CN114605540A - anti-CD 28 nano antibody, coding gene and application - Google Patents

Abstract

The application discloses an anti-CD 28 nano antibody, an encoding gene and application. The anti-CD 28 nano antibody can be efficiently and specifically combined with CD28, has the advantages of the nano antibody and the co-stimulation signal transduction capability of an immune system, can obtain a complete antibody sequence, can be produced stably with high quality through in vitro recombinant expression, and has wide application prospect.

Description

anti-CD 28 nano antibody, coding gene and application

Technical Field

The invention belongs to the technical field of biomedicine or biopharmaceutical, and relates to an anti-CD 28 nano antibody, a coding gene and application.

Background

The immune system is an organic whole of interactions between lymphocytes, organs, humoral and cytokines, and immunologically active substances. The leucocytes can completely recombine the cytoskeleton structure within a few minutes, and the plasticity promotes the migration and communication of the whole immune system and also enables the leucocytes to form a strong interaction network (1) for mutual recognition of cells and exertion of immune functions. The immune system is classified into innate immunity (also called nonspecific immunity) and adaptive immunity (also called specific immunity), wherein adaptive immunity is further classified into humoral immunity and cellular immunity. In cancer and infectious disease patients, there is a loss of essential functions of the immune system in host defense, hypoactivity leading to severe infections and tumors with defective immune regulation mechanisms, and even hyperactivity leading to allergic and autoimmune diseases (2-4). The reasons for the immune system to be less than dysfunctional are mainly as follows: (i) decreased or impaired recognition by immune cells due to variable expression of antigens or reduced expression of the major histocompatibility complex class I (MHC I) on the antigen surface (5); (ii) immunosuppressive microenvironments, which are adverse to the functioning of the immune system due to the secretion of multiple inhibitory cytokines (e.g., TGF- β, etc.) by immunosuppressive or tumor cells (6); (iii) decreased immune pathway signaling and decreased tumor immunogenicity due to lack of expression of co-stimulatory or signaling molecules on the surface of infected or tumor cells (7), leading to decreased T cell recognition and activation. The co-stimulatory molecules or the accessory factors provide co-stimulatory signals while immune cells play functions so as to maintain and start the recognition effect of the immune system on antigens such as tumors and the like and enhance the killing and monitoring functions, and the co-stimulatory molecules or the accessory factors are a feasible strategy for enhancing the function of the immune system.

CD28 is a 44kDa type I transmembrane protein expressed on the surface of most naive CD4+ and CD8+ T cells and is an Ig-V like domain protein assembled from extracellular homodimers. CD28 plays an important role in T cell activation as a costimulatory molecule expressed on the surface of T lymphocytes. Complete activation of naive T cells usually requires three signals, the first being the binding of antigen and antigen receptor, the second being the costimulatory signal, and the third being the cytokine signal. CD28 acts as a costimulatory molecule and is involved in the transduction of secondary signals. The linkage of the CD28 receptor on T cells together with the T Cell Receptor (TCR) linkage provides a critical secondary signal for initial T cell activation (8). CD28 binds to the B7 molecule, and the co-suppression pathway in the B7-CD28 family provides key signals that regulate immune homeostasis and defense and protect tissue integrity (9). For T cells, the co-stimulation transduction capability of CD28 can greatly enhance the proliferation and differentiation of T cells, CD28 plays an important role in the immune response process of T cells, and CD28 has multiple co-stimulation molecular effects: (1) promote the secretion of Th cell driving factor and cell factor; (2) inducing surface expression of IL-2R by the T cells to increase reception of the third signal; (3) up-regulation of other co-stimulatory and regulatory factors in the immune system. At present, no therapeutic drug taking CD28 as a target point is on the market, and the drug taking CD28 as the target point in clinical tests is mainly applied to the immunotherapy of various blood tumors and solid tumors such as melanoma and multiple myeloma.

In 1993, the Nature journal reported a specific natural light chain deleted antibody (10) present in the peripheral blood of alpaca. Unlike conventional antibodies, such antibodies consist of only two Heavy chains, with only one Heavy Chain Variable region (VHH) and two conventional CH2 and CH3 regions. The VHH purified by monoclonal expression can exist stably in vitro alone, and is called single domain antibody (SdAb) or nanobody. The molecular weight of the nano antibody is only 15kD, which is 1/10 of the molecular weight of the traditional antibody, but the nano antibody has complete antigen recognition capability. Unlike artificially modified scFv, nanobodies are not easily adhered to each other and aggregated into a block. In addition, the structural stability of VHH cloned and expressed separately and its antigen binding activity were comparable to those of the original heavy chain antibody. The nano antibody has a tiny structure and complete antigen binding capacity, so the nano antibody has the advantages of high affinity, high specificity, strong penetrability, easiness in modification and expression and the like.

Reference to the literature

1.Huse M.Mechanical forces in the immune system.Nat Rev Immunol.2017Nov；17(11):679-690.doi:10.1038/nri.2017.74.Epub 2017Jul 31.

2.Parkin J,Cohen B.An overview of the immune system.Lancet.2001Jun2；357(9270):1777-89.doi:10.1016/S0140-6736(00)04904-7.

3.Rosenberg SA,Progress in human tumour immunology and immunotherapy.Nature.2001May 17；411(6835):380-4.

4.Gu SS,et al.Therapeutically Increasing MHC-I Expression Potentiates Immune Checkpoint Blockade.Cancer Discov.2021Jun；11(6):1524-1541.

5.Antonia S,et al.Current developments of immunotherapy in the clinic.Curr Opin Immunol.2004Apr；16(2):130-6.

6.Yang L,et al.TGF-beta and immune cells:an important regulatory axis in the tumor microenvironment and progression.Trends Immunol.2010Jun；31(6):220-7.

7.Dong H,et al.B7-H1,a third member of the B7 family,co-stimulates T-cell proliferation and interleukin-10secretion.Nat Med.1999Dec；5(12):1365-9.

8.Esensten JH,Helou YA,Chopra G,Weiss A,Bluestone JA.CD28 Costimulation:From Mechanism to Therapy.Immunity.2016May 17；44(5):973-88.doi:10.1016/j.immuni.2016.04.020.

9.Schildberg FA,Klein SR,Freeman GJ,Sharpe AH.Coinhibitory Pathways in the B7-CD28 Ligand-Receptor Family.Immunity.2016May 17；44(5):955-72.doi:

10.1016/j.immuni.2016.05.002.

10.Hamers-Casterman C,et al.Naturally occurring antibodies devoid of light chains.Nature.1993Jun 3；363(6428):446-8.

Disclosure of Invention

Aiming at the problems and the defects, the application provides the nano antibody which has simple process and low cost and specifically targets CD28, and can be used in the fields of detection and pharmacy.

In order to solve the above problems, the present invention provides one of the technical solutions: providing an anti-CD 28 nanobody, which is a protein of the following a) or b):

a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

b) protein which is derived from a) and is related to the specific recognition of CD28 by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence of the sequence 1 in the sequence table.

Preferably, the protein derived from a) is a protein consisting of an amino acid sequence shown in a sequence 2-42.

Specifically, the invention divides the selected 42 sequences into 8 groups according to the sequence similarity comparison result, wherein the similarity among the groups is more than 93.68%, and the details are as follows.

The first group comprises 5 sequences, respectively: P2-6G, P1-1A, P1-8F, P2-2F, P2-7F respectively correspond to sequences 1-5 in the sequence table. The coding genes of the gene correspond to sequences 43 to 47 in the sequence table respectively.

The second group comprises 15 sequences, respectively:

P1-1C, P1-2F, P1-5A, P1-7F, P1-8H, P1-12C, P1-12E, P2-1H, P2-3G, P2-4B, P2-8D, P2-10B, P2-10F, P2-11G, P2-11H respectively correspond to sequences 6-20 in the sequence table. The coding genes of the gene correspond to sequences 48 to 62 in a sequence table respectively.

The third group comprises 2 sequences, respectively: P1-7C, P1-6E, corresponding to sequences 21 and 22 in the sequence list respectively. The coding genes of the gene correspond to sequences 63 and 64 in a sequence table respectively.

The fourth group comprises 16 sequences, respectively:

P1-3C, P2-1G, P1-4C, P1-6C, P1-6H, P1-8C, P1-8D, P1-9D, P1-11C, P2-2C, P2-2D, P2-2H, P2-4G, P2-5E, P2-5H, P2-8F, which respectively correspond to the sequences 23-38 in the sequence table. The coding genes of the gene correspond to sequences 65-80 in the sequence table respectively.

The fifth group comprises 1 sequence, which is P1-9E, corresponding to sequence 39 in the sequence table. The coding gene corresponds to a sequence 81 in a sequence table.

The sixth group comprises 1 sequence, is P1-10B, and corresponds to the sequence 40 in the sequence table. The coding gene corresponds to a sequence 82 in a sequence table.

The seventh group comprises 1 sequence, which is P2-5C, corresponding to sequence 41 in the sequence table. The coding gene corresponds to a sequence 83 in a sequence table.

The eighth group comprises 1 sequence which is P2-10A and corresponds to the sequence 42 in the sequence table. The coding gene corresponds to a sequence 84 in a sequence table.

The CD28 nanometer antibody skeleton designed by the invention is CD3-Fab, so that in an antibody function verification experiment, CD3-Fab is used as a negative control group, and all sequences in the group can replace a core sequence CD28 nanometer antibody part.

Preferably, the anti-CD 28 nanobody of the present invention further comprises SP34-LC region and SP34-HC region. The two regions jointly form the fusion construction of CD3-Fab, CD3-Fab and anti-CD 28 nano antibody, on one hand, the expression yield of the nano antibody is promoted, on the other hand, main TCR stimulation signals are provided, and the help is provided for the subsequent T cell activation verification. anti-CD 28 nanobody pass (G) of the present invention₄S)₃The connecting peptide is connected to the C terminal of SP34-LC to form a fused antibody structure taking CD3-Fab as a framework. The structure is beneficial to verifying the unique advantages of the anti-CD 28 nano antibody, and the CD3-Fab is taken as a control group in verification to illustrate the functional characteristics of the anti-CD 28 nano antibody. The SP34-LC region comprises SP34-VL and SP34-VL, the amino acid sequences of the two parts correspond to the sequences 85 and 86 in the sequence table, and the coding genes of the two parts correspond to the sequences 89 and 90 in the sequence table. (G)₄S)₃The amino acid sequence of the connecting peptide region corresponds to the sequence 87 in the sequence table, and the coding gene of the connecting peptide region is the sequence 91 in the corresponding sequence table. The amino acid sequence of SP34-HC region corresponds to sequence 88 in the sequence table, and the coding gene is corresponding to sequence 92 in the sequence table.

Some modifications and alterations, such as humanization, pegylation, or other alterations, were made to the anti-CD 28 nanobody to increase its activity.

The CDR area of the nano antibody is subjected to mutation modification, such as substitution and/or deletion and/or addition of one or more amino acids, so as to improve the affinity. In order to solve the above problems, the present invention provides the following two technical solutions: provides the coding gene of the anti-CD 28 nano antibody.

Preferably, the encoding gene is a gene of 1) or 2) or 3) as follows:

1) the nucleotide sequence is sequence 43 in the sequence table;

2) a DNA molecule which is hybridized with the DNA fragment defined by the sequence 43 under strict conditions and codes for a protein which specifically recognizes the CD28 related protein;

3) has more than 90 percent of homology with the gene of 1) or 2) and encodes a DNA molecule which can specifically recognize CD28 related protein.

Preferably, the nucleotide sequence of the gene in 2) or 3) is the sequence 44-84 in the sequence table.

In order to solve the above problems, the present invention provides a third technical solution: provides a recombinant expression vector or a recombinant strain containing the coding gene.

In order to solve the above problems, the fourth technical solution provided by the present invention is: provides the application of the anti-CD 28 nano antibody in preparing medicaments for preventing or treating tumors, immunodeficiency diseases, infectious diseases and the like.

The implementation of the invention has the following beneficial effects: the anti-CD 28 nano antibody provided by the invention can be efficiently and specifically combined with CD28, and has the affinity with a cell surface antigen up to 4.12nM which is 4.12-20.84 nM; the affinity with CD28 antigen is up to 0.12nM and is 0.12-19.4 nM. Because the complete antibody sequence can be obtained, the nano antibody can be produced stably with high quality through in vitro recombinant expression, and has wide application prospect.

The application of the invention comprises at least one of the following:

(1) the polypeptide is directly applied to the specific binding of CD28 antigen, such as the specific recognition and killing of tumor antigen taking CD28 as a target;

(2) the CD28 nano antibody is connected with effector molecules, such as chemical small molecule drugs, cytokines, toxic molecules or radioactive isotopes, and is used in the field of medicine;

(3) the CD28 nano antibody is used as a drug delivery carrier to target the antigenic parts such as tumor and the like at fixed points so as to realize the specific treatment at fixed points;

(4) adding chemiluminescent groups or fluorescent molecules on the CD28 nano antibody to prepare a specific probe;

(5) the CD28 nanobody is used for enhancing the curative effect of the immune system, such as CAR-T, TCR-T, CAR-NK, CAR-DC and the like, and is used in the field of immunotherapy.

Drawings

FIG. 1 results of alpaca anti-CD 28 serum titer test;

FIG. 2 first round PCR product identification results;

FIG. 3 second round PCR product identification;

FIG. 4 positive cloning efficiency colony PCR validation results;

FIG. 5 shows the results of a round of ELISA experiments with positive colonies, including FIGS. 5 a-5 o;

FIG. 6 shows the results of two rounds of ELISA experiments with positive colonies, including FIGS. 6 a-6 e;

FIG. 7 is a schematic representation of the sequence similarity of the anti-CD 28 nanobody, including FIGS. 7 a-7 e, with FIG. 7a showing the overall sequence similarity in the examples listed and FIGS. 7 b-7 e showing the sequence similarity between groups;

FIG. 8 shows ELISA assay results of CD3/CD28 fusion antibodies, including FIGS. 8a and 8b, wherein FIG. 8a shows assay results of the binding ability of CD3/CD28 fusion antibody and control CD3 antibody to target antigen CD28, and FIG. 8b shows assay results of the binding ability of CD3/CD28 fusion antibody and control CD3 antibody to unrelated antigen 4-1 BB;

FIG. 9 shows the results of experiments on the binding of the CD3/CD28 fusion antibody to K562 cell surface receptors, including FIGS. 9a and 9b, wherein FIG. 9a is a test of the binding ability of the CD3/CD28 fusion antibody and the control CD3 antibody to the target cells K562/CD28, and FIG. 9b is a test of the binding ability of the CD3/CD28 fusion antibody and the control CD3 antibody to the unrelated cells K562/4-1 BB;

FIG. 10 results of the activation of human PBMC by the CD3/CD28 fusion antibody.

Detailed Description

The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but the description of the embodiments is only a part of the embodiments of the present invention, and most of them are not limited thereto.

The nano antibody is superior to the traditional antibody in many aspects. Compared with the traditional antibody, the nano antibody is based on the special structure of the VHH single domain antibody of the alpaca heavy chain antibody, is the minimum unit known to be combined with a target antigen, has the advantages of the traditional antibody and a small molecular drug, almost perfectly overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody, and gradually becomes a new force in a new generation of therapeutic biological medicine and clinical diagnosis reagents. And the CD28 nano antibody has the advantages of the nano antibody and the capability of co-stimulating signal transduction of an immune system, and compared with the conventional antibody, the CD28 nano antibody has the advantages that:

(1) the molecular weight is small, the blood brain barrier can be penetrated, and the focus specific penetration is enhanced, so that the effect of targeted killing of antigen is improved;

(2) high expression in prokaryotic or eukaryotic systems;

(3) the stability is good, the specificity is strong, and the affinity is high;

(4) does not cause immunogenic reaction to human body.

The CD28 resistant nano antibody has the application advantages that the biological medicine research and development (gene engineering medicine research and development, ADC medicine research and development) is carried out; clinical in vitro diagnosis (colloidal gold method, enzyme-linked immunosorbent assay, electrochemiluminescence); basic researches such as tumor research and immunological therapy research, and specific target recognition and killing of probes.

Because the screening and obtaining technology of the monoclonal antibody is difficult and high in cost, the CD28 immune alpaca is subjected to nano antibody library obtaining, the high-affinity nano antibody is obtained through screening by a phage display library technology, and qualitative experiments are carried out on the CD28 protein through enzyme-linked adsorption reaction and the like. The antibody can be applied to the immunotherapy of tumors, immunodeficiency diseases and infectious diseases.

Example 1 expression and purification of CD28 protein

1.1 vector construction

A CD28-Fc fusion protein is constructed by a eukaryotic expression vector pFase, CD28 and human IgG1 Fc are connected in series in a PCR mode, and the complete molecule comprises a CD28 secretion signal peptide, a CD28 extracellular domain, a thrombin cleavage site, an IEGRMD short peptide, a human IgG1 hinge region, a CH2 domain and a CH3 domain. The CD28-Fc gene was double-digested with NcoI and NheI and then cloned into pFuse vector using T4 DNA ligase, or vector was constructed using a homologous recombination kit.

1.2 eukaryotic expression

Inoculation 1.5X 10⁶In 500mL shake flasks 200mL medium were used 293F cells/mL. Shaking at 37 deg.C and 165rpm in shaking incubator with 5% CO2 concentration for 24h, counting cells after 24h, and adjusting cell density to 3 × 10⁶and/mL, uniformly mixing the constructed plasmid with 10mL of opti-MEM, standing at room temperature for 5min, adding 500. mu.L (1:2.5) of PEI40000 to 10mL of opti-MEM, gently mixing, standing at room temperature for 5min, mixing the plasmid and PEI mixed solution, gently mixing, and standing at room temperature for 20 min. And dropwise adding the mixed solution into 200mL of 293F cell culture solution, gently shaking the culture bottle while dropwise adding, uniformly mixing, putting into a shaking table, and transfecting for 72 hours. After transfection, centrifugation is carried out for 5min at 100g, supernatant is taken, 100mL of culture medium is added into a culture flask, centrifugation is carried out for 5min at 3000rpm after suspension culture is carried out again, and supernatant is taken. The cell supernatants were mixed twice.

1.3 protein purification

400mL of the cell supernatant obtained in the step 1.1 above was centrifuged at 15000rpm at 4 ℃ for 30min, and the supernatant was collected, filtered through a 0.45 μm filter and placed on ice for further use. 4mL (20% ethanol/Protein G1: 1) of Protein G was loaded onto the column, washed 3 times with Binding buffer, and pressed against the surface of the resin using a pad. The Protein G column was equilibrated with 20mL Binding buffer. Samples were loaded every 10mL and run through the Protein G column at a constant rate (approximately 0.5 mL/min). The Protein G column was washed at 40mL Binding buffer constant speed (about 1 mL/min). First, 10% eluent volume Buffer was added to the collection tube of the Elution tube and the column was eluted 4 times with an Elution Buffer (5mL eluted once until the protein concentration could not be quantified). The collected protein samples were concentrated using an Amicon Ultra-15 centrifuge filter and centrifuged at 3000rpm for 20 minutes at 4 ℃ to quantify the protein concentration.

The protein samples were buffer exchanged. Put into a dialysis bag (microwave oven boiling with ultrapure water for ten minutes), 4L of dialysate (rotor stirring dialysis) is added at 4 ℃, and the dialysate is changed every 4 hours. After two times of fluid change, all protein fluid in the dialysis bag is collected, centrifuged at 8000rpm and 4 ℃ for 15 minutes, and the supernatant is collected and quantified.

Firstly, enzyme digestion efficiency test is carried out, a proper amount of protein solution after quantification is taken, the concentration is diluted to be 1mg/mL, and 100 mu L of protein sample is taken from each tube. Thrombin (Thrombin) was added at a concentration of 10IU/mg, diluted 2-fold (using dialysate), for 10 gradients. A control group without Thrombin was set. Shaking at room temperature for 16 hours. The enzyme digestion effect was checked by SDS-PAGE. Determining the appropriate Thrombin enzyme concentration, and completely digesting the residual protein sample. Shaking at room temperature for 16 hours. SDS-PAGE detects the enzyme digestion effect of the large system.

And (3) slowly passing the Protein sample after the thrombin digestion through a Protein G column at a constant speed, eluting according to the method of 1.2, and detecting by SDS-PAGE until no Fc exists in the sample and the purity of CD28 ECD is more than 90%.

The unlabeled CD28 ECD protein was concentrated to 1mg/mL and sampled for final characterization by SDS-PAGE (CD28 ECD-Fc molecular weight about 42kD, reduced SDS-PAGE should finally appear to be 55-65kD due to eukaryotic expressed glycosylation, Fc molecular weight about 30 kD).

Example 2 construction and screening of anti-CD 28 Nanobody phage display library

2.1 immunization of alpaca

Selecting one healthy adult alpaca, and marking the alpaca with an ear number. The CD28 protein and Freund's adjuvant are mixed uniformly in the ratio of 1:1, and stored at 4 ℃. The injection is divided into two parts, namely left and right injection, and each part is divided into 2 points, and 0.4mL of mixed adjuvant antigen is injected for each time. After the immunization, the alpaca is observed for half an hour, and the alpaca is confirmed to be in a good state and has no adverse reaction. Immunizing once every 2 weeks for 7 times, collecting alpaca peripheral blood from neck vein before immunization and after 1-4 times of immunization, and separating serum for serum titer detection. 5-7 days after 6 th and 7 th immunization, collecting alpaca peripheral blood by the jugular vein for constructing a phage display library.

2.2 alpaca serum titer detection

Antigen coated ELISA plates (2. mu.g/mL, 100. mu.L/well) were incubated overnight at 4 ℃. The antigen solution was discarded and the microplate was washed three times for 5 minutes each with 200. mu.L of PBST solution (1 XPBS plus 0.05% Tween20, the same below). The plate was blocked with 200. mu.L of blocking solution (2% BSA in PBST) at room temperature for 1.5 hours. The blocking solution was discarded and the microplate was washed three times with 5 minutes each time with 200. mu.L of PBST solution. And (3) performing gradient dilution on each batch of serum by using a sealing solution, adding 100 mu L of alpaca serum collected after the non-immunization and the 1-4 th immunization into a corresponding hole of the ELISA plate, and incubating for 2 hours at room temperature. The serum was discarded and the microplate was washed 4 times with 200. mu.L of PBST solution for 5 minutes each. To each well was added a detection antibody (goat anti-llama IgG, HRP conjugated, 1:10000 diluted in blocking solution, 100 μ Ι _ per well) for 1 hour at room temperature. The detection antibody was discarded and the microplate was washed 4 times with 200. mu.L of PBST solution for 5 minutes each. To each well 100. mu.L of TMB substrate solution was added and developed for 2-3 minutes. To each well was added 100. mu.L of a reaction stop solution. The absorbance at 450nm was measured using a microplate reader over 30 minutes. Positive well standard: immune serum sample OD₄₅₀The value is greater than the OD of the non-immune serum ₄₅₀3 times and reading greater than 0.5, the results are shown in figure 1.

2.3 alpaca-derived lymphocyte isolation

3mL of cell separation medium was added to a 15mL centrifuge tube, and 3mL of blood sample was added slowly. Precooling the sample in a centrifuge at 25 ℃, centrifuging the sample at 400g for 30min, and observing the separation condition of the blood sample. The middle cotton-like upper layer immune cells were carefully pipetted into a new 15mL centrifuge tube using a 200 μ L pipette, and the upper layer serum was transferred to a new centrifuge tube and stored at-80 ℃.10 mL of PBS buffer was added to the centrifuge tube containing the immune cells, centrifuged at 400g for 20min at 25 ℃ and the supernatant was removed. 5mL of PBS buffer was added to each tube, centrifuged at 400g for 20min at 25 ℃ and counted to remove the supernatant. The isolated lymphocytes were lysed using RNAioso Plus according to the cell count results to obtain a lysate, which was stored at-80 ℃.

2.4 RNA extraction

The peripheral blood lymphocytes preserved with Trizol were transferred to a 1.5mL centrifuge tube, and 1/5 volumes of chloroform were added and mixed well. After standing at room temperature for 5 minutes, the mixture was centrifuged at 12000g at 4 ℃ for 15 minutes. The centrifuged supernatant was carefully transferred to a new centrifuge tube. Add 0.5-1 volumes of isopropanol to the new centrifuge tube. After standing at room temperature for 10 minutes, the mixture was centrifuged at 12000g at 4 ℃ for 10 minutes. And (3) washing the precipitate by using 75% ethanol with the same volume as that of peripheral blood lymphocytes preserved by Trizol, centrifuging at 4 ℃ for 5 minutes, dissolving in a proper amount of water without RNase, and combining all samples to obtain the extracted total RNA.

2.5 Synthesis of cDNA by reverse transcription

The total RNA obtained was reverse transcribed using Takara reverse transcription kit. Dividing the total RNA sample into two parts, wherein one part uses Oligo dT Primer in a kit as a Primer, and the other part uses Random 6-mers in the kit as a Primer, and the total RNA obtained in the last step is reversely transcribed into cDNA according to the instruction of a reverse transcription kit and is respectively stored in 2 centrifuge tubes.

2.6 antibody variable region Gene amplification

Two rounds of PCR amplification were performed using Taq DNA Polymerase Hot Start enzyme using the cDNA obtained by reverse transcription as a template.

The first round of PCR reaction conditions and procedures were:

PCR amplification was performed using Taq DNA Polymerase Hot Start enzyme, and in order to determine the optimal template usage, 1. mu.L, 2. mu.L, 3. mu.L, 4. mu.L, 5. mu.L of oligo dT Primer and random 6-mer cDNAs were used as templates, respectively, and PCR reaction systems were prepared according to the following table:

the PCR reactions were performed according to the following configuration:

(1) after the reaction is finished, taking 20 mu LPCR product to carry out 1% agarose gel electrophoresis, finally selecting the quantity with single target band and 600bp fragment size in the electrophoresis result as the optimal template quantity, and carrying out PCR reaction on all cDNA according to the template quantity and by using the same condition;

(2) performing 1% agarose gel electrophoresis on all PCR products, cutting the gel and recovering a band with the target fragment size of about 600 bp;

(3) all the products of the first round of PCR purification and recovery are collected into a centrifuge tube to obtain the first round of PCR amplification products, and the products are stored at the temperature of minus 20 ℃.

The first round of PCR product identification is shown in figure 2.

The second round of PCR reaction conditions and procedures were:

the second round of PCR reaction was carried out using the first round of PCR amplification product as a template, and in order to determine the optimal amount of template used, 0.5. mu.L, 1. mu.L, 2. mu.L, 3. mu.L, and 4. mu.L were used as templates, and PCR reaction systems were prepared according to the following table:

the PCR reactions were performed according to the following configuration:

(1) after the reaction is finished, taking 20 mu L of PCR products to carry out 1% agarose gel electrophoresis, finally selecting the template quantity with single target band and 600bp fragment size in the electrophoresis result as the optimal template quantity, carrying out the PCR reaction on the obtained first round PCR amplification products with about 1/5 volumes according to the template quantity and using 4.1 (1) the same conditions, and then using a universal DNA purification recovery kit to carry out DNA purification on the PCR reaction solution;

(2) collecting the recovered product into a centrifuge tube to obtain a second round PCR amplification product, simultaneously detecting the concentration of the recovered product with a nucleic acid concentration measuring instrument by 2 μ L and recording, and storing the rest products at-20 ℃.

The second round of PCR product identification is shown in figure 3.

2.7 vector construction

(1) Using pADL-10b as a phage plasmid vector, digesting 10 mu g of pADL-10b vector and 5 mu g of second PCR amplification product by BglI respectively, and incubating for 4h at 37 ℃;

(2) the pADL-10b vector and the second round PCR amplification product were purified using a DNA recovery purification kit and stored at 4 ℃.

2.8 connection

(1) The vector and the fragment were ligated, and the ligation system was prepared according to the following table:

(2) the ligation reaction was incubated overnight (about 16h) at 4 ℃;

(3) and purifying the ligation reaction solution by using a universal DNA purification and recovery kit, detecting the concentration of the recovered product, and storing at 4 ℃.

2.9 validation of ligation product conversion

(1) Taking a 50 mu L of SS320 competent cells, and placing on ice for 5-10min to melt;

(2) adding 100ng of the ligation product, transferring the ligation product into a pre-cooled electric rotor cup with a distance of 1mm, and setting parameters in an electric rotor: 1800V, after 1mm, clicking a button to click and convert;

(3) after the electricity conversion is finished, 1mL of SOC culture solution preheated at 37 ℃ is immediately added, mixed evenly and then shaken at 37 ℃ and 200rpm for resuscitation for 1 h;

(4) taking 100 mu L of the recovered bacterial liquid from 1mL, carrying out 10-time gradient dilution and plate coating, and calculating the number of transformed colonies obtained by each reaction according to the dilution times and the number of single colonies, namely the transformation efficiency of the ligation product;

(5) meanwhile, 48 monoclonal bacteria are randomly selected for colony PCR, a single band of about 500bp of a PCR product is considered as a positive clone, and the positive rate of the monoclonal is estimated, and the result is shown in FIG. 4.

2.10 bacterial library construction

(1) Taking 25 groups of 100ng connecting systems, and carrying out electrotransformation reaction by using 25 tubes of competent cells according to the method;

(2) recovering at 37 deg.C for 1h, diluting 100 μ L of the extract with 10 gradient, plating, and culturing at 37 deg.C overnight;

(3) collecting all the remaining bacteria solution, uniformly spreading the bacteria solution on 5 square culture plates (2 XYT containing 100. mu.g/mL Amp and 2% glucose) with 245mm, and culturing at 37 ℃ for overnight;

(4) calculating the number of converted colonies obtained by reaction according to the dilution times and the number of single colonies, namely the library capacity of the bacterial library;

(5) simultaneously randomly selecting 48 monoclones from the gradient dilution plate to carry out colony PCR, and verifying the cloning positive rate of the bacterial library;

(6) overnight-cultured colonies of 245mm square-shaped plates were scraped off using 2 XYT liquid medium, placed in a 50mL centrifuge tube, and the OD measured₆₀₀Value, add 20% glycerol to final concentration-80 ℃ storage.

2.11 phage library preparation

According to OD of bacterial library₆₀₀Calculate the volume of the bacterial library to be added to 100mL of 2 XYT liquid medium, and inoculate the bacterial library to 100mL of 2 XYT liquid medium (containing 10. mu.g/. mu.L Tet and 100. mu.g/. mu.L Amp) according to the calculation, and culture at 37 ℃ and 250rpm until OD₆₀₀Is 0.5-0.55. According to helper phage titer, as 1: helper phage was added at a ratio of 20 (bacterial count: phage count) and incubated at 37 ℃ and 250rpm for 30-60 min. IPTG was added to a final concentration of 50. mu.g/mL and 200. mu.M, and incubated overnight at 250rpm at 30 ℃. The overnight-cultured broth was centrifuged at 8000rpm for 10min at 4 ℃ and the centrifuged supernatant was transferred to a new 50mL centrifuge tube. Adding 1/4 precooled PEG/NaCl stock solution, mixing well, and standing and incubating for 30min on ice. Centrifuging at 8000rpm for 10min at 4 deg.C, discarding supernatant, and inverting for 2 min. Add 5mL PBS to resuspend to fresh centrifugeTube, centrifuge at 8000rpm for 10min at 4 ℃. After centrifugation, the supernatant was transferred to a new centrifuge tube, and 1/4 volumes of pre-cooled PEG/NaCl stock solution were added again, mixed well, and incubated on ice for 10 min. Centrifuging at 4 ℃ and 8000rpm for 10min, discarding the supernatant, resuspending with 1mL PBS, centrifuging at 8000rpm for 10min, transferring the supernatant to a new centrifuge tube, and storing at-80 ℃ to obtain the purified phage library.

2.12 Positive phage library screening

First screening: taking out the screened antigen from a refrigerator at the temperature of-80 ℃, and standing and unfreezing the antigen on ice; the screening antigen CD28 and the negative screen antigen 41BB are coated on immune tubes (50. mu.g/tube, coating solution is coated overnight; PBS, 2 mL/tube) respectively, wherein the negative screen antigen coats two immune tubes, one tube is used as a negative screen, the other tube is used as a control, and the immune tubes are coated overnight at 4 ℃ by slow rotation. The overnight coated tubes were discarded and the tubes were washed 3 times with 5min each time by adding 2mL PBS buffer at room temperature. 2mL of blocking solution (3% skim milk powder) was added and spin-blocked at room temperature for 2 h. The liquid in the sealed negative-sieve immune tube is discarded, and 2mL of PBST buffer solution is added to wash the immune tube at room temperature for 3 times, and the immune tube is rotated for 5min each time. Discarding the washing solution in the negative-screen immune tube, adding 2mL PBS, calculating according to the following formula, adding 104 μ L of the prepared phage library as the first screening input phage library, and performing rotary incubation for 1h at room temperature:

where V is the volume of phage added (in. mu.L), T_libraryThe titer of the phage is obtained;

the blocking solution in the immune tubes coated with the antigen CD28 and the antigen 41BB as a control was discarded, and the tubes were washed 3 times with 5min each time by adding 2mL of PBST (1 XPBS plus 0.1% Tween20, the same applies below) buffer at room temperature. Discarding liquid in the immune tubes, dividing phage solution after negative screen incubation into two immune tubes, respectively adding the two immune tubes, and performing rotary incubation for 1h at room temperature; discard the tube, add 2mL of PBST buffer to wash the tube 20 times at room temperature, and rotate each time for 5 min. Discarding the liquid in the immune tube, removing the residual liquid as much as possible, adding 1mL of 0.25mg/mL Trypsin solution, and performing rotary elution at room temperature for 30 min. And adding 10 mu L of 10% AEBSF to terminate elution, and transferring the solution in the immune tube to a new 1.5mL centrifuge tube to obtain the first round of screened phage eluate.

Performing titer detection on the phage eluate in the first round: the SS320 strain stored at-80 ℃ in a refrigerator was streaked on 2 XYT solid medium (Tet-resistant), cultured overnight at 37 ℃ (stored at 4 ℃ for one week), one single colony was picked from the single colony plate to 5mL of 2 XYT medium containing 10. mu.g/mL Tet, and cultured overnight at 37 ℃. Transferring 500 μ L of overnight culture broth into 5mL of 2 XYT liquid culture medium containing 10 μ g/mL Tet, culturing at 37 deg.C and 50rpm for 45-60 min to OD₆₀₀Is 0.5-0.55. Taking 10 μ L of the first round of phage eluate to perform 10-fold gradient dilution in a 1.5mL centrifuge tube, diluting 10 gradients in total, namely diluting 10 μ L of the first round of phage eluate to 100 μ L, then diluting 10 μ L of the first round of phage eluate to 100 μ L, and so on, diluting 12 gradients to 10-¹⁰And oscillating and mixing uniformly. Add 90. mu.L of SS320 bacterial liquid into each dilution centrifuge tube, mix them evenly, incubate them for 30min at 37 ℃, take 5. mu.L from each dilution centrifuge tube and add them dropwise into 2 XYT solid medium (Amp), invert at 37 ℃ and culture overnight. Counting the number of single colonies on a plate capable of obviously distinguishing the dilution of the single colonies, and calculating the number of phagemids in each milliliter of thallus solution according to the following formula, namely the titer of a phage library:

T(pfu/ml)＝N×D×400

wherein T is the titer of the phage (unit pfu/mL), D is the dilution multiple, and N is the number of single colonies on the corresponding dilution multiple.

First round amplification of phage eluate: the SS320 strain stored in a refrigerator at-80 ℃ was streaked in advance on 2 XYT solid medium (Tet-resistant) for one colony, cultured overnight at 37 ℃ (stored at 4 ℃ for one week), one single colony was picked from the single colony plate to 5mL of 2 XYT medium containing 10. mu.g/mL of Tet, and cultured overnight at 37 ℃. Transferring 500 μ L of overnight culture broth into 5mL of 2 XYT liquid culture medium containing 10 μ g/mL Tet, culturing at 37 deg.C and 250rpm for 45-60 min to OD₆₀₀The value is 0.5-0.55. Adding 500 μ L phage eluate obtained after the first round of screening to OD₆₀₀0.5-0.55 (the remaining eluate is stored at 4 ℃). The incubation was continued at 37 ℃ and 250rpm for 30 min. The whole culture broth was spread evenly on a 245mm square plate of a medium containing 100. mu.g/mL Amp and 2% glucose in 2% agarose, and cultured overnight at 37 ℃. Taking a square plate cultured overnight, adding 6mL of 2 XYT liquid medium (containing 10 mu g/mL Tet) on the surface of the culture plate, gently scraping the colony of the square plate by using a coating rod, collecting bacterial liquid into a 15mL centrifuge tube to obtain an amplified bacterial sublibrary, and simultaneously measuring the OD of the bacterial liquid by using a spectrophotometer₆₀₀The value is the bacterial library OD of the eluent₆₀₀And adding glycerol with the final concentration of 20 percent to obtain a first round of bacterial library. The amount of the bacterial suspension was calculated from the eluate according to the following formula, and transferred to 100mL of 2 XYT liquid medium (containing 10. mu.g/mL Tet and 100. mu.g/mL Amp) to obtain the initial OD₆₀₀Is 0.1:

wherein V is the volume (unit μ L) of the transfer bacterium liquid, OD₆₀₀OD of bacterial library for construction of eluate₆₀₀。

Culturing at 37 deg.C and 250rpm until bacterial liquid OD₆₀₀To 0.5-0.55. The helper phage M13K07 was calculated and added to give a 1: 20 bacterial count to phage count according to the following equation:

wherein V is the volume (in mL) of the added helper phage, T_helper-phageFor the helper phage titer used, OD₆₀₀Is OD of bacterial liquid₆₀₀A value;

culturing at 37 deg.C and 250rpm for 30 min; kana was added to the culture medium at a final concentration of 50. mu.g/mL and IPTG was added at a final concentration of 0.2. mu.M, respectively, and the mixture was cultured overnight at 30 ℃ and 250 rpm.

First round phage purification: the overnight-cultured broth was transferred to a new 50mL centrifuge tube at 4000rpm and centrifuged at 4 ℃ for 10 min. The centrifuged supernatant was transferred to a new 50mL centrifuge tube, 1/4 volumes of 4 ℃ pre-cooled 20% PEG/2.5M NaCl were added, mixed well and placed on ice for 30 min. Centrifuge at 4000rpm for 20min at 4 ℃, discard the supernatant and invert on paper for 2 min. The pellet was resuspended by adding 1mL PBS and the resuspension was transferred to a new 1.5mL centrifuge tube, 13000rpm, and centrifuged at 4 ℃ for 20 min. The centrifuged supernatant was transferred to a new 1.5mL centrifuge tube, 1/4 volumes of pre-cooled 20% PEG/2.5M NaCl solution were added, mixed well and placed on ice for 10 min. 13000rpm, centrifuging for 10min at 4 ℃, discarding the supernatant, adding 1mL PBS for heavy suspension precipitation, centrifuging for 2min at 13000rpm and 4 ℃, transferring the supernatant into a new 1.5mL centrifuge tube, namely a first round screening phage sublibrary, subpackaging with 100 mu L/tube, storing at-80 ℃ for a long time, and storing at-20 ℃ for a short time (1-2 weeks). And performing titer detection on the phage sub-library in one round, wherein the method is as above.

And (3) second screening: the screening method is the same as the above, the phage is input for the first round of screening to obtain a phage sub-library, and the phage sub-library is input for the second round of screening to obtain a second round of screening phage eluate.

Performing titer detection on the phage eluate in the second round, amplifying the eluate, purifying, and screening titer detection on phage sublibraries by the same method.

Monoclonal ELISA detection: the SS320 strain stored in a refrigerator at-80 ℃ was streaked in advance on 2 XYT solid medium (Tet-resistant) for one colony, cultured overnight at 37 ℃ (stored at 4 ℃ for one week), one single colony was picked from the single colony plate to 5mL of 2 XYT medium containing 10. mu.g/mL of Tet, and cultured overnight at 37 ℃. Transferring 500 μ L of overnight culture broth into 5mL of 2 XYT liquid culture medium containing 10 μ g/mL Tet, culturing at 37 deg.C and 250rpm for 45-60 min to OD₆₀₀The value is 0.5-0.55. And taking 10 mu L of phage eluate after the second round of screening, diluting in a centrifugal tube of 1.5mL in a gradient manner by 10 times, diluting by 12 gradients in total, and shaking and mixing uniformly. Add 90. mu.L of OD to each dilution tube₆₀₀The bacterial liquid with the value of 0.5-0.55 is mixed evenly. The incubation was continued at 37 ℃ and 250rpm for 30 min. The bacterial suspension was spread evenly on a solid medium plate containing 100. mu.g/mLAmp, and cultured overnight at 37 ℃. Random selection of monoclonal colonies from overnight-cultured media plates in sterile 96-well cell culturesTo each well of the plate (P1-P2), 100. mu.L of 2 XYT medium (containing 100. mu.g/mL Amp and 10. mu.g/mL Tet) was added, and the plate was incubated overnight at 37 ℃. mu.L of overnight-cultured bacterial suspension was transferred to 250. mu.L of a new 96-well cell culture plate containing 2 XYT liquid medium (containing 100. mu.g/mL Amp and 10. mu.g/mL Tet) per well, and incubated at 37 ℃ for 3 hours, and the overnight-cultured bacterial suspension before transfer was stored at 4 ℃. The following equation was followed and helper phage M13K07 was added to each well to give a 1: 20 bacterial count to phage count:

wherein V is the volume (in mL) of the added helper phage, T_helper-phageThe helper phage titer used.

Incubation was carried out at 37 ℃ for 30min, Kana at a final concentration of 50. mu.g/mL and 0.2. mu.M IPTG were added, and the mixture was allowed to stand at 30 ℃ for overnight culture. The 96-well culture plate after overnight culture is centrifuged at 4000rpm for 10min at 4 ℃ and stored at 4 ℃ for later use. Screening antigen CD28 and negative screening antigen 41BB were coated on enzyme plates (1 ng/. mu.L, coating solution is CBS of pH9.4, 100. mu.L/well), BSA was coated in parallel as a control, and the plate was coated overnight at 4 ℃. The overnight coated microplate liquid was discarded, 200. mu.L PBS buffer was added to each well, and the microplate was washed 3 times for 10min each time at room temperature. Add 200. mu.L blocking solution (3% BSA) to each well to block the ELISA plate, and block for 1h at room temperature. The blocking solution was discarded, 200. mu.L of PBST (1 XPBS plus 0.1% Tween20, the same applies below) buffer was added to each well, and the microplate was washed 3 times for 10min each time at room temperature. mu.L of 3% BSA was added to each well followed by 80. mu.L of centrifuged supernatant and incubated at room temperature for 2 h. The liquid in the ELISA plate was discarded, and 200. mu.L of PBST buffer was added to each well and washed 3 times for 10min each time. M13 Bacteriophage Antibody (HRP), Mouse Mab, 1:8000 dilution in blocking solution was added per well, 100. mu.L/well, and incubated for 1h at room temperature. The ELISA plate was discarded and washed 3 times for 10min with 200. mu.L PBST buffer per well. Adding 100 μ L of TMB single-component color developing solution into each well, developing for 2-3min in dark, adding 100 μ L of 1M HCl into each well, stopping, and reading OD with enzyme-labeling instrument₄₅₀And (4) recording and storing the value. Negative control is carried out by negative screening antigens 41BB and BSA in one verification. The results are as followsFIGS. 5a to 5 o. By OD₆₀₀The positive cloning range is not less than 1.7133.

Secondary verification of positive clone ELISA: to eliminate false positive results, clones that were initially identified as positive were subjected to a secondary ELISA test, which was performed as above, with only the negative screen antigen 41BB control and no BSA control. Representative sequencing results the primary data for secondary verification of positive monoclonals were processed using GraphPad Prism and the results are shown in fig. 6a to 6 e. By OD₆₀₀The positive cloning range is not less than 0.9927.

Example 3 sequencing

3.1 Positive clone sequencing

And selecting positive monoclonals according to ELISA detection data and secondary verification data.

From the monoclonal ELISA plate, 5. mu.L of the positive clone was inoculated into 2mL of 2 XYT medium (containing 100. mu.g/mL Amp and 10. mu.g/mL Tet), cultured at 37 ℃ and 250rpm until OD₆₀₀And sequencing 1mL of bacterial liquid when the temperature is 0.8-1.0 (about 6-8h), and storing the rest of bacterial liquid at 4 ℃.

3.2 sequence analysis

The sequenced sequences were analyzed for sequence alignment using GENtle software, and the antibody sequences were translated into amino acids using GENtle software. The results are shown in FIGS. 7 a-7 e, where FIG. 7a shows the overall sequence similarity in the examples listed and FIGS. 7 b-7 e show the sequence similarity between groups.

Example 4 expression and purification of CD3/CD28 fusion antibodies

4.1 vector construction

SP34-HC and SP34-LC-CD28(Nanobody) fusion proteins were constructed with eukaryotic expression vector pFase. For SP34-HC, the complete molecule includes the IL2 secretion signal peptide and SP-34-VH and SP-34-CH 1; for the construction of SP34-LC-CD28(Nanobody), SP34-LC and anti-CD 28 Nanobody are connected in series by PCR, and the complete molecules comprise IL2 secretion signal peptide, SP34-LC, (G)₄S)₃Connecting peptide and anti-CD 28 nano antibody. SP34-HC or a different SP34-LC-CD28(Nanobody) gene was double digested with EcoRI and XhoI and cloned into pFase vector using T4 DNA ligase, or the vector was constructed using a homologous recombination kit.

4.2 eukaryotic expression

Inoculation 1.5X 10⁶In 250mL shake flask 50mL medium was placed 293F cells. Shaking at 37 deg.C and 165rpm in shaking incubator with 5% CO2 concentration for 24h, counting cells after 24h, and adjusting cell density to 3 × 10⁶mL, the plasmids SP34-HC and SP34-LC-CD28(Nanobody) (25. mu.g: 25. mu.g) constructed in the above 4.1 step were gently mixed in 5mL of opti-MEM, and then allowed to stand at room temperature for 5min, 125. mu.L (1:2.5) of PEI40000 was added to 10mL of opti-MEM, and then allowed to stand at room temperature for 5min, and the mixture of the plasmid and PEI was mixed, and then allowed to stand at room temperature for 20 min. And dropwise adding the mixed solution into 50mL of 293F cell culture solution, gently shaking the culture bottle while dropwise adding, uniformly mixing, putting into a shaking table, and transfecting for 72 hours. After transfection, centrifugation is carried out for 5min at 100g, supernatant is taken, 50mL of culture medium is added into a culture flask for re-suspension culture, centrifugation is carried out for 5min at 3000rpm, and supernatant is taken. The cell supernatants were mixed twice.

4.3 protein purification

The cell supernatant obtained in step 1.2 was centrifuged at 15000rpm for 30min at 4 ℃ for 50mL, and the supernatant was collected, filtered through a 0.45 μm filter and placed on ice for further use. 3mL (20% ethanol/Protein G1: 1) of Protein G was loaded onto the column, washed 3 times with Binding buffer, and pressed against the surface of the resin using a pad. The Protein G column was equilibrated with 20mL Binding buffer. Samples were loaded every 10mL and run through the Protein G column at a constant rate (approximately 0.5 mL/min). The Protein G column was washed at 40mL Binding buffer constant speed (about 1 mL/min). First, 10% eluent volume Buffer was added to the collection tube of the Elution tube and the column was eluted 4 times with an Elution Buffer (5mL eluted once until the protein concentration could not be quantified). The collected protein samples were concentrated using an Amicon Ultra-15 centrifuge filter, centrifuged at 3000rpm for 20 minutes at 4 ℃ and the protein concentration was measured using nanodrop.

Example 5 validation of the binding Capacity of the CD3/CD28 fusion antibody to the CD28 antigen

5.1 this round of experiments used two antigens, experiment group CD28-Fc, control group 4-1BB-Fc, both in parallel, each with a blank control, CD3-Fab as negative control.

5.2 coating: the coating solution was coated with CD28 antigen (10ug/mL) overnight at 4 ℃ and 2 controls of 2 parallel, 2 blank wells were included in each group. At the same time, antigen 4-1BB (10ug/mL) was coated as an irrelevant antigen control.

5.3 sealing: add blocking solution 200uL to each well, and block for 1h at 37 ℃.

5.4 sample adding: and setting a blank hole and a sample hole to be detected. All samples were diluted with blocking solution. The initial 5-fold gradient dilution of 200nM concentration was performed at 7 spots (100 uL/well) in the initial well at 550 uL followed by 6 wells at 440 uL of blocking solution per well, followed by 5-fold sequential back-gradient dilutions with blocking solution only in the blank control wells. Incubate at 37 ℃ for 1 h.

5.5 Secondary antibody action: the liquid was discarded (patted hard on paper) and 200 uL/well PBST wash was repeated 3 times. A secondary HRP-Anti kappa light chain antibody (100. mu.L, 1:5000) was added to each well and incubated at 37 ℃ for 1 hour.

5.6 color development: discarding liquid in the hole, spin-drying, washing the plate for 5 times with 200 uL/hole PBST, adding 100 μ L of chromogenic substrate TMB in each hole, adding a coating film on the ELISA plate, incubating at 37 deg.C in dark place, adding 2M H in each hole₂SO₄The reaction was stopped with 50. mu.L of stop solution, whereupon the blue color turned immediately yellow. The OD of each well was immediately measured at a wavelength of 450nm using a microplate reader. OD measurement data were obtained by GraphPad Prism treatment, and the results are shown in FIGS. 8a and 8b, in which FIG. 8a shows the measurement results of the binding ability of the CD3/CD28 fusion antibody and the control CD3 antibody to the target antigen CD28, and FIG. 8b shows the measurement results of the binding ability of the CD3/CD28 fusion antibody and the control CD3 antibody to the unrelated antigen 4-1 BB. The experimental group has obvious antigen binding capacity and is obviously different from the control group.

Example 6 binding experiments of the CD3/CD28 fusion antibody to K562/CD28 cells

6.1 two cell lines, K562/CD28 cells in the experimental group and K562/4-1BB cells in the control group, were used in the experiment, and the experiments were parallel, blank controls were set for each group, and CD3-Fab was used as a negative control.

6.2 two cells were collected, counted, adjusted to a cell density of 0.3 million/well and 100. mu.L/well for a concentration of 3million/mL, and samples were prepared in 7 wells of 100. mu.L cells per well.

6.3 sample preparation: in an EP tube, CD28 nano antibody is diluted to an initial concentration of 200nM, and five-fold gradient dilution is carried out for 7 points, namely, 110 mu L of antibody solution is sucked in the previous tube, 440 mu L of FACS is added backwards, gradient dilution is carried out sequentially, and 100 mu L of each sample is divided equally after configuration.

The cell tray is centrifuged at 6.42000 rpm for 5min, the supernatant medium is aspirated and then stained, namely, the diluted sample is added into a pore plate according to 100 mu L/pore for resuspending cells, the cell is incubated for 1h on ice, the tray is centrifuged at 2000rpm for 5min and then the cell is aspirated and washed twice, and a secondary antibody is prepared during centrifugation, wherein the secondary antibody is Goat anti-human kappa-AF467, and the final concentration of the secondary antibody is 1 mu g/mL.

6.5 add diluted Goat anti-human kappa-AF 467100. mu.L/well, resuspend cells on ice and incubate for 1h in dark. Add 200u L FACS washing free antibody, centrifugal to remove the washing twice, again 200u L FACS buffer heavy suspension.

6.6 flow detection: flow assay data were processed using GraphPad Prism and results are shown in fig. 9a and 9b, where fig. 9a is a measure of the binding capacity of the CD3/CD28 fusion antibody and the control CD3 antibody to target cells, K562/CD28 cells, and fig. 9b is a measure of the binding capacity of the CD3/CD28 fusion antibody and the control CD3 antibody to unrelated cells, K562/4-1 BB. The experimental group has obvious cell surface antigen binding capacity and is obviously different from the control group.

Example 7 validation of the activation Capacity of the CD3/CD28 fusion antibody on human PBMCs

7.1 this round of experiments was activated with PBMC, in parallel in two sets, each set was provided with a blank control, and CD3-Fab was used as a negative control.

7.2 cell recovery: the PBMC cells were removed from liquid nitrogen, put into a 37 ℃ water bath for rapid thawing, diluted with RPMI1640 medium (medium: cells > 10: 1), centrifuged at 1500rpm for 5min, the supernatant removed, the medium resuspended the PBMC cells, and the above washing was repeated once, adjusting the PBMC cell density to 1.5 milli-cells/mL, and the 48-well plate was used at 200. mu.L/well, i.e., 0.3 milli-cells/well.

7.3 CD3/CD28 fusion antibody: activator concentration gradients, three gradients, two in parallel, were configured to add 50nM, 5nM, 0.5nM and 0nM CD3/CD28 antibody per well, 100. mu.L/well.

7.4 cell volume 1/10 antibody (20. mu.L) was added per well at a concentration, and CD3/CD28 antibody was added.

Culturing at 7.537 deg.C for 24h, transferring cells to U-shaped plate, centrifuging at 2000rpm for 5min, removing supernatant, adding 0.5 μ g/mLCD25/CD69 double antibody to stain for 1h, resuspending negative control group with FACS, washing once at 2000rpm for 5min, and performing flow-type CD25/CD69 detection on 96-well plate. The streaming data was processed using GraphPad Prism, and the results are shown in fig. 10. The experimental group has obvious T cell activation capability and is obviously different from the control group.

The invention obtains 42 positive sequences by eukaryotic expression CD28 antigen, immune bank screening and antibody titer detection by CD28 immune alpaca and two rounds of colony enzyme-linked immunosorbent screening.

According to the sequence similarity comparison result, the invention divides the screened 42 sequences into 8 groups according to the similarity between groups being more than 93%, and each group selects a representative sequence for functional verification: (1) CD28 antigen binding capacity assay; (2) detecting the binding capacity of the cell surface antigen; (3) and (4) detecting the activation capacity of the T cells. The anti-CD 28 nano antibody designed by the invention takes CD3-Fab as a framework, so that the CD3-Fab is taken as a negative control in an antibody function verification experiment. The anti-CD 28 nano antibody designed by the invention has better CD28 antigen binding capacity, cell surface antigen binding capacity and activation capacity on human PBMC (peripheral blood mononuclear cell), and has obvious difference with a CD3-Fab negative control group.

The foregoing examples further illustrate the present invention but are not to be construed as limiting thereof. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Sequence listing

<110> Shenzhen institute of university of Beijing

<120> anti-CD 28 nano antibody, coding gene and application

<130>

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Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Arg Ile

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Leu Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 2

<211> 121

<212> PRT

<213> Artificial sequence

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Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Arg Ile

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Leu Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 3

<211> 121

<212> PRT

<213> Artificial sequence

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Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Arg Val

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Leu Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 4

<211> 121

<212> PRT

<213> Artificial sequence

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Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Arg Ile

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Leu Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 5

<211> 121

<212> PRT

<213> Artificial sequence

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Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Arg Ile

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Gly Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Leu Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 6

<211> 126

<212> PRT

<213> Artificial sequence

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Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 126

<212> PRT

<213> Artificial sequence

<400> 7

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 8

<211> 126

<212> PRT

<213> Artificial sequence

<400> 8

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 9

<211> 126

<212> PRT

<213> Artificial sequence

<400> 9

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Gly His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 126

<212> PRT

<213> Artificial sequence

<400> 10

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 11

<211> 126

<212> PRT

<213> Artificial sequence

<400> 11

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 12

<211> 126

<212> PRT

<213> Artificial sequence

<400> 12

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 13

<211> 126

<212> PRT

<213> Artificial sequence

<400> 13

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 14

<211> 126

<212> PRT

<213> Artificial sequence

<400> 14

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 15

<211> 126

<212> PRT

<213> Artificial sequence

<400> 15

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 16

<211> 126

<212> PRT

<213> Artificial sequence

<400> 16

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Ala Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 17

<211> 126

<212> PRT

<213> Artificial sequence

<400> 17

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Leu Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 18

<211> 126

<212> PRT

<213> Artificial sequence

<400> 18

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Val Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 19

<211> 126

<212> PRT

<213> Artificial sequence

<400> 19

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Thr

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu Tyr Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 20

<211> 126

<212> PRT

<213> Artificial sequence

<400> 20

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser His

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Leu His Ile Gly Asp Tyr Pro Gly Ser Tyr Gln

100 105 110

Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 21

<211> 129

<212> PRT

<213> Artificial sequence

<400> 21

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Ile Phe Gly Phe

20 25 30

Tyr Ala Met Gly Trp Tyr Arg Gln Ala Pro Glu Lys Gln Arg Glu Leu

35 40 45

Val Ala Thr Ile Thr Ser Gly Ser Ile Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Gln Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Arg Leu Ala Pro Leu Glu Ser Phe Asp Ser Thr Trp Arg Arg Val Gly

100 105 110

Gly Thr Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 22

<211> 129

<212> PRT

<213> Artificial sequence

<400> 22

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asn Ile Phe Gly Phe

20 25 30

Tyr Ala Met Gly Trp Tyr Arg Gln Ala Pro Glu Lys Gln Arg Glu Leu

35 40 45

Val Ala Thr Ile Thr Ser Gly Ser Ile Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Gln Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Arg Leu Ala Pro Leu Glu Ser Phe Asp Ser Thr Trp Arg Arg Val Gly

100 105 110

Gly Thr Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 23

<211> 121

<212> PRT

<213> Artificial sequence

<400> 23

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Gly Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 24

<211> 121

<212> PRT

<213> Artificial sequence

<400> 24

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 25

<211> 121

<212> PRT

<213> Artificial sequence

<400> 25

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Leu Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Glu Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 26

<211> 121

<212> PRT

<213> Artificial sequence

<400> 26

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 27

<211> 121

<212> PRT

<213> Artificial sequence

<400> 27

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Asn Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Ile Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 28

<211> 121

<212> PRT

<213> Artificial sequence

<400> 28

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 29

<211> 121

<212> PRT

<213> Artificial sequence

<400> 29

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Gly Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 30

<211> 121

<212> PRT

<213> Artificial sequence

<400> 30

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 31

<211> 120

<212> PRT

<213> Artificial sequence

<400> 31

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser

115 120

<210> 32

<211> 121

<212> PRT

<213> Artificial sequence

<400> 32

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 33

<211> 121

<212> PRT

<213> Artificial sequence

<400> 33

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Leu Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 34

<211> 121

<212> PRT

<213> Artificial sequence

<400> 34

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Gly Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 35

<211> 121

<212> PRT

<213> Artificial sequence

<400> 35

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Gly Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 36

<211> 121

<212> PRT

<213> Artificial sequence

<400> 36

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Ile Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 37

<211> 121

<212> PRT

<213> Artificial sequence

<400> 37

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile

20 25 30

Glu Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Val Leu

35 40 45

Val Ala Gly Ile Thr Ser Asn Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Ile Gly Gly Leu Pro Gly Gly Ser Trp Ala

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 38

<211> 121

<212> PRT

<213> Artificial sequence

<400> 38

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Ile Phe Ser Ile

20 25 30

Asn Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Asp Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Arg Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Ala Gln Thr Arg Arg Val Gly Gly Leu Pro Gly Gly Ser Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 39

<211> 116

<212> PRT

<213> Artificial sequence

<400> 39

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Pro Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Gly Thr Ser Gly Pro Ile Phe Gly His

20 25 30

Tyr Thr Met Asp Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu

35 40 45

Val Ala Gly Ile Thr Asp Gly Gly Leu Leu Asn Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

His Val Arg Val Leu Phe Ser His Tyr Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 40

<211> 134

<212> PRT

<213> Artificial sequence

<400> 40

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Arg Ala

20 25 30

Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Ser Pro Phe Phe Asp Ser Ser Trp Tyr Asp Gly Trp Ala

100 105 110

Pro Gly Gly Ala Gly Arg Gly Glu Tyr Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Gln Val Thr Val Ser Ser

130

<210> 41

<211> 125

<212> PRT

<213> Artificial sequence

<400> 41

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Trp Gly Thr Leu Ser Ser

20 25 30

Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Ala Glu Arg Glu Trp

35 40 45

Val Gly Gly Ile Ser Arg Arg Gly Asp Ser Ile Phe Tyr Gly Asp Ser

50 55 60

Val Lys Gly Arg Thr Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Ala Ser Glu Arg Leu Val Ala Arg Leu Ser Trp Glu Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 42

<211> 124

<212> PRT

<213> Artificial sequence

<400> 42

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser

20 25 30

Tyr Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Arg Phe Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Pro

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ala Arg Pro Phe Gly Trp Gly Thr Ala Gly Glu Pro Tyr Asn

100 105 110

Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 43

<211> 363

<212> DNA

<213> Artificial sequence

<400> 43

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGGCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTAG ATCTATCTTC AGAATCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGTATTACTA GTGGCGGTCT GACAAACTAT 180

GCAGGCTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGG ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCACAGACT 300

CGACGGCTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 44

<211> 363

<212> DNA

<213> Artificial sequence

<400> 44

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTAG ATCTATCTTC AGAATCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGTATTACTA GTGGCGGTCT GACAAACTAT 180

GCAGGCTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGG ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCACAGACT 300

CGACGGCTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 45

<211> 363

<212> DNA

<213> Artificial sequence

<400> 45

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTAG ATCTATCTTC AGAGTCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGTATTACTA GTGGCGGTCT GACAAACTAT 180

GCAGGCTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGG ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCACAGACT 300

CGACGGCTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 46

<211> 363

<212> DNA

<213> Artificial sequence

<400> 46

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTAG ATCTATCTTC AGAATCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGTATTACTA GTGGCGGTCT GACAAACTAT 180

GCAGGCTCCG TGAAGGGCCG ATTCGCCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGG ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCACAGACT 300

CGACGGCTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 47

<211> 363

<212> DNA

<213> Artificial sequence

<400> 47

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGTCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTAG ATCTATCTTC AGAATCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGTATTACTA GTGGCGGTCT GACAAACTAT 180

GCAGGCTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGG ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCACAGACT 300

CGACGGCTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 48

<211> 378

<212> DNA

<213> Artificial sequence

<400> 48

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 49

<211> 378

<212> DNA

<213> Artificial sequence

<400> 49

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC GGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 50

<211> 378

<212> DNA

<213> Artificial sequence

<400> 50

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACCCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 51

<211> 378

<212> DNA

<213> Artificial sequence

<400> 51

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC GGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 52

<211> 378

<212> DNA

<213> Artificial sequence

<400> 52

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAACCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 53

<211> 378

<212> DNA

<213> Artificial sequence

<400> 53

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACATGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 54

<211> 378

<212> DNA

<213> Artificial sequence

<400> 54

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGGAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCGGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 55

<211> 378

<212> DNA

<213> Artificial sequence

<400> 55

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGAGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 56

<211> 378

<212> DNA

<213> Artificial sequence

<400> 56

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCGGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAGCGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 57

<211> 378

<212> DNA

<213> Artificial sequence

<400> 57

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGGACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 58

<211> 378

<212> DNA

<213> Artificial sequence

<400> 58

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCGCATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 59

<211> 378

<212> DNA

<213> Artificial sequence

<400> 59

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA GGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCTGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 60

<211> 378

<212> DNA

<213> Artificial sequence

<400> 60

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGTAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 61

<211> 378

<212> DNA

<213> Artificial sequence

<400> 61

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACA CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTATACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 62

<211> 378

<212> DNA

<213> Artificial sequence

<400> 62

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGGG CTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACTCACCTTC AGTCACTATG CCATGGGCTG GTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTGG TAGCACATAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCAA GAACACGGTG 240

TTTCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACTG TGCAGCAGGA 300

GTCTTACACA TTGGCGACTA TCCGGGCTCC TACCAGTATG ACTACTGGGG CCAGGGGACC 360

CAGGTCACCG TCTCCTCA 378

<210> 63

<211> 387

<212> DNA

<213> Artificial sequence

<400> 63

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG TAGCCTCTGG AGACATCTTC GGGTTCTATG CCATGGGCTG GTACCGCCAG 120

GCTCCAGAGA AGCAGCGCGA GTTGGTCGCG ACCATTACAA GTGGTAGTAT AACAGACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCCAAATC TCCAGAGACA ACGCCAAGAA CACAGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAG GCTCGCACCC 300

CTTGAATCGT TCGATAGTAC CTGGCGTCGC GTAGGGGGGA CAGGCATGGA CTACTGGGGC 360

AAAGGGACCC AGGTCACCGT CTCCTCA 387

<210> 64

<211> 387

<212> DNA

<213> Artificial sequence

<400> 64

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG TAGCCTCTGG AAACATCTTC GGGTTCTATG CCATGGGCTG GTACCGCCAG 120

GCTCCAGAGA AGCAGCGCGA GTTGGTCGCG ACCATTACAA GTGGTAGTAT AACAGACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCCAAATC TCCAGAGACA ACGCCAAGAA CACAGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAG GCTCGCACCC 300

CTTGAATCGT TCGATAGTAC CTGGCGTCGC GTAGGGGGGA CAGGCATGGA CTACTGGGGC 360

AAAGGGACCC AGGTCACCGT CTCCTCA 387

<210> 65

<211> 363

<212> DNA

<213> Artificial sequence

<400> 65

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAAA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTG GTGGTGGTCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGGA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGACT ATTACTGTAA TGCCCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 66

<211> 363

<212> DNA

<213> Artificial sequence

<400> 66

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCATCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 67

<211> 363

<212> DNA

<213> Artificial sequence

<400> 67

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC TGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG AGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 68

<211> 363

<212> DNA

<213> Artificial sequence

<400> 68

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 69

<211> 363

<212> DNA

<213> Artificial sequence

<400> 69

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGAATTACTA GTAACGGCCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGGCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGATTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 70

<211> 363

<212> DNA

<213> Artificial sequence

<400> 70

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACG ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 71

<211> 363

<212> DNA

<213> Artificial sequence

<400> 71

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAAA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTG GTGGTGGTCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGACT ATTACTGTAA TGCCCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 72

<211> 363

<212> DNA

<213> Artificial sequence

<400> 72

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGCGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 73

<211> 360

<212> DNA

<213> Artificial sequence

<400> 73

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

<210> 74

<211> 363

<212> DNA

<213> Artificial sequence

<400> 74

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTCGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 75

<211> 363

<212> DNA

<213> Artificial sequence

<400> 75

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCTGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 76

<211> 363

<212> DNA

<213> Artificial sequence

<400> 76

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTCGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAAA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTG GTGGTGGTCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGACT ATTACTGTAA TGCCCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 77

<211> 363

<212> DNA

<213> Artificial sequence

<400> 77

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCGGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAAA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTG GTGGTGGTCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGACT ATTACTGTAA TGCCCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 78

<211> 363

<212> DNA

<213> Artificial sequence

<400> 78

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAGA TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGCATTACTA GTGGTGGTTT AACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGATGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 79

<211> 363

<212> DNA

<213> Artificial sequence

<400> 79

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG AAGCATCTTC AGTATCGAAG TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGT GTTGGTCGCA GGAATTACTA GTAATGGCCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCGAGAA CACGGTGTAT 240

CTGCAAATGA ACAGCCTGAA ACCTGAGGAC ACGGGCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGATTG GGGGTTTGCC CGGGGGTTCC TGGGCCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 80

<211> 363

<212> DNA

<213> Artificial sequence

<400> 80

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC AGCCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG TGGCATCTTC AGTATCAATC TCATGGGCTG GTACCGCCAG 120

GCTCCAGGGA AGCAGCGCGA GTTGGTCGCA GGTATTACTG ATGGTGGTCT CACAAACTAT 180

GCAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCGAATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTACTGTAA TGCGCAGACT 300

CGACGGGTTG GGGGTTTGCC CGGGGGTTCC TGGGGCCAGG GGACCCAGGT CACCGTCTCC 360

TCA 363

<210> 81

<211> 348

<212> DNA

<213> Artificial sequence

<400> 81

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGCTTGGTGC CCGCTGGGGG GTCTCTGAGA 60

CTCTCCTGTG GAACCTCTGG TCCTATCTTC GGGCACTATA CCATGGACTG GTACCGCCAG 120

GCTCCAGGGA ACCAGCGCGA ATTGGTCGCA GGGATTACTG ACGGTGGTCT CCTTAACTAT 180

GAAGACTCCG TGAAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CACGGTGTAT 240

CTGCAGATGA ACAGCCTGAA ACCTGAGGAC ACGGCCGTCT ATTATTGCCA TGTCAGAGTG 300

CTTTTCAGTC ACTATTGGGG CCAGGGGACC CAGGTCACCG TCTCCTCA 348

<210> 82

<211> 402

<212> DNA

<213> Artificial sequence

<400> 82

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGG GCTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACGCACCTTC AGGGCCTATG CCATGGGCT GGTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGCT GGAGTGGTG GTAGCACCTAC 180

TATGCAGACT CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCA AGAACACGGTG 240

TATCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACT GTGCAGCCTCC 300

CCTTTTTTTG ATAGTAGCTG GTACGATGGC TGGGCCCCCG GCGGGGCCG GGCGGGGGGAG 360

TATGACTACT GGGGCCAGGG GACCCAGGTC ACCGTCTCCT CA 402

<210> 83

<211> 375

<212> DNA

<213> Artificial sequence

<400> 83

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGG ACTCTCTGAGA 60

CTCTCTTGTG CAGCCTCTTG GGGCACCCTC AGTAGCTATT ACATGGGCT GGTTCCGCCAG 120

GCTCCAGGGG CGGAGCGTGA GTGGGTAGGG GGTATTAGCC GGAGGGGTG ATAGTATTTTC 180

TATGGAGACT CCGTGAAGGG CCGAACCACC ATCTCCAGAG ACAACGCCA AGAACACGGTG 240

TATCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACT GTGCAGCCGCG 300

AGTGAACGAC TCGTCGCACG ACTGTCGTGG GAGTATGACT ACTGGGGCC AGGGGACCCAG 360

GTCACCGTCT CCTCA 375

<210> 84

<211> 372

<212> DNA

<213> Artificial sequence

<400> 84

ATGGCGGTGC AGCTGGTGGA GTCTGGGGGA GGATTGGTGC AGGCTGGGG GCTCTCTGAGA 60

CTCTCCTGTG CAGCCTCTGG ACGTACCTTC AGTAGCTATA CCATGGCCT GGTTCCGCCAG 120

GCTCCAGGGA AGGAGCGTGA GTTTGTAGCA GCTATTAGGT TCAGTGGTG GTAGCACATAC 180

TATGCAGACC CCGTGAAGGG CCGATTCACC ATCTCCAGAG ACAACGCCA AGAACACGGTG 240

TATCTGCAAA TGAACAGCCT GAAACCTGAG GACACGGCCG TTTATTACT GTGCAGCTAGG 300

CCGTTCGGTT GGGGTACCGC GGGCGAGCCT TATAACTACT GGGGCCAGG GGACCCAGGTC 360

ACCGTCTCCT CA 372

<210> 85

<211> 109

<212> PRT

<213> Artificial sequence

<400> 85

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Asp His Leu Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Asp Lys Ala Ala Leu Thr Ile Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 86

<211> 108

<212> PRT

<213> Artificial sequence

<400> 86

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

1 5 10 15

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

20 25 30

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

35 40 45

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

50 55 60

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

65 70 75 80

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

85 90 95

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 87

<211> 15

<212> PRT

<213> Artificial sequence

<400> 87

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 88

<211> 228

<212> PRT

<213> Artificial sequence

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys

225

<210> 89

<211> 327

<212> DNA

<213> Artificial sequence

<400> 89

CAAGCTGTTG TGACCCAAGA ACCGAGTCTG ACCGTGTCTC CGGGCGGCAC CGTTACGCTG 60

ACCTGTCGCT CCTCTACCGG CGCAGTCACC ACGAGCAACT ATGCAAATTG GGTGCAGCAA 120

AAACCGGATC ATCTGTTTCG TGGCCTGATT GGCGGTACGA ACAAACGTGC GCCGGGGACC 180

CCGGCACGTT TTTCGGGCAG CCTGCTGGGC GATAAAGCAG CACTGACCAT CAGTGGTGCG 240

CAGCCGGAAG ATGAAGCAGA ATATTACTGC GCTCTGTGGT ATAGCAACCT GTGGGTCTTT 300

GGCGGTGGCA CGAAACTGAC CGTTCTG 327

<210> 90

<211> 324

<212> DNA

<213> Artificial sequence

<400> 90

AAACGAACTG TGGCTGCACC ATCTGTCTTC ATCTTCCCGC CATCTGATGA GCAGTTGAAA 60

TCTGGAACTG CCTCTGTCGT GTGCCTGCTG AATAACTTCT ATCCCAGAGA GGCCAAAGTA 120

CAGTGGAAGG TGGATAACGC CCTCCAATCG GGTAACTCCC AGGAGAGTGT CACAGAGCAG 180

GACAGCAAGG ACAGCACCTA CAGCCTCAGC AGCACCCTGA CGCTGAGCAA AGCAGACTAC 240

GAGAAACACA AAGTCTACGC CTGCGAAGTC ACCCATCAGG GCCTGTCCTC GCCCGTCACA 300

AAGAGCTTCA ACAGGGGAGA GTGT 324

<210> 91

<211> 45

<212> DNA

<213> Artificial sequence

<400> 91

GGTGGAGGCG GTTCAGGCGG AGGTGGCTCT GGCGGTGGCG GATCC 45

<210> 92

<211> 684

<212> DNA

<213> Artificial sequence

<400> 92

GAAGTCCAGC TGGTTGAATC TGGTGGCGGT CTGGTTCAAC CGGGCGGTTC GCTGAAACTG 60

AGCTGCGCAG CTTCTGGCTT TACGTTCAAC ACCTATGCGA TGAATTGGGT TCGCCAGGCC 120

TCAGGCAAAG GTCTGGAATG GGTCGCTCGT ATTCGCTCGA AATATAACAA TTACGCAACC 180

TATTACGCTG ATAGCGTGAA AGACCGTTTC ACCATCAGTC GCGATGACTC CAAAAACACG 240

CTGTATCTGC AAATGAATAG CCTGAAAACG GAAGATACCG CGGTCTATTA CTGCGTGCGT 300

CATGGCAACT TTGGTAATTC TTATGTGAGC TGGTTCGCCT ACTGGGGCCA GGGTACGCTG 360

GTTACCGTCA GCTCTGCTAG CACCAAGGGC CCATCGGTCT TCCCCCTGGC ACCCTCCTCC 420

AAGAGCACCT CTGGGGGCAC AGCGGCCCTG GGCTGCCTGG TCAAGGACTA CTTCCCCGAA 480

CCGGTGACGG TGTCGTGGAA CTCAGGCGCC CTGACCAGCG GCGTGCACAC CTTCCCGGCT 540

GTCCTACAGT CCTCAGGACT CTACTCCCTC AGCAGCGTGG TGACTGTGCC CTCTAGCAGC 600

TTGGGCACCC AGACCTACAT CTGCAACGTG AATCACAAGC CCAGCAACAC CAAGGTGGAC 660

AAGAAAGTTG AGCCCAAATC TTGT 684

Claims (8)

1. An anti-CD 28 nanobody, which is the protein of the following a) or b):

a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

b) protein which is derived from a) and is related to the specific recognition of CD28 by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence of the sequence 1 in the sequence table.

2. The anti-CD 28 nanobody according to claim 1, wherein the protein derived from a) is a protein consisting of the amino acid sequence shown in the sequence 2-42.

3. A gene encoding the anti-CD 28 nanobody of claim 1.

4. The encoding gene according to claim 3, wherein the encoding gene is a gene of 1) or 2) or 3) as follows:

1) the nucleotide sequence is sequence 43 in the sequence table;

2) a DNA molecule which is hybridized with the DNA fragment defined by the sequence 43 under strict conditions and codes for a protein which specifically recognizes the CD28 related protein;

3) has more than 90 percent of homology with the gene of 1) or 2) and encodes a DNA molecule which can specifically recognize CD28 related protein.

5. The encoding gene of claim 4, wherein the nucleotide sequence of the gene of 2) or 3) is the sequence 44-84 of the sequence listing.

6. A recombinant expression vector comprising the coding gene of claims 3-5.

7. A recombinant strain comprising the coding gene of claims 3-5.

8. The anti-CD 28 nanobody of claim 1 or 2, for the prevention or treatment of tumor, immunodeficiency diseases and infectious diseases.

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