Shark single-domain antibody targeting multi-species serum albumin as well as preparation method and application of shark single-domain antibody

Abstract

The invention belongs to the technical field of biology, and discloses a shark single-domain antibody targeting multi-species serum albumin as well as a preparation method and application of the shark single-domain antibody, a VNAR antibody is selected from protein expressed by amino acid sequences shown in SEQ ID NO.1-SEQ ID NO.6, and nucleotide sequences for encoding the amino acid sequences shown in SEQ ID NO.1-SEQ ID NO.6 are nucleotide sequences shown in SEQ ID NO.7-SEQ ID NO.12 respectively. The single-domain antibody targeting the human serum albumin is smaller than a traditional monoclonal antibody, good in stability, easier to store and transport than a conventional antibody and capable of being effectively combined with an antigen, expression protein of the sequence shown in SEQ ID NO.5 shows cross-species serum albumin combining capacity under the conditions of pH 6.0 and pH 7.4, and the single-domain antibody targeting the human serum albumin has the advantages of being high in specificity, high in specificity and high in specificity. The capability of prolonging the half-life period of the fusion protein is shown in a mouse model, and a foundation is laid for taking the fusion protein as a tool medicine for improving the half-life period of a small-molecule treatment medicine subsequently.

Description

Technical Field

[0001] The present invention relates to the field of preparation of biopharmaceutical antibodies, and specifically to shark single domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 targeting serum albumin of multiple species. In addition, the present invention also relates to a preparation method and use of the antibody. Background Art

[0002] Since recombinant biopharmaceuticals began to enter the therapeutic drug market, a major problem faced by many protein and peptide drugs is that their half-life is too short. Although these drugs have strong therapeutic potential, they are easily metabolized by the kidneys or degraded by proteases in the lysosomes due to their small molecular weight. This characteristic limits their application in chronic diseases and long-term treatment. In order to regulate the plasma half-life of these drugs, increase drug exposure, thereby reducing the frequency of dosing and increasing patient compliance, PEGylation, fusion of the Fc domain of traditional antibodies, and coupling or binding to serum albumin have all been explored as solutions by predecessors. Serum albumin is an abundant plasma protein that plays a vital role in osmotic pressure regulation and serves as a transporter of fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules. It has a molecular weight of 66 kDa and a half-life of up to 19 days in the human body. Coupled with its unique pharmacokinetic characteristics, it has become an ideal target protein for extending the half-life of short-lived proteins and peptides. In addition, serum albumin also participates in the FcRn-mediated endomembrane cycle, preventing it from being degraded by lysosomes through pH-dependent binding and dissociation mechanisms, thereby extending its half-life from another perspective. In the past, people have successfully used serum albumin as a drug carrier, prepared serum albumin fusion proteins, adhered serum albumin, and other methods to extend the half-life of peptide and protein drugs in vivo.

[0003] Monoclonal antibodies occupy a large market share in recombinant biopharmaceuticals. Shark single-domain antibodies (VNARs), discovered in 1995, are the variable regions of heavy-chain dimer antibodies (IgNARs) naturally present in cartilaginous fish such as sharks. They have the advantages of small molecular weight, high affinity, strong specificity, good solubility, and strong stability. Compared with traditional antibodies, the CDR2 region of VNAR is severely truncated and the CDR3 region is longer. The highly variable CDR3 domain not only makes VNAR more diverse, but its unique compact molecular structure also increases the accessibility of VNAR to hidden antigenic epitopes. Shark single-domain antibody VNAR has shown great potential to replace traditional antibodies as a therapeutic drug for clinical application. Shark single-domain antibodies, as a new type of antibody, have unique advantages. Developing a shark single-domain antibody that specifically binds to serum albumin and using it as a tool drug to extend the half-life of small molecule therapeutic drugs such as peptides is of great significance for solving the clinical problems of dosing frequency and long-term treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a shark single-domain antibody targeting serum albumin of multiple species and a preparation method and application thereof. The present invention obtains brand-new shark nanoantibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 through screening, which have high affinity and high specificity for serum albumin.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The present invention protects shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 targeting serum albumin of multiple species, and the amino acid sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.6.

[0006] Furthermore, the shark single domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, 4E11 comprise fixed amino acid sequences FR1, FR2, FR3a, FR3b and FR4, hypervariable regions HV2 and HV4, complementarity determining regions CDR1 and CDR3, and their CDR3 sequences are shown in SEQ ID NO.13 to SEQ ID NO.18.

[0007] The present invention also protects the coding gene of the shark single domain antibody targeting multi-species serum albumin, and the nucleotide sequence of the coding gene is shown as SEQ ID NO.7 to SEQ ID NO.12.

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

[0009] The present invention further protects a recombinant strain, the recombinant strain contains the above coding gene or the recombinant vector. The present invention also protects the use of the shark single domain antibody in the preparation of a serum albumin targeting agent.

[0010] The present invention also protects the use of the shark single-domain antibody in the preparation of auxiliary half-life extension products and long-acting drugs.

[0011] The present invention also protects the use of the shark single domain antibody in the preparation of an immune detection kit and a plasma protein detection kit.

[0012] Beneficial effects of the present invention: 1. The shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 prepared by the present invention have a small molecular weight of only about 15 kDa, which is smaller than traditional monoclonal antibodies; 2. The shark single domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 have high stability and good tissue permeability, are easier to store and transport than conventional antibodies, and have good affinity and specificity with serum albumin; 3. Easier to express and genetically engineer, 3A11, 3C11, 3F10, 3G3, 4A12, 4E11 are expressed using the E. coli prokaryotic expression system, and ultimately a large number of shark single-domain antibodies are obtained; 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.

[0013] Shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 can be used as vectors for extended half-life products and long-acting therapeutic drugs. The genes or recombinant plasmids encoding 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11, or recombinant cells containing the recombinant plasmids of the genes can be used to prepare immunoassay diagnostic kits and plasma protein detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the result of ELISA testing the titer of shark serum; Figure 2 PCR amplification of shark nanobody genes; lane description: M: DNA Maker, 1-12: PCR products, the red box is the VNAR specific band; Figure 3 The results of bacterial solution PCR to verify the insertion rate of the library; lane description: M: DNA Maker, 1-20: PCR product Figure 4 The results of random sequencing to verify the diversity of the library; Figure 5 This is the result of polyclonal ELISA; Figure 6 The results are monoclonal ELISA results; Figure 7 The results of SDS-PAGE of purified antibody protein; Figure 8 ELISA results to detect the binding ability of shark nanobodies to serum albumin; Fig. 9 BLI binding and dissociation curves to detect the affinity of shark nanobodies to serum albumin; Fig.10 To detect the ELISA results of the binding ability of VNAR-4A12 to serum albumin from different species; Fig.11The SDS-PAGE results of purified sfGFP and 4A12-sfGFP proteins; Fig.12 The blood concentration-time curves of sfGFP and 4A12-sfGFP after a single intravenous administration. DETAILED DESCRIPTION

[0015] 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.

[0016] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0017] 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.

[0018] Example 1 Immunization and titer detection of striped bamboo shark 1. Shark Immunity Sharks were given one initial immunization and four booster immunizations: the initial immunization used 250 µg HSA mixed with complete Freund's adjuvant and emulsified, which was injected subcutaneously through the lateral fin; the first booster immunization used 100 µg HSA mixed with incomplete Freund's adjuvant and emulsified, which was injected subcutaneously through the lateral fin; the subsequent three booster immunizations used 3 µg HSA injected into the blood through the lateral fin vein, with a one-month interval between each immunization.

[0019] Before immunization and 5 days after immunization, 5 mL of blood was collected from the tail vein, anticoagulated with heparin, centrifuged at 1000 rpm for 10 min, and the plasma and peripheral blood lymphocytes were collected.

[0020] 2. Enzyme-linked immunosorbent assay (ELISA) to detect plasma titer HSA was dissolved in PBS to 2 µg / mL, added to a 96-well ELISA plate for overnight coating at 4°C, blocked with 5% MPBS (skim milk powder dissolved in PBS) at 37°C for 1.5h, and then the plasma samples were diluted 10 times in a gradient, with the plasma before immunization as the negative control, incubated at 37°C for 1.5h, added with 1:1000 diluted rabbit anti-shark serum (prepared in this laboratory) and incubated at 37°C for 1h, then added with 0.1 µg / mL HRP-conjugated goat anti-rabbit IgG antibody, incubated at 37°C for 1h, and added with TMB colorimetric solution for color development at room temperature and in the dark for 5min. The color development was terminated with 1 M HCl, and the absorbance value at 450 nm was read by an ELISA reader.

[0021] The plasma titer obtained by ELISA test is about 1:100. Figure 1 shown.

[0022] Example 2 Construction of phage display library 1. Amplification of target gene The peripheral blood lymphocytes separated from the blood samples were used to extract total RNA using the Trizol method and reverse transcribed to synthesize cDNA. Through the structural and diversity analysis of the immunoglobulin gene loci of the striped bamboo shark, forward primers SEQ ID NO.19 to SEQ ID NO.20 were designed based on the partial conservative sequence upstream of VNAR, and reverse primers SEQ ID NO.21 to SEQ ID NO.22 were designed based on the partial conservative sequence downstream of the CDR3 domain to amplify the VNAR fragment.

[0023] SEQ ID NO. 19 NewVF1 CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC SEQ ID NO. 20 NewVF2 CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAAAAACACCG SEQ ID NO. 21 NewVR1 CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC SEQ ID NO. 22 NewVR2 CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC The 50 μL reaction system for PCR amplification was: 25 μL 2x PrimeSTAR® Max, 2.5 μL template cDNA, 2 μL upstream primer, 2 μL downstream primer, and 18.5 μL ddH2O.

[0024] The PCR amplification conditions were as follows: initial denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 5 s, repeated for 30 cycles; and final extension at 72°C for 5 min.

[0025] The results of nucleic acid electrophoresis of the VNAR region of the bamboo shark amplified by PCR are as follows: Figure 2 shown.

[0026] 2. Library Construction The amplified VNAR fragment and pComb3Xss vector were digested with restriction endonuclease SfiⅠ, and ligated with T4 ligase to construct a recombinant vector.

[0027] The 500 μL reaction system for SfiⅠ digestion is: SfiⅠ 10 μL, pComb3Xss / VNAR fragment 10 μg, 10× CutSmart Buffer 50 μL, ddH2O to 500 μL.

[0028] The ligation system of T4 ligase is: T4 DNA Ligase 10 µL, 10× T4 DNA Ligase Buffer 20 µL, pComb3Xss 5 µg, VNAR fragment 2.5 µg, ddH2O to 200 µL. The connected pComb3Xss-VNAR vector was transferred into Escherichia coli TG1 by electroporation, and the bacterial solution was applied to an ampicillin-resistant 2x YT solid plate and cultured overnight at 37°C. The bacteria on the plate were collected to obtain the original library strain. After the strain was cultured to the logarithmic phase, M13K07 auxiliary phage was added for superinfection, and cultured overnight at 30°C 200rpm. The supernatant was filtered with a 0.22um filter membrane, and a final volume of 20% PEG / NaCl solution was added. The mixture was ice-bathed for 30 minutes to precipitate the phage. The mixed solution was centrifuged at 4°C and 10000g for 15 minutes, and the precipitate was resuspended with PBS to obtain the original phage library storage solution.

[0029] Library size calculation: Final library size = initial library size x insertion rate x sequencing diversity.

[0030] The electroporated bacterial solution was diluted to 10 -6 , 10 -7 , 10-8 times, and spread them on 2x YT solid plates with ampicillin resistance and cultured at 37°C overnight. The initial library size = the number of single colonies grown on the plate x 10 6 , 10 7 , 10 8 . Randomly select 20 clones from the plate, use an equimolar mixture of upstream primers for VNAR amplification as upstream primers, and the conserved fragment on the vector as downstream primers for PCR identification. Insertion rate = number of target bands / total number of bacterial liquid PCR reactions × 100%. Randomly sequence the bacterial liquid that successfully obtains the target band by PCR, and the diversity = sequenced correctly / total number of sequenced samples × 100%.

[0031] The final library size used in the present invention is 1.53 x 10 8 .

[0032] The electrophoresis results of the inserted nucleic acid identified by bacterial liquid PCR are as follows Figure 3 The sequence alignment results of random sequencing are shown in Figure 4 The quality evaluation of the VNAR library is shown in Table 1.

[0033] Table 1: Evaluation of the quality of phage libraries

[0034] Example 3 Specific screening and identification for HSA The solid phase panning method was used. In the first round of panning, 10 µg / ml HSA and blocking solution were added to a 96-well ELISA plate and coated overnight at 4°C. On the second day, while blocking the HSA wells, 10 times the volume of phage preparation solution of the library was added to the wells coated with the blocking solution and incubated at 37°C for 1 h. After washing the plate, the phage preparation solution was added to the HSA wells and incubated at 37°C for 1 h. The plate was washed 15 times, and the TG1 bacterial solution grown to the logarithmic phase was directly added for infection 3 times. Each infection lasted for 30 min. The same method as the preparation of the library phage stock solution was used for superinfection and phage amplification. The coating concentrations for the second and third rounds of panning were reduced to 4 µg / ml and 2 µg / ml, and the plates were alternately blocked with 5% MPBS and gelatin blocking solution, and a total of three rounds of panning were performed. The phage solution amplified after each round of panning can be directly used for polyclonal ELISA to detect the enrichment after panning. The results are shown in Figure 5 shown.

[0035] 192 single clones were picked from the plates after the second and third rounds of panning, placed in a 96-well deep-well plate and cultured at 37°C with shaking until the logarithmic phase, and M13K07 helper phage was added for superinfection. The progeny phages were amplified using the same method as before. The plates were centrifuged at 500 xg for 20 min the next day, and the supernatant was directly used for monoclonal ELISA to detect the positive expression of the clones after panning. The results are shown in Figure 6 Through three rounds of screening, six VNAR sequences targeting HSA were obtained, namely the sequences shown in SEQ ID NO.1 to SEQ ID NO.6, the nucleotide sequences of their encoding genes are shown in SEQ ID NO.7 to SEQ ID NO.12, and their CDR3 sequences are shown in SEQ ID NO.13 to SEQ ID NO.18.

[0036] SEQ ID NO.1 3A11 QWVEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAFYSWDEGDVCRSWDSLSYEGGGTILTV SEQ ID NO.2 3C11 QWVEQTPTTTTKEAGESLTINCVLRDSSCALASTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPQLDEGDCVGWYPSEGGGTILSV SEQ ID NO.3 3F10 ARVEQTPTTTTKEAGESLTINCALRDSSCVLGRTYWYFTKKGAPKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYPLEFCDTHSWTEGGGTILTV SEQ ID NO.4 3G3 ARVEQTPTTTTKEAGESLTINCVLRDSSCALGKTYWYFTKKGATKKASLSTGGRYTETVNKASKSFSLRISDLRVEDSGTYHCKAYPLQLCSGHSWVEGGGTTLTV SEQ ID NO.5 4A12 ARVEQTPTTTTKEAGESLTINCVLRDSSCPLPSASTYWFFTKKGATKKESLSNGGRYAETMNKTSKSFSLRISDLRVEDSGTYHCKAYPGGYCDGIYYYEGGGTILTV SEQ ID NO.6 4E11 ARVEQTPTTTTKEAGESLTINCVLRDSSCALYSTYWYFTKKGATKKETLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPQLDEDCVSWKYYEGGGTIV SEQ ID NO.7 3A11 CAATGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTTTTACAGCTGGGATGAGGGAGACGTCTGCCGTAGCTGGGATTCTCTCTCTTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCCG SEQ ID NO.8 3C11 CAATGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGCTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCACAGCTGGATGAAGGGGACTGTGTAGGCTGGTATCCGTCTGAAGGAGGCGGCACCATTCTGAGTGTAAAACCC SEQ ID NO.9 3F10 GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGCCCTAAGAGACTCCAGCTGTGTATTGGGTAGGACGTACTGGTATTTCACAAAAAAGGGCGCGCCAAAGAAGGAGAGCTTATCGAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATTACTGTAAAGCGTATCCCCTGGAGTTCTGTGATACCCATAGTTGGACTGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGG SEQ ID NO.10 3G3 GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGGTAAAACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACACGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCGCTACAGCTCTGTTCCGGCCATAGCTGGGTTGAAGGAGGCGGCACCACTCTGACTGTG SEQ ID NO.11 4A12 GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTCCATTGCCTAGCGCTAGCACGTACTGGTTTTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAATGAACAAGACATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTACCCTGGGGGTTACTGTGATGGAATTTACTATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGG SEQ ID NO.12 4E11 GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGTATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAAACCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCTCAGCTGGATGAAGACTGTGTTAGCTGGAAGTATTATGAAGGAGGCGGCACCATTGTGACTGTGGG SEQ ID NO .13 3A11-CDR3 FYSWDEGDVCRSWDSLSYE SEQ ID NO .14 3C11-CDR3 YPQLDEGDCVGWYPSE SEQ ID NO .15 3F10-CDR3 YPLEFCDTHSWTE SEQ ID NO .16 3G3-CDR3 YPLQLCSGHSWVE SEQ ID NO. 17 4A12-CDR3 YPGGYCDGIYYYE SEQ ID NO. 18 4E11-CDR3 YPQLDEDCVSWKYYE Example 4 Recombinant expression and purification of shark nanobodies The plasmids were extracted from the positive clones screened by monoclonal ELISA and transformed into E. coli In HB2151, after the strain was cultured to the logarithmic phase, it was induced with 0.1 mM isopropyl-β-D-Thiogalactoside (IPTG) under 100 μg / ml ampicillin pressure at 30°C overnight. All proteins were expressed in soluble form. In order to increase the expression level, the sequence shown in SEQ ID NO.5 was cloned into the pET-28a (+) vector and transformed into E. coli The expression was induced with 0.1 mM IPTG under the pressure of 50 µg / ml kanamycin in BL21(DE3) at 37°C for 6 h and expressed as inclusion bodies.

[0037] After collecting the soluble expressed bacteria, they were ultrasonically disrupted, and the supernatant after disruption was purified by nickel column, impurities were removed with 20 mM imidazole, and eluted with 250 mM imidazole. The purified eluate was concentrated using a 3 K ultrafiltration column and replaced with PBS for storage. For inclusion bodies, the precipitate after ultrasonic disruption needs to be collected and dissolved with Tris buffer containing 8M urea. The nickel column purification process is the same as mentioned above, but 8M urea needs to be maintained in the system. The purified protein solution is replaced with 0 M urea by gradient dialysis, and then concentrated using a 3 K ultrafiltration column and replaced with PBS for storage. The antibody protein purified by the nickel column was further purified using a SuperdexTM 200 Increase 10 / 300GL column. The purified antibody protein was subjected to SDS-PAGE electrophoresis, and the bands after Coomassie Brilliant Blue staining were as shown below. Figure 7 shown.

[0038] Example 5 Affinity detection of shark nanobody protein and HSA 1. ELISA to detect binding affinity The 96-well ELISA plate was coated with 1 μg / mL HSA at 4°C overnight; the next day, 5% MPBS was used as a blocking solution and blocked at 37°C for 1 hour; the antibody protein was diluted with PBS to a certain concentration gradient and incubated in the plate at 37°C for 1 hour; then the HRP-coupled anti-HA antibody was diluted in the blocking solution at a ratio of 1:10000, added to the plate, and incubated at 37°C for 1.5 hours; finally, TMB color development solution was used for color development, and the absorbance at 450nm was detected after stopping the color development with 1M sulfuric acid. The results of the HSA binding ability of the sequences screened in Example 3 are as follows Figure 8 shown.

[0039] 2. Biolayer interferometry (BLI) to detect affinity The affinity of the target protein to HSA was detected using the Octet RED96e system. The purified protein solution was diluted to 5 μg / mL and detected using an anti-His probe. The probe was first pre-wetted in PBST containing 0.02% Tween-20 at 1000 rpm for 60 s, then immersed in the protein solution for solidification for 90 s, and then bound and dissociated with 100 nM HSA for 120 s and 180 s, respectively. The fitting binding and dissociation curves are shown in Figure 2. Fig. 9 shown.

[0040] The screened sequences all have a certain affinity for HSA, among which the sequence shown in SEQ ID NO.5 has the strongest binding ability to HSA.

[0041] Example 6 Detection of serum albumin binding capacity of VNAR-4A12 under multiple species and multiple pH conditions 1. ELISA to detect multi-species binding The 96-well ELISA plate was coated with 1 μg / mL mouse, rat, rabbit, and dog serum albumin at 4°C overnight; the next day, 5% MPBS was used as a blocking solution and blocked at 37°C for 1 hour; the antibody protein was diluted with PBS to a certain concentration gradient and incubated in the plate at 37°C for 1 hour; then HRP-coupled anti-His antibody was diluted in the blocking solution at a ratio of 1:10000, added to the plate, and incubated at 37°C for 1.5 hours; finally, TMB colorimetric solution was used for color development, and 1M sulfuric acid was used to stop the color development and then the absorbance at 450nm was detected. The binding results of the sequence shown in SEQ ID NO.5 to serum albumin of different species are shown in the figure. Fig.10 shown.

[0042] 2. Biolayer interferometry (BLI) to detect affinity under different pH conditions Purified VNAR-4A12 was diluted to 5 μg / mL, and serum albumin from different species was diluted to different concentration gradients: the concentration gradient of human and mouse serum albumin was 3.125, 6.25, 12.5, 25, 50, 100, 200 nM, and the concentration gradient of rabbit and rat serum albumin was 12.5, 25, 50, 100, 200, 400 and 800 nM. Anti-His probe was used for detection. The probe was first pre-wetted in PBST containing 0.02% Tween-20 at 1000 rpm for 60 s, then immersed in VNAR-4A12 solution at pH 7.4 for solidification, and then bound and dissociated with serum albumin from different species at pH 7.4 and pH 6.0, respectively. In the same experiment, the pH conditions of binding and dissociation were kept consistent. The affinity data of the sequence shown in SEQ ID NO.5 for serum albumin from different species are shown in Table 2.

[0043] Table 2: Affinity data of VNAR-4A12 for serum albumin from different species

[0044] Example 7 Testing of the ability of VNAR-4A12 to extend half-life in vivo 1. Construction, expression and purification of fusion protein The easily detectable green fluorescent protein sfGFP was selected as a control drug for prolonging the half-life. The sequence shown in SEQ ID NO.5, namely VNAR-4A12, was connected to sfGFP by three G4S linkers and inserted into the pET28a (+) expression vector through NcoⅠ and NotⅠ. Together with the sfGFP-pET28a vector, it was transformed into E. coli In BL21 (DE3), the expression was induced with 0.05 mM IPTG under 50 µg / ml kanamycin pressure at 30°C overnight. Both were soluble. The purification process of the two was the same as described in Example 4. The purified proteins were subjected to SDS-PAGE electrophoresis. The bands after Coomassie Brilliant Blue staining were as follows Fig.11 shown.

[0045] 2. In vivo half-life detection A total of 10 female 8-week-old BALB / c mice were divided into two groups (n=5), and each group was injected with a single tail vein injection of 100nmol / kg sfGFP or 4A12-sfGFP. 30 μL of blood was collected from the tail vein of mice at 0.25, 1, 3, 6, 9, 12, 24, 48, and 72 h, placed in an EP tube containing an anticoagulant, and centrifuged at 1500 xg at 4°C for 5 min. The upper plasma was diluted 5 times with PBS, and 30 μL of the diluted plasma was taken and placed in a black-bottom 384-well plate. The microplate reader was set to 495 nm excitation and 525 nm absorption to detect fluorescence intensity, and the blood drug concentration was calculated by fluorescence intensity until it was below the quantitative limit. The blood drug concentration data corresponding to different time points were processed using Phoenix WinNonlin Academic Version, and the pharmacokinetic parameters were calculated using the non-compartmental model (NCA). The blood drug concentration-time curve is shown in the figure. Fig.12 The calculated pharmacokinetic parameters are shown in Table 3.

[0046] Table 3: In vivo pharmacokinetic parameters of sfGFP and 4A12-sfGFP

[0047] λ z : Elimination rate constant; HL_λ z : terminal half-life; CL: clearance; AUC: 0→∞ : area under the curve; MRT: mean residence time; t 1 / 2 : Biological half-life, which can be calculated from 0.693*MRT in a single intravenous administration The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A shark single domain antibody targeting serum albumin of multiple species, characterized in that: The amino acid sequences thereof are shown in SEQ ID NO.1 to SEQ ID NO.

6.

2. The shark single domain antibody targeting multi-species serum albumin according to claim 1, characterized in that: The shark single-domain antibody targeting serum albumin of multiple species comprises fixed amino acid sequences FR1, FR2, FR3a, FR3b and FR4, hypervariable regions HV2 and HV4, and complementary determining regions CDR1 and CDR3.

3. The shark single domain antibody targeting multi-species serum albumin according to claim 2, characterized in that: The amino acid sequence of the CDR3 is shown in SEQ ID NO.13 to SEQ ID NO.

18.

4. The gene encoding the shark single domain antibody targeting serum albumin of multiple species according to claim 1, characterized in that: The nucleotide sequences of the coding genes are shown in SEQ ID NO.7 to SEQ ID NO.

12.

5. A recombinant vector, characterized in that: The recombinant vector contains the recombinant vector of the shark single domain antibody encoding gene targeting multi-species serum albumin as claimed in claim 4.

6. A recombinant strain, characterized in that: The recombinant strain contains the gene encoding the shark single domain antibody targeting multi-species serum albumin as claimed in claim 4 or the recombinant vector containing the gene encoding the shark single domain antibody targeting multi-species serum albumin as claimed in claim 5.

7. Use of the shark single domain antibody targeting multi-species serum albumin according to claim 1 in the preparation of serum albumin targeting preparations.

8. Use of the shark single-domain antibody targeting multi-species serum albumin according to claim 1 in the preparation of auxiliary half-life extension products and long-acting drugs.

9. Use of the shark single domain antibody targeting multi-species serum albumin according to claim 1 or the gene encoding the shark single domain antibody targeting multi-species serum albumin according to claim 4 in the preparation of an immunoassay kit and a plasma protein assay kit.

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