Anti-TNFα single domain antibody and its application

Abstract

The present invention provides an anti-TNFα single-domain antibody and its application. The single-domain antibody has antigen-antibody binding ability, which can further provide a basis for the development of TNFα blocking drugs and the diagnosis of related diseases, and has great prospects in TNFα-related research and the diagnosis and treatment of future diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-TNFα single-domain antibody and an application thereof. Background Art

[0002] Traditional antibodies have fallen into a development bottleneck due to their large molecular weight, complex structure, thermal instability, and high cost. Among them, the natural heavy chain antibodies found in cartilaginous fish are called immunoglobulin new antigen receptors (Immunoglobulin new antigen receptor, IgNAR). The variable domain of these immunoglobulin new antigen receptors (Variable domain of immunoglobulin new antigen receptor, VNAR) has been verified to have autonomous functions as single-domain antibodies, and has the advantages of good thermal stability, special structure, and small size. It has particularly outstanding advantages in size, solubility, thermal stability, and chemical stability, and is an ideal substitute for traditional antibodies.

[0003] Tumor necrosis factor (TNFα) is an important clinical detection factor and drug target that can trigger a series of autoimmune diseases and inflammation. TNFα plays a key role in resolving infections and tissue repair through signal transduction pathways. Sometimes, it may trigger a severe cytokine storm, leading to sepsis and autoimmune diseases. Studies have shown that biological blockers of TNFα (such as monoclonal antibodies) can treat many chronic inflammatory diseases, including vitiligo, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), etc., and can also be used as specific drugs to inhibit certain tumors. However, the large molecular weight, high immunogenicity and high production costs of these drugs limit their application. Summary of the invention

[0004] In order to solve the above problems, the present invention proposes an anti-TNFα single domain antibody and its application, which provides a basis for the development of TNFα blocking drugs and the diagnosis of related diseases.

[0005] In order to achieve the above objectives, the embodiments of the present invention provide, in the first aspect, an anti-TNFα single domain antibody, whose complementary determining region CDR comprises CDR1 and CDR3 with the amino acid sequences shown below:

[0006] (1) CDR1 shown in SEQ ID NO:6 and CDR3 shown in SEQ ID NO:38; or

[0007] (2) CDR1 shown in SEQ ID NO:7 and CDR3 shown in SEQ ID NO:39; or

[0008] (3) CDR1 shown in SEQ ID NO:7 and CDR3 shown in SEQ ID NO:40; or

[0009] (4) CDR1 shown in SEQ ID NO:8 and CDR3 shown in SEQ ID NO:40; or

[0010] (5) CDR1 shown in SEQ ID NO:9 and CDR3 shown in SEQ ID NO:40; or

[0011] (6) CDR1 shown in SEQ ID NO:10 and CDR3 shown in SEQ ID NO:40; or

[0012] (7) CDR1 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO:41; or

[0013] (8) CDR1 shown in SEQ ID NO:12 and CDR3 shown in SEQ ID NO:41.

[0014] According to an anti-TNFα single-domain antibody of an embodiment of the present invention, the antibody has a competitive effect on the TNFα receptor and has antigen-antibody binding ability, which can further provide a basis for the development of TNFα blocking drugs and the diagnosis of related diseases, and has great prospects in TNFα-related research and the diagnosis and treatment of future diseases.

[0015] In addition, the anti-TNFα single domain antibody proposed in the above embodiment of the present invention may also have the following additional technical features:

[0016] Optionally, the framework region FR includes FR1 to FR4 with the amino acid sequences shown below:

[0017] 1) FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 13, FR3a shown in SEQ ID NO: 25, FR3b shown in SEQ ID NO: 32, and FR4 shown in SEQ ID NO: 42; or

[0018] 2) FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 13, FR3a shown in SEQ ID NO: 26, FR3b shown in SEQ ID NO: 33, and FR4 shown in SEQ ID NO: 42; or

[0019] 3) FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 13, FR3a shown in SEQ ID NO: 26, FR3b shown in SEQ ID NO: 33, and FR4 shown in SEQ ID NO: 43; or

[0020] 4) FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 14, FR3a shown in SEQ ID NO: 26, FR3b shown in SEQ ID NO: 33, and FR4 shown in SEQ ID NO: 43; or

[0021] 5) FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 13, FR3a shown in SEQ ID NO: 26, FR3b shown in SEQ ID NO: 34, and FR4 shown in SEQ ID NO: 43; or

[0022] 6) FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 15, FR3a shown in SEQ ID NO: 26, FR3b shown in SEQ ID NO: 33, and FR4 shown in SEQ ID NO: 43; or

[0023] 7) FR1 shown in SEQ ID NO:2, FR2 shown in SEQ ID NO:16, FR3a shown in SEQ ID NO:27, FR3b shown in SEQ ID NO:35, and FR4 shown in SEQ ID NO:42; or

[0024] 8) FR1 shown in SEQ ID NO:3, FR2 shown in SEQ ID NO:17, FR3a shown in SEQ ID NO:27, FR3b shown in SEQ ID NO:36, and FR4 shown in SEQ ID NO:42; or

[0025] 9) FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:17, FR3a shown in SEQ ID NO:27, FR3b shown in SEQ ID NO:36, and FR4 shown in SEQ ID NO:42, or

[0026] 10) FR1 shown in SEQ ID NO:5, FR2 shown in SEQ ID NO:18, FR3a shown in SEQ ID NO:28, FR3b shown in SEQ ID NO:37, and FR4 shown in SEQ ID NO:42.

[0027] Optionally, the anti-TNFα single domain antibody comprises:

[0028] (a) a single domain antibody with an amino acid sequence as shown in any one of SEQ ID NOs: 44 to 58; or

[0029] (b) A single domain antibody having an amino acid sequence that is 80% or more identical to the sequence shown in any one of SEQ ID NOs: 44 to 58 and having the antibody function of (a).

[0030] According to an embodiment of the present invention, in a second aspect, an isolated polynucleotide is provided, encoding the above-mentioned anti-TNFα single domain antibody.

[0031] According to an embodiment of the present invention, a third aspect provides a construct comprising the isolated polynucleotide described above.

[0032] According to an embodiment of the present invention, a fourth aspect provides a pharmaceutical composition comprising the above-mentioned anti-TNFα single domain antibody.

[0033] Optionally, a pharmaceutically acceptable carrier is also included.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The mouse and human TNFα membrane extracellular protein sequences of the embodiments of the present invention;

[0036] Figure 2 The plasmid maps of TNFα-mouse-pET28a and TNFα-human-pET28a-3flag of the embodiments of the present invention;

[0037] Figure 3 It is the comparison result of the construction of TNFα-mouse-pET28a and TNFα-mouse-pET28a-3flag plasmids in the embodiment of the present invention;

[0038] Figure 4 The results of SDS-PAGE analysis of recombinant TNFα expression and purification in the embodiment of the present invention;

[0039] Figure 5 is the BSA protein standard curve of the embodiment of the present invention;

[0040] Figure 6 The first round of panning and the second round of panning dilution coating of the embodiment of the present invention;

[0041] Figure 7It is the second round of Phage ELISA of the embodiment of the present invention: detecting the binding ability of 96 VNARs to mouse TNFα;

[0042] Figure 8 15 anti-VNARs of the embodiment of the present invention TNFα Difference analysis of amino acid sequences;

[0043] Fig. 9 15 VNARs of the embodiment of the present invention TNFα Homology analysis of amino acid sequences;

[0044] Fig.10 The binding ability of the four specific CDR3s of the present invention to mouse and human TNFα;

[0045] Fig.11 This is the result of SDS-PAGE analysis of recombinant 3B11 expression and purification in the embodiment of the present invention;

[0046] Fig.12 It is the VNAR of the embodiment of the present invention 3B11 Affinity testing;

[0047] Fig.13 It is the VNAR of the embodiment of the present invention 3B11 Binding and dissociation curves of TNFα. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0049] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0050] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0051] Example 1 Fusion protein plasmid construction and induction purification

[0052] 1. By designing primers, add a homology arm to the TNFα sequence with the pET28a(+) plasmid, and connect the two together through T5 exonuclease-mediated seamless connection technology in a specific TEDA buffer system. The primer sequences are shown in Table 1:

[0053] Table 1

[0054]

[0055] 2. Preparation of competent E. coli

[0056] Take a small amount of bacteria and streak four lines on a 1.5% LB agar plate, and culture overnight in a 37℃ incubator for 10-16h. Pick a single clone and put it into a 50mL tube with 10mL TB culture medium, and first culture it at 37℃ until OD600 reaches 0.5-1.5. Inoculate it into 200mL TB culture medium in a 1L conical flask at 1:100 and culture it until OD600 reaches 0.5-1.5; when making DH5α competent state, it is: 18℃, 250-300rpm, more than 16h; when preparing BL21(DE3) competent state, it is: 26℃, 250–300rpm, 20-28h. The following steps are performed on ice. Centrifuge the bacteria into a 50mL tube, 2,600rpm, 5min centrifuge to collect the bacteria, and place the TB buffer at 4℃ in advance to cool. Discard the supernatant and add TB buffer to manually resuspend. Centrifuge at 2,600rpm, 5min to collect the bacteria, and discard the supernatant. Calculate the amount of TB buffer and DMSO required based on the previous OD600 value, OD600 reaches 5, and the final DMSO concentration is 7%. Manually resuspend, dispense into EP tubes with 100μL-500μL per tube, and place on ice for 0.5-1 h. After a batch is filled, place it in an ice box filled with liquid nitrogen for quick freezing for about 1 minute; remove the competent state from the liquid nitrogen, place it on ice for 10 seconds, and transfer it to other containers and store it in a -80℃ refrigerator for later use.

[0057] 3. pET-28a(+) plasmid digestion

[0058] The pET-28a(+) vector was double-digested with BamHI and XhoI restriction endonucleases.

[0059] The reaction system is shown in Table 2:

[0060] Table 2

[0061]

[0062] After digesting with the corresponding endonuclease in a 37°C water bath for 2 h, add 10× loading buffer to terminate the digestion reaction and wait for the next agarose gel electrophoresis and recovery.

[0063] 4. TNFα gene amplification

[0064] PCR system table 3:

[0065]

[0066] PCR amplification conditions Table 4:

[0067]

[0068] 5. Agarose gel electrophoresis DNA

[0069] Weigh 1.4g agarose and add 141mL TAE buffer solution, which can be purchased from Bio-Tech. Heat in a microwave oven for 2.5min until the solution is clear, add nucleic acid dye and pour into agarose gel electrophoresis plate. Mix PCR amplification product with loading buffer and load. Electrophoresis, constant voltage 120V, about 30min, stop electrophoresis when bromophenol blue line reaches the bottom. Illuminate the gel with UV light, and then use a scalpel blade to carefully cut the target band from the agarose gel for subsequent experiments. The gel can be temporarily stored in a -20℃ refrigerator away from light.

[0070] 6. DNA recovery by agarose gel

[0071] (1) Take out the gel from the refrigerator, weigh it, add the PC solution, and directly place it in a 50°C water bath until the colloid is completely dissolved.

[0072] (2) Equilibrate the adsorption column. Assemble the adsorption column and the collection tube, then add 500 μL of BL equilibration solution in turn. Centrifuge at a speed of 12,000 rpm for 1 min. After centrifugation, discard the transparent waste liquid in the lower collection tube.

[0073] (3) After the adsorption column is placed back into the collection tube, the orange-yellow liquid obtained in (2) is added to the adsorption column CB2 in batches.

[0074] (4) The centrifugal speed is 13,000 rpm, and centrifugation is repeated for 1 min. At this time, the orange-yellow waste liquid enters the collection tube, and the DNA fragments have been adsorbed on the column.

[0075] (5) Put the adsorption column back and add 500 μL of rinsing solution PW into the adsorption column.

[0076] (6) The centrifugal speed was 12,000 rpm and the centrifugation time was 1.5 min. The PW waste liquid in the collection tube was discarded and the adsorption column was returned.

[0077] (7) Repeat steps (6) and (7).

[0078] (8) Place the adsorption column into the collection tube again and centrifuge at 12,000 rpm for 2 min to remove as much clean rinse solution as possible.

[0079] (9) Remove the adsorption column from the collection tube and place it in a clean working environment with low wind for 3 minutes to ensure that the alcohol in the adsorption column CB2 is completely dried.

[0080] (10) Preheat the eluent EB in a 65-70℃ water bath.

[0081] (11) Place the dried adsorption column into a 1.5 mL labeled centrifuge tube, add 35-50 μL of elution EB, and place it in a cool room temperature for 2-5 min. Centrifuge it at 12,000 rpm for 2-3 min to completely centrifuge the liquid and collect the DNA solution.

[0082] (12) Take 2 μL of the liquid and measure the DNA concentration using a spectrophotometer ND-100.

[0083] 7. Ligation of target fragment and vector

[0084] Take 5×TEDA pre-prepared T5 exonuclease solution and establish a ligation system (20 μL), wherein the molar ratio of the target fragment to the vector is about 4:1, and the ligation is carried out at 30°C.

[0085] Connection system table 5:

[0086]

[0087] E. coli transformation:

[0088] (1) When the above system is about to end, take out the DH5α competent cells prepared in step 2 from the -80°C refrigerator. After taking it out of the refrigerator, quickly put it into ice and let it thaw on ice for about 6 minutes.

[0089] (2) Transfer the product after T5 ligase ligation into the thawed competent medium, gently tap the bottom of the EP tube with your hand, and then place it on ice for 10 minutes.

[0090] (3) Heat shock at 42°C in a water bath for 90 seconds, then quickly return to ice and let stand for 5 minutes.

[0091] (4) Spread the plate onto a solid LB plate containing kanamycin, completely dry the surface in a clean bench, and then culture at 37°C overnight.

[0092] 8. Recombinant plasmid colony PCR verification

[0093] The single clone grown in the above plate was picked and put into 5 mL of LB liquid medium containing 30 μg / mL kanamycin, and cultured at 37°C for 5 hours. 1 μL of bacterial solution was taken out (the remaining bacterial solution was centrifuged and placed in a -20°C refrigerator) for colony PCR verification. The upstream primer used T7 universal primer, and the downstream primer used the downstream primer of the corresponding target fragment.

[0094] The reaction system is shown in Table 6:

[0095]

[0096] After the colony PCR is completed, 10 μL of the product is directly taken for agarose gel electrophoresis without adding loading buffer, and the results are observed under ultraviolet light.

[0097] 9. DNA sequencing identification

[0098] (1) Add anhydrous alcohol to the rinse solution PW until the required level is reached. Add the RNase in the kit to P1.

[0099] (2) Take the above colony PCR to verify the correct colony, and use 5 mL LB liquid culture medium to culture until the stationary phase.

[0100] (3) Use a 1.5 mL centrifuge tube to collect the bacteria. During the last collection, use a 100 μL pipette to remove the culture medium and remove the upper culture medium.

[0101] (4) Slowly add 250 μL of solution P1 to the above centrifuge tube and resuspend the E. coli cells at the bottom using a 1 mL pipette or vortex mixer.

[0102] (5) Slowly add 250 μL of solution P2 to the centrifuge tube and gently shake the tube several times to allow the bacteria to be completely lysed.

[0103] (6) Slowly add 350 μL of solution P3 to the centrifuge tube and immediately shake it gently from side to side several times until it is completely mixed. Place the centrifuge tube in a centrifuge and centrifuge at 12,000 rpm for 10 min at room temperature.

[0104] (7) During the 10-min centrifugation in the previous step, balance the adsorption column: After taking out the adsorption column CP3, put it into the collection tube. After marking the number with a marker pen, add 500 μL of balancing solution, centrifuge at 13,800 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back.

[0105] (8) After the mixture in (5) has been completely centrifuged to the bottom, if it has not, centrifuge again.

[0106] (9) Use a suitable pipette to add the supernatant after high-speed centrifugation to the pre-equilibrated adsorption column CP3 according to the number. Do not let the pipette tip touch the milky white precipitate below. Centrifuge at 13,800 rpm for 45 seconds, discard the waste liquid, and put the adsorption column back.

[0107] (10) After ensuring that all the supernatant has passed through the adsorption column, add 600 μL of rinse solution PW and centrifuge at 13,800 rpm for 60 s. Pour out the liquid in the collection tube and put the adsorption column back.

[0108] (11) Repeat the above operation (9).

[0109] (12) Completely remove the remaining alcohol rinse solution in the adsorption column, return the washed adsorption column to the collection tube, and centrifuge at 13,800 rpm for 2 min.

[0110] (13) Use a marker to mark the corresponding parameters on the lid of a clean centrifuge tube, take out the adsorption column and place it in it. Open the adsorption column and add 35 μL of eluent to the center, taking care not to touch the adsorption membrane.

[0111] (14) After the adsorption column was placed in a cool place at room temperature for 2-10 min, it was centrifuged at a maximum speed of 13,800 rpm for 2 min, and the resulting plasmid solution was collected for subsequent use.

[0112] (15) Take an appropriate amount and send it to Xiamen Borui Biotechnology Co., Ltd. for sequencing. The remaining markers should be stored in a -20℃ refrigerator.

[0113] 10. Expression and purification of TNFα fusion protein

[0114] Select the plasmid with the correct sequence alignment and transform it into BL21 (DE3) Escherichia coli. The next day, select a single colony until it is cultured in 5mL LB medium for at least about 12h. Then take 1ml of the bacterial solution and inoculate it into 100mL LB or 2×YT medium with kana resistance, and culture it in a shaker at 37℃ and 220rpm for about 4h. When the OD600 value reaches about 0.45, add IPTG with a final concentration of 0.1mM and continue to induce at 30℃ overnight. 6,000rpm, 4℃, centrifuge for 5min to collect the bacteria, wash once with 1×PBS, and freeze at -80℃ for 1h. Add 50mL lysis buffer, vortex and place on ice for 30min. 35% power, ultrasonic for 5s, intermittent for 3s, total ultrasonic time for 10min, stop when the liquid is clear, centrifuge at 10,000rpm, 4℃ for 30min. The supernatant was filtered through a 0.45μM filter, mixed with 1mL of Ni-NTA resin, and bound at 4℃ for 2h. Balance buffer was used for washing 3 times, and 5mL was passed through the column each time. 25mM imidazole Elution buffer, 5mL was passed through the column each time. 50mM imidazole Elution buffer, 5mL was passed through the column each time. 75mM imidazole Elution buffer, 5mL was passed through the column each time. 100mM imidazole Elution buffer, 5mL was passed through the column each time. 200mM imidazole Elution buffer, 5mL was passed through the column each time. 300mM imidazole Elution buffer, 5mL was passed through the column each time. 500mM imidazole Elution buffer, 5mL was passed through the column each time, and eluted for 15min. Take 60μL of the above imidazole eluate and add it to loading buffer, boil it in 100℃ boiling water for 10min, and then proceed to the next step of SDS-polyacrylamide gel electrophoresis.

[0115] 11. SDS-polyacrylamide gel electrophoresis

[0116] Sample preparation: Collect 60μL of the eluate, add protein 4× loading buffer, and heat in a water bath at 100℃ for 10min. Centrifuge at 12,000 for 1min and place on ice for later use. Prepare separation gel with a concentration of 15%; mix the separation gel as shown in the table above and carefully inject it into the gap between the plates. Use a pipette to add 1mL of isopropanol to keep the pages between the concentrated gel and the separation gel flat. Prepare 3mL of 5% concentrated gel, carefully and slowly insert the comb dedicated to protein gel, try to avoid mixing with air, and place it in a cool place at room temperature for 1-2h. Wash the colloidal glass plate, load it into the electrophoresis tank, and add 1×SDS electrophoresis solution. Load the sample. Pour the electrophoresis solution into the middle of the two plates, and run the electrophoresis at a constant voltage of 70-80V. When the protein marker is observed to be stratified, increase the voltage to 135V and continue electrophoresis. Remove the colloid, add an appropriate amount of Coomassie Brilliant Blue dye, heat it in a microwave oven on high heat for 30s, and place it on a shaker at room temperature for staining for 2h. The dye solution is recovered and eluted with a decolorizing solution.

[0117] Protein dialysis and quantification:

[0118] The target protein was dialyzed overnight using an 8kDa dialysis bag, during which the solution was changed 3 times. 10μL of the protein solution in the dialysis bag was taken for BCA quantification. Thrombin was added to remove the His tag. Protein immunoblotting was used for verification.

[0119] 12. BCA protein quantification:

[0120] Take out 2mg / mL BSA. Since BSA protein is stable, melt it at room temperature for later use. Take several 1.5mL centrifuge tubes and mark them as 2mg, 1mg, 0.5mg, 0.25mg, and 0.125mg. Perform gradient dilution of BSA according to protein concentration. Mix A and B solutions in a ratio of 50 to 1 as the detection solution. Add 200μL of the detection solution to a 96-well plate. Add the standard solution and sample in sequence. Leave at room temperature for 30min. Perform colorimetric analysis on an enzyme reader (Thermo).

[0121] 13. WB Verification

[0122] 2 hours in advance, put the transfer solution into the refrigerator to pre-cool, and put the Immobilon-P SQ The membrane was placed in methanol for activation for 5 minutes. The pre-cooled transfer solution was slowly poured into a large enamel dish, and a transfer clip, a black and white sponge pad, a glass test tube, a thick filter paper for WB, and an Immobilon-P activated with methanol were placed on it. SQMembrane. Open the clamp and place the black side close to the enamel basin. Place a black sponge pad on it and use it to roll out the bubbles inside. Add thick filter paper to remove the bubbles. Add protein glue, glass test tube, or use transfer solution to flush or gently push out the bubbles inside. Then add membrane, filter paper and sponge in turn. Finally, clamp the clamp. Put the clamp in the transfer tank. Put an ice pack on it and add pre-cooled transfer solution. Put the entire electrophoresis tank in the basin, pour in ice cubes, and transfer the membrane at a constant voltage of 80V for 1 hour. After the transfer is completed, put the membrane in TBST buffer solution and decolorize on a shaker for 2 minutes, twice. Remove the washing solution, add the blocking solution, block at room temperature for 2 hours, or let it stand in a refrigerator at 4℃ overnight. Dilute the His-tag antibody with TBST, 1 to 1,000 to prepare 10mL. After the blocking is completed, take the plastic protective film and spread it on the laboratory table; take out the Immobilon-P from the blocking solution. SQ Place the membrane in a plastic protective film, seal the edges, add the antibody, and seal. Bind for 2 hours at room temperature, or overnight in a refrigerator at 4°C. Wash with TBST three times, 10 minutes each time. Dilute the goat anti-mouse secondary antibody with TBST at a ratio of 1:5,000 to prepare 10mL. Prepare the secondary antibody as above and incubate at room temperature for 1 hour. After the incubation, wash with TBST for 10 minutes, and repeat three times. Take out the plastic wrap and lay it flat on the table. Place the membrane on the plastic wrap with the protein side on top. Mix the color developing solution reagent A and reagent B and add them to the membrane, ensuring that every corner of the membrane is in contact with the reagent. After 1 minute, move the membrane to a new plastic wrap, gently touch the edge with a clean paper towel, wrap it, and clamp it in an X-ray film clip. In a dark room, prepare the color developing solution and decolorizing solution. Take out two X-ray films and cut them in half with scissors; open the X-ray film clip, place the 4-layer X-ray film on the membrane, close the film clip, and expose for 15 minutes. When the time is up, take out the X-ray film and put it into the developer. After the obvious stripes are observed, immerse the film in the fixer for 15 minutes. After the fixer is rinsed with tap water, dry it in the oven.

[0123] Result description:

[0124] The human TNFα and mouse TNFα membrane protein sequences were selected (the nucleic acid sequences are shown in SEQ ID NO: 59 and SEQ ID NO: 60). The mouse TNFα membrane protein sequence of 175 amino acids and the human TNFα membrane protein sequence of 177 amino acids were selected. After comparing the sequences of the two, it can be seen that the mouse TNFα membrane protein sequence we selected is highly homologous to the human TNFα membrane protein sequence (as shown in SEQ ID NO: 59 and SEQ ID NO: 60). Figure 1 shown).

[0125] The plasmid SnapGene map of the final fusion protein is as follows: Figure 2The left side shows the TNFα-mouse-pET28a plasmid map, and the right side shows the TNFα-human-pET28a-3flag plasmid map.

[0126] like Figure 3 As shown, after the plasmid is successfully constructed, a single clone is picked to extract the plasmid and sent to Xiamen Borui Biological Company for DNA sequencing. The T7 universal primer is used to sequence 800bp of the plasmid in the forward direction. The obtained results are analyzed and compared, and the similarity between the sequences is 100%. The TNFα fusion protein plasmid is successfully constructed.

[0127] like Figure 4 As shown, a clear band at about 20 kDa can be clearly observed, indicating that the protein purity is significantly higher than 90% and can be used for subsequent immune experiments. Figure 4 (a) is the mouse TNFα protein after the His-tag was removed by thrombin, in order to facilitate the subsequent ELISA detection of VNAR affinity; (b) is the mouse TNF with His tag; (c) is the human TNF with His tag. After WB verification, it was confirmed that it was indeed a protein fused with a His tag of TNFα.

[0128] The concentration of purified TNFα protein was determined by BCA method, and the standard curve was drawn with BSA protein as the standard. Figure 5 The absorbance values ​​of mouse TNFα and human TNFα were 0.2530 and 0.4610, respectively. Combined with the fitting equation, the concentrations of mouse TNFα and human TNFα were measured to be approximately 1.40 mg / mL and 0.56 mg / mL.

[0129] Example 2 Panning of phage library

[0130] 1. TNFα antigen immunization of striped bamboo shark:

[0131] The striped bamboo sharks required for the experiment were raised at the Third Institute of Oceanography, Ministry of Natural Resources.

[0132] The conventional immunization method was used to immunize bamboo sharks by subcutaneous injection of TNFα protein (mouse TNFα protein obtained in Example 1) to ensure that shark serum already contained TNFα antibodies.

[0133] Specifically, the immunization plan for the striped bamboo shark is shown in Table 7:

[0134]

[0135] For each immunization, the TNFα protein was diluted and dissolved in a sterilized PBS buffer solution, and an equal volume of adjuvant was added, mixed evenly with a homogenizer, and then injected subcutaneously at multiple points. After the fourth immunization, when the shark produced sufficient antibodies, 3-aminobenzoic acid ethyl ester methane sulfonate anesthesia was used in seawater at about 0.1% (w / v). Blood was drawn from the tail vein, an equal volume of heparin sodium salt was added, and then rotated at 2,000 rpm for 10 minutes to separate blood cells. The shark spleen was removed, crushed and homogenized in TRIZOL solution, and packaged in a test tube. Sufficient TRIZOL was added to all products and stored in a -80°C refrigerator.

[0136] 2. Extraction of mRNA

[0137] Take the blood cell homogenate out of the -80℃ freezer. Keep the homogenate sample away from UV light as much as possible and place it at room temperature (15~30℃) to completely dissolve, and then separate the nucleic acid and protein. If 1mL TRIZOL is used in the homogenate, add 0.2mL chloroform, and increase or decrease in proportion. After shaking for 15 seconds, place it at room temperature for 5min. Centrifuge at 2-8℃, 10,000rpm for 15 minutes. It can be clearly observed that the sample is divided into three layers. Transfer the upper aqueous phase to a new centrifuge tube, and generally 550μL and up to 600μL can be taken from it. 1If 1mL TRIZOL is used in the homogenate, add 0.5mL isopropanol and place it at room temperature for 10 minutes. After RNA is precipitated, centrifuge at 4℃, 10,000rpm for 10min. After centrifugation, a precipitate can be seen on the side and bottom of the tube. Remove the supernatant. Resuspend in 75% ethanol prepared with RNase-free water. Add at least 1 mL of 75% alcohol per 1 mL of TRIZOL at 4°C, centrifuge at 6,000 rpm for 10 minutes, and discard the supernatant. Blow dry the RNA precipitate on a clean bench for about 7.5 minutes. Add RNase-free water to completely dissolve the precipitate. Immediately take 5 μL for agarose gel electrophoresis.

[0138] 3. Reverse transcription Total RNA Table 8

[0139]

[0140] The first strand synthesis system was prepared as follows.

[0141] Incubate in a 70℃ water bath for 10 min, cool on ice for 2 min. Centrifuge to collect the liquid. Prepare the following reverse transcription reaction components and mix well.

[0142] Table 9

[0143]

[0144] After the above solution was kept at 42°C for 15 min, a small amount was taken to run agarose gel for verification.

[0145] 4. PCR to obtain VNAR sequence from cDNA:

[0146] The PCR reaction was performed according to the following conditions, Table 10:

[0147]

[0148] Table 11:

[0149]

[0150] Primers used in PCR experiments, SfiI restriction endonuclease cleavage sites are underlined.

[0151] Table 12

[0152]

[0153] 5. VNAR-pComb3XSS plasmid library ligation and transformation

[0154] The PCR product was recovered; the bacteriophage pComb3XSS was digested with SfiI restriction endonuclease; the two were ligated overnight at 4°C using T4 ligase, and the system is shown in Table 13:

[0155]

[0156] Electroporation: TG1 streaking culture, pick single clones into 5mL TB liquid medium, culture at 37℃200rpm overnight. 1:100 inoculated into 100mL TB medium, culture at 18℃250rpm for 18-19h to make OD600 reach 0.28-3. 25mL bacterial solution is used in a 50mL centrifuge tube, centrifuged at 3,000g, 4℃ for 10min, gently remove the supernatant, centrifuged at 3,000g, 4℃ for 1min, and remove the remaining medium with a pipette. Add 30mL, 10% pre-cooled glycerol to each tube, gently resuspend the bacteria, centrifuged at 3,000g, 4℃ for 10min. Gently remove the supernatant, centrifuged at 3,000g, 4℃ for 1min, and remove the remaining liquid with a pipette. Add 20ml, 10% pre-cooled glycerol to each tube, gently resuspend the bacteria, rotate vertically for 5min, and then combine the 4 tubes into 2 tubes. Centrifuge at 3,000g, 4℃ for 10min. Gently remove the supernatant, centrifuge at 3,000g, 4℃ for 1min, and remove the remaining liquid with a pipette. Add 20mL of 10% pre-cooled glycerol to each tube, gently resuspend the bacteria, rotate vertically for 5min, and then combine the two tubes into 1 tube. Centrifuge at 3,000g, 4℃ for 10min. Gently remove the supernatant, centrifuge at 3,000g, 4℃ for 1min, and remove the remaining liquid with a pipette. Add 0.5mL of 10% pre-cooled glycerol and gently resuspend the bacteria. Dilute to OD600 of about 10% pre-cooled glycerol. Take 1.5mL of electroporation competent medium and add the ligation product, and place on ice. Add 50μL 1.25KKV to the pre-cooled 1mm electroporation cup and electrotransform. Quickly add to 1mL of TB medium, and finally pipette into a clean Erlenmeyer flask, shake at 37℃ for 1h, and apply to a large plate. The next day, scrape the bacteria, mix well in a 1:1 ratio, add 50% glycerol, and freeze at -80℃ for later use.

[0157] 6. Amplification of phage library

[0158] Take out the phage library from the refrigerator and put it into 100mL 2×YT ampicillin-resistant liquid culture medium. The initial OD value is about 0.1. Cultivate at 37℃ and 200rpm. When the OD value reaches 0.5, add 100μL helper phage (MOI=10) and continue to culture for 1h. Add kanamycin and shake at 25℃ and 200rpm overnight. Centrifuge 100mL of bacterial solution at 9,000rpm and 4℃ for 20min. Take the supernatant and add 1 / 5 volume of about 20mL PEG6000, place it in a 50mL centrifuge tube and shake on ice for 60min. Centrifuge at 10,000rpm for 30min at 4℃. After resuspending the precipitate with PBS, centrifuge it at 13,800rpm and 4℃ for 2min. Take the supernatant and place it in a new EP tube and centrifuge it at 13,800rpm and 4℃ for 2min. Measure the concentration: every 1OD=5×10 12 cfu / mL.

[0159] 7. VNAR selection

[0160] On the TG1 plate, pick a single clone and add it to 5mL 2×YT medium with ampicillin, culture at 250rpm, 37℃ for more than 13h. Take 1mL TG1 bacterial liquid and inoculate it to 100mL 2×YT medium with ampicillin, and culture it until OD600 is about 0.5. Take two immunotubes A and B, immunotube A as the experimental group, and immunotube B as the control group. Add 400μL of 1.4mg / mL mouse TNFα to immunotube A, dilute it to 5mL with PBS, and incubate it overnight at 4℃. Immunotube B: 5% skimmed milk powder, incubate it overnight at 4℃. The next day, pour out the liquid in immunotube A, add 5mL of blocking solution, and block it at 37℃ for 2h. Immunotube B, wash once with PBST. Take 100μL of Anti-TNF shark phage library (10 12 pfu / mL) add 2mL 5% skim milk powder + PBS and shake for 1h. Immunotube B: add the above phage to tube B and invert for 1h. Immunotube A: pour out the supernatant, aspirate dry, add all the phage in immunotube B to tube A, let it stand for 1h, invert at room temperature for 1h, add 5mL PBS to immunotube B and let it stand. Pour out the supernatant from tubes A and B at the same time, aspirate dry, add PBST to seal, invert for 5min and repeat three times, increase the number of washes by 3 times after each round. Add 1mL 0.1M HCl to tubes A and B and invert for 8min. Immediately add 500μL 1M Tris-HCl with a pH of 7.4 to tubes A and B. Take the supernatant of tubes A and B and add 10mL TG1 at 37℃ respectively. Spread on a large plate, take an appropriate amount of bacterial solution, and incubate at 10 -4 and 10 -5 Dilution count.

[0161] 8. Phage ELISA selection of specific VNAR monoclones

[0162] Add 800μL of 2×YT medium with ampicillin to a 96-well deep-well plate. Pick one clone in each well with a single-head toothpick treated at 121℃. Apply sealing film. Incubate at 250rpm in a 37℃ incubator for 4 hours. Add 110μL of helper phage to 11mL of 2×YT and mix well. Add 103μL to each well of the 96-well deep-well plate. Seal the lid and return to the 37℃ incubator at 250rpm for another 1h. Add 110μL of kanamycin sulfate to 11mL of 2×YT medium and mix well. Add 103μL to each well of the 96-well deep-well plate. Incubate overnight at 25℃ and 250rpm. The first ELISA plate has 500ng of antigen / well, which is diluted with 1×PBS. Take 50μg of antigen and dilute it to 10mL of 1×PBS, and add 100μL to each well. The second plate is also a control plate, 5% skim milk powder, diluted with 1× PBS, 300 μL per well. Incubate at room temperature overnight. Wash once with 1× PBST, add 15% skim milk powder to the ELISA plate, 300 μL per well, and add 5% skim milk powder to the ELISA plate coated with 5% skim milk powder, 300 μL per well, incubate at 37℃ for 2h. Centrifuge the 96-well plate at 4℃ 4,500rpm for 5min, and store at 4℃ after centrifugation. Wash both ELISA plates once with 1× PBST, and coat each well with 100 μL of the centrifuged phage supernatant, and incubate at room temperature for 2h. Wash 9 times with 1× PBST, and add 1:20,000 anti-M13 (HRP) antibody prepared with milk blocking solution to each well of the ELISA plate. Wash 9 times with 1×PBST, add 100μL TMB to each well for color development, and immediately add 50μL 0.1M sulfuric acid to terminate the reaction when a significant color change occurs. Detect the results using the OD450 nm absorbance value of the microplate reader. Select the correct bacterial solution, extract the plasmid, and send it to a biological company for sequencing and data analysis.

[0163] Result description:

[0164] The VNAR-pComb3XSS vector was transferred into E. coli TG1 by electroporation to construct a phage library. The phages were then selected using 5 mL of 100 μg / mL coated immunotubes. A small amount was taken and diluted to 10 -4 and 10 -5 , and the plate was counted. The first round used 3.45×10 12 The phages selected were approximately 1.62×10 6 cfu / mL( Figure 6 ).

[0165] Then, randomly place the Figure 6) Pick 96 monoclones and shake out a small amount of phage in a 96-well plate. Verify the VNAR-displaying phage released by the monoclones on an ELISA plate coated with 5 μg / mL mouse TNFα antigen, and read the plate using an absorbance value of 450 nM on an ELISA reader. A positive clone is identified when the experimental value is more than 5 times the control value. After analysis, 13 positive clones were verified.

[0166] The second time, 1.04×10 12 The phages selected were approximately 2.59×10 7 cfu / mL( Figure 6 ). The 96 monoclonal clones were verified again using an ELISA plate coated with 5 μg / mL TNFα antigen, and 64 positive clones were generated (Figure 7). Compared with the first time, the binding ability was significantly enhanced, and the ratio of most clones to the control was more than 60 times.

[0167] The positive clones detected above were subjected to plasmid extraction and sent to a biological company for DNA sequencing. The nucleic acid results returned were compared, translated into amino acid sequences and analyzed for differences. The results showed that 15 strains had different antibodies (see Figure 8 ). Through the analysis of the differences in the amino acid sequences of the 15 VNARs and the specificity of the CDR3 framework region, these 15 sequences can be roughly divided into four families (see Fig. 9), the differences mainly occur in the CDR3 region. In family 1, family 2, and family 3, the cysteine ​​residues in the CDR3 region and the CDR1 region form disulfide bonds, and the cysteine ​​residues in FR3 and FR1 form disulfide bonds, which are consistent with the type II VNAR subtype structure. Unlike the other three families, in family 4, the cysteine ​​in CRR1 is replaced by tyrosine and histidine. This VNAR subtype does not conform to the currently recognized 5 VNAR subtypes. Compared with type I, the 1D11 sequence lacks a pair of cysteines in CDR3 and FR4, and lacks the cysteine ​​motif that anchors CDR3 in the framework region, which is similar to type II, but its cysteine ​​position is in the FR2 region instead of the CDR1 region. 2B11, 2D2, and 2E9 are also similar to type II, but lack the cysteine ​​in the CDR1 region, making it impossible to form a disulfide bond with the cysteine ​​residues in the CDR3 region. This new VNAR skeleton structure has only a pair of cysteines, which makes its structure more variable, but at the same time easier to deform. The additional cysteine ​​residues in the CDR3 variable region make it easier to bind to the antigen to form hydrogen bonds, increasing the affinity stability of the antigen and antibody. Analyzing the structures of the above four families of VNARs, it was found that the 3B11 VNAR, which represents the family, has a competitive binding effect on the TNFα receptor because its CDR3 region just coincides with the binding site of the TNFα receptor; and the CDR1 region of 3B11 is very close to the site of the TNFα receptor and TNFα, and has a weak steric effect.

[0168] Typical VNAR sequences from the four families were selected and Phage ELISA experiments were performed again using mouse TNFα and human TNFα. The results are as follows: Fig.10 The ratio of VNARs numbered 1D11, 2F3, and 3B7 to the control showed that mouse TNFα was significantly greater than that of human TNFα, while 3B11 showed no significant difference between the two.

[0169] The software ExPASy was used to analyze the amino acid sequences of 15 strains of VNAR and collect their data; the total hydrophilicity average coefficient (GRAVY), including molecular weight, theoretical isoelectric point (pI) information, as shown in Table 14, the molecular weight of the 15 strains of VNAR proteins are all between 11.17 and 12.77 kDa, which is consistent with the distribution characteristics of the molecular weight of shark VNAR. Among them, 14 isoelectric points are between 7.87 and 9.42, higher than 7, and only one is lower than 7, indicating that VNAR is positively charged in the physiological environment, while the antigen is the TNFα membrane outer end protein pI value of 4.96, which is secondary charged in the physiological environment. Therefore, the binding between the two may be affected by electrostatic effects. The pI value of human TNFα is 7, so VNAR's binding ability to human TNFα is relatively weak, which is consistent with the experimental results of Phage ELISA. In addition, according to the GRAVY values ​​of the 15 VNARs shown in the table, these 15 VNAR proteins are hydrophilic proteins.

[0170] Table 14

[0171]

[0172]

[0173] The amino acid sequences involved in the 15 strains of VNAR are shown in Table 15

[0174]

[0175]

[0176] Example 3 Purification and affinity detection of VNAR

[0177] 1. Since the FR3 end of VNAR is its conserved sequence, the primers required for constructing the recombinant expression plasmids of all VNARs are shown in Table 16 below:

[0178] Table 16

[0179]

[0180] The steps of plasmid construction and protein purification of VNAR are the same as those in Example 1.

[0181] 2. ELISA to verify VNAR affinity

[0182] Antigen was coated in PBS buffer at 4°C in a 96-well ELISA plate at about 100-200 ng per well. The second plate was also a control plate, 5% skim milk powder, diluted with 1×PBS, and 300 μL was added to each well. Incubate at room temperature overnight. The next day, the well surface was blocked with blocking buffer (PBS, 5% skim milk powder) at 37°C for 2 hours. Both plates were incubated at room temperature overnight, and the next day, they were washed once with 1×PBST, and 15% skim milk powder was added to the protein-coated plate, 300 μL per well, and 5% skim milk powder was added to the plate coated with 5% skim milk powder, 300 μL per well, and incubated at 37°C for 2 hours. The 96-well deep-well plate was centrifuged at 45,00 rpm for 5 minutes at 4°C, and the centrifugation was temporarily stored in the refrigerator. Wash once with 1×PBST, add VNAR to each well of the coated plate, dilute it in gradients, start from 1uM and dilute it 13 times to 0.8pM, prepare it with 2.5% skim milk powder, add 100μL to each well of the coated plate, and incubate it at room temperature for 2h. Wash 9 times with 1×PBST, add His-tag mAb antibody prepared with 2.5% skim milk powder to each well of the ELISA plate, incubate it at room temperature for 2h. Wash 9 times with 1×PBST, add goat anti-mouse IgG antibody (1:5,000) prepared with 2.5% skim milk powder to each well of the ELISA plate, and incubate it at room temperature for 1h. Wash 9 times with 1×PBST, add 100μL TMB to each well for color development, wait for obvious color change, about 5-10min, and immediately add 50μL 0.1M sulfuric acid to stop. Detect the results of absorbance value OD450 of the microplate reader.

[0183] 3. BLI-based TNFα binding capacity assay

[0184] Take the desalted TNFα protein and dilute it to 1 mg / mL with PBS. Then add 10 μL of 10 mg / mL biotin-NHS prepared with DMSO. Incubate at room temperature for 30 minutes. Dialyze with PBS overnight, changing the eluent at least 3 times. Edit the Octet program as shown in Table 17. Add the solutions in sequence to the 96-well plate. After the experiment, use Octet Alanysis analysis to normalize the response data.

[0185] Table 17

[0186]

[0187] Result description:

[0188] VNAR 3B11The fragment gene was connected with the expression plasmid pET28a plasmid to construct a recombinant plasmid. After PCR colony verification and plasmid sequencing, the band position and DNA nucleic acid sequence were consistent with expectations. The sequencing results were consistent with the expected map, and the VNAR fusion protein plasmid was successfully constructed. The recombinant expression vector was transferred to BL21 Escherichia coli for expression, and the bacteria were collected for protein purification. SDS-PAGE analysis showed that ( Fig.11 ), VNAR 3B11 The protein was successfully purified and expressed, and the concentration reached 0.24 mg / mL.

[0189] When inducing protein expression, the VNAR is very easy to form soluble proteins. When the IPTG concentration ranges from 0.1mM to 0.5mM and the induction temperature ranges from 18℃ to 25℃, most proteins exist in the cytoplasm as soluble proteins. Because of this, the purified protein contains certain miscellaneous bands. When BL21 Escherichia coli is used to induce protein purification, a relatively large miscellaneous band will be formed at around 35bp. Since the expression amount of VNAR is relatively large and almost all the expressed proteins are soluble proteins, we choose to use more concentration gradient imidazole elution buffer solutions for elution, or use Akta protein purification system for purification. After SDS-PAGE electrophoresis, it can be clearly seen that when the gradient elution is about 75mM or above 400mM imidazole concentration, a relatively single band protein can be obtained. At the same time, it was also found that the stability of VNAR in Tris-HCl with imidazole buffer system is much greater than that of PBS buffer system. It can be stored in Tris-HCl solution at -80℃ ultra-low temperature refrigerator for a long time and dialyzed with 5kDa dialysis membrane when used.

[0190] After coating the ELISA plate with 200 ng / mL, the plate was blocked with milk for 2 hours. 3B11 Continuously dilute 5 times, from 1μM to 0.8192fM, 13 dilution gradients, negative control using HSA VNAR, incubate for 2 hours, wash the plate 9 times with PBST. Incubate with 1:1000 His antibody for 2 hours, wash the plate 9 times with PBST. Finally, incubate with goat anti-mouse secondary antibody for 1 hour, then use TMB for color development, and read the plate at OD450 to find VNAR 3B11 The specific binding capacity for mouse TNFα was calculated by Prism and the EC50 reached the sub-nanomolar level of 0.36nM ( Fig.12 ).

[0191] Table 18 VNAR 3B11 ELISA test

[0192]

[0193] exist R2 shows the determination of TNFα dissociation constant Kd based on BLI. The BLI technology method is used to verify the specificity of the screened antibodies. Different concentration gradients of antigens are set. Preliminary experimental results show that obvious binding signals can be detected above 25nM, so four concentration gradients of 500nM, 250nM, 100nM, and 50nM are set. Fig.13 It can be seen that VNAR 3B11 It has a slow binding and dissociation with TNFα, and has a high affinity. Its equilibrium dissociation constant (KD) value also reaches 16.7nM.

[0194] In summary, according to the embodiments of the present invention, an anti-TNFα phage library was established by immunizing striped bamboo sharks, and 15 anti-TNFα VNARs were selected by panning. These 15 VNARs can be divided into four families according to the difference in CDR3 region, and one of the families does not belong to any of the five subtypes of VNARs currently recognized; their structures were analyzed, and it was found that this VNAR had a competitive effect on the TNFα receptor; and the most representative one was purified, and a set of purification systems most suitable for VNAR was explored; finally, the purified VNAR was tested by ELISA and BLI, and it was found that it reached the affinity of sub-nanomolar level. The anti-TNFα gene selected from the established striped bamboo shark VNAR library can efficiently express soluble proteins from the Escherichia coli prokaryotic expression system, and has antigen-antibody binding ability, which can further provide a basis for the development of TNFα blocking drugs and the diagnosis of related diseases, and has great prospects in TNFα related research and diagnosis and treatment of future diseases.

[0195] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0196] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention. SEQUENCE LISTING <110> The Third Institute of Oceanography, Ministry of Natural Resources, China Ocean Mineral Resources Research and Development Association (China Ocean Affairs Administration) <120> Anti-TNFα single domain antibody and its application <130> none <160> 60 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> PRT <213> Artificial sequence <400> 1 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys 20 25 <210> 2 <211> 25 <212> PRT <213> Artificial sequence <400> 2 Gln Arg Val Glu Gln Thr Pro Arg Thr Ile Thr Lys Glu Thr Gly Glu 1 5 10 15 Ser Pro Thr Ile Asn Cys Val Leu Lys 20 25 <210> 3 <211> 25 <212> PRT <213> Artificial sequence <400> 3 Gln Arg Val Glu Gln Thr Pro Arg Thr Ile Thr Lys Glu Thr Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys 20 25 <210> 4 <211> 25 <212> PRT <213> Artificial sequence <400> 4 Gln Arg Val Glu Gln Thr Leu Arg Thr Ile Thr Lys Glu Thr Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys 20 25 <210> 5 <211> 25 <212> PRT <213> Artificial sequence <400> 5 Gln Arg Val Glu Gln Thr Pro Thr Asn Asn Asn Glu Gly Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys 20 25 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <400> 6 Gly Ser Ser Asn Ala Leu Cys Asn 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <400> 7 Gly Ser Ser Cys Ala Leu Gly Ser 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <400> 8 Gly Ser Ser Cys Ala Leu Gly Thr 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial sequence <400> 9 Gly Ser Ser Cys Ala Leu Ala Ser 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <400> 10 Gly Ser Asp Cys Ala Leu Gly Ser 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <400> 11 Asp Ala Ser Tyr Ala Leu Ser Gly 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <400> 12 Gly Ser Ser His Ala Leu Ser Gly 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial sequence <400> 13 Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial sequence <400> 14 Thr Asn Trp Tyr Phe Thr Lys Lys Gly Ala Thr 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <400> 15 Thr His Trp Tyr Phe Thr Lys Lys Gly Ala Thr 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial sequence <400> 16 Thr Tyr Cys Asn Leu Thr Lys Leu Asp Ala Thr 1 5 10 <210> 17 <211> 11 <212> PRT <213> Artificial sequence <400> 17 Thr Tyr Trp Tyr Leu Thr Lys Leu Asp Ala Thr 1 5 10 <210> 18 <211> 11 <212> PRT <213> Artificial sequence <400> 18 Thr Tyr Trp Tyr Leu Thr Ser Leu Asp Ala Thr 1 5 10 <210> 19 <211> 8 <212> PRT <213> Artificial sequence <400> 19 Lys Lys Glu Ser Leu Thr Asn Gly 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial sequence <400> 20 Lys Lys Ala Ser Leu Ser Thr Gly 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial sequence <400> twenty one Lys Lys Ala Ser Leu Ser Thr Gly 1 5 <210> twenty two <211> 8 <212> PRT <213> Artificial sequence <400> twenty two Lys Lys Ala Ser Val Ser Thr Gly 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial sequence <400> twenty three Lys Trp Asp Arg Ile Ser Ile Gly 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial sequence <400> twenty four Asn Trp Asp Arg Ile Ser Met Gly 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <400> 25 Gly Arg Tyr Ser Val Thr Met 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <400> 26 Gly Arg Tyr Ser Asp Thr Lys 1 5 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <400> 27 Gly Arg Tyr Ser Glu Thr Val 1 5 <210> 28 <211> 7 <212> PRT <213> Artificial sequence <400> 28 Gly Gln Tyr Ser Glu Thr Val 1 5 <210> 29 <211> 5 <212> PRT <213> Artificial sequence <400> 29 Asn Lys Ala Ser Lys 1 5 <210> 30 <211> 5 <212> PRT <213> Artificial sequence <400> 30 Asn Thr Ala Ser Lys 1 5 <210> 31 <211> 5 <212> PRT <213> Artificial sequence <400> 31 Asn Lys Gly Ser Lys 1 5 <210> 32 <211> twenty one <212> PRT <213> Artificial sequence <400> 32 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 1 5 10 15 Tyr His Cys Lys Val 20 <210> 33 <211> twenty one <212> PRT <213> Artificial sequence <400> 33 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 1 5 10 15 Tyr His Cys Glu Ala 20 <210> 34 <211> twenty one <212> PRT <213> Artificial sequence <400> 34 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 1 5 10 15 Tyr His Cys Gln Ala 20 <210> 35 <211> twenty one <212> PRT <213> Artificial sequence <400> 35 Ser Phe Ser Leu Arg Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Thr 1 5 10 15 Tyr His Cys Glu Ala 20 <210> 36 <211> twenty one <212> PRT <213> Artificial sequence <400> 36 Ser Phe Ser Leu Arg Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Ala 1 5 10 15 Tyr His Cys Glu Ala 20 <210> 37 <211> twenty one <212> PRT <213> Artificial sequence <400> 37 Ser Phe Ser Leu Arg Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Ala 1 5 10 15 Tyr His Cys Glu Ala 20 <210> 38 <211> 15 <212> PRT <213> Artificial sequence <400> 38 Tyr Ser Ser Thr Val Gly Tyr Cys Asp Trp Asp Ser Asp Tyr Tyr 1 5 10 15 <210> 39 <211> 20 <212> PRT <213> Artificial sequence <400> 39 Tyr Arg Asp Gly Trp Asp Val Val Gly His Tyr Cys Tyr Ser Trp Gly 1 5 10 15 Tyr Tyr Ser Tyr 20 <210> 40 <211> 13 <212> PRT <213> Artificial sequence <400> 40 Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu Gly Leu 1 5 10 <210> 41 <211> 20 <212> PRT <213> Artificial sequence <400> 41 Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr Glu Ala Gly Ile Pro 1 5 10 15 His Asn Tyr Ile 20 <210> 42 <211> 10 <212> PRT <213> Artificial sequence <400> 42 Glu Gly Gly Gly Thr Ile Leu Thr Val Lys 1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial sequence <400> 43 Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 1 5 10 <210> 44 <211> 108 <212> PRT <213> Artificial sequence <400> 44 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 45 <211> 108 <212> PRT <213> Artificial Sequence <400> 45 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Thr Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 46 <211> 115 <212> PRT <213> Artificial Sequence <400> 46 Gln Arg Val Glu Gln Thr Pro Arg Thr Ile Thr Lys Glu Thr Gly Glu 1 5 10 15 Ser Pro Thr Ile Asn Cys Val Leu Lys Asp Ala Ser Tyr Ala Leu Ser 20 25 30 Gly Thr Tyr Cys Asn Leu Thr Lys Leu Asp Ala Thr Lys Trp Asp Arg 35 40 45 Ile Ser Ile Gly Gly Arg Tyr Ser Glu Thr Val Asn Lys Gly Ser Lys 50 55 60 Ser Phe Ser Leu Arg Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr 85 90 95 Glu Ala Gly Ile Pro His Asn Tyr Ile Glu Gly Gly Gly Thr Ile Leu 100 105 110 Thr Val Lys 115 <210> 47 <211> 108 <212> PRT <213> Artificial Sequence <400> 47 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Arg 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 48 <211> 108 <212> PRT <213> Artificial Sequence <400> 48 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Asn Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 49 <211> 115 <212> PRT <213> Artificial Sequence <400> 49 Gln Arg Val Glu Gln Thr Pro Arg Thr Ile Thr Lys Glu Thr Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Asp Ala Ser Tyr Ala Leu Ser 20 25 30 Gly Thr Tyr Trp Tyr Leu Thr Lys Leu Asp Ala Thr Lys Trp Asp Arg 35 40 45 Ile Ser Ile Gly Gly Arg Tyr Ser Glu Thr Val Asn Lys Gly Ser Lys 50 55 60 Ser Phe Ser Leu Arg Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Ala 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr 85 90 95 Glu Ala Gly Ile Pro His Asn Tyr Ile Glu Gly Gly Gly Thr Ile Leu 100 105 110 Thr Val Lys 115 <210> 50 <211> 115 <212> PRT <213> Artificial Sequence <400> 50 Gln Arg Val Glu Gln Thr Leu Arg Thr Ile Thr Lys Glu Thr Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Asp Ala Ser Tyr Ala Leu Ser 20 25 30 Gly Thr Tyr Trp Tyr Leu Thr Lys Leu Asp Ala Thr Lys Trp Asp Arg 35 40 45 Ile Ser Ile Gly Gly Arg Tyr Ser Glu Thr Val Asn Lys Gly Ser Lys 50 55 60 Ser Phe Ser Leu Arg Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Ala 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr 85 90 95 Glu Ala Gly Ile Pro His Asn Tyr Ile Glu Gly Gly Gly Thr Ile Leu 100 105 110 Thr Val Lys 115 <210> 51 <211> 108 <212> PRT <213> Artificial Sequence <400> 51 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Ala 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 52 <211> 108 <212> PRT <213> Artificial Sequence <400> 52 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Val Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 53 <211> 115 <212> PRT <213> Artificial sequence <400> 53 Gln Arg Val Glu Gln Thr Pro Thr Asn Asn Asn Glu Gly Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser His Ala Leu Ser 20 25 30 Gly Thr Tyr Trp Tyr Leu Thr Ser Leu Asp Ala Thr Asn Trp Asp Arg 35 40 45 Ile Ser Met Gly Gly Gln Tyr Ser Glu Thr Val Asn Lys Gly Ser Lys 50 55 60 Ser Phe Ser Leu Gln Leu Arg Asp Leu Arg Val Glu Asp Ser Gly Ala 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Ser Ser Thr Ala Gly Ser Tyr Cys Tyr 85 90 95 Glu Ala Gly Ile Pro His Asn Tyr Ile Glu Gly Gly Gly Thr Ile Leu 100 105 110 Thr Val Lys 115 <210> 54 <211> 108 <212> PRT <213> Artificial sequence <400> 54 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Asp Cys Ala Leu Gly 20 25 30 Ser Thr His Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Val Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 55 <211> 115 <212> PRT <213> Artificial Sequence <400> 55 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Arg Asp Gly Trp Asp Val Val Gly His Tyr 85 90 95 Cys Tyr Ser Trp Gly Tyr Tyr Ser Tyr Glu Gly Gly Gly Thr Ile Leu 100 105 110 Thr Val Lys 115 <210> 56 <211> 108 <212> PRT <213> Artificial Sequence <400> 56 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Val Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Glu Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 57 <211> 108 <212> PRT <213> Artificial Sequence <400> 57 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Cys Ala Leu Gly 20 25 30 Ser Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Ala Ser 35 40 45 Leu Ser Thr Gly Gly Arg Tyr Ser Asp Thr Lys Asn Thr Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Gln Ala Tyr Ser Trp Gln Leu Gly Cys Arg Tyr Pro Leu 85 90 95 Gly Leu Gln Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 <210> 58 <211> 110 <212> PRT <213> Artificial sequence <400> 58 Gln Arg Val Glu Gln Thr Pro Thr Thr Thr Thr Lys Glu Ala Gly Glu 1 5 10 15 Ser Leu Thr Ile Asn Cys Val Leu Lys Gly Ser Ser Asn Ala Leu Cys 20 25 30 Asn Thr Tyr Trp Tyr Phe Thr Lys Lys Gly Ala Thr Lys Lys Glu Ser 35 40 45 Leu Thr Asn Gly Gly Arg Tyr Ser Val Thr Met Asn Lys Ala Ser Lys 50 55 60 Ser Phe Ser Leu Arg Ile Ser Asp Leu Arg Val Glu Asp Ser Gly Thr 65 70 75 80 Tyr His Cys Lys Val Tyr Ser Ser Thr Val Gly Tyr Cys Asp Trp Asp 85 90 95 Ser Asp Tyr Tyr Glu Gly Gly Gly Thr Ile Leu Thr Val Lys 100 105 110 <210> 59 <211> 159 <212> PRT <213> Artificial sequence <400> 59 Thr Leu Thr Leu Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val 1 5 10 15 Ala His Val Val Ala Asn His Gln Val Glu Glu Gln Leu Glu Trp Leu 20 25 30 Ser Gln Arg Ala Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp 35 40 45 Asn Gln Leu Val Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln 50 55 60 Val Leu Phe Lys Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His 65 70 75 80 Thr Val Ser Arg Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu 85 90 95 Ser Ala Val Lys Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu 100 105 110 Leu Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu 115 120 125 Glu Lys Gly Asp Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu 130 135 140 Asp Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu 145 150 155 <210> 60 <211> 157 <212> PRT <213> Artificial Sequence <400> 60 Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 145 150 155

Claims

1. An anti-TNFα single domain antibody, wherein the complementary determining regions (CDRs) are CDR1 and CDR3 with the amino acid sequences shown below: CDR1 shown in SEQ ID NO:6 and CDR3 shown in SEQ ID NO:38; Also included are HV2 with an amino acid sequence as shown in SEQ ID NO:19 and HV4 with an amino acid sequence as shown in SEQ ID NO:

29.

2. The anti-TNFα single domain antibody according to claim 1, characterized in that Its framework region FR includes FR1 to FR4 with the amino acid sequences shown below: FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 13, FR3a shown in SEQ ID NO: 25, FR3b shown in SEQ ID NO: 32, and FR4 shown in SEQ ID NO:

42.

3. The anti-TNFα single domain antibody according to claim 1, characterized in that The anti-TNFα single-domain antibody is a single-domain antibody having an amino acid sequence as shown in SEQ ID NO:

58.

4. An isolated polynucleotide encoding the anti-TNFα single domain antibody according to any one of claims 1 to 3.

5. A construct, characterized in that Comprising the isolated polynucleotide as described in claim 4.

6. A pharmaceutical composition, characterized in that It comprises the anti-TNFα single domain antibody as described in any one of claims 1-3.

7. The pharmaceutical composition according to claim 6, characterized in that Also included are pharmaceutically acceptable carriers.

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