeba. Summary of the Invention

[0004] Based on the screening of a natural antibody library of alpacas, the present invention obtained a nanobody against Acanthamoeba, which has a unique antigenic determinant recognition site, to solve the problem of the lack of nanobodies with high specificity and affinity against Acanthamoeba in the prior art. The present invention adopts the following technical solutions:

[0005] A nanobody against Acanthamoeba, the nanobody against Acanthamoeba comprises complementary determining regions CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of CDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.3.

[0006] Optionally, the acanthamoeba nanobody has an amino acid sequence with at least 95% identity to SEQ ID NO: 4.

[0007] Optionally, the acanthamoeba nanobody has the amino acid sequence shown in SEQ ID NO: 4.

[0008] The present invention also provides a method for screening acanthamoeba nanobodies, comprising the following steps:

[0009] Step 1) Isolate lymphocytes from alpaca blood, extract total RNA from the lymphocytes, and then reverse transcribe the RNA into cDNA; then amplify the heavy chain variable region gene to obtain the VHH target fragment;

[0010] Step 2) Construct a phage library using the VHH target fragment; then screen for nanobodies that can bind to acanthamoeba cysts by co-incubating the phage library with acanthamoeba cysts;

[0011] Step 3) Screen the nanobodies that can bind to acanthamoeba cysts by ELISA (enzyme-linked immunosorbent assay) to obtain highly specific nanobodies;

[0012] Step 4) Detect the binding affinity of the highly specific nanobodies to acanthamoeba cysts, and screen for acanthamoeba nanobodies with strong binding affinity to acanthamoeba cysts.

[0013] Preferably, in the ELISA screening, positive screening uses three genotypes of amoeba cysts, T3, T4, and T11, and negative screening uses 3% FBS / PBS and Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus oralis, Fusarium, Aspergillus fumigatus, and Candida albicans.

[0014] The present application also provides a nucleic acid encoding the above acanthamoeba nanobody.

[0015] The present application also provides a vector containing the above nucleic acid.

[0016] The present application also provides a host cell containing the above nucleic acid.

[0017] Optionally, the host cell is obtained by infecting Escherichia coli with a phage containing the above nucleic acid.

[0018] The method for preparing the above acanthamoeba nanobody comprises the following steps:

[0019] Culture the above-mentioned host cells and recover the Acanthamoeba nanobody from the cells or the cell culture.

[0020] This application also provides the use of the above-mentioned Acanthamoeba nanobody, nucleic acid, vector, and host cell in detecting Acanthamoeba.

[0021] This application also provides the use of the above-mentioned Acanthamoeba nanobody, nucleic acid, vector, and host cell in preparing products for preventing or treating Acanthamoeba infection.

[0022] Compared with the prior art, the present invention has the following technical effects:

[0023] Based on the alpaca natural antibody library, the present invention screened a nanobody against Acanthamoeba. The antibody has excellent solubility, stability, resistance to aggregation, refolding ability, and expression yield, and the expression cost is low. It can be widely applied to the pathogen detection of tap water, swimming pools, contact lens cases, and clinical patient specimens.

[0024] The present invention proves through experiments that this nanobody still has strong affinity when expressed in fusion with other proteins. Therefore, developing targeted drugs based on this antibody can fill the gap in the current treatment of Acanthamoeba keratitis and has important clinical value and market potential. Description of the Drawings

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is the ELISA test result of Example 1

[0027] Figure 2 It is the flow cytometry detection of the affinity of the Acanthamoeba antibody and the Acanthamoeba-targeted drug to Acanthamoeba in Example 1; in the figure, A is the blank control, B is the negative control, C is the detection result of antibody No. 18, D is the detection result of antibody No. 48, E is the detection result of antibody No. 86, F is the detection result of antibody No. 87, and G is the detection result of antibody No. 95.

[0028] Figure 3 It is the detection result of the antibody affinity screened in Example 1.

[0029] Figure 4 It is the plasmid schematic diagram of the nanobody-CXCL1 chimeric protein designed in Example 2.

[0030] Figure 5 The affinity detection results of the nanobody-CXCL1 chimeric protein synthesized in Example 2.

[0031] Figure 6 The observation results under a fluorescence microscope of the co-incubation of the nanobody-CXCL1 chimeric protein synthesized in Example 2 with amoeba cysts. Detailed implementation manners

[0032] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention. It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not used to limit the present invention.

[0033] In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention.

[0035] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are meant to include but not be limited to.

[0036] The present invention has screened a nanobody against Acanthamoeba protozoa from an alpaca natural antibody library. This antibody has a unique antigenic determinant recognition site to solve the problem of the lack of nanobodies with high specificity and affinity for Acanthamoeba protozoa in the prior art.

[0037] The screening of the above antibody includes the following steps:

[0038] Step 1) Isolate lymphocytes from alpaca blood, extract total RNA from the lymphocytes, and then reverse transcribe the RNA into cDNA; then amplify the heavy chain variable region gene to obtain the VHH target fragment;

[0039] Step 2) Construct a phage library using the VHH target fragment; then screen for nanobodies that can bind to Acanthamoeba cysts by co-incubating the phage library with Acanthamoeba cysts;

[0040] Step 3) ELISA detection and screening of the nanobody that can bind to Acanthamoeba cyst is performed to obtain a highly specific nanobody;

[0041] Step 4) The binding ability between the highly specific nanobody and Acanthamoeba cyst is detected, and a nanobody of Acanthamoeba with strong binding ability to Acanthamoeba cyst is screened.

[0042] Finally, an antibody AcAb95 with high specificity and high affinity is screened. This kind of nanobody of Acanthamoeba contains complementary determining regions CDR1, CDR2 and CDR3;

[0043] The amino acid sequence of its CDR1 is shown as SEQ ID NO.1: GSPFSTYFMG;

[0044] The amino acid sequence of its CDR2 is shown as SEQ ID NO.2: AIGWIGGTTS;

[0045] The amino acid sequence of its CDR3 is shown as SEQ ID NO.3: LGNSWPGGYDYW;

[0046] The overall amino acid sequence is shown as SEQ ID NO.4: DVQLQESGGGLVQAGGSLRLSCLASGSPFSTYFMGWFRQAPGEERKFVAAIGWIGGT TSYTDAVKGRFIISRDTAKNTLYLQMNRLKLEDTAVYYCMLLGNSWPGGYDYWGQG TQVTVSS.

[0047] The nucleic acid sequence encoding the antibody is shown in SEQ ID NO:5: GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTCTGGCCTCTGGATCCCCCTTCAGTACCTACTTCATGGGCTGGTTCCGCCAGGCTCCAGGCGAGGAGCGTAAGTTTGTAGCAGCGATTGGCTGGATTGGTGGTACTACATCCTATACAGACGCCGTAAAGGGCCGATTCATCATCTCCAGGGACACCGCCAAGAACACACTGTATCTGCAAATGAACCGCCTGAAACTTGAGGACACGGCCGTCTATTACTGTATGCTCTTAGGTAATAGCTGGCCAGGGGGCTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0048] Example 1

[0049] In this example, a nanobody against Acanthamoeba was proposed, and the screening method of the nanobody is as follows:

[0050] I. Screening of nanobodies with affinity for Acanthamoeba from the phage library

[0051] 1. Select 10 male alpacas over 2 years old, collect 20 mL of peripheral blood from their jugular veins, mix with an equal volume of sample diluent according to the instructions of the camel peripheral blood lymphocyte separation kit, separate lymphocytes, extract total RNA using an RNA extraction kit, and reverse transcribe the RNA into cDNA using a reverse transcription kit. Perform nested PCR amplification on the heavy chain variable region VHH gene according to the instructions of the PCR kit to obtain the VHH target fragment.

[0052] 2. Double-digest the pMES4 vector and the VHH target fragment with the restriction enzymes PstⅠ and Bst EⅡ, then ligate the vector and the target fragment with T4 ligase, and then electrotransfer them into TG1 competent cells. Incubate at 37 °C with shaking at 200 r / min for 1.5 h; take 100 μL of the resuscitated bacterial solution, and sequentially dilute the bacterial solution concentration in gradients to 10 5 、10 6 and 10 7, 100 μL of the diluted bacterial solution was taken and spread on an LB solid culture plate resistant to AG, and cultured overnight at 37 °C to calculate the library capacity. 24 clones were randomly selected from the culture plates with less than 300 positive clones for PCR identification to determine the positive rate. Then, the bacterial solution after electroporation and recovery was cultured until OD600 = 0.5. After adding the M13K07 helper phage, a phage library with a capacity of 1×10 12 pfu / ml was obtained.

[0053] 3. After blocking T4 genotype amoeba cysts with 3% FBS / PBS, they were co-incubated with the phage library for 1 h. After incubation, they were repeatedly centrifuged and washed. After the centrifugation and washing were completed, they were resuspended and incubated with Gly-HCl (pH = 2.2), and neutralized with Tris to a final pH of 7.0. The supernatant was retained for the next screening. TG1 monoclonal colonies were shaken to the logarithmic growth phase. After gradient dilution of the phage, TG1 bacterial solution was infected with phages at different concentrations. After infection, the TG1 bacterial solution was plated on a 2YT-A plate and cultured overnight at 37 °C. The next day, the bacteria were lysed to release the phage library, and the above processes of binding to cysts, incubation, and infection were repeated three times.

[0054] 4. After the last round of screening, monoclonal colonies were randomly selected on the plate and the CDR regions of the antibodies were sequenced to screen for non-repeated antibody sequences.

[0055] The antibody nucleic acid sequences obtained by screening are shown in Table 1:

[0056] Table 1 Antibody nucleic acid sequences obtained by screening

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Then, the above antibody sequences were detected for specificity and affinity, and antibodies with high specificity and high affinity were selected.

[0069] II. ELISA detection for screening high-specificity antibodies:

[0070] For positive screening, three genotypes of amoeba cysts, namely T3, T4, and T11, were used. For negative screening, 3% FBS / PBS and common ophthalmic pathogenic microorganisms such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus oralis, Fusarium, Aspergillus fumigatus, and Candida albicans were used. The same phage supernatant was added to the positive and negative screening wells, incubated, and then washed. Anti-M13-HRP antibody was added for incubation, and TMB was used for color development. The OD values were detected at 450 nm and 630 nm. A ratio of the OD value of positive screening to that of negative screening greater than 2 was considered to indicate affinity. The screening results are as Figure 1 shown. Among the phages with 47 non-repetitive sequences, 43 phage sequences that could bind to T3, T4, and T11 genotype amoeba cysts were screened out. Through reverse selection of the sequences of bacteria and fungi, finally, five phages with high specificity for binding to amoeba cysts, numbered 18, 48, 86, 87, and 95, were selected.

[0071] III. Flow cytometry detection for screening high-affinity antibodies:

[0072] The above-mentioned 5 phages screened by ELISA were further detected for affinity. Before detection, the phages were amplified to 10 11 / mL, co-incubated with T4 genotype amoeba cysts on ice, and then repeatedly washed with flow staining buffer. Anti-M13-PE antibody was added for incubation (M13K07 helper phage was added in the negative control), and the binding of different phages to amoeba cysts was analyzed using FlowJo7 software.

[0073] Figure 2 It was shown that the PE fluorescence signals in the phage group were all increased compared with the negative control group, indicating their binding. After incubation of the phages with amoeba cysts, the antibodies numbered 18, 48, 86, and 87 had poor binding ability. Finally, the antibody of the phage numbered 95 with high affinity was obtained and named AcAb95.

[0074] The amino acid sequence of its CDR1 is shown in SEQ ID NO.1: GSPFSTYFMG.

[0075] The amino acid sequence of its CDR2 is shown in SEQ ID NO.2: AIGWIGGTTS.

[0076] The amino acid sequence of its CDR3 is shown in SEQ ID NO.3: LGNSWPGGYDYW.

[0077] Its overall amino acid sequence is shown in SEQ ID NO.4: DVQLQESGGGLVQAGGSLRLSCLASGSPFSTYFMGWFRQAPGEERKFVAAIGWIGGTTSYTDAVKGRFIISRDTAKNTLYLQMNRLKLEDTAVYYCMLLGNSWPGGYDYWGQG TQVTVSS.

[0078] After expressing the selected antibodies, affinity detection was carried out: When using flow cytometry for affinity detection, 1×10 6 amoeba cysts were added to each tube. After co-incubating the antibody protein and the chimeric protein with the cysts, they were repeatedly washed with flow staining buffer, and then incubated with anti-His-AF647 antibody (purchased from abcam, containing red fluorescent protein). The binding of the antibody protein to the amoeba cysts was analyzed using Flow Jo7 software.

[0079] The detection results are as Figure 3 shown. Compared with the negative control, the expressed antibody protein has 100% affinity for amoeba cysts.

[0080] Example 2

[0081] Design a nanobody-CXCL1 fusion protein, which includes the AcAb95 antibody screened in Example 1, and connect mouse chemokine CXCL1 to the AcAb95 antibody. The protein sequence of mouse chemokine CXCL1 is as follows: MIPATRSLLCAALLLLATSRLATGAPIANELRCQCLQTMAGIHLKNIQSLKVLPSGPHCTQTEVIATLKNGREACLDPEAPLVQKIVQKMLKGVPK. A flexible linker peptide GSGGGGSGSGGSGGGGSSGGGGS is used to connect the AcAb95 antibody and mouse chemokine CXCL1 to form a chimeric protein. After further expression, affinity function verification is carried out, which specifically includes the following steps:

[0082] According to Figure 4 Design a fusion protein plasmid for expressing the AcAb95 nanobody and CXCL1 chemokine, and carry out protein expression in the E. coli system. The protein is purified by a nickel affinity chromatography column to obtain the chimeric protein.

[0083] Affinity detection 1: When using flow cytometry, 1×10 6Amoeba cysts were incubated with antibody proteins and chimeric proteins, and then repeatedly washed with flow cytometry staining buffer. Anti-His-AF647 antibody was added for incubation, and the binding of antibody proteins to amoeba cysts was analyzed using Flow Jo7 software.

[0084] Affinity detection 2: After incubating the chimeric protein with amoeba cysts, anti-His-AF488 antibody (purchased from abcam, containing green fluorescent protein) was used for incubation, and the amoeba was stained with DAPI and observed under a fluorescence microscope. The results are as Figure 6 shown. In the figure, the blue color is amoeba stained with DAPI, and the green color is the chimeric protein binding to His-AF488 antibody. It can be seen that the chimeric protein binds to the surface of the amoeba.

[0085] According to Figure 5 and Figure 6 the results, the fusion protein of AcAb95 nanobody and CXCL1 chemokine still has good affinity. Therefore, targeted drugs can be prepared based on the AcAb95 nanobody in the present invention.

[0086] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An Acanthamoeba nanobody, characterized in that: The Acanthamoeba nanobody comprises complementary determining regions CDR1, CDR2 and CDR3; wherein the amino acid sequence of the CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.

3.

2. The Acanthamoeba nanobody according to claim 1, characterized in that The Acanthamoeba Nanobody has an amino acid sequence that is at least 95% identical to SEQ ID NO:

4.

3. The Acanthamoeba nanobody according to claim 1, characterized in that The Acanthamoeba nanobody has an amino acid sequence as shown in SEQ ID NO:

4.

4. A nucleic acid, characterized in that The nucleic acid encodes the Acanthamoeba Nanobody according to any one of claims 1-3.

5. A carrier, characterized in that The vector comprises the nucleic acid of claim 4.

6. A host cell, characterized in that The host cell comprises the nucleic acid of claim 4.

7. A method for screening Acanthamoeba nanobodies, characterized in that: The steps include: Step 1) separating lymphocytes from alpaca blood, extracting total RNA from the lymphocytes, and then reverse transcribing the RNA into cDNA; then amplifying the heavy chain variable region gene to obtain the VHH target fragment; Step 2) constructing a phage library using the VHH target fragment; Then, the phage library is co-incubated with Acanthamoeba cysts to screen and obtain nanobodies that can bind to Acanthamoeba cysts; Step 3) performing ELISA detection and screening on the nanoantibodies that can bind to Acanthamoeba cysts to obtain highly specific nanoantibodies; Step 4) detecting the binding force between the highly specific nano-antibody and the Acanthamoeba cyst, and screening for the Acanthamoeba nano-antibody with strong binding force to the Acanthamoeba cyst.

8. A method for preparing an Acanthamoeba nanobody, characterized in that: The steps include: Cultivate the host cell of claim 6, and recover the Acanthamoeba nanobody from the cell or the culture of the cell.

9. Use of the Acanthamoeba nanobody according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5 or the host cell according to claim 6 in detecting Acanthamoeba.

10. Use of the Acanthamoeba nanobody according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5 or the host cell according to claim 6 in the preparation of a product for preventing or treating Acanthamoeba infection.

Images