Preparation method and application of shark nano antibody targeting IgM (Immunoglobulin M)

Abstract

The invention belongs to the technical field of biology, and discloses a preparation method and application of a shark nano antibody targeting IgM (Immunoglobulin M). The shark nano antibody is characterized in that the amino acid sequences of the shark nano antibody are 1-1D, 1-3G and 1-5E as shown in SEQ ID NO. 1-SEQ ID NO. 3. Compared with the traditional antibody, the VNAR prepared by the invention has the characteristics of small molecular weight, strong physical and chemical stability, low production cost and the like. The invention provides a preparation method of targeted IgM, namely immunizing sharks in the earlier stage, then constructing a phage display library with high storage capacity, performing three rounds of panning by utilizing a phage display technology to obtain high-affinity VNAR, finally purifying to obtain three VNAR proteins, and detecting that the IgM and the VNAR can be effectively combined by BLI. The screened VNAR can be used as a raw material of an ELISA detection kit, a colloidal gold method detection kit and the like, and a foundation is laid for 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 of shark nano antibodies targeting IgM and application thereof. Background Art

[0002] Immunoglobulin M (IgM) plays an important role in disease intervention. IgM can improve kidney pathology and reduce organ damage. In cancer treatment, it is related to the recognition and elimination of precancerous cells. Natural IgM without natural sugars 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 low immunoglobulin G, such as high IgM syndrome and common variable immunodeficiency (CVID), can be diagnosed by measuring serum immunoglobulin (IG) levels. They are characterized by abnormally high IgM levels and changes in the levels of IgA and IgG in the serum. Therefore, detecting changes in IgA, IgM and IgG in the serum can distinguish them from other primary immunodeficiency diseases. IG testing can determine infection by detecting antibodies in serological experiments. Since IgM is the first antibody produced when the body has a humoral immune response, its detection is particularly important for the early diagnosis of acute infection. For some viruses, such as hepatitis E virus, cytomegalovirus, measles virus, and adenovirus, the detection of IgM content can determine the infection status and make a correct diagnosis in time. Enzyme-linked immunosorbent assay (ELISA) and other methods usually detect immune responses by directly or indirectly monitoring IgM levels.

[0003] Traditional antibodies have been widely used in the field of diagnosis and treatment, but despite many successful examples, they also have many limitations, such as 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 effect of Fc. However, these forms show poor stability and even loss of antigen activity. Based on this, in 1993, scientists isolated homodimers containing only heavy-chain antibodies from the serum of camelids. This dimer is called heavy-chain antibody (HCAb). Later, in 1995, immunoglobulin new antigen receptors (IgNAR) with a structure similar to HcAb were also found in shark serum. The variable antigen binding regions at their N-termini were cloned and expressed, and the resulting antigen-binding domains were single-domain antibodies, also known as nanobodies. 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 physical and chemical stability (resistance to salt, acid, alkali, organic solvents, and high temperature), strong tissue penetration, and low production cost. Compared with camel-derived nanoantibodies, shark nanoantibodies have a smaller molecular weight and higher physical and chemical stability. Currently, shark nanoantibodies have received extensive attention in the fields of drug development, in vitro diagnosis, and immunoassays. Based on this, the present invention proposes a new type of shark nanoantibody with stable physical and chemical properties, laying the foundation for the development of virus detection and kits. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing and using shark nanobodies targeting IgM. The present invention obtains brand-new shark nanobodies 1-1D, 1-3G, and 1-5E through screening, which have high affinity and high specificity for IgM.

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

[0006] Furthermore, the shark nanobody comprises a framework region, a complementary determining region and a hypervariable region; The framework regions are FR1, FR2, FR3a, FR3b and FR4; The amino acid sequence of FR1 is shown in SEQ ID No.4 to SEQ ID No.6; The amino acid sequence of FR2 is shown in SEQ ID No.7 to SEQ ID No.9; The amino acid sequence of FR3a is shown in SEQ ID No.10 to SEQ ID No.12; The amino acid sequence of FR3b is shown in SEQ ID No.13 to SEQ ID No.15; The amino acid sequence of FR4 is shown in SEQ ID No.16 to SEQ ID No.18; The complementary determining regions are CDR1 and CDR3; The amino acid sequence of the CDR1 is shown in SEQ ID No. 19 to SEQ ID No. 21; The amino acid sequence of the CDR3 is shown in SEQ ID No. 22 to SEQ ID No. 24; The hypervariable regions are HV2 and HV4; The amino acid sequence of HV2 is shown in SEQ ID No. 25 to SEQ ID No. 27; The amino acid sequence of HV4 is shown in SEQ ID No.28 to SEQ ID No.30.

[0007] The present invention also protects the genes encoding the shark nanobodies 1-1D, 1-3G, and 1-5E, and the nucleotide sequences of the genes are SEQ ID No.31 to SEQ ID No.33.

[0008] The present invention further protects a recombinant vector, which contains the above-mentioned coding gene.

[0009] The present invention further protects a recombinant strain, which contains the above-mentioned encoding gene or the above-mentioned recombinant vector.

[0010] The present invention also protects the use of the shark nano antibodies 1-1D, 1-3G, and 1-5E in the preparation of IgM protein targeting agents.

[0011] The present invention also protects the use of the shark nano antibodies 1-1D, 1-3G and 1-5E in virus detection kits.

[0012] Beneficial effects of the present invention: 1. The molecular weight of the shark nanoantibodies 1-1D, 1-3G, and 1-5E prepared by the present invention is smaller than that of traditional monoclonal antibodies, which is only about 15 kDa.

[0013] 2. The shark nanoantibody prepared by the present invention has high stability and strong physical and chemical stability. It is still stable in extreme environments (high temperature, strong acid and strong alkali), and has good affinity and specificity with IgM.

[0014] 3. The shark nanoantibodies prepared by the present invention can be used as raw materials for ELISA detection kits and colloidal gold detection kits for early diagnosis of diseases.

[0015] 4. The present invention selects the striped bamboo shark as a model animal for antibody preparation to prepare VNAR. The bamboo shark is not an endangered shark species, is small in size, and is easy to breed artificially, making it suitable for antibody development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The gel electrophoresis results of the PCR amplification products of the VNAR fragment; lane description: M. DNA Maker, 1. Negative control, 2-13. PCR products; Figure 2 The results of polyclonal ELISA assay of specific phages; Figure 3 The results of monoclonal ELISA assay of specific phages; Figure 4 This is the SDS-PAGE result of VNAR protein purification; Lane description: M. Protein Marker, 1.1~1D, 2.1~3G, 3.1~5E; Figure 5 These are the 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 DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments.

[0019] The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art and are performed according to the techniques described in the literature in the art or according to the product instructions; the various biological materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0020] Example 1: Construction of IgM shark-targeted nanoantibody immune library 1. Immunity of the bamboo shark The bamboo shark is cultured in a fish tank with a suitable salinity of about 1.023 and a suitable temperature of 23 ℃-25 ℃. Fresh shrimps are fed every two days, and each fish is fed about 2-3 shrimps. Food residues are removed on the day of feeding. Vitamins are added once a week, and protein skimmers and sewage are cleaned in time to ensure water quality. When immunizing bamboo sharks, IgM is used as an antigen for immunization. The immunization injection sites of bamboo sharks are selected in the dorsal fin and lateral fin of the shark. For the first immunization, 100 μg of mixed antigens are emulsified with Freund's complete adjuvant. For the second and subsequent immunizations, 100 μg of mixed antigens are emulsified with Freund's incomplete adjuvant. Immunization is performed at sites close to the previous round of immunization sites. The total immunization period is about 3 months, and immunization is performed every 2 weeks.

[0021] 2. Construction of shark nanoantibody immune library Peripheral blood lymphocytes were extracted from the immunized bamboo sharks. The peripheral blood lymphocytes were obtained by centrifugation of the shark whole blood. Total RNA of the peripheral blood lymphocytes was then extracted according to the operating instructions of the RNA extraction kit, and the RNA was reverse transcribed into cDNA.

[0022] Using cDNA as a template, PCR amplification was performed with the following reaction system (see Table 1) to amplify the VNAR fragment (see Figure 1 ), the amplified product was recovered by gel, wherein the sequence of the primer combination is as follows: Upstream primer NewVF1 (SEQ ID NO.34): CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC.

[0023] Upstream primer NewVF2 (SEQ ID NO.35): CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAAAAACACCG.

[0024] Upstream primer NewVF3 (SEQ ID NO.36): CAATTTGATTGGGCCCAGGCGGCCCAATGGGTTGAACAAACACCGA.

[0025] Primer NewVR1 (SEQ ID NO.37): CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC.

[0026] Primer NewVR2 (SEQ ID NO.38): CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC.

[0027] Primer NewVR3 (SEQ ID NO.39): CAATTTGATTGGGCCGGCCTGCCAGGTTTCACAGTCAGAATGGTG The PCR amplification system is as follows: Table 1 PCR reaction system

[0028] The reaction conditions are as follows: Table 2 PCR reaction procedure

[0029] At the same time, the amplified VNAR fragment and the pcomb3XSS vector were double-digested and recovered. The enzyme digestion system is as follows: Table 3 Enzyme digestion reaction system

[0030] The purified VNAR fragment was then ligated into the pcomb3XSS vector using T4 ligase. The ligation system is as follows: Table 4 T 4 Connection system

[0031] The ligation product was electrotransformed into E. coli TG1 competent cells, and then part of the bacterial solution was gradient diluted and spread on 2×YT plates, inverted and cultured at 37°C overnight. The library capacity of the constructed shark nanoantibody phage library was calculated based on the number of colonies on the plate, and then the monoclonal clone was sent for sequencing for sequence comparison. Finally, according to the calculation, the library capacity of the shark nanoantibody phage library was 3×10 9 CFU, sequence diversity was 100%.

[0032] Example 2: Panning of IgM shark nanobody phage (three-stage panning) 1. Three rounds of panning of shark IgM-targeted nanoantibodies 1. Streak TG1 two days in advance and incubate overnight at 37°C (LB solid, no antibodies).

[0033] 2. (1) The next day, pick a single clone and transfer it to 10 mL 2×YT (without antibodies) and incubate at 37°C, 180 rpm overnight.

[0034] (2) Prepare 50 mL of PEG / NaCl and stir magnetically overnight.

[0035] (3) Encapsulation.

[0036] a. 96-well plate, 4 wells of IgM package, 10 ug / mL, 100 uL / well, overnight at 4℃. (1st round 10 ug / mL, 2nd round 4ug / mL, 3rd round 2 ug / mL).

[0037] b. Pack 4 wells with gelatin buffer, 200 uL / well, and incubate at 4°C overnight.

[0038] 3. After washing the plate 3 times, block IgM, add phage library, and incubate at 37℃ for 1-1.5 hours (1) Wash the plate with PBST (0.1% Tween-20 in PBS), wash the wells coated with IgM, fill the entire well (about 300uL), and tap the plate (you can use a plate washer to wash).

[0039] (2) Add 200 uL / well of 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, and block alternately).

[0040] (3) At the same time, add diluted phage into the blocking solution wells, 100 uL / well (diluted with gelatin blocking solution).

[0041] Calculation method: Make the added 400 uL phage contain 1000 times the library capacity to ensure complete panning (1000 times the library capacity in the first round, 100 times the library capacity in the second and third rounds) 4. Activate TG1 and incubate (1) Take 100 μL of the TG1 strain that has been shaken overnight and add it to 5 mL of fresh 2x YT medium. Incubate at 37°C, 190 rpm for 1-1.5 h until the culture reaches the logarithmic growth phase (OD600 = 0.5-0.8).

[0042] (2) Take out the phages in the blocking solution wells and put them into EP tubes. Wash the plate three times and add the phages to the IgM wells. Incubate at 37°C for 1-1.5 h.

[0043] 5. Wash the plate 10-15 times (9 times in the first round, 12 times in the second round, and 12 times in the third round) 6. Infection Add activated TG1 to the IgM wells, 100 uL / well, and let stand at 37°C for 30 min. Collect the infected TG1 and replace with fresh TG1. Repeat this process 3 times (all infection steps require standing).

[0044] 7. Coating verification The bacterial solution was diluted to 100, 1000, and 10000 times (1 mL volume) by limiting dilution, and 100uL was taken from each solution to be plated (Amp + ), then dilute 1000, 10000, 100000 times, culture at 37℃ overnight, and observe the number of clones grown, which is the TG1 that has been successfully infected. When the input TG1 is the same, a significant increase in the number of clones grown indicates enrichment, and the following experiment can be performed.

[0045] 8. Helper phage superinfection Add 20 times the amount of M13K07 to the remaining infected TG1 and incubate at 37°C for 1 h (mix once every 30 min).

[0046] 9. Progeny phage amplification Incubate the bacterial solution at 5000 rpm and 25 ℃ for 10 min, discard the supernatant, resuspend in 10 mL of fresh 2x YT, add Kana (50 ug / mL) and Amp (100 ug / mL), and culture overnight at 30 ℃ and 180 rpm.

[0047] 10. Collecting phage The bacterial solution (10 mL) shaken overnight in the previous round was centrifuged at 5000 rpm for 25 min, the supernatant was filtered through 0.45 um and 0.22 um filter membranes, PEG / NaCl (2.5 mL) with 1 / 4 volume of the bacterial solution was added, and ice bathed for 0.5-1 h (white flocs were present); 5000 rpm at 4 ℃ for 30 min, the supernatant was discarded, inverted on absorbent paper to dry, and resuspended with PBS (500 uL) with 1 / 20 volume of the original supernatant, and OD280 was measured (OD280 = 1 is equivalent to 2.5x10 12 cfu / mlL) 2. Polyclonal ELISA 1. Coating Coated with IgM, concentration is 1 ug / mL, 100 uL / well.

[0048] Coat with gelatin blocking buffer, 100 uL / well, at 4°C overnight.

[0049] 2. Wash the plate 3 times 3. Closure Gelatin blocking buffer (same as coating) 300 uL / well, block at 37 ℃ for 1-2 h.

[0050] 4. Sample Preparation Prepare the original library, the first, second, and third rounds of panning phages, measure OD280, and dilute them to 10 with gelatin blocking buffer. 10 cfu.

[0051] 5. Wash the plate 3 times 6. Incubation Add the diluted phage to the plate, 100 uL / well, and incubate at 37 °C for 1 h.

[0052] 7. Wash the plate 3 times 8. Secondary Antibody Incubation HRP-linked anti-M13 antibody (1:10,000) was diluted in gelatin blocking buffer, 100 uL / well, and incubated at 37°C for 1 h.

[0053] 9. Wash the plate 3 times 10. Color rendering Mix 100 uL / well TMB colorimetric solution A and solution B in a ratio of 1:1 for 2-15 min.

[0054] 11. Termination Use 0.1 MH 2 SO 4 Stop by adding 50 uL to each well.

[0055] 12. Reading OD450 was read by microplate reader.

[0056] The results of polyclonal ELISA phage detection are as follows Figure 2 As shown, specific phages were enriched with increasing panning times.

[0057] 3. Monoclonal ELISA 1. Pick a single clone (1) Prepare a 96-well U-bottom plate, add 2x YT (Amp+) 200 uL / well, select the plate diluted during the second or third round of identification, pick a single clone and inoculate it into the 96-well plate, and culture at 37°C 220 rpm for 3 h until the logarithmic growth phase.

[0058] (2) Add new 2x YT[M13K07 (20 times the amount of bacteria) and Amp + ], 100uL / well, add 100uL of bacterial solution shaken to logarithmic growth phase to each well, incubate at 37℃ for 30min for infection, and culture overnight at 30℃ 220rpm.

[0059] 2.ELISA Identification (1) Coating Coated with IgM, coating concentration is 1 ug / mL, 100 uL / well, incubate at 4 ℃ overnight.

[0060] (2) Wash the plate 3 times (3) Closed Block with gelatin blocking buffer, 300 uL / well, incubate at 37 ℃ for 1-2 h.

[0061] (4) Sample preparation After picking the single clone, the overnight cultured 96-well U-bottom plate bacterial solution was centrifuged at 2000 rpm for 10 min.

[0062] (5) Wash the plate 3 times (6) Incubation After centrifugation, the supernatant in the 96-well U-bottom plate was added to the ELISA plate, 100 uL / well, and incubated at 37 °C for 1 h.

[0063] (7) The remaining steps are the same as those of polyclonal ELISA.

[0064] The results of monoclonal ELISA detection of phage are as follows Figure 3 As shown, positive phages with an absorbance value greater than 1.5 at 450 nm were selected for sequencing. The NCBI-BLAST tool was used to perform homology comparison of the sequencing results, the NCBI-ORFfinder tool was used to translate the amino acid sequence, and the amino acid sequence diversity was analyzed in BioEdit. Finally, three different VNAR sequences targeting IgM were obtained, named 1-1D, 1-3G, and 1-5E, and their amino acid and nucleotide sequences are as follows: GWVDQTPTTTTKEAGESLTINCVLKGSRYALCDTYWYFTKKGATKKERLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKASRRTSTVSWNCRPIHYEGGGTILTVKP (SEQ ID NO.1) GWVDQTPTTTTKEAGESLTINCVLKGSRYALCDTYWYFTKKGATKKERLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKASRRTGTVSWNCRPIHYEGGGTILTVKP (SEQ ID NO.2) AWVDQTPRTITKETGESLTINCVLKDASYALSGTYWYLTKLDATKWDRISIGGRYSETVNKGSKSFSLRLRDLRVEDSGAYHCEAYGTAAMTGRGGRGTFLAVKP (SEQ ID NO.3) GGTTGGGTGGACCAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGTGTCCTAAAAGGTTCCAGATATGCATTGTGTGACACGTATTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGGTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTCTCGTCGGACGAGCACCGTCAGCTGGAATTGCCGGCCTATTCATTATGAGGGAGGCGGCACCATTCTGACTGTGAAACCT(SEQ ID NO.31) GGTTGGGTGGACCAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGATATGCATTGTGTGACACGTATTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGGTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTCTCGTCGGACGGGCACCGTCAGCTGGAATTGCCGGCCTATTCATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCT(SEQ ID NO.32) GCATGGGTTGACCAAACACCGAGAACAATAACGAAGGAGACAGGCGAATCACTGACCATCAACTGTGTCCTAAAAGATGCTAGCTATGCATTGAGTGGCACGTACTGGTATCTGACAAAATTGGATGCAACAAAGTGGGACCGCATATCAATTGGTGGA CGATACTCTGAAACAGTGAACAAGGGATCAAAGTCCTTTTCTTTGCGACTTCGTGATCTGAGAGTTGAAGACAGTGGTGCATATCACTGTGAAGCGTATGGCACAGCTGCGATGACGGGACGGGGAGGAAGAGGCACTTTTCTGGCAGTGAAACCT (SEQ ID NO.33) Shark nanobodies 1-1D, 1-3G, and 1-5E include framework regions FR, complementary determining regions CDR, and hypervariable regions, wherein the framework region FR includes the amino acid sequences of FR1, FR2, FR3a, FR3b, and FR4, which are as follows: 1-1D: FR1: GWVDQTPTTTTKEAGESLTINCVLK (SEQ ID NO.4); FR2: TYWYFT (SEQ ID NO. 7); FR3a: GRYAETV (SEQ ID NO.10); FR3b: FSLRISDLRVEDSGTYHC (SEQ ID NO.13); FR4:EGGGTILTVKP (SEQ ID NO. 16).

[0065] HV2: KKGATKKERLSNG (SEQ ID NO. 19); HV4:NKASKS(SEQ ID NO.22); CDR1: GSRYALCD (SEQ ID NO.25); CDR3:KASRRTSTVSWNCRPIHY (SEQ ID NO. 28).

[0066] 1-3G: FR1: GWVDQTPTTTTKEAGESLTINCVLK (SEQ ID NO.5); FR2: TYWYFT (SEQ ID NO. 8); FR3a: GRYAETV (SEQ ID NO.11); FR3b: FSLRISDLRVEDSGTYHC (SEQ ID NO.14); FR4:EGGGTILTVKP (SEQ ID NO. 17).

[0067] HV2: KKGATKKERLSNG (SEQ ID NO. 20); HV4:NKASKS(SEQ ID NO.23); CDR1: GSRYALCD (SEQ ID NO.26); CDR3:KASRRTGTVSWNCRPIHY (SEQ ID NO. 29).

[0068] 1-5E: FR1:AWVDQTPRTITKETGESLTINCVLK (SEQ ID NO.6); FR2: TYWYLT (SEQ ID NO. 9); FR3a:GRYSETV (SEQ ID NO.12); FR3b: FSLRLRDLRVEDSGAYHC (SEQ ID NO. 15); FR4: GGGRGTFLAVKP (SEQ ID NO. 18).

[0069] HV2: KLDATKWDRISIG (SEQ ID NO. 21); HV4:NKGSKS(SEQ ID NO.24); CDR1:DASYALS (SEQ ID NO.27); CDR3: EAYGTAAMTGR (SEQ ID NO. 30).

[0070] Example 3: Prokaryotic expression and purification of shark nanobodies 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 connected using a homologous recombination kit.

[0071] The plasmid successfully sequenced was transformed into E. coli BL21 (DE3) competent cells for protein expression. The next day after transformation, single clones were picked up and shaken overnight. After shaking, the bacterial solution was transferred to 200 mL 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 induction, the cells were collected by centrifugation, the supernatant was discarded, and resuspended with 20 mL PBS. After adding protease inhibitors at a ratio of 1:100, ultrasonic crushing (20% power, ultrasonic on for 3s, stop for 6s) was performed until the solution was clear. After ultrasonication, centrifugation was performed at 12000 rpm for 15 min at 4°C. The shark nanobody protein was purified by nickel column affinity chromatography, and the imidazole eluate of each component was collected. The eluate of each component was detected by SDS-PAGE, and the eluate containing shark nanoantibodies was concentrated by ultrafiltration. After concentration, the concentration of shark nanoantibodies was detected by BCA method, and finally three VNAR proteins were successfully obtained. The SDS-PAGE results are as follows: Figure 4 shown.

[0072] Example 4: BLI detection of affinity between human IgM, IgG and shark nanobodies Human IgM and IgG were diluted to 300 nM with 0.02% PBST and fixed on the nickel (Ni) biosensor for 90 s. After that, the baseline was stabilized in basiline buffer for 120 s. Then, they were combined with 10 μg / mL shark nanoantibodies and then combined and dissociated. The samples were added to 96-well black bottom plates at 200 μL / well. All steps were carried out at room temperature. The affinity determination results of human IgM and shark nanoantibodies are shown in Figure 2. Figure 5 As shown in A, VNAR protein has affinity with human IgM, and the affinity determination results of human IgG and shark nanoantibodies are shown in Figure 5 As shown in B, the VNAR protein has almost no affinity for human IgG, indicating that the screened targeted IgM protein has good specificity.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed 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 sequences thereof are shown in SEQ ID No.1 to SEQ ID No.

3.

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

30.

3. The shark nanobody 1-1D, 1-3G, 1-5E according to claim 1, characterized in that: The nucleotide sequences encoding the genes are SEQ ID No.31 to SEQ ID No.

33.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 3.

5. A recombinant strain, characterized in that: The recombinant strain contains the gene according to claim 3 or the recombinant vector according to claim 4.

6. Use of the shark nanobodies 1-1D, 1-3G, and 1-5E according to claim 1 in the preparation of IgM protein targeting agents.

7. Use of the shark nanobodies 1-1D, 1-3G, and 1-5E according to claim 1 in a virus detection kit.

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