A method for preparing shark nanobodies targeting IgM and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a method for preparing shark nanobodies targeting IgM and its application. The amino acid sequences of the shark nanobodies are 1-1D, 1-3G, and 1-5E shown in SEQ ID NO.1-SEQ ID NO.3. Compared with traditional antibodies, the VNARs prepared in the present invention have the characteristics of small molecular weight, strong physicochemical stability, and low production cost. The present invention provides a method for preparing targeting IgM, that is, immunizing sharks in the early stage, then constructing a phage display library with a high library capacity, using phage display technology for three rounds of panning to obtain VNARs with high affinity, and finally purifying three VNAR proteins. BLI detection shows that IgM and VNAR can effectively bind. The screened VNARs can be used as raw materials for ELISA detection kits and colloidal gold method detection kits, etc., laying a foundation for the early diagnosis of diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical antibody preparation, and particularly relates to a preparation method and application of a shark nanobody targeting IgM. Background Art

[0002] Immunoglobulin M (IgM) plays an important role in disease intervention. IgM can improve renal pathology and reduce organ damage. In cancer treatment, it is related to the recognition and clearance of precancerous cells. Native IgM without detectable natural glycosylation can be used as a diagnostic and prognostic marker, especially for breast cancer. IgM is also a target molecule for diagnosing various diseases. Some hereditary immunodeficiency diseases characterized by hypogammaglobulinemia, such as hyper-IgM syndrome and common variable immunodeficiency (CVID), can be diagnosed by measuring the levels of serum immunoglobulins (IG) classes, which are characterized by abnormally high IgM levels and changes in the contents of IgA and IgG in serum. Therefore, detecting the changes of IgA, IgM, and IgG in serum can distinguish them from other primary immunodeficiency diseases. IG detection can be used to determine whether there is an infection by detecting antibodies in serological experiments. Since IgM is the first antibody produced during the body's humoral immune response, its detection is particularly important for the early diagnosis of acute infections. For some viruses, such as hepatitis E virus, cytomegalovirus, measles virus, and adenovirus, detecting the IgM content of these viruses can determine the infection status and make a correct diagnosis in a timely manner. Methods such as enzyme-linked immunosorbent assay (ELISA) usually detect immune responses by directly or indirectly monitoring the IgM content level.

[0003] Traditional antibodies have been widely used in the fields of diagnosis and treatment. However, despite many successful examples, they also have many limitations, such as their large size, complex structure, and long production time. With the development of genetic engineering technology, recombinant antibody engineering can reduce conventional IgG antibodies to their smallest antigen-binding fragments, which can not only reduce the production cost of recombinant proteins but also reduce the off-target effects of Fc. However, these forms have shown poor stability and even loss of antigen activity. Based on this, in 1993, scientists isolated a homodimer containing only heavy-chain antibodies from the serum of camelids. This dimer is called a heavy-chain antibody (HCAb). Later, in 1995, a new antigen receptor of immunoglobulin (IgNAR) with a structure similar to HcAb was also found in shark serum. By cloning and expressing the antigen variable binding region at their N-terminus, the obtained antigen-binding domain is a single-domain antibody, also known as a nanobody. Camel-derived single-domain antibodies and shark-derived single-domain antibodies are called VHH and VNAR, respectively. Single-domain antibodies have a small molecular weight, strong physicochemical stability (salt tolerance, acid tolerance, alkali tolerance, organic solvent tolerance, high temperature tolerance), strong tissue penetration, and low production cost. Moreover, compared with camel-derived nanobodies, shark nanobodies have a smaller molecular weight and higher physicochemical stability. Currently, shark nanobodies have received extensive attention in the fields of drug development, in vitro diagnosis, and immunoassay. Based on this, the present invention proposes novel shark nanobodies with stable physicochemical properties, laying a foundation for the development of virus detection and test kits. Summary of the Invention

[0004] The object of the present invention is to provide a preparation method and application of shark nanobodies targeting IgM. The present invention has screened out brand-new shark nanobodies 1-1D, 1-3G, and 1-5E, which have high affinity and high specificity for IgM.

[0005] The present invention protects shark nanobodies 1-1D, 1-3G, and 1-5E targeting IgM, and their amino acid sequences are shown in SEQ ID No.1 - SEQ ID No.3.

[0006] Furthermore, the shark nanobody comprises a framework region, a complementary determining region, and a hypervariable region;

[0007] The framework region is FR1, FR2, FR3a, FR3b, and FR4;

[0008] The amino acid sequence of FR1 is shown in SEQ ID No.4 - SEQ ID No.6;

[0009] The amino acid sequence of said FR2 is shown in SEQ ID No. 7 - SEQ ID No. 9;

[0010] The amino acid sequence of said FR3a is shown in SEQ ID No. 10 - SEQ ID No. 12;

[0011] The amino acid sequence of said FR3b is shown in SEQ ID No. 13 - SEQ ID No. 15;

[0012] The amino acid sequence of said FR4 is shown in SEQ ID No. 16 - SEQ ID No. 18;

[0013] The complementarity-determining regions are CDR1 and CDR3;

[0014] The amino acid sequence of said CDR1 is shown in SEQ ID No. 19 - SEQ ID No. 21;

[0015] The amino acid sequence of said CDR3 is shown in SEQ ID No. 22 - SEQ ID No. 24;

[0016] The hypervariable regions are HV2 and HV4;

[0017] The amino acid sequence of said HV2 is shown in SEQ ID No. 25 - SEQ ID No. 27;

[0018] The amino acid sequence of said HV4 is shown in SEQ ID No. 28 - SEQ ID No. 30.

[0019] The present invention also protects the genes encoding the shark nanobodies 1-1D, 1-3G, 1-5E, and the nucleotide sequences of said genes are SEQ ID No. 31 - SEQ ID No. 33.

[0020] The present invention also protects a recombinant vector, and said recombinant vector contains the above-mentioned encoding genes.

[0021] The present invention also protects a recombinant strain, and said recombinant strain contains the above-mentioned encoding genes or the above-mentioned recombinant vector.

[0022] The present invention also protects the use of the shark nanobodies 1-1D, 1-3G, 1-5E in the preparation of an IgM protein targeting agent.

[0023] The present invention also protects the use of the shark nanobodies 1-1D, 1-3G, 1-5E in a virus detection kit.

[0024] Advantages of the present invention:

[0025] 1. The molecular weights of the shark nanobodies 1-1D, 1-3G, and 1-5E prepared by the present invention are smaller compared to traditional monoclonal antibodies, only about 15 kDa.

[0026] 2. The shark nanobodies prepared by the present invention have high stability and strong physical and chemical stability. They remain stable in extreme environments (high temperature, strong acid and strong alkali), and have good affinity and specificity with IgM.

[0027] 3. The shark nanobodies prepared by the present invention can be used as raw materials for ELISA test kits and colloidal gold test kits, etc., for the early diagnosis of diseases.

[0028] 4. The present invention selects the bamboo shark as a model animal for antibody preparation to prepare VNAR. It does not belong to endangered shark species, has a small body size, is easy to breed artificially, and is suitable for antibody development. Description of the Drawings

[0029] Figure 1 Gel electrophoresis results of PCR amplification products of VNAR fragments; Lane description: M. DNA Marker, 1. Negative control, 2-13. PCR products;

[0030] Figure 2 Polyclonal ELISA assay results of specific phages;

[0031] Figure 3 Monoclonal ELISA assay results of specific phages;

[0032] Figure 4 SDS-PAGE result diagram of VNAR protein purification; Lane description: M. Protein Marker, 1. 1-1D, 2. 1-3G, 3. 1-5E;

[0033] Figure 5 BLI detection results of the binding of human IgM, IgG and VNAR protein; A. BLI detection results of the binding of human IgM and VNAR protein; B. BLI detection results of the binding of human IgG and VNAR protein. Detailed Embodiments

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] To better illustrate the purpose, technical solution and advantages of the present invention, the technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0036] The experimental methods in the following embodiments are all conventional methods in the art unless otherwise specified, and are carried out according to the techniques described in the literature in the art or according to the product specifications; various biological materials, reagents, etc. used in the embodiments can be obtained from commercial sources unless otherwise specified.

[0037] Example 1: Construction of a targeted IgM shark nanobody immune library

[0038] 1. Immunization of Chiloscyllium plagiosum

[0039] Chiloscyllium plagiosum is cultured in an aquarium with an appropriate salinity of about 1.023 and an appropriate temperature of 23°C - 25°C. Fresh shrimps are fed once every two days, and each fish is fed about 2 - 3 shrimps. The food residues are fished out on the day after feeding. Vitamins are added once a week, and the protein skimmer and sewage are cleaned in time to ensure the water quality. When immunizing Chiloscyllium plagiosum, IgM is used as the antigen for immunization. The immunization injection sites of Chiloscyllium plagiosum are selected at the dorsal fin and lateral fin parts of the shark. For the first immunization, 100 μg of the mixed antigen is emulsified with Freund's complete adjuvant. For the second and subsequent immunizations, 100 μg of the mixed antigen is emulsified with Freund's incomplete adjuvant, and the immunization is carried out at a site close to the previous immunization site. The immunization lasts for about 3 months, and the immunization is carried out once every 2 weeks.

[0040] 2. Construction of a shark nanobody immune library

[0041] Peripheral blood lymphocytes are extracted from the immunized Chiloscyllium plagiosum. Peripheral blood lymphocytes can be obtained by centrifuging the whole blood of the shark, and then the total RNA of the peripheral blood lymphocytes is extracted according to the operation instructions of the RNA extraction kit, and the RNA is reverse transcribed into cDNA.

[0042] Using cDNA as a template, through PCR reaction amplification, the reaction system is as follows (see Table 1), and the VNAR fragment is amplified (see Figure 1 ), and the amplified product is recovered by gel electrophoresis. Among them, the sequences of the primer combinations are as follows:

[0043] Forward primer NewVF1 (SEQ ID NO.34):

[0044] CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC.

[0045] Forward primer NewVF2 (SEQ ID NO.35):

[0046] CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAACAAACACCG。

[0047] Upstream primer NewVF3 (SEQ ID NO.36):

[0048] CAATTTGATTGGGCCCAGGCGGCCCAATGGGTTGAACAAACACCGA。

[0049] Primer NewVR1 (SEQ ID NO.37):

[0050] CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC。

[0051] Primer NewVR2 (SEQ ID NO.38):

[0052] CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC。

[0053] Primer NewVR3 (SEQ ID NO.39):

[0054] CAATTTGATTGGGCCGGCCTGGCCAGGTTTCACAGTCAGAATGGTG

[0055] The PCR amplification system is as follows:

[0056] Table 1 PCR reaction system

[0057]

[0058] ddH2O up to 50μL

[0059] 2×Phanta Max Master Mix 25μL

[0060] Upstream primer (10μM) 2μL

[0061] Downstream primer (10μM) 2μL

[0062] Template DNA* 1.0μL

[0063] The reaction conditions are as follows:

[0064] Table 2 PCR reaction program

[0065]

[0066] Meanwhile, the amplified VNAR fragments and the pComb3XSS vector were digested with two restriction enzymes and recovered. The digestion system was as follows:

[0067] Table 3 Restriction Enzyme Reaction System

[0068]

[0069] Then, the purified VNAR fragments were ligated into the pComb3XSS vector using T4 ligase. The ligation system was as follows:

[0070] Table 4 T4 Ligation System

[0071]

[0072]

[0073] The ligation products were then electrotransformed into competent E. coli TG1 cells. Then, a portion of the bacterial solution was serially diluted and spread on 2×YT plates, and incubated overnight at 37°C in an inverted position. According to the number of colonies on the plates, the library capacity of the constructed shark nanobody phage library was calculated. Then, monoclonal colonies were sent for sequencing for sequence alignment. Finally, it was calculated that the library capacity of the shark nanobody phage library was 3×10 9 CFU, and the sequence diversity was 100%.

[0074] Example 2: Panning of Shark Nanobody Phages Targeting IgM (Three Rounds of Panning)

[0075] I. Three Rounds of Panning of Shark Nanobodies Targeting IgM

[0076] 1. Streak TG1 on an LB solid plate without antibiotics two days in advance and incubate overnight at 37°C, then let it stand.

[0077] 2. (1) The next day, pick a monoclonal colony and inoculate it into 10 mL of 2×YT medium without antibiotics, and incubate overnight at 37°C with shaking at 180 rpm.

[0078] (2) Prepare 50 mL of PEG / NaCl solution and stir magnetically overnight.

[0079] (3) Coating.

[0080] a. Coat 4 wells of a 96-well plate with IgM at a concentration of 10 μg / mL, 100 μL per well, and incubate overnight at 4°C. (10 μg / mL for the first round, 4 μg / mL for the second round, and 2 μg / mL for the third round).

[0081] b. Coat 4 wells with gelatin buffer, 200 μL per well, and incubate overnight at 4°C.

[0082] 3. After washing the plate 3 times, block the IgM, add the phage library, and incubate at 37°C for 1 - 1.5 h

[0083] (1) Wash the plate with PBST (0.1% Tween-20 in PBS), wash the wells coated with IgM, fill the whole well (about 300 uL), and tap the plate (it can be washed with a plate washer).

[0084] (2) Add gelatin blocking buffer to the IgM wells (use gelatin blocking buffer in the first round, protein-free blocking buffer in the second round, and gelatin blocking buffer in the third round, alternating blocking) 200 uL / well.

[0085] (3) At the same time, add the diluted phage to the blocking solution wells, 100 uL / well (diluted with gelatin blocking solution).

[0086] Calculation method: Make the added 400 uL phage contain 1000 times the library capacity to ensure complete panning (input 1000 times the library capacity in the first round, and 100 times the library capacity in the second and third rounds).

[0087] 4. Activate TG1 and incubate

[0088] (1) For the stored TG1 strain shaken overnight, take 100 uL and add it to 5 mL of fresh 2xYT medium, and incubate at 37 °C at 190 rpm for 1 - 1.5 h until the logarithmic growth phase is reached (OD600 = 0.5 - 0.8).

[0089] (2) Take out the phage in the blocking solution wells into an EP tube, wash the plate 3 times, add the above phage to the IgM wells, and incubate at 37 °C for 1 - 1.5 h.

[0090] 5. Wash the plate 10 - 15 times (wash 9 times in the first round, 12 times in the second round, and 12 times in the third round)

[0091] 6. Infect

[0092] Add the activated TG1 to the IgM wells, 100 uL / well, let it stand at 37 °C for 30 min, collect the infected TG1, replace it with fresh TG1, and repeat this 3 times (as long as it is the infection step, it needs to stand).

[0093] 7. Plate coating and verification

[0094] Dilute the bacterial solution to 100, 1000, and 10000 times (1 mL volume) by the method of limited dilution, and then take 100 uL from each and plate it (Amp + ), then it is diluted 1000, 10000, and 100000 times, culture overnight at 37 °C, and observe the number of colonies grown, which is the successfully infected TG1. When the input TG1 is the same, a significant increase in the number of colonies grown indicates enrichment and the following experiment can be carried out.

[0095] 8. Helper phage superinfection

[0096] Add M13K07 with a volume 20 times that of the remaining TG1 after infection, and let it stand at 37 °C for 1 h (mix well at 30 min).

[0097] 9. Amplification of progeny phages

[0098] Centrifuge the bacterial solution at 5000 rpm at 25 °C for 10 min, discard the supernatant, resuspend it in 10 mL of fresh 2xYT, add Kana (50 μg / mL) and Amp (100 μg / mL), and culture overnight at 30 °C with shaking at 180 rpm.

[0099] 10. Harvest phages

[0100] Centrifuge the bacterial solution (10 mL) that was shaken overnight in the previous round at 5000 rpm for 25 min, filter the supernatant through 0.45 μm and 0.22 μm filters, add PEG / NaCl (2.5 mL) with a volume of 1 / 4 of the bacterial solution volume, and incubate on ice for 0.5 - 1 h (until white flocculates appear); centrifuge at 5000 rpm at 4 °C for 30 min, discard the supernatant, invert and dry on absorbent paper, resuspend with PBS (500 μL) with a volume of 1 / 20 of the original supernatant volume, and measure OD280 (OD280 = 1 is equivalent to 2.5x10 12 cfu / mlL) II. Polyclonal ELISA

[0101] 1. Coating

[0102] Coat with IgM at a concentration of 1 μg / mL, 100 μL / well.

[0103] Coat with gelatin blocking buffer, 100 μL / well, overnight at 4 °C.

[0104] 2. Wash the plate 3 times

[0105] 3. Blocking

[0106] Gelatin blocking buffer (the same as the coating one), 300 μL / well, block at 37 °C for 1 - 2 h.

[0107] 4. Sample preparation

[0108] Prepare the original library, phages after the first, second, and third rounds of panning, measure OD280, and dilute them to 10 10 cfu with gelatin blocking buffer.

[0109] 5. Wash the plate 3 times

[0110] 6. Incubation

[0111] Add the diluted phages to the plate, 100 μL / well, incubate at 37 °C for 1 h.

[0112] 7. Wash the plate 3 times

[0113] 8. Secondary antibody incubation

[0114] Dilute the HRP linked anti - M13 antibody (1:10000) with gelatin blocking buffer, 100 μL per well, incubate at 37 °C for 1 h.

[0115] 9. Wash the plate 3 times

[0116] 10. Color development

[0117] Mix 100 μL / well of Solution A and Solution B of TMB color development solution at a ratio of 1:1 for 2 - 15 min.

[0118] 11. Termination

[0119] Terminate with 0.1 M H2SO4, add 50 μL per well.

[0120] 12. Reading

[0121] Read the OD450 with an ELISA reader.

[0122] The results of polyclonal ELISA for phage detection are as Figure 2 shown. With the increase of panning times, specific phages are enriched.

[0123] III. Monoclonal ELISA

[0124] 1. Picking monoclonal

[0125] (1) Prepare a 96 - well U - bottom plate, add 2xYT (Amp+) 200 μL per well, select the plate diluted during the second or third round of identification for plating, pick monoclonal and inoculate it into the 96 - well plate, culture at 37 °C and 220 rpm for 3 h until the logarithmic growth phase.

[0126] (2) Add new 2xYT [M13K07 (20 - fold bacterial volume), Amp + to another 96 - well U - bottom plate, 100 μL per well, add 100 μL of the bacterial solution that has been shaken to the logarithmic growth phase to each well, incubate at 37 °C for 30 min for infection, and culture overnight at 30 °C and 220 rpm.

[0127] 2. ELISA identification

[0128] (1) Coating

[0129] Coat with IgM, coating concentration is 1 μg / mL, 100 μL per well, overnight at 4 °C.

[0130] (2) Wash the plate 3 times

[0131] (3) Blocking

[0132] Block with gelatin blocking buffer, 300 uL / well, incubate at 37 °C for 1 - 2 h.

[0133] (4) Sample preparation

[0134] Centrifuge the bacterial liquid in the 96-well U-bottom plate after overnight culture of monoclonal colonies at 2000 rpm for 10 min.

[0135] (5) Wash the plate 3 times

[0136] (6) Incubation

[0137] Take the supernatant in the 96-well U-bottom plate after centrifugation and add it to the ELISA plate, 100 uL / well, incubate at 37 °C for 1 h.

[0138] (7) The remaining steps are the same as those for polyclonal ELISA.

[0139] The results of monoclonal ELISA detection of phages are as Figure 3 shown. Take the positive phages with an absorbance value greater than 1.5 at 450 nm for sequencing. Use the NCBI-BLAST tool for homology comparison of the sequencing results, use the NCBI-ORFfinder tool for translation of the amino acid sequence, and perform analysis of amino acid sequence diversity in BioEdit. Finally, 3 different VNAR sequences targeting IgM are obtained, named 1-1D, 1-3G, 1-5E, and their amino acid and nucleotide sequences are as follows:

[0140] GWVDQTPTTTTKEAGESLTINCVLKGSRYALCDTYWYFTKKGATKKERL SNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKASRRTSTVSWNCRPIHYE GGGTILTVKP (SEQ ID NO.1)

[0141] GWVDQTPTTTTKEAGESLTINCVLKGSRYALCDTYWYFTKKGATKKERL SNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKASRRTGTVSWNCRPIHYE GGGTILTVKP (SEQ ID NO.2)

[0142] AWVDQTPRTITKETGESLTINCVLKDASYALSGTYWYLTKLDATKWDRIS IGGRYSETVNKGSKSFSLRLRDLRVEDSGAYHCEAYGTAAMTGRGGRGTFLA VKP (SEQ ID NO.3)

[0143] GGTTGGGTGGACCAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGTGTCCTAAAAGGTTCCAGATATGCATTGTGTGACACGTATTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGGTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTCTCGTCGGACGAGCACCGTCAGCTGGAATTGCCGGCCTATTCATTATGAGGGAGGCGGCACCATTCTGACTGTGAAACCT(SEQ ID NO.31)

[0144] GGTTGGGTGGACCAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGATATGCATTGTGTGACACGTATTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGGTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTCTCGTCGGACGGGCACCGTCAGCTGGAATTGCCGGCCTATTCATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCT(SEQ ID NO.32)

[0145] GCATGGGTTGACCAAACACCGAGAACAATAACGAAGGAGACAGGCGAATCACTGACCATCAACTGTGTCCTAAAAGATGCTAGCTATGCATTGAGTGGCACGTACTGGTATCTGACAAAATTGGATGCAACAAAGTGGGACCGCATATCAATTGGTGGACGATACTCTGAAACAGTGAACAAGGGATCAAAGTCCTTTTCTTTGCGACTTCGTGATCTGAGAGTTGAAGACAGTGGTGCATATCACTGTGAAGCGTATGGCACAGCTGCGATGACGGGACGGGGAGGAAGAGGCACTTTTCTGGCAGTGAAACCT(SEQ ID NO.33)

[0146] The shark nanobodies 1-1D, 1-3G, and 1-5E include framework regions FR, complementarity-determining regions CDR, and hypervariable regions. The amino acid sequences of the framework regions FR, including FR1, FR2, FR3a, FR3b, and FR4, are as follows:

[0147] 1-1D:

[0148] FR1: GWVDQTPTTTTKEAGESLTINCVLK(SEQ ID NO.4);

[0149] FR2: TYWYFT(SEQ ID NO.7);

[0150] FR3a: GRYAETV(SEQ ID NO.10);

[0151] FR3b: FSLRISDLRVEDSGTYHC(SEQ ID NO.13);

[0152] FR4: EGGGTILTVKP(SEQ ID NO.16).

[0153] HV2: KKGATKKERLSNG(SEQ ID NO.19);

[0154] HV4: NKASKS(SEQ ID NO.22);

[0155] CDR1: GSRYALCD(SEQ ID NO.25);

[0156] CDR3: KASRRTSTVSWNCRPIHY(SEQ ID NO.28).

[0157] 1-3G:

[0158] FR1: GWVDQTPTTTTKEAGESLTINCVLK (SEQ ID NO.5);

[0159] FR2: TYWYFT (SEQ ID NO.8);

[0160] FR3a: GRYAETV (SEQ ID NO.11);

[0161] FR3b: FSLRISDLRVEDSGTYHC (SEQ ID NO.14);

[0162] FR4: EGGGTILTVKP (SEQ ID NO.17).

[0163] HV2: KKGATKKERLSNG (SEQ ID NO.20);

[0164] HV4: NKASKS (SEQ ID NO.23);

[0165] CDR1: GSRYALCD (SEQ ID NO.26);

[0166] CDR3: KASRRTGTVSWNCRPIHY (SEQ ID NO.29).

[0167] 1-5E:

[0168] FR1: AWVDQTPRTITKETGESLTINCVLK (SEQ ID NO.6);

[0169] FR2: TYWYLT (SEQ ID NO.9);

[0170] FR3a: GRYSETV (SEQ ID NO.12);

[0171] FR3b: FSLRLRDLRVEDSGAYHC (SEQ ID NO.15);

[0172] FR4: GGRGTFLAVKP (SEQ ID NO.18).

[0173] HV2: KLDATKWDRISIG (SEQ ID NO.21);

[0174] HV4: NKGSKS (SEQ ID NO.24);

[0175] CDR1: DASYALS (SEQ ID NO.27);

[0176] CDR3: EAYGTAAMTGR (SEQ ID NO.30).

[0177] Example 3: Prokaryotic Expression and Purification of Shark Nanobody

[0178] The vector pET28a was double digested with two restriction endonucleases, Nco1 and Not1. The VNAR fragment was amplified by PCR, and the vector and the fragment were ligated using a homologous recombination kit.

[0179] The plasmid with successful sequencing was transformed into E. coli BL21(DE3) competent cells for protein expression. The next day after transformation, a single clone was picked for overnight shaking culture. After the culture, the bacterial solution was transferred to 200 mL of BL21 medium (containing kanamycin) at a ratio of 1:100. When the OD value reached 0.5 - 0.8, IPTG with a final concentration of 0.1 mM was used for induction at 18 °C for 24 h. After the induction was completed, the bacteria were collected by centrifugation, the supernatant was discarded, and the bacteria were resuspended with 20 mL of PBS. Protease inhibitor was added at a ratio of 1:100, and then ultrasonic crushing (20% power, ultrasonic on for 3 s, off for 6 s) was performed until the solution became clear. After ultrasonic treatment, centrifugation was carried out at 12,000 rpm for 15 min at 4 °C. The shark nanobody protein was purified by nickel column affinity chromatography, and the imidazole eluate of each fraction was collected. The eluate of each fraction was detected by SDS-PAGE. The eluate containing shark nanobody was ultrafiltered and concentrated. After the concentration was completed, the concentration of shark nanobody was detected by the BCA method. Finally, 3 VNAR proteins were successfully obtained. The SDS-PAGE results are as Figure 4 shown.

[0180] Example 4: Detection of the Affinity between Human IgM, IgG and Shark Nanobody by BLI

[0181] Human IgM and IgG were diluted to 300 nM with 0.02% PBST and immobilized on a nickel (Ni) biosensor for 90 s. Then, basiline was performed for 120 s in the buffer to make the baseline stable, and then it was combined with 10 μg / mL shark nanobody, and then dissociation was carried out. The samples were added to a 96-well black bottom plate at 200 μL / well. All steps were carried out at room temperature. The results of the affinity determination between human IgM and shark nanobody are as Figure 5 shown in A. The VNAR protein has an affinity for human IgM. The results of the affinity determination between human IgG and shark nanobody are as Figure 5 shown in B. The VNAR protein has almost no affinity for human IgG, indicating that the screened IgM-targeting protein has good specificity.

[0182] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A shark nanobody 1-1D, 1-3G, 1-5E targeting IgM, characterized in that: The amino acid sequence of 1-1D is shown in SEQ ID No.1, the amino acid sequence of 1-3G is shown in SEQ ID No.2, and the amino acid sequence of 1-5E is shown in SEQ ID No.

3.

2. The shark nanobody 1-1D, 1-3G, 1-5E according to claim 1, characterized in that: The three shark nanobodies comprise a framework region, a complementarity determining region and a hypervariable region; The framework regions are FR1, FR2, FR3a, FR3b and FR4; The amino acid sequence of the FR1 region of 1-1D is shown in SEQ ID No.4, the amino acid sequence of the FR1 region of 1-3G is shown in SEQ ID No.5, and the amino acid sequence of the FR1 region of 1-5E is shown in SEQ ID No.6; The amino acid sequence of the FR2 region of 1-1D is shown in SEQ ID No.7, the amino acid sequence of the FR2 region of 1-3G is shown in SEQ ID No.8, and the amino acid sequence of the FR2 region of 1-5E is shown in SEQ ID No.9; The amino acid sequence of the FR3a region of 1-1D is shown in SEQ ID No.10, the amino acid sequence of the FR3a region of 1-3G is shown in SEQ ID No.11, and the amino acid sequence of the FR3a region of 1-5E is shown in SEQ ID No.12; The amino acid sequence of the FR3b region of 1-1D is shown in SEQ ID No.13, the amino acid sequence of the FR3b region of 1-3G is shown in SEQ ID No.14, and the amino acid sequence of the FR3b region of 1-5E is shown in SEQ ID No.15; The amino acid sequence of the FR4 region of 1-1D is shown in SEQ ID No.16, the amino acid sequence of the FR4 region of 1-3G is shown in SEQ ID No.17, and the amino acid sequence of the FR4 region of 1-5E is shown in SEQ ID No.18; The hypervariable regions are HV2 and HV4; The amino acid sequence of the HV2 region of 1-1D is shown in SEQ ID No. 19, the amino acid sequence of the HV2 region of 1-3G is shown in SEQ ID No. 20, and the amino acid sequence of the HV2 region of 1-5E is shown in SEQ ID No. 21; The amino acid sequence of the HV4 region of 1-1D is shown in SEQ ID No. 22, the amino acid sequence of the HV4 region of 1-3G is shown in SEQ ID No. 23, and the amino acid sequence of the HV4 region of 1-5E is shown in SEQ ID No. 24; The complementary determining regions are CDR1 and CDR3; The amino acid sequence of the CDR1 region of 1-1D is shown in SEQ ID No.25, the amino acid sequence of the CDR1 region of 1-3G is shown in SEQ ID No.26, and the amino acid sequence of the CDR1 region of 1-5E is shown in SEQ ID No.27; The amino acid sequence of the CDR3 region of 1-1D is shown in SEQ ID No.28, the amino acid sequence of the CDR3 region of 1-3G is shown in SEQ ID No.29, and the amino acid sequence of the CDR3 region of 1-5E is shown in SEQ ID No.

30.

3. The gene encoding the shark nanobody 1-1D, 1-3G, 1-5E according to claim 1, characterized in that: The nucleotide sequence of the 1-1D gene is shown in SEQ ID No.31, the nucleotide sequence of the 1-3G gene is shown in SEQ ID No.32, and the nucleotide sequence of the 1-5E gene is shown in SEQ ID No.

33.

4. A recombinant vector, characterized in that: The recombinant vector contains any one of the three genes described in claim 3.

5. A recombinant strain, characterized in that: The recombinant strain contains any one of the three genes described in claim 3 or the recombinant vector described in claim 4.

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