CN111057148B - Single-domain antibody aiming at bovine serum albumin BSA and derivative protein thereof - Google Patents

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

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CN111057148B
CN111057148B CN201911238185.9A CN201911238185A CN111057148B CN 111057148 B CN111057148 B CN 111057148B CN 201911238185 A CN201911238185 A CN 201911238185A CN 111057148 B CN111057148 B CN 111057148B
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CN111057148A (en
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苏志鹏
赵泽英
姚尧
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Nanjing Rongjiekang Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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) NaHCO diluted 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) phage specifically bound to BSA protein were lysed with trypsin at a final concentration of 25mg/mLThe E.coli TG1 cells in logarithmic growth phase were detached and infected, cultured at 37 ℃ for 1h, phage were generated and collected for the next round of screening, the same screening process was repeated for 3 rounds, and enrichment was achieved step by step, as 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 had a very significant enrichment against 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 (Mouse anti-HA antibody, Thermo Fisher) diluted at 1:2000 was added, and incubated at room temperature for 1 h; (5) unbound antibody was washed away with PBST, 100ul of Anti-Rabbit HRP conjugate (goat Anti-Rabbit horseradish peroxidase labeled antibody, purchased from Thermo Fisher) diluted 1:20000 was added, and incubated 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
Figure BDA0002305451930000081
Figure BDA0002305451930000091
CDR3 sequences of the 238 single domain antibodies
Figure BDA0002305451930000092
Figure BDA0002305451930000101
FR1 and FR2 sequences of 338 single-domain antibodies
Figure BDA0002305451930000102
Figure BDA0002305451930000111
FR3 sequences of Table 438 Single Domain antibodies
Figure BDA0002305451930000112
Figure BDA0002305451930000121
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 OD600nmAdding 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 example 3 was subcloned into eukaryotic expression vectors: (1) the antibody screened out in the example 3 is subjected to Sanger sequencing to obtain a nucleotide sequence; (2) the nucleotide sequences (SEQ ID NO.39-76) after codon optimization are respectively cloned into a carrier RJK-V4-hFC designed and modified by the company by a sequence synthesis mode (namely, the SEQ ID NO.39-76 is respectively inserted into a multi-cloning site MCS of the carrier RJK-V4-hFC, and the construction process of the RJK-V4-hFc carrier is described in example 12); (3) transforming a recombinant eukaryotic expression vector constructed by a company into DH5 alpha escherichia coli, culturing, carrying out plasmid macro-extraction, and removing endotoxin; (4) carrying out sequencing identification on the greatly extracted plasmid; (5) and preparing the recombinant vector which is determined to be error-free for subsequent eukaryotic cell transfection 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-STMCells, 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-STMCell 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 inversionTMCHO reagent, 3.7ml OptiPROTMDilution of Expifeacylamine in culture MediumTMCHO reagent, swirling or mixing; (6) with refrigerated 4ml OptiPROTMDiluting 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 addedTMCHO Enhancer and 24ml ExpicHO feed. (10) At approximately 8 days post transfection (cell viability below 70%) harvestAnd (5) clearing.
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 (NCBI Accession No.: AB776838.1) of human IgG to 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 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 5 10 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 5 10 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
Phe His 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 cagc 384
<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 c 381
<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 Gly Gly 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 Ala Gly 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 5 10 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 Gln Met 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
Thr Ala 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 (8)

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 sequence of the CDR of the single domain antibody is (1) or (2) as follows:
(1) CDR1 shown in SEQ ID NO. 86, CDR2 shown in SEQ ID NO. 118, CDR3 shown in SEQ ID NO. 131;
(2) CDR1 shown in SEQ ID NO. 81, CDR2 shown in SEQ ID NO. 124 and CDR3 shown in SEQ ID NO. 200.
2. The single domain antibody aiming at bovine serum albumin BSA is characterized by being shown as SEQ ID NO.8 or SEQ ID NO. 28.
3. The single domain antibody against bovine serum albumin BSA according to claim 2, wherein the coding sequences of the single domain antibody are as set forth in SEQ ID NOs: 46. SEQ ID NO: as shown at 66.
4. The single domain antibody against bovine serum albumin BSA according to any one of claims 1 to 3, characterised in that the single domain antibody is VHH.
5. A nucleotide molecule encoding the single domain antibody against bovine serum albumin BSA according to any one of claims 1 to 3, having the nucleotide sequence shown in SEQ ID NO: 46 or SEQ ID NO: as shown at 66.
6. An expression vector comprising the nucleotide molecule of claim 5.
7. A host cell capable of expressing a single domain antibody against bovine serum albumin BSA according to any one of claims 1 to 3 or comprising an expression vector according to claim 6.
8. An Fc fusion antibody according to any one of claims 1 to 3 to a single domain antibody of bovine serum albumin BSA.
CN201911238185.9A 2019-12-06 2019-12-06 Single-domain antibody aiming at bovine serum albumin BSA and derivative protein thereof Active CN111057148B (en)

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CN113831411B (en) * 2021-09-18 2023-05-02 南京融捷康生物科技有限公司 Single-domain antibody for L1CAM and derived protein and application thereof
CN116925215A (en) * 2022-03-31 2023-10-24 普米斯生物技术(珠海)有限公司 Antiserum albumin nanobody and derivatives thereof

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