CN111057148A - Single-domain antibody aiming at bovine serum albumin BSA and derivative protein thereof - Google Patents

Abstract

The invention relates to the technical field of biotechnology or immunology, and relates to a single domain antibody aiming at bovine serum albumin BSA and a derivative protein thereof. The amino acid sequence of the CDR1 of the single domain antibody is shown as SEQ ID NO:77-SEQ ID NO: 95; the amino acid sequence of the CDR2 is shown in SEQ ID NO:96-SEQ ID NO: 124; the amino acid sequence of the CDR3 is shown in SEQ ID NO. 125-SEQ ID NO. 141 and SEQ ID NO. 195-SEQ ID NO. 206. The invention has the beneficial effects that: the single-domain antibody is used as a BSA detection antibody, and has higher affinity compared with the traditional monoclonal antibody; compared with the polyclonal antibody in the existing detection kit, the kit has the advantages of single component, better batch stability and easier quality control, thereby ensuring higher reliability of the actual BSA content detection result.

Description

Single-domain antibody aiming at bovine serum albumin BSA and derivative protein thereof

Technical Field

The invention relates to the technical field of biotechnology or immunology, and relates to a single domain antibody aiming at bovine serum albumin BSA and a derivative protein thereof.

Background

Antibodies (abs), i.e., immunoglobulins (immunoglobulins, igs), are glycoproteins in blood and tissue fluid, are produced by plasma cells generated by proliferation and differentiation of B cells after stimulation by antigens, are mainly present in body fluids such as serum, can be specifically bound to the corresponding antigens, and are important effector molecules for mediating humoral immunity. In addition to antibodies which mediate specific humoral immune responses as important effector molecules, antibodies play an important role in the prevention and treatment of diseases, particularly infectious diseases, and Behring created serotherapy has thus gained medical and physiological nobel. Thereafter, antibodies artificially prepared by polyclonal, monoclonal and genetically engineered antibody techniques are gradually applied to clinical use. In 2011 alone, antibody drugs are getting across the global drug market at a sales of $ 480 billion, accounting for 34.4% of the entire biopharmaceutical market. By 2017, 76 therapeutic antibody drugs approved by FDA and EMA of the european union in the united states are mainly used for the treatment of diseases such as tumors and autoimmune diseases.

It is also obvious that an antibody, which is a molecule capable of specifically recognizing a certain protein (antigen), has a strong effect in the prevention and treatment of diseases, and is also useful as a detection tool in the field of examination and scientific research. Enzyme-linked immunosorbent assay (ELISA) is an important method for detecting by using antibody/antigen combination. At present, the common ELISA detection methods include a direct method, an indirect method, a competition method and the like, and the basic principles are antigen-antibody binding reaction. The ELISA kit based on the method has wide application in inspection and quarantine and other occasions. The affinity and stability of the antibody in the partial inspection and quarantine method are very strict, so that the used antibody has very high affinity and very good stability with the antigen specifically bound with the antibody, and the application and storage environment of the antibody is more extensive so as to be more beneficial to the popularization of the detection method. Traditional monoclonal antibodies perform well in terms of antigen affinity, and although the stability of the monoclonal antibodies can be improved by adding a protective agent or changing a buffer solution, the storage conditions of the monoclonal antibodies are still relatively strict, and the monoclonal antibodies are generally transported or stored at low temperature. The single-domain antibody is more excellent in affinity and stability, the affinity of the single-domain antibody with an antigen can reach a pM level before affinity maturation is carried out on the single-domain antibody, the room temperature stability of the single-domain antibody is good, and the transportation and storage cost can be reduced. In addition, the single-domain antibody can be expressed in a large amount of solubility in a prokaryotic expression system, the production cost is reduced, and the stability of antibody batches is improved.

Bovine Serum Albumin (BSA), also known as the fifth component of bovine serum, contains 583 amino acid residues, has a molecular weight of 66.430kDa, and an isoelectric point of 4.7. Bovine serum albumin has wide application in biochemical experiments, such as in western blotting (western blot) as a blocking agent; BSA is added into the enzyme digestion buffer solution, and the enzyme is protected by increasing the concentration of protein in the solution. Prevent enzyme decomposition and nonspecific adsorption, and reduce the denaturation of some enzymes and adverse environmental factors such as heat, surface tension and chemical factors. Bovine serum is an important additive component in a mammalian cell culture medium, and the proportion of bovine serum in the culture medium can reach 10% or even higher, for example, Vero cells used for producing rabies vaccines for human use need to be added with 10% of fetal bovine serum during culture. BSA is an important component in fetal calf serum, so the content of BSA in the culture medium is very high, an important quality control link of a rabies vaccine for human use is to detect the content of BSA in a vaccine product, and the BSA belongs to a foreign body for the human body and can cause strong immune reaction of an injector so as to cause other adverse reactions, so that the minimization of BSA in the vaccine product becomes a necessary standard for vaccine manufacturers, and the quantitative or qualitative analysis of the specific content of BSA in the vaccine product is a necessary way for the process. At present, the lowest limit of detection of BSA content in biological products at home and abroad is 5ng/mL, and antibodies used in the detection reagents are all polyclonal antibodies (rabbit source), and compared with the murine monoclonal antibody, the rabbit polyclonal antibody has the advantages of short preparation period and slightly strong affinity, and has obvious defects: 1) the single batch preparation amount is limited, and the antibody with good effect on the single batch cannot be obtained for a long time; 2) the inter-batch difference is too large; 3) the components are relatively complex, and the quality control is difficult to control. Therefore, the method can ensure the quality of the antibody and the quality control of components, and can ensure the affinity of the antibody and the antigen to reach the current BSA detection lower limit, thereby becoming two important targets for the development of the BSA detection antibody at present.

Disclosure of Invention

In order to overcome the defects, the invention aims to provide a single domain antibody specifically aiming at BSA protein and establish an anti-BSA single domain antibody and a derivative protein thereof, firstly, the single domain antibody with high specificity can be obtained through prokaryotic expression or yeast expression, the yield and the purification efficiency are high, the batch quality is stable, secondly, the affinity of the single domain antibody has certain advantages compared with the traditional antibody, the antigen capture capacity and the detection effect can be improved, the detection sensitivity is improved, in addition, the single domain antibody belongs to a monoclonal antibody, the unicity and the controllability of a detection reagent can be ensured, and the detection standard of a detection kit can be better controlled.

The single domain antibody is a monoclonal antibody, has generally higher affinity and better stability than the traditional monoclonal antibody, and also has the advantages of the traditional monoclonal antibody. Therefore, the present invention utilizes the above advantages to obtain antibodies against BSA, and these obtained antibodies can be used as a detection tool in common ELISA experiments to qualitatively analyze BSA in various biological products, and can also be developed into ELISA detection kits to quantitatively analyze BSA in human biological products (including but not limited to human rabies vaccines).

In order to solve the technical problems, the invention adopts the following technical scheme: a single domain antibody directed against bovine serum albumin BSA, wherein the sequence of the single domain antibody comprises complementarity determining regions CDRs; the complementarity determining region CDRs include the amino acid sequences of CDR1, CDR2, and CDR 3; the amino acid sequence of the CDR1 is shown as any one of SEQ ID NO:77-SEQ ID NO: 95; the amino acid sequence of the CDR2 is shown in any one of SEQ ID NO:96-SEQ ID NO: 124; the amino acid sequence of the CDR3 is shown in any one of SEQ ID NO 125-SEQ ID NO 141 and SEQ ID NO 195-SEQ ID NO 206.

All the above sequences may be replaced with a sequence having "at least 80% homology" with the sequence or a sequence having only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

A single domain antibody against bovine serum albumin BSA, the sequence of said single domain antibody comprising complementarity determining regions CDRs; the complementarity determining region CDRs include the amino acid sequences of CDR1, CDR2, and CDR 3; the sequence of the CDR of the single domain antibody is one of the following (1) to (34):

(1) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 113, CDR3 shown in SEQ ID NO. 127 (corresponding to SEQ ID NO. 1);

(2) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 109, CDR3 shown in SEQ ID NO. 126 (corresponding to SEQ ID NO. 2);

(3) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 113, CDR3 shown in SEQ ID NO. 197 (corresponding to SEQ ID NO. 3);

(4) CDR1 shown in SEQ ID NO:93, CDR2 shown in SEQ ID NO:117, CDR3 shown in SEQ ID NO:197 (corresponding to SEQ ID NO. 4);

(5) CDR1 shown in SEQ ID NO:94, CDR2 shown in SEQ ID NO:106, CDR3 shown in SEQ ID NO:128 (corresponding to SEQ ID NO. 5);

(6) CDR1 shown in SEQ ID NO:88, CDR2 shown in SEQ ID NO:105, CDR3 shown in SEQ ID NO:129 (corresponding to SEQ ID NO. 6);

(7) CDR1 shown in SEQ ID NO:88, CDR2 shown in SEQ ID NO:104, CDR3 shown in SEQ ID NO:130 (corresponding to SEQ ID NO. 7);

(8) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:118, CDR3 shown in SEQ ID NO:131 (corresponding to SEQ ID NO. 8-9);

(9) CDR1 shown in SEQ ID NO:92, CDR2 shown in SEQ ID NO:96, CDR3 shown in SEQ ID NO:132 (corresponding to SEQ ID NO. 10);

(10) CDR1 shown in SEQ ID NO:92, CDR2 shown in SEQ ID NO:98, CDR3 shown in SEQ ID NO:133 (corresponding to SEQ ID NO. 11);

(11) CDR1 shown in SEQ ID NO:92, CDR2 shown in SEQ ID NO:123, CDR3 shown in SEQ ID NO:134 (corresponding to SEQ ID NO. 12);

(12) CDR1 shown in SEQ ID NO:84, CDR2 shown in SEQ ID NO:121, CDR3 shown in SEQ ID NO:125 (corresponding to SEQ ID NO. 13);

(13) CDR1 shown in SEQ ID NO:77, CDR2 shown in SEQ ID NO:120, CDR3 shown in SEQ ID NO:135 (corresponding to SEQ ID NO. 14);

(14) CDR1 shown in SEQ ID NO:77, CDR2 shown in SEQ ID NO:114, CDR3 shown in SEQ ID NO:135 (corresponding to SEQ ID NO. 15);

(15) CDR1 shown in SEQ ID NO:83, CDR2 shown in SEQ ID NO:122, CDR3 shown in SEQ ID NO:136 (corresponding to SEQ ID NO. 16);

(16) CDR1 shown in SEQ ID NO. 91, CDR2 shown in SEQ ID NO. 110, CDR3 shown in SEQ ID NO. 137 (corresponding to SEQ ID NO. 17);

(17) CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:99, CDR3 shown in SEQ ID NO:138 (corresponding to SEQ ID NO. 18);

(18) CDR1 shown in SEQ ID NO:87, CDR2 shown in SEQ ID NO:116, CDR3 shown in SEQ ID NO:139 (corresponding to SEQ ID NO. 19);

(19) CDR1 shown in SEQ ID NO:93, CDR2 shown in SEQ ID NO:117, CDR3 shown in SEQ ID NO:196 (corresponding to SEQ ID NO:20, 22);

(20) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 112, CDR3 shown in SEQ ID NO. 195 (corresponding to SEQ ID NO. 21);

(21) CDR1 shown in SEQ ID NO:93, CDR2 shown in SEQ ID NO:115, CDR3 shown in SEQ ID NO:197 (corresponding to SEQ ID NO.23, 24);

(22) CDR1 shown in SEQ ID NO:93, CDR2 shown in SEQ ID NO:113, CDR3 shown in SEQ ID NO:198 (corresponding to SEQ ID NO.25, 26);

(23) CDR1 shown in SEQ ID NO. 95, CDR2 shown in SEQ ID NO. 108, CDR3 shown in SEQ ID NO. 199 (corresponding to SEQ ID NO. 27);

(24) CDR1 shown in SEQ ID NO:81, CDR2 shown in SEQ ID NO:124, CDR3 shown in SEQ ID NO:200 (corresponding to SEQ ID NO. 28);

(25) CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:100, CDR3 shown in SEQ ID NO:201 (corresponding to SEQ ID NO: 29);

(26) CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:101, CDR3 shown in SEQ ID NO:138 (corresponding to SEQ ID NO. 30);

(27) CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:101, CDR3 shown in SEQ ID NO:202 (corresponding to SEQ ID NO. 31);

(28) CDR1 shown in SEQ ID NO:80, CDR2 shown in SEQ ID NO:102, CDR3 shown in SEQ ID NO:202 (corresponding to SEQ ID NO. 32);

(29) CDR1 shown in SEQ ID NO:82, CDR2 shown in SEQ ID NO:107, CDR3 shown in SEQ ID NO:203 (corresponding to SEQ ID NO. 33);

(30) CDR1 shown in SEQ ID NO:92, CDR2 shown in SEQ ID NO:103, CDR3 shown in SEQ ID NO:204 (corresponding to SEQ ID NO. 34);

(31) CDR1 shown in SEQ ID NO:85, CDR2 shown in SEQ ID NO:97, CDR3 shown in SEQ ID NO:205 (corresponding to SEQ ID NO. 35);

(32) CDR1 shown in SEQ ID NO. 89, CDR2 shown in SEQ ID NO. 111, CDR3 shown in SEQ ID NO. 206 (corresponding to SEQ ID NO. 36);

(33) CDR1 shown in SEQ ID NO:90, CDR2 shown in SEQ ID NO:113, CDR3 shown in SEQ ID NO:140 (corresponding to SEQ ID NO. 37);

(34) CDR1 shown in SEQ ID NO:77, CDR2 shown in SEQ ID NO:119, and CDR3 shown in SEQ ID NO:141 (corresponding to SEQ ID NO. 38).

Preferably, the single domain antibody is a single domain antibody of SEQ ID NO 1-38 of bovine serum albumin BSA.

The coding sequences of the single domain antibody chains are respectively shown in SEQ ID NO: 39-76.

The antibody sequence further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the FR regions of the framework regions are:

FR1 shown in any one of SEQ ID NOs 142-156; FR2 shown in any one of SEQ ID NO: 157-167; FR3 shown in any one of SEQ ID NO: 168-192; FR4 shown in any one of SEQ ID NOS 193-194. The sequences of all of FR1, FR2, FR3 and FR4 above may be replaced by conservative sequence variants thereof.

The single domain antibody is a VHH comprising only antibody heavy chains and no antibody light chains.

It is still another object of the present invention to provide a nucleotide molecule encoding the single domain antibody against bovine serum albumin BSA, which has a nucleotide sequence shown in SEQ ID NO: 39-76, respectively.

The invention also provides an expression vector which comprises the nucleotide molecule.

The invention also relates to a host cell which can express the single-domain antibody aiming at the bovine serum albumin BSA, or comprises the expression vector.

The invention also provides an Fc fusion antibody of the single-domain antibody of bovine serum albumin BSA.

The invention also discloses the application of the single-domain antibody specifically aiming at the BSA protein in a BSA-ELISA detection kit, and a semi-quantitative and quantitative detection method for detecting the BSA content in a human biological product by using the single-domain antibody aiming at the BSA protein.

The invention finally discloses the application of the single-domain antibody aiming at the BSA protein in the separation and purification of the BSA protein fusion protein.

Compared with the prior art, the invention has the beneficial effects that: the single-domain antibody is used as a BSA detection antibody, and has higher affinity compared with the traditional monoclonal antibody; compared with the polyclonal antibody in the existing detection kit, the kit has the advantages of single component, better batch stability and easier quality control, thereby ensuring higher reliability of the actual BSA content detection result.

In the subsequent research, researchers can also fuse the target protein and the BSA tag protein by a genetic engineering method, then the fusion protein is expressed or secreted in cells, the BSA fusion protein is detected by using the single-domain antibody specific to the BSA protein in the invention, and the target protein in the cells or the culture medium is further purified, so that the high-purity BSA fusion protein is obtained.

Drawings

FIG. 1 is an electrophoresis diagram of PCR products from left to right showing the detection of the insertion rate of a target fragment in a constructed single domain antibody phage display library, wherein lanes 1 and 27 are DNA molecule markers, and lanes 3-26 and 28-37 are PCR products of different clones randomly picked from a constructed single domain antibody library against BSA protein;

FIG. 2 shows the results of biopanning the constructed library of single domain antibodies, where P/N is the number of monoclonal bacteria grown after phage eluted from positive wells infected with TG1 bacteria/the number of monoclonal bacteria grown after phage eluted from negative wells infected with TG1 bacteria, which parameter gradually increases after enrichment has occurred; I/E is the total amount of phage added to the positive well in each round of biopanning/the total amount of phage eluted from the positive well in each round of biopanning, and the parameter gradually approaches to 1 after enrichment occurs;

FIG. 3 shows SDS-PAGE of crude protein solutions purified by nickel column affinity chromatography, from left to right, lanes 1, 15, 25 and 39 represent protein markers, and lanes 2-14, 16-24, 26-37 and 40-42 represent single domain antibodies purified by nickel ion-coupled agarose resin one-step affinity chromatography; wherein VHH1-38 corresponds to SEQ ID NO.1-38 in sequence;

FIG. 4 shows the results of ELISA experiments with human Fc fused single domain antibodies expressed in mammalian expression systems, wherein rabbit monoclonal antibodies are obtained from Cell Signaling Technology under the designation 23053S, which are positive reference antibodies for the entire experiment; CN201810002538 is a nanobody sequence against BSA in the patent with application number 201810002538.4; in the experiment, except for the rabbit-derived monoclonal antibody as a positive reference, other recombinant single-domain antibodies are expressed, identified and purified according to the specific schemes in the embodiments 5, 6 and 7; the results of the figure prove that under the same experimental conditions, compared with the nano antibody in the 201810002538.4 patent, the single domain antibody and the derived protein thereof disclosed by the invention are better in the binding capacity with BSA;

FIG. 5 shows the results of ELISA experiments with single domain antibodies against BSA protein expressed in prokaryotic expression system, all of which were expressed and purified according to example 4;

FIG. 6 is a schematic representation of the sequence around the multiple cloning site of the company's vector No. RJK-V4-hFc.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

Nanobodies against bovine serum albumin BSA

Single domain antibodies (sdabs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Thus, single domain antibodies comprise a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which do not naturally contain a light chain), single domain antibodies derived from conventional antibodies, and engineered antibodies.

Single domain antibodies may be derived from any species, including mouse, human, camel, llama, goat, rabbit and cow. For example, naturally occurring VHH molecules may be derived from antibodies provided by species in the family camelidae (e.g. camel, dromedary, llama and guanaco). Like intact antibodies, single domain antibodies are capable of selectively binding to a particular antigen. Single domain antibodies may contain only the variable domains of immunoglobulin chains, with CDR1, CDR2 and CDR3, and the framework regions. The molecular weight of the nanobody is only about 12-15kDa, which is much smaller than the molecular weight of a normal antibody consisting of two heavy chains and two light chains (150-160 kDa).

It is worth mentioning that, in the present invention, the nanobody against bovine serum albumin BSA can be obtained from the sequence with high sequence homology with CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" to the sequences in (1) - (34), or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" can all achieve the objectives of the invention (i.e., to derive proteins).

In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in (1) - (34), e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, may also achieve the objects of the invention. Indeed, in determining the degree of sequence identity between two amino acid sequences or in determining the CDR1, CDR2, and CDR3 combination in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution, which may generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art, for example conservative amino acid substitutions are those in which one or a few amino acids within the following groups (a) - (d) are replaced by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: asp, Asn, Glu, Gln; (b) polar positively charged residues: his, Arg, Lys; (c) aromatic residue: phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val and Cys. Particularly preferred conservative amino acid substitutions are as follows: asp substituted by Glu; asn is replaced by Gln or His; glu is substituted with Asp; gln is substituted by Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys substituted by Arg, Gln; phe is replaced by Met, Leu, Tyr; trp is substituted by Tyr; tyr is substituted by Phe, Trp; ala substituted by Gly or Ser; ser substituted by Thr; thr is substituted by Ser; gly by Ala or Pro; met is substituted by Leu, Tyr or Ile; leu is substituted by Ile or Val; ile is substituted by Leu or Val; val is substituted by Ile or Leu; cys is substituted with Ser. In addition, the skilled person knows that the creativity of single domain antibodies is found in the CDR1-3 region, whereas the framework region sequence FR1-4 is not unalterable and the sequence of FR1-4 may take the form of conservative sequence variants of the sequences disclosed in the present invention.

The method adopts BSA protein with the purity of 98% purchased from Sigma-Aldrich company to immunize the Alexan bactrian camel, extracts the bactrian camel peripheral blood lymphocytes after 7 times of immunization, and establishes a single-domain antibody library of the BSA protein. In the antibody screening process, BSA polypeptide is coupled with Biotin (BSA-Biotin), neutravidin protein is coupled on an enzyme label plate, the BSA polypeptide is indirectly displayed on the surface of the enzyme label plate by utilizing the characteristic of combination of the Biotin and the neutravidin, so that the antigen epitope of the BSA polypeptide is exposed, then a single domain antibody gene library (camel heavy chain antibody phage display gene library) after BSA protein immunization is screened by utilizing a phage display technology, and a single domain antibody strain which can be efficiently expressed in escherichia coli is obtained.

And (3) expressing the single domain antibody obtained by screening in escherichia coli, purifying through agarose coupled with nickel ions, and analyzing the purified nano antibody.

The invention will be further illustrated with reference to the following specific examples.

Example 1: construction of a single domain antibody library against BSA protein:

(1) mixing 1mg BSA antigen and Freund's adjuvant in equal volume, immunizing one inner Mongolia alashana humped camel once per week for 7 times, and stimulating B cells to express specific nano antibody in the immunization process; (2) after the immunization is finished, extracting 100ml of camel peripheral blood lymphocytes and extracting total RNA; (3) synthesizing cDNA and amplifying VHH by using nested PCR; (4) digesting 20 mu g of pMECS phage display vector and 10 mu g of VHH by using restriction enzymes Pst I and Not I and connecting the two fragments; (5) the ligation products were transformed into electroporation competent cells TG1, and a BSA protein phage display library was constructed and the library volume was determined, the size of the library volume being about 2X 10⁹(ii) a Meanwhile, the correct insertion rate of the target fragment in the created library is detected through colony PCR, the result of colony PCR is shown in figure 1, 34 clones are randomly selected to be used as colony PCR, and the result shows that the insertion rate reaches 90%.

Example 2: screening for single domain antibodies to BSA protein:

(1) culturing 200 μ L of recombinant TG1 cells in 2 × TY culture medium, adding 40 μ L of helper phage VCSM13 to infect TG1 cells, culturing overnight to amplify phage, precipitating phage with PEG/NaCl the next day, centrifuging, and collecting amplified phage; (2) will diluteNaHCO released at 100mM pH 8.3₃500ug of the neutral avidin protein is coupled on an enzyme label plate, is placed at 4 ℃ overnight, and is provided with a negative control hole at the same time; (3) the next day 100. mu.L of Biotin-labeled BSA protein (BSA-Biotin) was added, incubated at room temperature for 2 hours, and 100. mu.L of PBS was added to the negative control wells; (4) after 2 hours, 100 mu L of 3% skim milk is added, and the mixture is sealed for 2 hours at room temperature; (5) after the end of blocking, 100. mu.l of the amplified phage library (approx.2X 10) was added¹¹Individual phage particles), and reacting for 1h at room temperature; (6) after 1 hour of action, wash 5 times with PBS + 0.05% Tween-20 to wash away unbound phage; (7) the phage specifically bound to BSA protein was dissociated with trypsin at a final concentration of 25mg/mL and infected with E.coli TG1 cells in logarithmic growth phase, cultured at 37 ℃ for 1h, phage was generated and collected for the next round of screening, and the same screening process was repeated for 3 rounds to gradually obtain enrichment, the enrichment effect is shown in FIG. 2, the P/N value was about 10000 from 2.2 to the third round of the first round, and I/E substance was about 13 from 13605 to the third round of the first round, demonstrating that the library has a very significant enrichment for BSA.

Example 3: screening of specific positive clones by phage enzyme-linked immunosorbent assay (ELISA):

(1) carrying out 3 rounds of screening on BSA protein according to the single-domain antibody screening method, after screening is finished, aiming at that the phage enrichment factor of the BSA protein reaches more than 10 (about 10000), selecting 400 single colonies from positive clones obtained by screening, respectively inoculating the single colonies into a 96-deep-well plate of a TB culture medium containing 100 mu g/mL ampicillin, setting a blank control, culturing at 37 ℃ until the logarithmic phase, adding IPTG with the final concentration of 1mM, and culturing at 28 ℃ overnight; (2) obtaining a crude antibody by using a permeation cracking method; neutral avidin protein was diluted to 100mM NaHCO, pH 8.3₃Neutralizing, coating 100 mu g of neutral avidin protein in an enzyme label plate at 4 ℃ overnight, and adding 100ug of BSA-Biotin protein into the enzyme label plate the next day; (3) taking 100uL of the crude antibody extract obtained in the step, transferring the crude antibody extract to an ELISA plate added with an antigen, and incubating for 1h at room temperature; (4) unbound antibody was washed away with PBST, 100ul of Mouse anti-HA tag antibody (murine anti-HA antibody, ThermoFisher) diluted at 1:2000 was added, and incubated at room temperature for 1 h; (5) washing unbound with PBSTAdding 100ul of Anti-Rabbit HRP conjugate (goat Anti-Rabbit horseradish peroxidase labeled antibody, purchased from ThermoFisher) diluted at a ratio of 1:20000 to the antibody, and incubating at room temperature for 1 h; (6) washing away unbound antibodies by PBST, adding horseradish peroxidase developing solution, reacting at 37 ℃ for 15min, adding a stop solution, and reading an absorption value at a wavelength of 450nm on an enzyme-labeling instrument; (7) when the OD value of the sample hole is more than 5 times of that of the control hole, judging the sample hole as a positive cloning hole; (8) the bacteria of the positive cloning wells were shaken in LB medium containing 100. mu.g/. mu.l ampicillin to extract plasmids and to sequence.

Gene sequences of the respective clones were analyzed by Vector NTI according to sequence alignment software, and strains having the same CDR1, CDR2, and CDR3 sequences were regarded as the same clones, and strains having different sequences were regarded as different clones, and finally single domain antibodies (SEQ ID nos. 1 to 38) specific to BSA protein were obtained. The amino acid sequence of the antibody is in a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the whole VHH is formed. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally single domain antibody protein is obtained.

Wherein SEQ ID NO.1-38 are antibody numbers 1A5, 2C9, 1B7, 1D8, 1B4, 1D10, 2D8, 1C6, 1D6, 1B2, 1C2, 2C11, 2C6, 2D10, 2D11, 2D6, 1C9, 2A11, 2C10, 1A10, 2B4, 2A7, 2B9, 2D2, 1B1, 1B6, 2A4, 1A2, 1C1, 1C3, 2C5, 2D5, 1C10, 1B3, 2B3, 1D11, 2B1, 1D3 in sequence.

The sequences of CDR1-3 of the 38 single domain antibody sequences are shown in Table 1-2, and the sequences of FR1-3 of the 38 single domain antibodies are shown in Table 3-4. Among the 38 single-domain antibodies, the FR4 sequences of the 36 antibodies were all SEQ ID NO:194(GQGTQVTVSS) except that the FR4 sequences of the antibodies 2C6 and 2B3 were SEQ ID NO:193 (GRGTQVTVSS).

CDR1-2 sequences of Table 138 Single Domain antibodies

CDR3 sequences of the 238 single domain antibodies

FR1 and FR2 sequences of 338 single-domain antibodies

FR3 sequences of Table 438 Single Domain antibodies

Example 4: purification and expression of specific single-domain antibody of BSA protein in host bacterium escherichia coli

(1) The plasmids (pMECS-VHH) of the different clones obtained by the above sequencing analysis were electrically transformed into E.coli HB2151, spread on LB + amp + glucose, i.e., a culture plate containing ampicillin and glucose, and cultured overnight at 37 ℃; (2) selecting a single colony to be inoculated in 5mL LB culture solution containing shore penicillin, and carrying out shake culture at 37 ℃ overnight; (3) inoculating 1mL of overnight cultured strain to 330mL of TB culture medium, shake culturing at 37 deg.C, and culturing to OD_600nmAdding 1M IPTG when the value reaches 0.6-0.9, and carrying out shake culture at 28 ℃ overnight; (4) centrifuging, collecting Escherichia coli, and obtaining crude antibody extractive solution by use of osmotic bursting method; (5) the antibody was purified by nickel column affinity chromatography, and the purified single domain antibody was shown in FIG. 3.

Example 5: construction of Fc fusion antibody eukaryotic expression vector of specific single domain antibody of BSA protein

The target sequence obtained in the example 3 is subcloned into a eukaryotic expression vector, (1) the antibody screened in the example 3 is subjected to Sanger sequencing to obtain a nucleotide sequence thereof, (2) the nucleotide sequence (SEQ ID NO.39-76) after codon optimization is respectively cloned into a vector RJK-V4-hFC designed and modified by the company in a sequence synthesis mode (namely, the SEQ ID NO.39-76 is respectively inserted into a multi-cloning site MCS of the vector RJK-V4-hFC, and the construction process of the RJK-V4-hFc vector is as described in the example 12), (3) the recombinant eukaryotic expression vector constructed by the company is transformed into Escherichia coli 5 α to be cultured for plasmid amplification and endotoxin removal, (4) the plasmid after amplification is subjected to sequencing sequence identification, and (5) the recombinant vector after error correction is prepared for subsequent cell transfection and eukaryotic expression.

Example 6: fc fusion antibody of specific single domain antibody of BSA protein expressed in suspension ExpicHO-S cells

(1) 3 days before transfection at 2.5X 10⁵/ml cell passage and expanded culture ExpCHO-S^TMCells, calculated required cell volume transferred to ExpCHO filled with fresh preheated 120ml (final volume)^TMIn a 500ml shake flask of expression medium; to achieve a cell concentration of about 4X 10⁶-6×10⁶Viable cells/mL; (2) one day before transfection, ExpicHO-S^TMCell dilution to 3.5X 10⁶Viable cells/mL, cells were cultured overnight; (3) on the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 7X 10⁶-10×10⁶Viable cells/mL; (4) with fresh ExpiCHO preheated to 37 ℃^TMExpression media cells were diluted to 6X 10⁶Viable cells/mL. The calculated required cell volume was transferred to ExpicHO containing fresh preheated 100ml (final volume)^TMIn a 500ml shake flask of expression medium; (5) expifeacmine was mixed by gentle inversion^TMCHO reagent, 3.7ml OptiPRO^TMDilution of Expifeacylamine in culture Medium^TMCHO reagent, swirling or mixing; (6) with refrigerated 4ml OptiPRO^TMDiluting plasmid DNA with a culture medium, and mixing uniformly; (7) incubating the Expifactamine CHO/plasmid DNA complex for 1-5 minutes at room temperature, then gently adding the Expifactamine CHO/plasmid DNA complex into the prepared cell suspension, and gently swirling the shake flask in the adding process; (8) cells were incubated at 37 ℃ with 8% CO₂Carrying out shake culture in humidified air; (9) day 1 post transfection (18-22 hours later) 600ul Expifeacylamine was added^TMCHO Enhancer and 24ml ExpicHO feed. (10) Supernatants were collected approximately 8 days after transfection (cell viability below 70%).

Example 7: expression of Fc fusion antibodies of specific single domain antibodies of BSA proteins in 293F cells in suspension

Recombinant single domain antibody expression experimental protocol (taking 500ml shake flask as an example):

1. 3 days before transfection at 2.5X 10⁵The 293F cells were passaged and expanded in culture and the calculated required cell volume was transferred to a 500ml shake flask containing fresh pre-warmed 120ml (final volume) OPM-293CD05 Medium. The cell concentration is about 2X 10⁶-3×10⁶Viable cells/mL.

2. On the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 2X 10⁶-3×10⁶Viable cells/mL.

3. Cells were diluted to 1X 10 with pre-warmed OPM-293CD05 Medium⁶Viable cells/mL. The required cell volume was calculated and transferred to a 500ml shake flask containing fresh pre-warmed 100ml (final volume) of medium.

4. Diluting PEI (1mg/ml) reagent with 4ml of Opti-MEM medium, and swirling or blowing to mix evenly; the plasmid DNA was diluted with 4ml Opt-MEM medium, vortexed, mixed well, and filtered through a 0.22um filter tip. Incubate at room temperature for 5 min.

5. Diluted PEI reagent was added to the diluted DNA and mixed by inversion. The PEI/plasmid DNA complex was incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, with gentle swirling of the flask during the addition.

6. Cells were incubated at 37 ℃ with 5% CO₂And shake culturing at 120 rpm.

7. 5ml OPM-CHO PFF05 feed was added at 24h, 72h post transfection.

8. Supernatants were collected approximately 7 days after transfection (cell viability below 70%).

Example 8: purification of human Fc recombinant Single Domain antibodies

1. Filtering the protein expression supernatant obtained in example 6 or 7 with a 0.45 μm disposable filter to remove insoluble impurities;

2. performing affinity chromatography purification on the filtrate by using a Protein purifier, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human-derived Fc and Protein A;

3. passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is bound to the packing;

4. washing the impurity protein bound on the column by low-salt and high-salt buffer solutions;

5. eluting the target protein bound on the column by using a low pH buffer solution;

6. adding the eluent into Tris-HCl solution with pH9.0 rapidly for neutralization;

7. dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein has a purity of 95% or more and a concentration of 0.5mg/mL or more, and storing at low temperature for later use.

Example 9: primary detection of binding capacity of prokaryotic expression nano antibody and BSA (bovine serum albumin)

Primarily detecting the affinity of the single-domain antibody purified in the example 4 to BSA; firstly, the affinity of the recombinant single-domain antibody and BSA is measured by using a quantitative standard substance, and the steps are as follows;

(1) coating 100ng/100 μ L of standard BSA sample on ELISA plate;

(2) sealing the coated plate by using skimmed milk powder;

(3) adding the single domain antibody against BSA obtained in example 4;

(4) adding a monoclonal antibody of a mouse anti-HA label marked by HRP;

(5) adding a chromogenic substrate TMB;

(6) adding a stop solution to terminate the reaction;

(7) by measuring the OD450 values (the results are shown in FIG. 5), it can be seen that all 38 single-domain antibodies against BSA have stronger affinity with BSA.

Example 10: ELISA detection of affinity of recombinant single-domain antibody specifically aiming at BSA protein and BSA and drawing of detection standard curve

The affinity of the recombinant antibody obtained by purification in example 8 (the Fc fusion antibody of the specific single domain antibody prepared in example 6 was purified in example 8) for BSA was preliminarily determined; firstly, the affinity of the recombinant single-domain antibody and BSA is measured by using a quantitative standard substance, and the steps are as follows;

(1) coating 100ng/100 μ L of standard BSA sample on ELISA plate;

(2) sealing the coated plate by using skimmed milk powder;

(3) adding the recombinant single domain antibody against BSA obtained in example 8;

(4) adding a detection antibody of mouse anti-human Fc marked by HRP;

(5) adding a chromogenic substrate TMB;

(6) adding a stop solution to terminate the reaction;

(7) measuring the OD450 value (the result is shown in FIG. 4);

the results of this figure demonstrate that under the same experimental conditions, the single domain antibody and its derived protein of the present invention have better binding ability with BSA than the nano antibody of the' 201810002538.4 patent.

And then selecting the recombinant antibody with the best ELISA binding force to draw a BSA content standard curve.

(1) Coating the standard BSA sample of 0-100ng/100 μ L on the ELISA plate;

(2) sealing the coated plate in the step (1) by using skimmed milk powder;

(3) adding the recombinant single-domain antibody with the best effect (namely, the ELISA binding force is the best, and 2D6 is selected) aiming at the BSA;

(4) adding a detection antibody (HRP mark) specific to human Fc;

(5) adding a chromogenic substrate TMB;

(6) adding a stop solution to terminate the reaction;

(7) measuring OD450 value, drawing a curve graph of the relation between BSA content and OD450 according to the result, selecting a linear range section of the curve graph to perform linear regression calculation, and using the linear range section as a standard curve to determine the BSA content in other samples.

Example 11: application of specific single-domain antibody of BSA protein in BSA-ELISA kit (double antibody sandwich method)

Detecting the content of BSA in a target sample by a double-antibody sandwich method through the single-domain antibodies obtained in the example 8 and the example 4;

firstly, a standard substance is used for measuring and drawing a standard curve of BSA content and OD450, and the steps are as follows;

(1) coating the single-domain antibody obtained by identification and purification in the embodiment 8 on an ELISA plate as a capture antibody;

(2) sealing the coated plate by using skimmed milk powder;

(3) adding a standard sample for incubation;

(4) adding the single-domain antibody obtained in example 4 as a detection antibody (with an HA tag), and incubating;

(5) incubation with anti-HA antibody (HRP labeled);

(6) adding a chromogenic substrate TMB;

(7) adding a stop solution to terminate the reaction;

(8) measuring an OD450 value, and drawing a standard curve;

and then detecting the sample to be detected based on the standard curve obtained in the step.

Example 12: construction of nano antibody eukaryotic expression vector RJK-V4-hFc

The target vector RJK-V4-hFC for the general use of the nano-antibody is modified by fusing an Fc segment in a heavy chain coding sequence (NCBIAccess No.: AB776838.1) of human IgG on the basis of an invitrogen commercial vector pcDNA3.4 (vector data link https:// Assets. thermofisher. com/TFS-Assets/LSG/vitamins/pcdna 3-4 _ topo _ ta _ cloning _ kit _ man. pdf), namely the vector comprises a Hinge region (Hinge) CH2 and a CH3 region of the IgG heavy chain. The specific modification scheme is as follows:

(1) selecting restriction sites XbaI and AgeI on pcDNA3.4;

(2) introducing a Multiple Cloning Site (MCS) and a 6 XHis tag at the 5 'end and the 3' end of the Fc fragment coding sequence, respectively, by means of overlapping PCR, as shown in FIG. 6;

(3) amplifying the fragment by using a pair of primers with XbaI and AgeI enzyme cutting sites respectively in a PCR mode;

(4) the recombinant DNA fragments in pcDNA3.4 and (3) are digested with restriction enzymes XbaI and AgeI respectively;

(5) and (3) connecting the vector and the insert after enzyme digestion under the action of T4 ligase, then transforming the connection product into escherichia coli, amplifying, sequencing and verifying to obtain the recombinant plasmid.

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.

Sequence listing

<110> Nanjing Congjiekang Biotech Co., Ltd

<120> Single Domain antibody against bovine serum Albumin BSA and derived proteins thereof

<130>GXM2019120201

<141>2019-12-02

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Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

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

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

50 55 60

Lys Gly Arg Phe Thr Val 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 Met Tyr Tyr Cys

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Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Pro Leu Asp Pro Arg

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Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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

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Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

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

50 55 60

Lys Gly Arg Phe Thr Val 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 Met Tyr Tyr Cys

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Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Gln Leu Asp Pro Arg

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Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

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

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

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

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Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Pro Leu Asp Pro Arg

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Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

Ala Val Ile Tyr Thr Thr Gly Gly Arg Thr Tyr 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 Met Tyr Tyr Cys

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Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Pro Leu Asp Pro Arg

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Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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

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

20 25 30

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

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Ala Ala Ile Tyr Ile Ala Ser Gly Asn Ser Gly Ser Thr Tyr Tyr Ala

50 55 60

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

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

85 90 95

Tyr Tyr Cys Ala Ser Asp Leu Ser Arg Arg Asn Trp Leu Pro Pro Leu

100 105 110

Val Pro Gly Lys Tyr Ser Leu Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser

130

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Tyr Gly Tyr Gly Gly Ser Thr Tyr 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 Ile Pro Glu Asp Thr Ala Ser Tyr Tyr Cys

85 90 95

Ala Ser Asp Gln Ser Arg Arg Ser Trp Leu Ser Pro Leu Val Pro Gly

100 105 110

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

115 120 125

<210>7

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<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Tyr Thr Gly Ser Val Asn

20 25 30

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

35 40 45

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

50 55 60

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

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

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Ala Ser Asp Gln Ser Arg Arg Ser Trp Phe Pro Pro Leu Val Pro Gly

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

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<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

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Ala Ala Val Arg Arg Pro Trp Arg Leu Asp Ser Leu Asp Pro Ser Ser

100 105 110

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

115 120 125

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<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Gly Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Ala Val Arg Arg Pro Trp Arg Leu Asp Ser Leu Asp Pro Ser Ser

100 105 110

Phe Arg Tyr Trp Gly GlnGly Thr Gln Val Thr Val Ser Ser

115 120 125

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

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Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Asn Tyr Met

20 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala

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

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Arg Phe Thr Ile Ser Gln Asp Asn Gly Lys Asn Thr Leu His Leu Gln

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

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Gly Ser His Tyr Ser Gly Tyr Ser Leu Ser Pro Gly Arg Tyr Lys Tyr

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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

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<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Asn Tyr Met

20 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala

35 40 45

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Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Thr Asn Leu Gln

65 70 75 80

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

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Gly Ser His Tyr Ala Gly Tyr Ser Met Ser Pro Gly Arg Tyr Lys Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

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<212>PRT

<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Asn Tyr Met

20 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val

35 40 45

Met His Thr Gly Ser Gly Arg Ala Tyr Tyr Val Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His Leu Gln

65 70 75 80

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

85 90 95

Gly Ser His Tyr Ser Gly Tyr Ser Met Ser Pro Gly Arg Tyr Lys Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>13

<211>128

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ala Asp Arg Arg Asn

20 25 30

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

35 40 45

Ala Ala Leu Gly Ile Val Ser Gly Phe Thr His Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Arg Met Ile Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Arg Thr Arg Val Leu Ile Ser Trp Phe Leu Asn Pro

100 105 110

Asp Lys Tyr Asn Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>14

<211>133

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Val Gln Leu GlnGlu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Thr Pro Phe Ser Leu Ser Lys Thr Thr Arg Leu Ala Lys Phe

100 105 110

Gly Ser Leu Asn Glu Asn Glu Tyr Asp Leu Trp Gly Gln Gly Thr Gln

115 120 125

Val Thr Val Ser Ser

130

<210>15

<211>133

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Thr Pro Phe Ser Leu Ser Lys Thr Thr Arg Leu Ala Lys Phe

100 105 110

Gly Ser Leu Asn Glu Asn Glu Tyr Asp Leu Trp Gly Gln Gly Thr Gln

115 120 125

Val Thr Val Ser Ser

130

<210>16

<211>123

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1 5 10 15

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

20 25 30

Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>17

<211>130

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>17

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

1 510 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Val Glu Arg Arg Gly Gly Ser Cys Tyr Thr Arg Tyr

100 105 110

Lys Tyr Ala Ala Phe Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser

130

<210>18

<211>129

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

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

1 510 15

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

20 25 30

Asp Leu Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val

35 40 45

Ser Ile Ile Arg Arg Asp Gly Ser Pro Tyr His Thr Tyr Tyr Pro Asp

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Ala Ala Asp Pro Ser Arg Tyr Tyr Ser Asp Tyr Gly Ala Pro

100 105 110

Gly Cys Gly Phe Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210>19

<211>130

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>19

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Phe Ser Ser Asp

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu Gly Val

35 40 45

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

85 90 95

Ala Ala Arg Arg Ser Ser Gly Gly Tyr Cys Tyr Ile Gly Ser Asp Phe

100 105 110

Gln Ile Ser Leu Tyr Asn Asn Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser

130

<210>20

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>20

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

Ala Val Ile Tyr Thr Thr Gly Gly Arg Thr Tyr 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 Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Gln Leu Asp Pro Arg

100 105 110

Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>21

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>21

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

Ala Val Ile Tyr Thr Phe Gly Gly Arg Thr Tyr 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 Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Gln Leu Asp Pro Arg

100 105 110

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

115 120 125

<210>22

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>22

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

Ala Val Ile Tyr Thr Thr Gly Gly Arg Thr Tyr 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 Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Gln Leu Asp Pro Arg

100 105 110

Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>23

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>23

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Val 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 Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Pro Leu Asp Pro Arg

100 105 110

Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>24

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>24

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Val 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 Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Pro Leu Asp Pro Arg

100 105 110

Lys Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>25

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>25

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Gln Leu Asp Pro Arg

100 105 110

Lys Tyr Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>26

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>26

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Gln Leu Asp Pro Arg

100 105 110

Lys Tyr Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>27

<211>127

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>27

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Tyr Ser Gly Gly Gly Ser Thr Tyr 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 Ile Pro Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Asp Gln Ser Arg Arg Ser Trp Phe Ser Pro Leu Val Pro Gly

100 105 110

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

115 120 125

<210>28

<211>126

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>28

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Val Val Arg Ser Asp Gly Thr Thr Tyr Tyr Pro Pro Ser Val Arg

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Pro Ser Pro Tyr Tyr Arg Asp Tyr Gly Ala Pro Gly Cys Gly

100 105 110

Phe Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>29

<211>126

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>29

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Val Ile Arg Ser Asp Gly Arg Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Pro Ser Arg Arg Tyr Ser Asp Tyr Gly Ala Pro Gly Cys Gly

100 105 110

Phe Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>30

<211>126

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>30

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Val Ile Arg Ser Asp Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Pro Ser Arg Tyr Tyr Ser Asp Tyr Gly Ala Pro Gly Cys Gly

100 105 110

Phe Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>31

<211>126

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>31

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Val Ile Arg Ser Asp Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Pro Ser Arg Tyr Tyr Ser Asp Tyr Gly Ala Pro Gly Cys Gly

100 105 110

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

115 120 125

<210>32

<211>126

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>32

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Val Ile Arg Ser Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val Lys

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asp Pro Ser Arg Tyr Tyr Ser Asp Tyr Gly Ala Pro Gly Cys Gly

100 105 110

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

115 120 125

<210>33

<211>129

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>33

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

1 5 10 15

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

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Ser Ser Gly Gly Tyr Cys Tyr Ile Gly Ser Asp Phe Gln

100 105 110

Ile Ser Leu Phe Asn Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210>34

<211>123

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>34

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

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Asn Tyr Met

20 25 30

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala

35 40 45

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

50 55 60

Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Thr Tyr Leu Gln

65 70 75 80

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

85 90 95

Gly Ser His Tyr Ser Gly Tyr Ser Ile Ser Pro Gly Arg Tyr Lys Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>35

<211>128

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>35

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Arg Met Ile Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Arg Thr Arg Val Ile Ile Gly Trp Phe Leu Asn Pro

100 105 110

Asp Lys Tyr Asn Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210>36

<211>121

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>36

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr His Arg Met Ala

20 25 30

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile

35 40 45

Tyr Thr Asp Val Gly His Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg

50 55 60

Phe Thr Ile Ala Gln Asp Asn Ala Asn Asn Thr Ala Tyr Leu Gln Met

65 70 75 80

Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Ala

85 90 95

Arg Gly Gly Tyr Ser Trp Arg Lys Val Asn Glu Tyr Asn Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210>37

<211>128

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>37

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr Leu Ser Asn Lys

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Arg Gly Tyr Gly Gln Asn Trp Tyr Arg Thr Leu Arg Thr

100 105 110

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

115 120 125

<210>38

<211>133

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>38

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Thr Pro Phe Ser Leu Ser Lys Ala Thr Arg Leu Ala Lys Phe

100 105 110

Gly Ser Leu Asn Glu Asn Glu Tyr Asp Leu Trp Gly Gln Gly Thr Gln

115 120 125

Val Thr Val Ser Ser

130

<210>39

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggatc cctgagactg 60

gcctgtgccg cctccggcta cacctacagc tccagctaca tgggctggtt caggcaggcc 120

cctggcaagg agagagaggg cgtggccgtg atctacaccg gcggcggaag aacctactac 180

gccgattccg tgaagggcag gttcacagtg tccagggata atgccaagaa cacagtgtac 240

ctgcagatga atagcctgaa gcctgaggac accgccatgt actactgcgc cgccgacggc 300

agcaggagga actggttctc ccccctggac cccaggaagt acaactactg gggccagggc 360

acccaggtga ccgtgtccag c 381

<210>40

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

caggtgcagc tgcaggagag cggcggaggc agcgtgcagg ctggaggaag cctgagactg 60

gcctgtgccg cctccggcta cacatacagc tccagctaca tgggctggtt caggcaggcc 120

cctggcaagg agagggaggg cgtggctgtg atctacacag ccggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccgtg agcagagaca atgccaagaa caccgtgtac 240

ctgcagatga actccctgaa gcccgaggac acagccatgt actactgtgc cgccgatggc 300

agcaggagga actggttcag ccagctggac cccaggaagt acaactactg gggccagggc 360

acacaggtga ccgtgtcctc c 381

<210>41

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggagagag cctgagactg 60

agctgtgccg cctccggcta cacatactcc tcctcctaca tgggctggtt caggcaggcc 120

cccggcaagg agagagaggg cgtggctgtg atctacaccg gcggcggcag aacctactac 180

gccgacagcg tgaagggcag attcacaatc agcagagata acgccgagaa cacagtgtac 240

ctgaggatga atagcctgaa gcctgaggat accgccatgt actactgcgc cgccgatggc 300

tccagaagaa attggtacag ccccctggac cccagaaagt acaattactg gggccagggc 360

acccaggtga ccgtgagctc c 381

<210>42

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggaag cctgagactg 60

tcctgcgccg cctccggcta cacctactcc tcctcctaca tgggctggtt caggcaggcc 120

cctggcaagg agagagaggg cgtggccgtg atctacacca ccggcggcag aacatactac 180

gccgatagcg tgaagggcag gttcaccatc tccagagata acgccaagaa taccgtgtac 240

ctgcagatga atagcctgaa gcctgaggac accgccatgt actactgtgc cgccgatggc 300

agcaggagga attggtacag ccctctggat cccagaaagt acaactactg gggccagggc 360

acacaggtga cagtgagctc c 381

<210>43

<211>390

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

caggtgcagc tgcaggagtc cggcggaggc agcgtgcagg ctggaggatc cctgaggctg 60

tcctgcgtgg ccagcaggta catgggctcc acatactaca tggcctggtt caggcaggcc 120

cccggcaagg agagggaggg agtggctgcc atctacatcg ccagcggcaa cagcggcagc 180

acatactacg ccgactccgt gaagggcagg ttcacaatct ccagagataa tgccaagaat 240

accgtgtacc tgcagatgaa tagcctgatc cccgaggata ccgcctccta ctactgtgcc 300

agcgacctga gcagaaggaa ttggctgcct cccctggtgc ccggcaagta cagcctgtgg 360

ggccagggca cccaggtgac agtgtccagc 390

<210>44

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggaag cctgaggctg 60

agctgtgccg cctccggcta caccggcagc gtgaactaca tggcctggtt caggcaggcc 120

cctggcaagg agagggaggg cgtggctgcc atctacggct acggcggcag cacctactac 180

gccgactccg tgaagggcag gttcacaatc agcagggata atgccaagaa caccgtgtac 240

ctgcagatga acagcctgat ccctgaggat acagcctcct actactgtgc cagcgaccag 300

tccaggaggt cctggctgag ccccctggtg cccggaaagt actccctgtg gggccagggc 360

acacaggtga ccgtgagcag c 381

<210>45

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

caggtgcagc tgcaggagtc cggcggaggc tccgtgcagg ctggaggaag cctgagactg 60

tcctgtgagg cctccggcta cacaggcagc gtgaattaca tggcctggtt cagacagcct 120

cctggcaagg agagagaggg cgtggccgcc atctacggcg gcggaggaag cacatactac 180

gccgacgccg tgaagggcag gttcaccatc tccagggata acgccaagaa tacagtgtac 240

ctgcagatga acagcctgat ccccgaggat acagccagct actactgcgc ctccgatcag 300

tccaggagga gctggttccc tcccctggtg cccggcaagt acagcctgtg gggccagggc 360

acccaggtga cagtgagctc c 381

<210>46

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

caggtgcagc tgcaggagtc cggcggaggc agcgtgcagg ctggaggaag cctgaggctg 60

agctgtgtgg tgtccggcta cgcctacagc acaaactaca tgggctggtt cagacaggcc 120

cccggcaagg agagagaggg cgtggctgtg atctacacca cagtgggcac cacatactac 180

gccgattccg tgaagggcag gttcaccatc agccaggaca acgccaagaa tacagtgtcc 240

ctgcagatga atagcctgca gcccgaggac acagccatgt actactgcgc cgccgtgagg 300

aggccttgga ggctggattc cctggatcct tcctccttca ggtactgggg ccagggcaca 360

caggtgaccg tgagcagc 378

<210>47

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

caggtgcagc tgcaggagtc cggcggaggc agcgtgcagg ctggaggaag cctgaggctg 60

tcctgtgtgg tgagcggcta cgcctactcc acaaactaca tgggctggtt caggcaggcc 120

cctggcaagg agagagaggg cgtggccgtg atctacacca ccgtgggcac cacatactac 180

gccgacagcg tgaagggcag gttcaccatc agccaggaca atgccaagaa cacagtgagc 240

ctgcagatga acggcctgca gcctgaggat acagccatgt actactgtgc cgccgtgaga 300

aggccctgga ggctggacag cctggatccc tcctccttca gatactgggg ccagggcaca 360

caggtgacag tgtccagc 378

<210>48

<211>369

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

caggtgcagc tgcaggagag cggcggaggc acagtgcagg ccggaggatc cctgaccctg 60

agctgtgccg ccagcggcta cacctacaat tacatgggct ggttcagaca ggcccctggc 120

aaggagaggg agggcgtggc tgccatcgcc accagctccg gaagggccta ctacgccgac 180

agcgtgaagg gcaggttcac catctcccag gacaacggca agaataccct gcacctgcag 240

atgaacagcc tgaagcctga ggacaccgcc atgtactact gcgccgccgg ctcccactac 300

tccggctact ccctgagccc tggcaggtac aagtactggg gccagggcac acaggtgacc 360

gtgtccagc 369

<210>49

<211>369

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

caggtgcagc tgcaggagtc cggcggaggc acagtgcagg ccggaggatc cctgacactg 60

tcctgcgccg ccagcggcta cacatacaat tacatgggct ggttcagaca ggcccctggc 120

aaggagaggg agggcgtggc tgccatccac acaggcagcg gcaggaccta ctacatcgac 180

tccgtgaagg gcagattcac catcagccag gacaacgcca agaacacaac caacctgcag 240

atgacaagcc tgaagcccga ggacaccgcc atgtactact gtgccgccgg cagccactac 300

gccggctact ctatgtcccc tggcaggtac aagtactggg gccagggcac acaggtgaca 360

gtgagcagc 369

<210>50

<211>369

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

caggtgcagc tgcaggagtc cggcggaggc acagtgcagg ccggaggatc cctgacactg 60

agctgcgccg cctccggcta cacctacaac tacatgggct ggttcagaca ggcccccggc 120

aaggagagag agggcgtggc tgtgatgcac accggcagcg gcagagccta ctacgtggat 180

tccgtgaagg gcagattcac aatcagcaga gacaatgcca agaacaccgt gcacctgcag 240

atgaactccc tgaagcccga ggataccgcc atgtactact gtgccgccgg cagccactac 300

tccggctact ccatgagccc cggcagatac aagtactggg gccagggcac acaggtgaca 360

gtgagcagc 369

<210>51

<211>384

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggatc cctgagactg 60

agctgtgccg cctccggcag cgccgatagg agaaattacg tggcctggtt cagacaggcc 120

cccggcaagg agagagaggg cgtggctgcc ctgggcatcg tgtccggatt cacacactac 180

gccgacagcg tgaagggcag attcacaatc tcccaggata acgccagaaa cacagtgtac 240

ctgcagatga acaggatgat ccctgaggat acagccatgt actactgcgc cgccgattcc 300

aggaccaggg tgctgatctc ctggttcctg aatcctgata agtacaacta ctggggcaga 360

ggcacccagg tgaccgtgag cagc384

<210>52

<211>399

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggaag cctgaggctg 60

tcctgcgccg ccagcgagta caccagctcc gtgacattca tgggctgggt gagacaggcc 120

cccggcaagg agagggaggg cgttgctacc atctacaccg tgaccggcag gacctactac 180

gccgactccg tgaagggcag attcaccatc agccaggaca aggccaagaa taccgtgtac 240

ctgcagatgg actccctgaa gcccgaggac accgccatgt actactgtgc cgccaccccc 300

ttctccctga gcaagaccac aaggctggcc aagttcggca gcctgaacga gaatgagtac 360

gatctgtggg gccagggcac acaggtgacc gtgagcagc 399

<210>53

<211>399

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggaag cctgaggctg 60

tcctgtgccg cctccgagta caccagcagc gtgacattca tgggctgggt gaggcaggcc 120

cccggcaagg aaagggaggg cgtggctacc atctacacac ccaccggcag gacctactac 180

gccgattccg tgaagggcag attcaccatc agccaggaca aggccaagaa tacagtgtac 240

ctgcagatga atagcctgaa gcctgaggac accgccatgt actactgtgc cgccacaccc 300

ttcagcctgt ccaagacaac aaggctggcc aagttcggct ccctgaacga gaacgagtac 360

gacctgtggg gccagggcac ccaggtgacc gtgagcagc 399

<210>54

<211>369

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

caggtgcagc tgcaggagtc cggcggaggc agcgtgcaga ccggaggatc cctgagactg 60

agctgtgccg cctccggcaa cagcgacggc gcttggtcca tgggctggtt caggcaggcc 120

cctggcaagg agagggaggg cgtggctgcc ctgtacgtgt tcacaggcat cacatactac 180

gccggctccg tgaagggcag gttcacaatc tcccaggata aggccaagaa cacagtgtac 240

ctgcagatga acagcctgaa gcctgaggac accgccatgt actactgtgc cgccggcaca 300

gccttcccca ccctgaacag gaataagtac aactactggg gccagggcac ccaggtgacc 360

gtgagcagc 369

<210>55

<211>390

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

caggtgcagc tgcaggagag cggcggaggc agcgtgaggg ctggaggatc cctgaggctg 60

tcctgtgccg ccagcggcta cacctcctcc agctactgta tgggctggtt cagacaggcc 120

cctggcaagg agagagaggg cgtggccgcc atctacaccg atacaggcgg cggctccaca 180

tactacgccg attccgtgaa gggcagattc accatcagcc agggcaatgc caagaacaca 240

ctgtacctgc agatgaatag cctgaagccc gaggacacag ccatgtacta ctgtgccgcc 300

gtggagagaa gaggcggctc ctgctacacc agatacaagt acgccgcctt cgcctactgg 360

ggccagggca cacaggtgac cgtgtcctcc 390

<210>56

<211>387

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>56

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggaag cctgaggctg 60

tcctgcacag ccagcggctt caccctggac gattccgatc tgggctggta caggcaggcc 120

cccggcaatg agtgtgagct ggtgagcatc atcaggagag atggcagccc ctaccacaca 180

tactaccctg atagcgtgaa gggcaggttc accatcagcc aggataatgc caagaatacc 240

ctgtacctgc agatgaatag cctgaagcct gaggacacag ccgtgtacta ctgcgccgcc 300

gatcctagca ggtactactc cgattacggc gcccctggct gtggcttcaa ttactggggc 360

cagggcacac aggtgaccgt gtcctcc 387

<210>57

<211>390

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>57

caggtgcagc tgcaggagag cggcggaggc tccgtgcagg ctggaggatc cctgaggctg 60

tcctgcgtgg ccagcggcta caccttcagc tccgattgca tgggctggtt caggcaggcc 120

cctggcaaga agagagaggg cgtggccgcc atctacacat ccggctcctc cacctactac 180

gccgactccg tgaagggcag gttcacaatc tccagagata atgccaagaa taccgtgtac 240

ctggagatga actccctgaa gcctgaggat acagccatct actactgcgc cgccagaagg 300

agctccggcg gctactgtta catcggctcc gacttccaga tctccctgta caataactgg 360

ggccagggca cccaggtgac agtgtccagc 390

<210>58

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>58

caggtgcagc tgcaggagag cggcggcggc ctggtgcagg ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agcagctaca tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacacca ccggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccgtgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgacggc 300

agcaggagga actggtacag ccagctggac cccaggaagt acaactactg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>59

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>59

caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agcagctaca tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacacct tcggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccgtgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgacggc 300

agcaggagga actggtacag ccagctggac cccaggaagt acctgtactg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>60

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agcagctaca tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacacca ccggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccgtgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgacggc 300

agcaggagga actggtacag ccagctggac cccaggaagt acaactactg gggccagggc 360

acccaggtga ccgtgagcag c381

<210>61

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

gcctgcgccg ccagcggcta cacctacagc agcagctaca tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacacca gcggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccgtg agcagggaca acgccaagaa caccgtgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgacggc 300

agcaggagga actggtacag ccccctggac cccaggaagt acaactactg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>62

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>62

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

gcctgcgccg ccagcggcta cacctacagc agcagctaca tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacacca gcggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccgtg agcagggaca acgccaagaa caccgtgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgacggc 300

agcaggagga actggtacag ccccctggac cccaggaagt acaactactg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>63

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>63

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agcagctaca tgggctactt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacaccg gcggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaacaa caccctgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccgtgt actactgcgc cgccgacggc 300

agcaggagga actggttcag ccagctggac cccaggaagt acatctactg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>64

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>64

caggtgcagc tgcaggagag cggcggcggc cccgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agcagctaca tgggctactt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgtg atctacaccg gcggcggcag gacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaacaa caccctgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccgtgt actactgcgc cgccgacggc 300

agcaggagga actggttcag ccagctggac cccaggaagt acatctactg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>65

<211>381

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>65

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg ccagcagcta caccggcagc gtgaactaca tggcctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgcc atctacagcg gcggcggcag cacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccgtgtac 240

ctgcagatga acagcctgat ccccgaggac accgccacct actactgcgc cagcgaccag 300

agcaggagga gctggttcag ccccctggtg cccggcaagt acagcctgtg gggccagggc 360

acccaggtga ccgtgagcag c 381

<210>66

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>66

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcaccg ccagcggctt caccagcgac gacagcgaca tgggctggta caggcaggcc 120

cccggcaacg agtgcgagct ggtgagcgtg gtgaggagcg acggcaccac ctactacccc 180

cccagcgtga ggggcaggtt caccatcagc caggacaacg ccaagaacac cgtgagcctg 240

cagatgaaca gcctgaagcc cgaggacacc gccgtgtact actgcgccgc cgaccccagc 300

ccctactaca gggactacgg cgcccccggc tgcggcttca actactgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210>67

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>67

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcaccg ccagcggctt cagcctggac gacagggaca tgggctggta caggcaggcc 120

cccggcaacg agtgcgagct ggtgagcgtg atcaggagcg acggcaggac ctactacccc 180

gacagcgtga agggcaggtt caccatcagc caggacaacg ccaagaggac cctgtacctg 240

cagatgaaca gcctgaaggc cgaggacacc gccgtgtact actgcgccgc cgaccccagc 300

aggaggtaca gcgactacgg cgcccccggc tgcggcttca actactgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210>68

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>68

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcaccg ccagcggctt cagcctggac gacagggaca tgggctggta caggcaggcc 120

cccggcaacg agtgcgagct ggtgagcgtg atcaggagcg acggcagcac ctactacccc 180

gacagcgtga agggcaggtt caccatcagc caggacaacg ccaagaacac cctgtacctg 240

cagatgaaca gcctgaagcc cgaggacacc gccgtgtact actgcgccgc cgaccccagc 300

aggtactaca gcgactacgg cgcccccggc tgcggcttca actactgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210>69

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>69

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcaccg ccagcggctt cagcctggac gacagggaca tgggctggta caggcaggcc 120

cccggcaacg agtgcgagct ggtgagcgtg atcaggagcg acggcagcac ctactacccc 180

gacagcgtga agggcaggtt caccatcagc caggacaacg ccaagaacac cctgtacctg 240

cagatgaaca gcctgaagcc cgaggacacc gccgtgtact actgcgccgc cgaccccagc 300

aggtactaca gcgactacgg cgcccccggc tgcggcttcc actactgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210>70

<211>378

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>70

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcaccg ccagcggctt cacccccgac ggcagcgaca tgggctggta caggcaggcc 120

cccggcaacg agtgcgagct ggtgagcgtg atcaggagcg gcggcagcac ctactacacc 180

gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240

cagatgaaca gcctgaagcc cgaggacacc gccgtgtact actgcgccgc cgaccccagc 300

aggtactaca gcgactacgg cgcccccggc tgcggcttcc actactgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210>71

<211>387

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>71

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg tgagcggcct gacctacagc agcaactgca tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgcc atctacaacg gcggcaacac ctactacgcc 180

gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cgtgtacctg 240

gagatgaaca gcctgaagcc cgaggacacc gccatctact actgcgccgc caggaggagc 300

agcggcggct actgctacat cggcagcgac ttccagatca gcctgttcaa caactggggc 360

cagggcaccc aggtgaccgt gagcagc 387

<210>72

<211>369

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>72

caggtgcagc tgcaggagag cggcggcggc accgtgcagg ccggcggcag cctgaccctg 60

agctgcgccg ccagcggcta cacctacaac tacatgggct ggttcaggca ggcccccggc 120

aaggagaggg agggcgtggc cgccatcagc accagcagcg gcagggccta ctacgtggac 180

agcgtgaagg gcaggttcac catcagccag gacaacgcca agaacaccac ctacctgcag 240

atgaacagcc tgaagcccga ggacaccgcc atgtactact gcgccgccgg cagccactac 300

agcggctaca gcatcagccc cggcaggtac aagtactggg gccagggcac ccaggtgacc 360

gtgagcagc 369

<210>73

<211>384

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>73

caggtgcagc tgcaggagag cggcggcggc agcgtgcaga ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcag caccgacagg aggaactaca tggcctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgcc atcggcacca tcagcggctt cacccactac 180

gccgacagcg tgaagggcag gttcaccatc agccaggaca acgccaggaa caccgtgtac 240

ctgcagatga acaggatgat ccccgaggac accgccatgt actactgcgc cgccgacagc 300

aggaccaggg tgatcatcgg ctggttcctg aaccccgaca agtacaacta ctggggcagg 360

ggcacccagg tgaccgtgag cagc 384

<210>74

<211>363

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>74

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcaccg ccagcggcta cacccacagg atggcctggt tcaggcaggc ccccggcaag 120

gagagggagg gcgtggccgc catctacacc gacgtgggcc acacctacta cgccgccagc 180

gtgaagggca ggttcaccat cgcccaggac aacgccaaca acaccgccta cctgcagatg 240

aacagcctga agcccgagga caccgccatc tactactgcg ccgccgccag gggcggctac 300

agctggagga aggtgaacga gtacaactac tggggccagg gcacccaggt gaccgtgagc 360

agc 363

<210>75

<211>384

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>75

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg tgagcggcta caccctgagc aacaagtgga tgggctggtt caggcaggcc 120

cccggcaagg agagggaggg cgtggccgcc atctacaccg gcggcggcag gaccgactac 180

gccgacagcg tgaagggcag gttcaccatc agccaggaca acgcccagaa caccctgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccgccagg 300

ggctacggcc agaactggta caggaccctg aggaccgagg cctacgacat ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210>76

<211>399

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>76

caggtgcagc tgcaggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60

agctgcgccg ccagcgagta caccagcagc gtgaccttca tggcctgggt gaggcaggcc 120

cccggcaagg agagggaggg cgtggccacc atctacaccg tgaccggcag gagctactac 180

gccgacagcg tgaagggcag gttcaccatc agccaggaca aggccaagaa caccgtgtac 240

ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccaccccc 300

ttcagcctga gcaaggccac caggctggcc aagttcggca gcctgaacga gaacgagtac 360

gacctgtggg gccagggcac ccaggtgacc gtgagcagc 399

<210>77

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>77

Glu Tyr Thr Ser Ser Val Thr Phe

1 5

<210>78

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>78

Gly Phe Ser Leu Asp Asp Arg Asp

1 5

<210>79

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>79

Gly Phe Thr Leu Asp Asp Ser Asp

1 5

<210>80

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>80

Gly Phe Thr Pro Asp Gly Ser Asp

1 5

<210>81

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>81

Gly Phe Thr Ser Asp Asp Ser Asp

1 5

<210>82

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>82

Gly Leu Thr Tyr Ser Ser Asn Cys

1 5

<210>83

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>83

Gly Asn Ser Asp Gly Ala Trp Ser

1 5

<210>84

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>84

Gly Ser Ala Asp Arg Arg Asn Tyr

1 5

<210>85

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>85

Gly Ser Thr Asp Arg Arg Asn Tyr

1 5

<210>86

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>86

Gly Tyr Ala Tyr Ser Thr Asn Tyr

1 5

<210>87

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>87

Gly Tyr Thr Phe Ser Ser Asp Cys

1 5

<210>88

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>88

Gly Tyr Thr Gly Ser Val Asn Tyr

1 5

<210>89

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>89

Gly Tyr Thr His Arg

1 5

<210>90

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>90

Gly Tyr Thr Leu Ser Asn Lys Trp

1 5

<210>91

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>91

Gly Tyr Thr Ser Ser Ser Tyr Cys

1 5

<210>92

<211>6

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>92

Gly Tyr Thr Tyr Asn Tyr

1 5

<210>93

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>93

Gly Tyr Thr Tyr Ser Ser Ser Tyr

1 5

<210>94

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>94

Arg Tyr Met Gly Ser Thr Tyr Tyr

1 5

<210>95

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>95

Ser Tyr Thr Gly Ser Val Asn Tyr

1 5

<210>96

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>96

Ile Ala Thr Ser Ser Gly Arg Ala

1 5

<210>97

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>97

Ile Gly Thr Ile Ser Gly Phe Thr

1 5

<210>98

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>98

Ile His Thr Gly Ser Gly Arg Thr

1 5

<210>99

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>99

Ile Arg Arg Asp Gly Ser Pro Tyr His Thr

1 5 10

<210>100

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>100

Ile Arg Ser Asp Gly Arg Thr

1 5

<210>101

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>101

Ile Arg Ser Asp Gly Ser Thr

1 5

<210>102

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>102

Ile Arg Ser Gly Gly Ser Thr

1 5

<210>103

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>103

Ile Ser Thr Ser Ser Gly Arg Ala

1 5

<210>104

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>104

Ile Tyr Gly Gly Gly Gly Ser Thr

1 5

<210>105

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>105

Ile Tyr Gly Tyr Gly Gly Ser Thr

1 5

<210>106

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>106

Ile Tyr Ile Ala Ser Gly Asn Ser Gly Ser Thr

1 5 10

<210>107

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>107

Ile Tyr Asn Gly Gly Asn Thr

1 5

<210>108

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>108

Ile Tyr Ser Gly Gly Gly Ser Thr

1 5

<210>109

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>109

Ile Tyr Thr Ala Gly Gly Arg Thr

1 5

<210>110

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>110

Ile Tyr Thr Asp Thr Gly Gly Gly Ser Thr

1 5 10

<210>111

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>111

Ile Tyr Thr Asp Val Gly His Thr

1 5

<210>112

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>112

Ile Tyr Thr Phe Gly Gly Arg Thr

1 5

<210>113

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>113

Ile Tyr Thr Gly Gly Gly Arg Thr

1 5

<210>114

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>114

Ile Tyr Thr Pro Thr Gly Arg Thr

1 5

<210>115

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>115

Ile Tyr Thr Ser Gly Gly Arg Thr

1 5

<210>116

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>116

Ile Tyr Thr Ser Gly Ser Ser Thr

1 5

<210>117

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>117

Ile Tyr Thr Thr GlyGly Arg Thr

1 5

<210>118

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>118

Ile Tyr Thr Thr Val Gly Thr Thr

1 5

<210>119

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>119

Ile Tyr Thr Val Thr Gly Arg Ser

1 5

<210>120

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>120

Ile Tyr Thr Val Thr Gly Arg Thr

1 5

<210>121

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>121

Leu Gly Ile Val Ser Gly Phe Thr

1 5

<210>122

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>122

Leu Tyr Val Phe Thr Gly Ile Thr

1 5

<210>123

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>123

Met His Thr Gly Ser Gly Arg Ala

1 5

<210>124

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>124

Val Arg Ser Asp Gly Thr Thr

1 5

<210>125

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>125

Ala Ala Asp Ser Arg Thr Arg Val Leu Ile Ser Trp Phe Leu Asn Pro

1 5 10 15

Asp Lys Tyr Asn Tyr Trp

20

<210>126

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>126

Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Gln Leu Asp Pro Arg

1 5 10 15

Lys Tyr Asn Tyr Trp

20

<210>127

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>127

Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Pro Leu Asp Pro Arg

1 5 10 15

Lys Tyr Asn Tyr Trp

20

<210>128

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>128

Ala Ser Asp Leu Ser Arg Arg Asn Trp Leu Pro Pro Leu Val Pro Gly

1 5 10 15

Lys Tyr Ser Leu Trp

20

<210>129

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>129

Ala Ser Asp Gln Ser Arg Arg Ser Trp Leu Ser Pro Leu Val Pro Gly

1 5 10 15

Lys Tyr Ser Leu Trp

20

<210>130

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>130

Ala Ser Asp Gln Ser Arg Arg Ser Trp Phe Pro Pro Leu Val Pro Gly

1 5 10 15

Lys Tyr Ser Leu Trp

20

<210>131

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>131

Ala Ala Val Arg Arg Pro Trp Arg Leu Asp Ser Leu Asp Pro Ser Ser

1 5 10 15

Phe Arg Tyr Trp

20

<210>132

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>132

Ala AlaGly Ser His Tyr Ser Gly Tyr Ser Leu Ser Pro Gly Arg Tyr

1 5 10 15

Lys Tyr Trp

<210>133

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>133

Ala Ala Gly Ser His Tyr Ala Gly Tyr Ser Met Ser Pro Gly Arg Tyr

1 5 10 15

Lys Tyr Trp

<210>134

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>134

Ala Ala Gly Ser His Tyr Ser Gly Tyr Ser Met Ser Pro Gly Arg Tyr

1 5 10 15

Lys Tyr Trp

<210>135

<211>27

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>135

Ala Ala Thr Pro Phe Ser Leu Ser Lys Thr Thr Arg Leu Ala Lys Phe

1 5 10 15

Gly Ser Leu Asn Glu Asn Glu Tyr Asp Leu Trp

20 25

<210>136

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>136

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

1 5 10 15

Trp

<210>137

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>137

Ala Ala Val Glu Arg Arg Gly Gly Ser Cys Tyr Thr Arg Tyr Lys Tyr

1 5 10 15

Ala Ala Phe Ala Tyr Trp

20

<210>138

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>138

Ala Ala Asp Pro Ser Arg Tyr Tyr Ser Asp Tyr Gly Ala Pro Gly Cys

1 5 10 15

Gly Phe Asn Tyr Trp

20

<210>139

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>139

Ala Ala Arg Arg Ser Ser Gly Gly Tyr Cys Tyr Ile Gly Ser Asp Phe

1 5 10 15

Gln Ile Ser Leu Tyr Asn Asn Trp

20

<210>140

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>140

Ala Ala Ala Arg Gly Tyr Gly Gln Asn Trp Tyr Arg Thr Leu Arg Thr

1 5 10 15

Glu Ala Tyr Asp Ile Trp

20

<210>141

<211>27

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>141

Ala Ala Thr Pro Phe Ser Leu Ser Lys Ala Thr Arg Leu Ala Lys Phe

1 5 10 15

Gly Ser Leu Asn Glu Asn Glu Tyr Asp Leu Trp

20 25

<210>142

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>142

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>143

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>143

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser

20 25

<210>144

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>144

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>145

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>145

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>146

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>146

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>147

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>147

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser

20 25

<210>148

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>148

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Glu Ala Ser

20 25

<210>149

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>149

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Val Ala Ser

20 25

<210>150

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>150

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

1 5 10 15

Ser Leu Arg Leu Ala Cys Val Val Ser

20 25

<210>151

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>151

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>152

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>152

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser

20 25

<210>153

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>153

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser

20 25

<210>154

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>154

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>155

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>155

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>156

<211>25

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>156

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

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser

20 25

<210>157

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>157

Leu Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser

1 5 10 15

Ile

<210>158

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>158

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

1 5 10 15

Ala

<210>159

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>159

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

1 5 10 15

Ala

<210>160

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>160

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

1 5 10 15

Thr

<210>161

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>161

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

1 5 10 15

Ala

<210>162

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>162

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

1 5 10 15

Val

<210>163

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>163

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

1 5 10 15

Ala

<210>164

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>164

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

1 5 10 15

Thr

<210>165

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>165

Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser

1 5 10 15

Val

<210>166

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>166

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

1 5 10 15

Val

<210>167

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>167

Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala

1 510 15

Ala

<210>168

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>168

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>169

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>169

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

1 5 10 15

Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Arg Met Ile Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>170

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>170

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

1 5 10 15

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

20 25 30

Thr Ala Ile Tyr Tyr Cys

35

<210>171

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>171

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

1 5 10 15

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

20 25 30

Thr Ala Ser Tyr Tyr Cys

35

<210>172

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>172

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

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu GlnMet Asp Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>173

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>173

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>174

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>174

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>175

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>175

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>176

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>176

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

1 5 10 15

Gly Lys Asn Thr Leu His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>177

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>177

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>178

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>178

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>179

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>179

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

1 5 10 15

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

20 25 30

Thr Ala Ile Tyr Tyr Cys

35

<210>180

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>180

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

1 5 10 15

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

20 25 30

Thr Ala Ser Tyr Tyr Cys

35

<210>181

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>181

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

1 5 10 15

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

20 25 30

Thr Ala Thr Tyr Tyr Cys

35

<210>182

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>182

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>183

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>183

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

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210>184

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>184

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>185

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>185

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>186

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>186

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>187

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>187

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

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210>188

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>188

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

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210>189

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>189

Tyr Tyr Pro Pro Ser Val Arg Gly Arg Phe Thr Ile Ser Gln Asp Asn

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210>190

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>190

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

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210>191

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>191

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

1 5 10 15

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

20 25 30

ThrAla Met Tyr Tyr Cys

35

<210>192

<211>38

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>192

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210>193

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>193

Gly Arg Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>194

<211>10

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>194

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>195

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>195

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Gln Leu Asp Pro Arg

1 5 10 15

Lys Tyr Leu Tyr Trp

20

<210>196

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>196

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Gln Leu Asp Pro Arg

1 5 10 15

Lys Tyr Asn Tyr Trp

20

<210>197

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>197

Ala Ala Asp Gly Ser Arg Arg Asn Trp Tyr Ser Pro Leu Asp Pro Arg

1 5 10 15

Lys Tyr Asn Tyr Trp

20

<210>198

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>198

Ala Ala Asp Gly Ser Arg Arg Asn Trp Phe Ser Gln Leu Asp Pro Arg

1 5 10 15

Lys Tyr Ile Tyr Trp

20

<210>199

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>199

Ala Ser Asp Gln Ser Arg Arg Ser Trp Phe Ser Pro Leu Val Pro Gly

1 5 10 15

Lys Tyr Ser Leu Trp

20

<210>200

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>200

Ala Ala Asp Pro Ser Pro Tyr Tyr Arg Asp Tyr Gly Ala Pro Gly Cys

1 5 10 15

Gly Phe Asn Tyr Trp

20

<210>201

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>201

Ala Ala Asp Pro Ser Arg Arg Tyr Ser Asp Tyr Gly Ala Pro Gly Cys

1 5 10 15

Gly Phe Asn Tyr Trp

20

<210>202

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>202

Ala Ala Asp Pro Ser Arg Tyr Tyr Ser Asp Tyr Gly Ala Pro Gly Cys

1 5 10 15

Gly Phe His Tyr Trp

20

<210>203

<211>24

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>203

Ala Ala Arg Arg Ser Ser Gly Gly Tyr Cys Tyr Ile Gly Ser Asp Phe

1 5 10 15

Gln Ile Ser Leu Phe Asn Asn Trp

20

<210>204

<211>19

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>204

Ala Ala Gly Ser His Tyr Ser Gly Tyr Ser Ile Ser Pro Gly Arg Tyr

1 5 10 15

Lys Tyr Trp

<210>205

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>205

Ala Ala Asp Ser Arg Thr Arg Val Ile Ile Gly Trp Phe Leu Asn Pro

1 5 10 15

Asp Lys Tyr Asn Tyr Trp

20

<210>206

<211>18

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>206

Ala Ala Ala Arg Gly Gly Tyr Ser Trp Arg Lys Val Asn Glu Tyr Asn

1 5 10 15

Tyr Trp

Claims (10)

1. A single domain antibody directed against bovine serum albumin BSA, wherein the sequence of the single domain antibody comprises complementarity determining regions CDRs; the complementarity determining region CDRs include the amino acid sequences of CDR1, CDR2, and CDR 3; the amino acid sequence of the CDR1 is shown as any one of SEQ ID NO:77-SEQ ID NO: 95; the amino acid sequence of the CDR2 is shown as any one of SEQ ID NO. 96-SEQ ID NO. 124; the amino acid sequence of the CDR3 is shown in any one of SEQ ID NO 125-SEQ ID NO 141 and SEQ ID NO 195-SEQ ID NO 206.

2. A single domain antibody directed against bovine serum albumin BSA, wherein the sequence of the single domain antibody comprises complementarity determining regions CDRs; the complementarity determining region CDRs include the amino acid sequences of CDR1, CDR2, and CDR 3; the sequence of the CDR of the single domain antibody is one of the following (1) to (34):

(1) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 113, CDR3 shown in SEQ ID NO. 127;

(2) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 109, CDR3 shown in SEQ ID NO. 126;

(3) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 113, CDR3 shown in SEQ ID NO. 197;

(4) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 117, CDR3 shown in SEQ ID NO. 197;

(5) CDR1 shown in SEQ ID NO. 94, CDR2 shown in SEQ ID NO. 106, CDR3 shown in SEQ ID NO. 128;

(6) CDR1 shown in SEQ ID NO. 88, CDR2 shown in SEQ ID NO. 105, CDR3 shown in SEQ ID NO. 129;

(7) CDR1 shown in SEQ ID NO:88, CDR2 shown in SEQ ID NO:104, CDR3 shown in SEQ ID NO: 130;

(8) CDR1 shown in SEQ ID NO. 86, CDR2 shown in SEQ ID NO. 118, CDR3 shown in SEQ ID NO. 131;

(9) CDR1 shown in SEQ ID NO. 92, CDR2 shown in SEQ ID NO. 96, CDR3 shown in SEQ ID NO. 132;

(10) CDR1 shown in SEQ ID NO. 92, CDR2 shown in SEQ ID NO. 98, CDR3 shown in SEQ ID NO. 133;

(11) CDR1 shown in SEQ ID NO. 92, CDR2 shown in SEQ ID NO. 123, CDR3 shown in SEQ ID NO. 134;

(12) CDR1 shown in SEQ ID NO:84, CDR2 shown in SEQ ID NO:121, CDR3 shown in SEQ ID NO: 125;

(13) CDR1 shown in SEQ ID NO. 77, CDR2 shown in SEQ ID NO. 120, CDR3 shown in SEQ ID NO. 135;

(14) CDR1 shown in SEQ ID NO. 77, CDR2 shown in SEQ ID NO. 114, CDR3 shown in SEQ ID NO. 135;

(15) CDR1 shown in SEQ ID NO:83, CDR2 shown in SEQ ID NO:122, CDR3 shown in SEQ ID NO: 136;

(16) CDR1 shown in SEQ ID NO. 91, CDR2 shown in SEQ ID NO. 110, CDR3 shown in SEQ ID NO. 137;

(17) CDR1 shown in SEQ ID NO. 79, CDR2 shown in SEQ ID NO. 99, CDR3 shown in SEQ ID NO. 138;

(18) CDR1 shown in SEQ ID NO. 87, CDR2 shown in SEQ ID NO. 116, CDR3 shown in SEQ ID NO. 139;

(19) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 117, CDR3 shown in SEQ ID NO. 196;

(20) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 112, CDR3 shown in SEQ ID NO. 195;

(21) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 115, CDR3 shown in SEQ ID NO. 197;

(22) CDR1 shown in SEQ ID NO. 93, CDR2 shown in SEQ ID NO. 113, CDR3 shown in SEQ ID NO. 198;

(23) CDR1 shown in SEQ ID NO. 95, CDR2 shown in SEQ ID NO. 108, CDR3 shown in SEQ ID NO. 199;

(24) CDR1 shown in SEQ ID NO. 81, CDR2 shown in SEQ ID NO. 124, CDR3 shown in SEQ ID NO. 200;

(25) CDR1 shown in SEQ ID NO. 78, CDR2 shown in SEQ ID NO. 100, CDR3 shown in SEQ ID NO. 201;

(26) CDR1 shown in SEQ ID NO. 78, CDR2 shown in SEQ ID NO. 101, CDR3 shown in SEQ ID NO. 138;

(27) CDR1 shown in SEQ ID NO. 78, CDR2 shown in SEQ ID NO. 101, CDR3 shown in SEQ ID NO. 202;

(28) CDR1 shown in SEQ ID NO. 80, CDR2 shown in SEQ ID NO. 102, CDR3 shown in SEQ ID NO. 202;

(29) CDR1 shown in SEQ ID NO. 82, CDR2 shown in SEQ ID NO. 107, CDR3 shown in SEQ ID NO. 203;

(30) CDR1 shown in SEQ ID NO. 92, CDR2 shown in SEQ ID NO. 103, CDR3 shown in SEQ ID NO. 204;

(31) CDR1 shown in SEQ ID NO. 85, CDR2 shown in SEQ ID NO. 97, CDR3 shown in SEQ ID NO. 205;

(32) CDR1 shown in SEQ ID NO. 89, CDR2 shown in SEQ ID NO. 111, CDR3 shown in SEQ ID NO. 206;

(33) CDR1 shown in SEQ ID NO. 90, CDR2 shown in SEQ ID NO. 113, CDR3 shown in SEQ ID NO. 140;

(34) CDR1 shown in SEQ ID NO. 77, CDR2 shown in SEQ ID NO. 119, and CDR3 shown in SEQ ID NO. 141.

3. A single domain antibody against bovine serum albumin BSA, characterized in that the single domain antibody is an antibody chain represented by any one of SEQ ID NOS: 1-38.

4. The single domain antibody against bovine serum albumin BSA according to claim 3, wherein the coding sequences of the antibody chains are as set forth in SEQ ID NOs: 39-76.

5. The single domain antibody against bovine serum albumin BSA according to any one of claims 1 to 4, characterised in that the antibody sequence further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the FR regions of the framework regions are:

FR1 shown in any one of SEQ ID NOs 142-156;

FR2 shown in any one of SEQ ID NO: 157-167;

FR3 shown in any one of SEQ ID NO: 168-192;

FR4 shown in any one of SEQ ID NOS 193-194.

6. The single domain antibody against bovine serum albumin BSA according to any of claims 1-5, wherein said single domain antibody is VHH.

7. A nucleotide molecule encoding the single domain antibody against bovine serum albumin BSA according to any of claims 1-5, having the nucleotide sequence shown in SEQ ID NO: 39-76, respectively.

8. An expression vector comprising the nucleotide molecule of claim 7.

9. A host cell capable of expressing a single domain antibody against bovine serum albumin BSA according to any one of claims 1 to 5 or comprising an expression vector according to claim 8.

10. An Fc fusion antibody according to any one of claims 1 to 5 against a single domain antibody of bovine serum albumin BSA.

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