Shark single-domain antibody targeting multi-species serum albumin, its preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and discloses a shark single-domain antibody 4A12 targeting multi-species serum albumin, its preparation method and application. The VNAR-4A12 antibody is selected from the protein expressed by the amino acid sequence shown in SEQ ID NO.5, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.5 is the nucleotide sequence shown in SEQ ID NO.11. The shark single-domain antibody 4A12 targeting multi-species serum albumin of the present invention is smaller than traditional monoclonal antibodies, has good stability, is easier to store and transport than conventional antibodies, and can effectively bind to antigens. The expressed protein of the sequence shown in SEQ ID NO.5 exhibits cross-species serum albumin binding ability under the conditions of pH 6.0 and pH 7.4, and shows the ability to increase the half-life of fusion proteins in a mouse model, laying a foundation for subsequent use as a tool drug to improve the half-life of small molecule therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical antibody preparation, and particularly relates to shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 targeting multi-species serum albumin. In addition, the present invention also relates to the 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 polypeptide drugs is their too short half-life. Although these drugs have strong therapeutic potential, due to their small molecular weight, they are extremely easily metabolized by the kidneys quickly or degraded by proteases in lysosomes. 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 dosing frequency and increasing patient compliance, polyethylene glycolylation, fusing the Fc domain of traditional antibodies, conjugating or binding to serum albumin have all been explored as solutions by predecessors. Serum albumin is an abundant plasma protein that plays a crucial role in osmotic regulation and serves as a transporter for fatty acids, bilirubin, amino acids, steroid hormones, metal ions, and many therapeutic molecules. It has a molecular weight of 66 kDa and a half-life in the human body that can reach 19 days. 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 endomembrane recycling mediated by FcRn, and through a pH-dependent binding and dissociation mechanism, it avoids being degraded by lysosomes, extending its half-life from another perspective. In the past, people have used various methods such as using serum albumin as a drug carrier, preparing serum albumin fusion proteins, and adhering serum albumin to utilize serum albumin to extend the in vivo half-life of polypeptide and protein drugs and achieved success.

[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 dimeric 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 while the CDR3 region is longer. The highly variable CDR3 domain not only makes the diversity of VNAR more abundant, but its unique compact molecular structure also increases the accessibility of VNAR to hidden antigenic epitopes. Shark single-domain antibody VNAR shows great potential to replace traditional antibodies and be used as a therapeutic drug in clinical practice. As a new type of antibody, shark single-domain antibody has 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 polypeptides is of great significance for solving the problems of dosing frequency and long-term treatment in clinical practice. Summary of the Invention

[0004] The object of the present invention is to provide a shark single-domain antibody targeting multi-species serum albumin, its preparation method and application. Through screening, the present invention has obtained brand-new shark nanobodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11, which have high affinity and high specificity for serum albumin.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention protects a shark single-domain antibody 3A11, 3C11, 3F10, 3G3, 4A12, 4E11 targeting multi-species serum albumin, and its amino acid sequences are as shown in SEQ ID NO.1 to SEQ ID NO.6.

[0007] Furthermore, the shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, 4E11 include fixed amino acid sequences FR1, FR2, FR3a, FR3b and FR4, hypervariable regions HV2 and HV4, and complementary determining regions CDR1 and CDR3, and its CDR3 sequences are as shown in SEQ ID NO.13 to SEQ ID NO.18.

[0008] 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 as shown in SEQ ID NO.7 to SEQ ID NO.12.

[0009] The present invention further protects a recombinant vector, and the recombinant vector contains the above coding gene.

[0010] The present invention further protects a recombinant strain, and the recombinant strain contains the above coding gene or the above recombinant vector. The present invention also protects the application of the shark single-domain antibody in the preparation of a serum albumin targeting agent.

[0011] The present invention also protects the application of the shark single-domain antibody in the preparation of products for assisting in prolonging the half-life and long-acting drugs.

[0012] The present invention also protects the application of the shark single-domain antibody in the preparation of an immunoassay kit and a plasma protein detection kit.

[0013] Advantages of the present invention:

[0014] 1. The shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, 4E11 prepared by the present invention have a small molecular weight, only about 15 kDa, which is smaller than traditional monoclonal antibodies;

[0015] 2. The shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 have high stability, good tissue permeability, are easier to store and transport than conventional antibodies, and have good affinity and specificity for serum albumin.

[0016] 3. They are easier to express and genetically engineered. 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 are expressed using the Escherichia coli prokaryotic expression system, and a large number of shark single-domain antibodies are finally obtained.

[0017] 4. In this invention, the bamboo shark (Chiloscyllium plagiosum) is selected as the 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.

[0018] The shark single-domain antibodies 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11 can be applied to carriers for products with extended half-lives and long-acting therapeutic drugs. Genes encoding 3A11, 3C11, 3F10, 3G3, 4A12, and 4E11, recombinant plasmids, or recombinant cells containing the gene can be applied to the preparation of immunoassay diagnostic kits and plasma protein detection kits. Description of the Drawings

[0019] Figure 1 Results of ELISA for detecting the titer of shark serum;

[0020] Figure 2 PCR amplification of shark nanobody genes; Lane description: M: DNA Maker, 1 - 12: PCR products, and the specific VNAR band is within the red box;

[0021] Figure 3 Results of colony PCR for verifying the library insertion rate; Lane description: M: DNA Maker, 1 - 20: PCR products

[0022] Figure 4 Results of random sequencing for verifying library diversity;

[0023] Figure 5 Results of polyclonal ELISA;

[0024] Figure 6 Results of monoclonal ELISA;

[0025] Figure 7 Results of SDS-PAGE of the purified antibody protein;

[0026] Figure 8 Results of ELISA for detecting the binding ability of shark nanobodies to serum albumin;

[0027] Figure 9 BLI binding and dissociation curve for detecting the affinity between shark nanobody and serum albumin;

[0028] Figure 10 ELISA results for detecting the binding ability of VNAR-4A12 to serum albumins of different species;

[0029] Figure 11 SDS-PAGE results of purified sfGFP and 4A12-sfGFP proteins;

[0030] Figure 12 Plasma concentration-time curve after single intravenous administration of sfGFP and 4A12-sfGFP. Specific implementation manners

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying 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 the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] To further understand the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0033] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods in the art, 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, unless otherwise specified, can be obtained from commercial channels.

[0034] Example 1 Immunization of Chiloscyllium plagiosum and Titer Detection

[0035] 1. Shark immunization

[0036] The shark was immunized a total of 1 time for primary immunization and 4 times for booster immunization: for primary immunization, 250 μg of HSA was mixed and emulsified with complete Freund's adjuvant and injected subcutaneously via the lateral fin; for the first booster immunization, 100 μg of HSA was mixed and emulsified with incomplete Freund's adjuvant and injected subcutaneously via the lateral fin; for the subsequent 3 times of booster immunization, 3 μg of HSA was injected into the blood via the lateral fin vein, and the interval between each two immunizations was 1 month.

[0037] 5 mL of blood was collected via the caudal vein 5 days before immunization and after immunization was completed. The blood sample was anticoagulated with heparin, centrifuged at 1000 rpm for 10 min for stratification, and plasma and peripheral blood lymphocytes were collected.

[0038] 2. Detection of plasma titer by enzyme-linked immunosorbent assay (ELISA)

[0039] Dissolve HSA in PBS to 2 μg / mL, add it to a 96-well ELISA plate and coat overnight at 4°C. Block with 5% MPBS (skim milk powder dissolved in PBS) at 37°C for 1.5 h. Then dilute the plasma samples in a 10-fold gradient, using pre-immune plasma as a negative control, incubate at 37°C for 1.5 h, add rabbit anti-shark serum diluted 1:1000 (prepared in our laboratory) and incubate at 37°C for 1 h. Next, add 0.1 μg / mL HRP-conjugated goat anti-rabbit IgG antibody and incubate at 37°C for 1 h. Add TMB chromogenic solution and develop color at room temperature in the dark for 5 min. Stop the color development with 1 M HCl and read the absorbance at 450 nm using an ELISA reader.

[0040] The plasma titer obtained by ELISA was around 1:100, and the results are as Figure 1 shown.

[0041] Example 2 Construction of Phage Display Library

[0042] 1. Amplification of target gene

[0043] Total RNA was extracted from peripheral blood lymphocytes isolated from blood samples using the Trizol method and reverse transcribed into cDNA. By analyzing the structure and diversity of the immunoglobulin gene locus of the striped bamboo shark, forward primers SEQ ID NO.19 - SEQ ID NO.20 were designed based on partial conserved sequences upstream of VNAR, and reverse primers SEQ ID NO.21 - SEQ ID NO.22 were designed based on partial conserved sequences downstream of the CDR3 domain to amplify the VNAR fragment.

[0044] SEQ ID NO.19

[0045] NewVF1

[0046] CAATTTGATTGGGCCCAGGCGGCCGCCSMACGGSTTGAACAAACACC

[0047] SEQ ID NO.20

[0048] NewVF2

[0049] CAATTTGATTGGGCCCAGGCGGCCGCCGCACGGGTTGAACAAACACCG

[0050] SEQ ID NO.21

[0051] NewVR1

[0052] CTTAATCGACTGGCCGGCCTGGCCCACAGTCASARKGGTSCC

[0053] SEQ ID NO.22

[0054] NewVR2

[0055] CTTAATCGACTGGCCGGCCTGGCCCACAGTCAGAGGGGTGCCGCCTCC The 50 μL reaction system for PCR amplification is: 2x Max 25 μL, template cDNA 2.5 μL, upstream primer 2 μL, downstream primer 2 μL, ddH2O 18.5 μL.

[0056] The amplification conditions for PCR are: pre-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; final extension at 72°C for 5 min.

[0057] The nucleic acid electrophoresis results of the VNAR region of Chiloscyllium plagiosum amplified by the PCR method are as Figure 2 shown.

[0058] 2. Library construction

[0059] The amplified VNAR fragment and the pComb3Xss vector were digested with the restriction endonuclease SfiⅠ respectively and ligated with T4 ligase to construct a recombinant vector.

[0060] 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.

[0061] The ligation system for 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.

[0062] The ligated pComb3Xss-VNAR vector was transformed into Escherichia coli TG1 by electroporation. The bacterial solution was spread on an ampicillin-resistant 2xYT solid plate and cultured overnight at 37°C. The bacteria on the plate were collected to obtain the original library strains. After the strains were cultured to the logarithmic phase, M13K07 helper phage was added for superinfection, and the culture was continued overnight at 30°C with shaking at 200 rpm. The supernatant was filtered through a 0.22-μm filter membrane, and a PEG / NaCl solution with a final volume of 20% was added. The mixture was incubated on ice for 30 min to precipitate the phages. The mixture was centrifuged at 4°C and 10,000 g for 15 min, and the precipitate was resuspended in PBS to obtain the storage solution of the original phage library.

[0063] Calculation of library size: Final library size = Initial library size × Insertion rate × Sequencing diversity.

[0064] The electroporated bacterial solution was serially diluted to 10 -6 、10 -7 、10 -8 times, and then spread on an ampicillin-resistant 2xYT solid plate and cultured overnight at 37°C. Initial library size = Number of single colonies growing on the plate × 10 6 、10 7 、10 8 . Twenty clones were randomly selected from the plate. The upstream primers of VNAR amplification were mixed in equimolar amounts as the upstream primer, and the conserved fragment on the vector was used as the downstream primer for PCR identification. Insertion rate = Number of target bands / Total number of PCR reactions of the bacterial solution × 100%. The bacterial solutions that successfully obtained the target bands by PCR were randomly sequenced. Diversity = Number of correctly sequenced sequences / Total number of sequenced samples × 100%.

[0065] The final library size applied in the present invention is 1.53x10 8 .

[0066] The nucleic acid electrophoresis results of the inserted fragments identified by bacterial solution PCR are shown in Figure 3 , and the sequence alignment results of the random sequencing are shown in Figure 4 . The quality evaluation of the VNAR library is shown in Table 1.

[0067] Table 1: Quality evaluation of the phage library

[0068] Initial library capacity Insertion rate Diversity Final library capacity <![CDATA[2.10×10 8 > 90％ 81％ <![CDATA[1.53x10 8 >

[0069] Example 3 Specific screening and identification for HSA

[0070] Using the method of solid-phase panning, in the first round of panning, 10 μg / ml of HSA and the blocking solution were respectively added to a 96-well ELISA plate and coated overnight at 4 °C. The next day, while blocking the HSA wells, a phage preparation solution 10 times the library volume 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 then directly added with TG1 bacterial solution grown to the logarithmic phase for infection 3 times, with each infection lasting 30 min. The same method as for preparing the library phage stock solution was used for superinfection and amplification of phages. The coating concentrations in the second and third rounds of panning were reduced to 4 μg / ml and 2 μg / ml, and were alternately blocked with 5% MPBS and gelatin blocking solution. A total of three rounds of panning were carried out. The phage solution amplified after each round of panning could be directly used for polyclonal ELISA to detect the enrichment after panning. The results are as Figure 5 shown.

[0071] 192 monoclonal colonies were picked from the out-of-library plates after the second and third rounds of panning and placed in a 96-well deep-well plate. They were cultured with shaking at 37 °C until the logarithmic phase, and then M13K07 helper phage was added for superinfection. The same method as before was used to amplify the progeny phages. The next day, the plate was centrifuged at 500 x g for 20 min, and the supernatant could be directly used for monoclonal ELISA to detect the positive expression of the clones after panning. The results are as Figure 6 shown. Six VNAR sequences targeting HSA were obtained through three rounds of screening, namely the sequences shown in SEQ ID NO.1 to SEQ ID NO.6. The nucleotide sequences of their encoding genes are as shown in SEQ ID NO.7 to SEQ ID NO.12, and their CDR3 sequences are as shown in SEQ ID NO.13 to SEQ ID NO.18.

[0072] SEQ ID NO.1

[0073] 3A11

[0074] QWVEQTPTTTTKEAGESLTINCVLKGSSCALGSTYWYFTKKGATKKASLSTGGRYSDTKN

[0075] TASKSFSLRISDLRVEDSGTYHCEAFYSWDEGDVCRSWDSLSYEGGGTILTV

[0076] SEQ ID NO.2

[0077] 3C11

[0078] QWVEQTPTTTTKEAGESLTINCVLRDSSCALASTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPQLDEGDCVGWYPSEGGGTILSV

[0079] SEQ ID NO.3

[0080] 3F10

[0081] ARVEQTPTTTTKEAGESLTINCALRDSSCVLGRTYWYFTKKGAPKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYPLEFCDTHSWTEGGGTILTV

[0082] SEQ ID NO.4

[0083] 3G3

[0084] ARVEQTPTTTTKEAGESLTINCVLRDSSCALGKTYWYFTKKGATKKASLSTGGRYTETVNKASKSFSLRISDLRVEDSGTYHCKAYPLQLCSGHSWVEGGGTTLTV

[0085] SEQ ID NO.5

[0086] 4A12

[0087] ARVEQTPTTTTKEAGESLTINCVLRDSSCPLPSASTYWFFTKKGATKKESLSNGGRYAETMNKTSKSFSLRISDLRVEDSGTYHCKAYPGGYCDGIYYYEGGGTILTV

[0088] SEQ ID NO.6

[0089] 4E11

[0090] ARVEQTPTTTTKEAGESLTINCVLRDSSCALYSTYWYFTKKGATKKETLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYHCKAYPQLDEDCVSWKYYEGGGTIV

[0091] SEQ ID NO.7

[0092] 3A11

[0093] CAATGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTGCATTGGGTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTTTTACAGCTGGGATGAGGGAGACGTCTGCCGTAGCTGGGATTCTCTCTCTTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCCG

[0094] SEQ ID NO.8

[0095] 3C11

[0096] CAATGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGCTAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCACAGCTGGATGAAGGGGACTGTGTAGGCTGGTATCCGTCTGAAGGAGGCGGCACCATTCTGAGTGTAAAACCC

[0097] SEQ ID NO.9

[0098] 3F10

[0099] GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGCCCTAAGAGACTCCAGCTGTGTATTGGGTAGGACGTACTGGTATTTCACAAAAAAGGGCGCGCCAAAGAAGGAGAGCTTATCGAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATTACTGTAAAGCGTATCCCCTGGAGTTCTGTGATACCCATAGTTGGACTGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGG

[0100] SEQ ID NO.10

[0101] 3G3

[0102] GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGGTAAAACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGCGAGCTTATCAACTGGCGGACGATACACGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCGCTACAGCTCTGTTCCGGCCATAGCTGGGTTGAAGGAGGCGGCACCACTCTGACTGTG

[0103] SEQ ID NO.11

[0104] 4A12

[0105] GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTCCATTGCCTAGCGCTAGCACGTACTGGTTTTTCACAAAAAAGGGCGCAACAAAGAAGGAGAGCTTATCAAATGGCGGACGATACGCGGAAACAATGAACAAGACATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTACCCTGGGGGTTACTGTGATGGAATTTACTATTATGAAGGAGGCGGCACCATTCTGACTGTGAAACCTGG

[0106] SEQ ID NO.12

[0107] 4E11

[0108] GCACGGGTTGAACAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGTATAGCACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAGAAGGAAACCTTATCAAATGGCGGACGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTAAAGCGTATCCTCAGCTGGATGAAGACTGTGTTAGCTGGAAGTATTATGAAGGAGGCGGCACCATTGTGACTGTGGG

[0109] SEQ ID NO.13

[0110] 3A11-CDR3

[0111] FYSWDEGDVCRSWDSLSYE

[0112] SEQ ID NO.14

[0113] 3C11-CDR3

[0114] YPQLDEGDCVGWYPSE

[0115] SEQ ID NO.15

[0116] 3F10 - CDR3

[0117] YPLEFCDTHSWTE

[0118] SEQ ID NO.16

[0119] 3G3 - CDR3

[0120] YPLQLCSGHSWVE

[0121] SEQ ID NO.17

[0122] 4A12 - CDR3

[0123] YPGGYCDGIYYYE

[0124] SEQ ID NO.18

[0125] 4E11 - CDR3

[0126] YPQLDEDCVSWKYYE

[0127] Example 4 Recombinant Expression and Purification of Shark Nanobodies

[0128] Plasmids were extracted from the positive clone strains screened by monoclonal ELISA and transformed into E. coli HB2151. After the strains were cultured to the logarithmic phase, they were induced to express with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) under the pressure of 100 μg / ml ampicillin at 30 °C overnight, and all proteins were expressed in a soluble form. 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 BL21(DE3). It was induced to express with 0.1 mM IPTG under the pressure of 50 μg / ml kanamycin at 37 °C for 6 h, and the expression was in the form of inclusion bodies.

[0129] After collecting the soluble-expressed bacterial cells, they were ultrasonically disrupted. The supernatant after disruption was purified using a nickel column, impurities were removed with 20 mM imidazole, and eluted with 250 mM imidazole. The purified eluate was concentrated using a 3K ultrafiltration column and exchanged with PBS for storage. For inclusion bodies, the precipitate after ultrasonic disruption needed to be collected and dissolved in Tris buffer containing 8 M urea. The nickel column purification process was the same as described above, but 8 M urea needed to be maintained in the system all the time. The urea in the purified protein solution was replaced with 0 M through gradient dialysis, and then concentrated using a 3K ultrafiltration column and exchanged with PBS for storage. The antibody protein after nickel column purification was further purified using a SuperdexTM 200 Increase 10 / 300 GL column. The purified antibody protein was subjected to SDS-PAGE electrophoresis, and the bands after Coomassie Brilliant Blue staining were as Figure 7 shown.

[0130] Example 5 Detection of the Affinity of Shark Nanobody Protein to HSA

[0131] 1. Detection of Binding Ability by Enzyme-Linked Immunosorbent Assay (ELISA)

[0132] A 96-well enzyme-linked immunosorbent assay (ELISA) plate was coated with 1 μg / mL HSA at 4 °C overnight. The next day, 5% MPBS was used as the blocking solution and blocked at 37 °C for 1 hour. The antibody protein was diluted to a certain concentration gradient with PBS and incubated in the well plate at 37 °C for 1 hour. Then, the HRP-conjugated anti-HA antibody was diluted 1:10000 in the blocking solution, added to the well plate, and incubated at 37 °C for 1.5 hours. Finally, TMB chromogenic solution was used for color development, and the color development was terminated with 1 M sulfuric acid, and the absorbance at 450 nm was measured. The results of the binding ability of the sequences screened in Example 3 to HSA were as Figure 8 shown.

[0133] 2. Detection of Affinity by Biolayer Interferometry (BLI)

[0134] The Octet RED96e system was used to detect the affinity of the target protein to HSA. The purified protein solution was diluted to 5 μg / mL and detected using an anti-His probe. First, the probe was pre-wetted at 1000 rpm in PBST containing 0.02% Tween-20 for 60 s, then the probe was immersed in the protein solution for 90 s for immobilization, and then combined and dissociated with 100 nM HSA for 120 s and 180 s respectively. The fitted binding and dissociation curves were as Figure 9 shown.

[0135] The screened sequences all had a certain affinity for HSA, and the sequence shown in SEQ ID NO.5 had the strongest binding ability to HSA.

[0136] Example 6 Detection of Serum Albumin Binding Ability of VNAR-4A12 under Multi-Species and Multi-pH Conditions

[0137] 1. Detection of the binding ability to multiple species by enzyme-linked immunosorbent assay (ELISA)

[0138] Coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate with mouse, rat, rabbit, and canine serum albumin at a concentration of 1 μg / mL and incubate overnight at 4°C. The next day, use 5% MPBS as a blocking solution and block at 37°C for 1 hour. Dilute the antibody protein to a certain concentration gradient with PBS and incubate in the well plate at 37°C for 1 hour. Then, dilute the HRP-conjugated anti-His antibody at a ratio of 1:10,000 in the blocking solution, add it to the well plate, and incubate at 37°C for 1.5 hours. Finally, develop the color with TMB chromogenic solution, terminate the color development with 1 M sulfuric acid, and detect the absorbance at 450 nm. The binding ability results of the sequence shown in SEQ ID NO.5 to serum albumin of different species are as Figure 10 shown.

[0139] 2. Detection of the affinity under different pH conditions by biolayer interferometry (BLI)

[0140] Dilute the purified VNAR-4A12 to 5 μg / mL and dilute the serum albumin of different species to different concentration gradients: the concentration gradients of human and mouse serum albumin are 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM, and the concentration gradients of rabbit and rat serum albumin are 12.5, 25, 50, 100, 200, 400, and 800 nM. Use an anti-His probe for detection. First, pre-wet the probe at 1000 rpm in PBST containing 0.02% Tween-20 for 60 s, then immerse the probe in the VNAR-4A12 solution at pH 7.4 for immobilization, and then bind and dissociate with the serum albumin of different species at pH 7.4 and pH 6.0, respectively. In the same experiment, the pH conditions for binding and dissociation are kept consistent. The affinity data of the sequence shown in SEQ ID NO.5 to serum albumin of different species are shown in Table 2.

[0141] Table 2: Affinity data of VNAR-4A12 to serum albumin of different species

[0142]

[0143] Example 7 Detection of the ability of VNAR-4A12 to extend the half-life in in vivo experiments

[0144] 1. Construction, expression, and purification of the fusion protein

[0145] The easily detectable green fluorescent protein sfGFP was selected as the control drug for extending 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 BL21(DE3). Expression was induced with 0.05 mM IPTG under the pressure of 50 μg / ml kanamycin at 30 °C overnight, and both were soluble expressions. The purification processes of both were the same as those described in Example 4. The purified proteins were subjected to SDS-PAGE electrophoresis, and the bands after Coomassie brilliant blue staining were as Figure 11 shown.

[0146] 2. In vivo half-life detection

[0147] A total of 10 female 8-week-old BALB / c mice were divided into 2 groups (n = 5). Each group was intravenously injected with 100 nmol / kg of sfGFP or 4A12-sfGFP via the tail vein once. At 0.25, 1, 3, 6, 9, 12, 24, 48, and 72 h, 30 μL of blood was collected from the tail vein of the mice and placed in an EP tube containing anticoagulant. After centrifugation at 1500 xg for 5 min at 4 °C, the upper plasma was diluted 5-fold with PBS. 30 μL of the diluted plasma was taken and placed in a black-bottom 384-well plate. The fluorescence intensity was detected with an enzyme-labeled instrument by setting an excitation wavelength of 495 nm and an absorption wavelength of 525 nm, and the blood drug concentration was calculated from the fluorescence intensity until it was lower than the lower limit of quantification. The blood drug concentration data corresponding to different time points were processed with Phoenix WinNonlin Academic Version, and the pharmacokinetic parameters were calculated using the non-compartmental model (NCA). The drawn blood drug concentration-time curve was as Figure 12 shown, and the calculated pharmacokinetic parameters are shown in Table 3.

[0148] Table 3: In vivo pharmacokinetic parameters of sfGFP and 4A12-sfGFP

[0149] Parameter <![CDATA[λ z > <![CDATA[HL_λ z > CL <![CDATA[AUC 0→∞ > MRT <![CDATA[t 1 / 2 > Unit 1 / h h L / h*kg h*nM h h sfGFP 0.229 3.026 0.012 144.077 3.945 2.734 4A12-sfGFP 0.024 28.688 0.001 1236.708 37.619 26.070

[0150] λ z : Elimination rate constant; HL_λ z : Terminal half-life; CL: Clearance rate; AUC 0→∞ : Area under the curve; MRT: Mean residence time; t 1 / 2: Biological half-life can be calculated by 0.693 * MRT in a single intravenous administration. The above description is only a preferred embodiment of the present invention and is not a limitation of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, modifications, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Shark single domain antibody 4A12 targeting serum albumin of multiple species, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

5.

2. The shark single domain antibody 4A12 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 4A12 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.

17.

4. The gene encoding the shark single domain antibody 4A12 targeting multi-species serum albumin according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

11.

5. A recombinant vector, characterized in that: The recombinant vector contains the gene encoding the shark single-domain antibody 4A12 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 4A12 targeting multi-species serum albumin as claimed in claim 4 or the recombinant vector containing the gene encoding the shark single domain antibody 4A12 targeting multi-species serum albumin as claimed in claim 5.

7. Use of the shark single-domain antibody 4A12 targeting multi-species serum albumin according to claim 1 in the preparation of a serum albumin targeting preparation.

8. Use of the shark single-domain antibody 4A12 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 4A12 targeting multi-species serum albumin according to claim 1 or the encoding gene of the shark single domain antibody 4A12 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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