Proteins for inhibiting biological activity of venezuelan equine encephalitis virus and uses thereof

Abstract

The application discloses a protein for inhibiting biological activity of Venezuelan equine encephalitis virus and application thereof, wherein the protein is an LDLRAD3 mutant, and the protein is: P1) a protein obtained by mutating at least one of positions 30, 34, 39, 41 and 56 of the amino acid in SEQ ID No. 51 in the sequence listing; or P2) a fusion protein obtained by connecting an amino acid sequence at the N terminal or / and C terminal of P1). Compared with a wild-type protein, the protein of the application improves the binding force with VLP and the activity of inhibiting VEEV. The infection of VEEV can be effectively inhibited by using only a low dose of the protein of the application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a variety of bioactive proteins that can effectively inhibit Venezuelan equine encephalitis virus and their applications. Background Technology

[0002] Avira are enveloped, positive-sense RNA viruses transmitted by arthropods, belonging to the genus Avira in the family Cotyledonidae. Based on geographical distribution, members of the genus Avira are divided into two main categories: Old World viruses and New World viruses. While most Avira infections cause only mild illness in humans and animals, many Old World Avira infections, such as Chikungunya virus (CHIKV), Ross River virus (RRV), Mayaro virus (MAYV), Semliki Forest virus (SFV), and O'nyong-nyong virus (ONNV), can cause fever, rash, headache, vomiting, diarrhea, and acute inflammation of the musculoskeletal system and joints, and can also lead to severe chronic joint disease. New World viruses, such as Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV), can cause brain and nervous system diseases, leading to encephalitis and, in severe cases, death. To date, no vaccine or drug has been approved for the prevention or treatment of alphavirus infection.

[0003] VEEV is a deadly pathogen causing Venezuelan equine encephalitis, frequently occurring in equines such as horses, donkeys, mules, zebras, and humans. Outbreaks are widespread and can last for months or years. Sporadic outbreaks often occur in Central and South America. VEEV is a zoonotic pathogen. Due to its aerosol transmission characteristics, VEEV could be used as a biological weapon. In late 2020, Nature reported the discovery that LDLRAD3 is the host receptor protein for VEEV. LDLRAD3 binds to VEEV through its N-terminal domain 1 (LDLRAD3-D1). Summary of the Invention

[0004] The purpose of this invention is to provide a series of VEEV protein inhibitors with high biological activity.

[0005] This invention provides a protein, which is an LDLRAD3 mutant, and the amino acid sequence of the protein is as follows:

[0006] P1) A protein obtained by mutating at least one of the following positions: position 30, position 34, position 39, position 41, and position 56 of the amino acids described in positions 1-70 of SEQ ID No. 51 in the sequence listing;

[0007] P2) is a fusion protein obtained by linking an amino acid sequence to the N-terminus and / or C-terminus of P1).

[0008] Furthermore, the protein described in P1) can be from A1) to A25):

[0009] A1) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 26 in the sequence listing;

[0010] A2) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 27 in the sequence listing;

[0011] A3) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 28 in the sequence listing;

[0012] A4) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 29 in the sequence listing;

[0013] A5) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 30 in the sequence listing;

[0014] A6) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 31 in the sequence listing;

[0015] A7) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 32 in the sequence listing;

[0016] A8) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 33 in the sequence listing;

[0017] A9) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 34 in the sequence listing;

[0018] A10) A protein whose amino acid sequence is the amino acid described in positions 1-70 of SEQ ID No. 35 in the sequence listing;

[0019] A11) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 36 in the sequence listing;

[0020] A12) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 37 in the sequence listing;

[0021] A13) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 38 in the sequence listing;

[0022] A14) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 39 in the sequence listing;

[0023] A15) A protein whose amino acid sequence is the amino acid described in positions 1-70 of SEQ ID No. 40 in the sequence listing;

[0024] A16) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 41 in the sequence listing;

[0025] A17) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 42 in the sequence listing;

[0026] A18) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 43 in the sequence listing;

[0027] A19) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 44 in the sequence listing;

[0028] A20) is a protein whose amino acid sequence is the amino acid described in positions 1-70 of SEQ ID No. 45 in the sequence listing;

[0029] A21) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 46 in the sequence listing;

[0030] A22) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 47 in the sequence listing;

[0031] A23) A protein whose amino acid sequence is the amino acid sequence of positions 1-70 of SEQ ID No. 48 in the sequence listing;

[0032] A24) A protein whose amino acid sequence is the amino acid described in positions 1-70 of SEQ ID No. 49 in the sequence listing;

[0033] A25) A protein whose amino acid sequence is the amino acid described in positions 1-70 of SEQ ID No. 50 in the sequence listing;

[0034] Further, the fusion protein described in P2) is a protein with the amino acid from positions 71 to 315 of SEQ ID No. 26 in the sequence listing added to the end of any of the proteins described in A1)-A25); the fusion protein has any of the amino acid sequences described in SEQ ID No. 26-SEQ ID No. 50 in the sequence listing.

[0035] This invention provides a biomaterial, wherein the biomaterial is:

[0036] B1) The nucleic acid molecule that encodes the protein;

[0037] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0038] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0039] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0040] B5) A transgenic animal cell line containing the nucleic acid molecule described in B1), or a transgenic animal cell line containing the expression cassette described in B2).

[0041] B1) The nucleic acid molecule is the gene encoding the protein shown in b1)-b4) below:

[0042] b1) The coding sequence is the cDNA molecule or DNA molecule of nucleotides 52-210 of any of the sequences described in sequences 1 to 25 in the sequence listing;

[0043] b2) A nucleotide is a cDNA molecule or DNA molecule consisting of nucleotides 52-210 of any of the sequences described in sequences 1 to 25 in the sequence listing;

[0044] b3) The coding sequence is a cDNA molecule or DNA molecule whose nucleotides 52-915 are any of the sequences described in sequences 1 to 25 in the sequence listing;

[0045] b4) A nucleotide is a cDNA molecule or DNA molecule of any of the nucleotides described in sequences 1 through 25 of the sequence listing.

[0046] B3) The recombinant vector containing the nucleic acid molecule described in B1) is a recombinant expression vector obtained by cloning the nucleic acid molecule described in B1) into a eukaryotic expression vector or a prokaryotic expression vector.

[0047] B3) The recombinant vector containing the nucleic acid molecule described in B1) is a recombinant expression vector obtained by cloning the nucleic acid molecule described in B1) into the expression vector pCMV.

[0048] The present invention also provides a method for preparing a fusion protein, comprising the following steps: introducing the recombinant vector described in B3) into mammalian cells, followed by cell culture, thereby obtaining the fusion protein;

[0049] The present invention also provides a method for preparing a fusion protein, comprising the following steps: obtaining the inclusion body protein described herein, and obtaining the corresponding soluble active protein through denaturation and renaturation.

[0050] A VEEV protein inhibitor comprising the protein or a fusion protein.

[0051] The following applications should also be covered by this law:

[0052] Q1. The use of any of the biomaterials described in claims 4-7 in the preparation of the protein described in claim 1;

[0053] Q2. The use of the protein described in any one of claims 1-3 or the biomaterial described in any one of claims 4-7 in the preparation of products for the prevention or treatment of diseases caused by VEEV infection.

[0054] The product may be a medicine, vaccine or test kit used to prevent or treat diseases caused by VEEV infection.

[0055] The beneficial effects of this invention are as follows: Compared with wild-type proteins, the fusion proteins of this invention all exhibit improved binding affinity to VLP. Specifically, N39D, R41E, R41Q, N39D-R41E, N39D-R41M, N30E-N39D-F56Y, and N39D-R41M-F56Y show a binding affinity to VLP that is more than 100-fold higher. The fusion proteins of this invention can effectively inhibit VEEV virus activity. As shown in Table 2, protein 41E exhibits the best activity against VEEV virus, with an IC50 value of 19 ng / ml, which is approximately 6-fold higher than the IC50 of WT. Only low doses of the proteins of this invention are needed to effectively inhibit VEEV infection. Attached Figure Description

[0056] Figure 1 This is a schematic diagram of the domain structure of the LDLARD3 mutant fusion protein.

[0057] Figure 2 The results were used to detect the inhibitory activity of the LDLRAD3 mutant fusion protein on the virus. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] Unless otherwise specified, the buffer solutions used in the following examples are as follows: containing 20 mM HEPES and 150 mM NaCl, with the balance being water, pH 8.0. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged.

[0061] The pCMV carriers used in the following examples were purchased from Thermo Fisher Scientific.

[0062] The HEK293F cells used in the following examples were purchased from Sinocare Biotechnology Co., Ltd.

[0063] The SMM 293-TII culture medium used in the following examples was purchased from Sinocare Biotechnology Co., Ltd.

[0064] The SINV-VEEV chimeric virus in the following examples is referenced in Paessler, S., Fayzulin, RZ, Anishchenko, M., Greene, IP, Weaver, SC, and Frolov, I. (2003). Recombinants indbis / Venezuelan equine encephalitis virus is highly attenuated and immunogenic. J Virol 77, 9278-9286. The recombinant SINV-VEEV virus possesses the non-structural protein of the SINV Girdwood strain and the structural protein of the VEEV TC-83 strain, with green fluorescent protein inserted into nsP3; 3B4C-4 antibody Porta, J., Jose, J., Roehrig, JT, Blair, CD, Kuhn, RJ, and Rossmann, MG (2014). Locking and blocking the viral landscape of an alphavirus with neutralizing antibodies. J Virol 88, 9616-9623.; VEEV virus-like particle reference: Ko, SY, Akahata, W., Yang, ES, Kong, WP, Burke, CW, Honnold, SP, Nichols, DK, Huang, YS, Schieber, GL, Carlton, K., et al. (2019). A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates. Sci Transl Med11.; Plasmid RAP reference: Ma, H., Kim, AS, Kafai, NM, Earnest, JT, Shah, AP, Case, JB, Basore, K., Gilliland, TC, Sun, C., Nelson, CA, et al. (2020). LDLRAD3 is a receptor for Venezuelan equine encephalitis virus. Nature The gene sequence corresponding to RAP in the literature (588, 308-314) is cloned into the pCMV vector to obtain plasmid RAP; it can be obtained by the public from Tsinghua University School of Medicine.

[0065] Example 1: Preparation of LDLRAD3 mutant fusion protein

[0066] I. Construction of recombinant plasmids

[0067] The small fragment between XbaI and BamHI in the pCMV vector was replaced by the double-stranded DNA molecules shown in SEQ ID No. 1 to SEQ ID No. 25 respectively. After sequencing verification, the recombinant plasmids pcMV-vLDLRAD3-Fc-1 to pcMV-vLDLRAD3-Fc-25 were obtained. The pcMV-vLDLRAD3-Fc-1 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 1, while keeping the other sequences of the pCMV vector unchanged; the pcMV-vLDLRAD3-Fc-2 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 2, while keeping the other sequences of the pCMV vector unchanged; the pcMV-vLDLRAD3-Fc-3 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 3, while keeping the other sequences of the pCMV vector unchanged; the pcMV-vLDLRAD3-Fc-4 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 3. The recombinant plasmids described in SEQ ID No. 4 are: pcMV-vLDLRAD3-Fc-5, pcMV-vLDLRAD3-Fc-6, pcMV-vLDLRAD3-Fc-7, and pcMV-vLDLRAD3-Fc-8. pcMV-vLDLRAD3-Fc-8 are recombinant plasmids that replace the shorter sequence between XbaI and BamHI in the pCMV vector with the double-stranded DNA molecule shown in SEQ ID No. 5, while maintaining the other sequences of the pCMV vector. The double-stranded DNA molecule shown in No. 8 is a recombinant plasmid that retains the other sequences of the pCMV vector unchanged; the pcMV-vLDLRAD3-Fc-9 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with SEQ ID No.The recombinant plasmids described in SEQ ID No. 9 are: pcMV-vLDLRAD3-Fc-10, pcMV-vLDLRAD3-Fc-11, pcMV-vLDLRAD3-Fc-12, and pcMV-vLDLRAD3-Fc-13. pcMV-vLDLRAD3-Fc-13 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 10, while maintaining the other sequences of the pCMV vector. The recombinant plasmids described in SEQ ID No. 9 are: pcMV-vLDLRAD3-Fc-10, pcMV-vLDLRAD3-Fc-13, pcMV-vLDLRAD3-Fc-14, pcMV-vLDLRAD3-Fc-15, pcMV-vLDLRAD3-Fc-16, pcMV-vLDLRAD3-Fc-17, pcMV-vLDLRAD3-Fc-18, pcMV-vLDLRAD3-Fc-19, pcMV-vLDLRAD3-Fc-10, pcMV-vLDLRAD3-Fc-12, pcMV-vLDLRAD3-Fc-13, pcMV-vLDLRAD3-Fc-15, pcMV-vLDLRAD3-Fc-16, pcMV-vLDLRAD3-Fc-19, pcMV-vLDLRAD3-Fc-10 ... The recombinant plasmids described in SEQ ID No. 13 are: pcMV-vLDLRAD3-Fc-14, pcMV-vLDLRAD3-Fc-15, pcMV-vLDLRAD3-Fc-16, and pcMV-vLDLRAD3-Fc-17. pcMV-vLDLRAD3-Fc-17 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 14, while maintaining the other sequences of the pCMV vector. The recombinant plasmids described in SEQ ID No. 13 are: pcMV-vLDLRAD3-Fc-14, pcMV-vLDLRAD3-Fc-15, pcMV-vLDLRAD3-Fc-16, pcMV-vLDLRAD3-Fc-17, and pcMV-vLDLRAD3-Fc-18. The recombinant plasmid pcMV-vLDLRAD3-Fc-18 is a double-stranded DNA molecule shown in SEQ ID No. 17, with the other sequences of the pCMV vector remaining unchanged; pcMV-vLDLRAD3-Fc-19 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 18, with the other sequences of the pCMV vector remaining unchanged;The recombinant plasmids described in SEQ ID No. 19 are: pcMV-vLDLRAD3-Fc-20, pcMV-vLDLRAD3-Fc-21, pcMV-vLDLRAD3-Fc-22, and pcMV-vLDLRAD3-Fc-23. pcMV-vLDLRAD3-Fc-23 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 20, while maintaining the other sequences of the pCMV vector. The recombinant plasmids described in SEQ ID No. 23 and SEQ ID No. 24 are double-stranded DNA molecules, with the other sequences of the pCMV vector remaining unchanged. SEQ ID No. 24 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 24, while maintaining the other sequences of the pCMV vector unchanged. SEQ ID No. 25 is a recombinant plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 25, while maintaining the other sequences of the pCMV vector unchanged.

[0068] The first 51 positions of SEQ ID No. 1 to SEQ ID No. 25 are the coding regions of the signal peptide; the 52nd to 210th positions are the coding regions of LDLRAD3 D1; the 211th to 240th positions are the coding regions of the 3C restriction site; and the 241st to 918th positions are the coding regions of the Fc protein.

[0069] The double-stranded DNA molecules shown in SEQ ID No. 1 to SEQ ID No. 25 encode the LDLRAD3 mutant fusion proteins shown in SEQ ID No. 26 to SEQ ID No. 50, respectively (see schematic diagram of the domains). Figure 1 In the mutant fusion proteins with amino acid sequences SEQ ID No. 26 to SEQ ID No. 50, positions 1-17 are signal peptides; positions 18-70 are LDLRAD3 mutant proteins (where positions 28-67 are the core region binding to VEEV); positions 71-80 are 3C restriction sites; and positions 81-305 are Fc proteins.

[0070] II. Preparation of vLDLRAD3-Fc

[0071] The 25 recombinant plasmids (pcMV-vLDLRAD3-Fc-1 to pcMV-vLDLRAD3-Fc-25) from step one were transformed into HEK293F cells, further cultured, and purified to obtain 25 proteins LDLRAD3-D1-Fc. The protein obtained after transformation of pcMV-vLDLRAD3-Fc-1 was named N30A; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-2 was named N30D; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-3 was named N30E; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-4 was named N30R; and the protein obtained after transformation of p... The protein obtained after transformation of cMV-vLDLRAD3-Fc-5 was named N30S; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-6 was named N30T; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-7 was named N34A; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-8 was named N34L; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-9 was named N39D; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-10 was named N39E; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-11 was named R41A; the protein obtained after transformation of p The protein obtained after transformation with cMV-vLDLRAD3-Fc-12 was named R41E; the protein obtained after transformation with pcMV-vLDLRAD3-Fc-13 was named R41G; the protein obtained after transformation with pcMV-vLDLRAD3-Fc-14 was named R41K; the protein obtained after transformation with pcMV-vLDLRAD3-Fc-15 was named R41L; the protein obtained after transformation with pcMV-vLDLRAD3-Fc-16 was named R41M; the protein obtained after transformation with pcMV-vLDLRAD3-Fc-17 was named R41N; and the protein obtained after transformation with pcMV-vLDLRAD3-Fc-18 was named R41Q. The protein obtained after transformation of pcMV-vLDLRAD3-Fc-19 was named R41W; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-20 was named R41Y; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-21 was named N39D-R41E; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-22 was named N39D-R41M; the protein obtained after transformation of pcMV-vLDLRAD3-Fc-23 was named N30E-N39D-F56Y; and the protein obtained after transformation of pcMV-vLDLRAD3-Fc-24 was named N39D-R41E-F56Y.The protein obtained after transforming pcMV-vLDLRAD3-Fc-25 was named N39D-R41M-F56Y. The specific steps are as follows:

[0072] 1. HEK293F cells were cultured in SMM 293-TII medium until the logarithmic growth phase (density reached 1.5 × 10⁻⁶). 6 -2.0×10 6 The cells were cultured in a 1000-cell / ml culture system. Then, the recombinant plasmid pCMV-vLDLRAD3-Fc (2 μg recombinant plasmid / ml culture system) and plasmid RAP (1 μg RAP plasmid / ml culture system) were transfected using PEI MAX (Polysciences, 24765-2), and then cultured for another 60 h.

[0073] 2. After completing step 1, centrifuge at 1000g for 10 minutes and collect the supernatant.

[0074] 3. Take the supernatant obtained in step 2, centrifuge at 4000g for 30 minutes, and collect the supernatant.

[0075] 4. Take the supernatant obtained in step 3 and pass it through a gravity flow column containing Protein A agarose beads, repeating twice. Then wash away any extraneous proteins with buffer.

[0076] 5. Elute the Protein A agarose beads from step 4 with elution buffer containing 0.1M Glycine, 20mM HEPES, and 150mM NaCl, pH 3.0. Transfer the eluted protein to a 15ml centrifuge tube containing 1M Tris-HCl, pH 9.0 (5ml of elution buffer corresponds to approximately 250ul of neutralization buffer, maintaining the pH of the entire solution between 7.6 and 8.0).

[0077] 6. Take the solution obtained in step 5, use nanodrop to detect the protein concentration, aliquot and flash freeze in liquid nitrogen at -80℃.

[0078] III. Preparation of Control Samples

[0079] The recombinant plasmid WT was obtained by replacing the short fragment between XbaI and BamHI in the pCMV vector with the double-stranded DNA molecule shown in SEQ ID No. 52 and verifying its correctness through sequencing. The recombinant plasmid WT is a plasmid in which the shorter sequence between XbaI and BamHI in the pCMV vector is replaced with the double-stranded DNA molecule shown in SEQ ID No. 52, while keeping the other sequences of the pCMV vector unchanged.

[0080] The recombinant plasmid WT was transformed into HEK293F cells according to the steps described in step two. After further cultivation and purification, protein WT was obtained. The amino acid sequence of the protein WT is shown in SEQ ID No. 51.

[0081] Example 2: Evaluation of VEEV binding activity

[0082] LDLRAD3 is the receptor for VEEV. LDLRAD3 binds to VEEV through its N-terminal first domain. The binding affinity of the purified protein to VEEV can be detected using biolayer interferometry (BLI). BLI experiments were performed at 25°C using a ForteBio OctetRed instrument. The biosensor used was a streptavidin sensor (ForteBio, 18-5019). The binding activity of the 25 fusion proteins prepared in Example 1 of this invention (N30A, N30D, N30E, N30R, N30S, N30T, N34A, N34L, N39D, N39E, R41A, R41E, R41G, R41K, R41L, R41M, R41N, R41Q, R41W, R41Y, N39D-R41E, N39D-R41M, N30E-N39D-F56Y, N39D-R41E-F56Y, and N39D-R41M-F56Y) and the control protein WT to VEEV virus-like particles was detected according to the following steps:

[0083] 1. The 3B4C-4 antibody and biotin (EZ-Link-NHS-LC-LC-Biotin, Thermo Fisher) were mixed at a molar ratio of 1:3 and incubated at room temperature for 30 min. Then, excess biotin was removed using a desalting column (2 ml Zeba Spin 7K MWCO, Thermo Fisher).

[0084] 2. Take the biotinylated 3B4C-4 antibody from step 1 and adjust its concentration to 50 μg / ml using buffer. Immobilize it on the streptavidin sensor for 3 minutes.

[0085] 3. Adjust the concentration of VEEV virus-like particles to 50 μg / ml using buffer solution, and then set a 30-minute program to coat the VEEV virus-like particles onto the streptavidin sensor processed in step 2.

[0086] 4. Perform binding and dissociation experiments on the streptavidin sensor processed in step 3. Specifically, the different LDLRAD3 mutant fusion proteins obtained in Example 1 are serially diluted 2-fold with buffer containing 0.1% BSA, and 200 μl is added to each well of a 96-well plate. The specific parameters for the program are: equilibrate the streptavidin sensor with 0.1% BSA buffer for 3 min; then move the sensor to wells containing different protein concentrations and read the values ​​for 5 min; finally, move the sensor to wells containing only 0.1% BSA buffer and read the values ​​for 5 min.

[0087] 5. Data processing was performed using ForteBio's Octet software, version 9.0. The data were fitted using a standard 1:1 binding model to obtain the binding constant Kd value for each mutant fusion protein (Table 1).

[0088] Table 1. Binding constants of LDLRAD3-D1 mutant fusion protein to VEEV VLP

[0089]

[0090] Table 1 shows that all 25 mutant fusion proteins exhibited improved binding affinity to VLP compared to the wild-type protein. Specifically, N39D, R41E, R41Q, N39D-R41E, N39D-R41M, N30E-N39D-F56Y, and N39D-R41M-F56Y showed over 100-fold increased binding affinity to VLP. Even the less effective mutants N39E, R41K, N30R, R41G, and R41N showed at least a 2-4 fold increase in VLP binding affinity.

[0091] Example 3: Evaluation of VEEV virus inhibitory activity

[0092] The purified LDLRAD3 mutant fusion protein can be used to inhibit VEEV virus infection. Because VEEV is a level 3 pathogen, for safety reasons, the virus used in this experiment is a SINV-VEEV chimeric virus. This virus genome contains a non-structural protein genome encoding the alphavirus Sindbis virus (SINV) and a structural protein genome encoding VEEV. A gene encoding green fluorescent protein was inserted between the SINV non-structural protein genomes. The recombinant SINV-VEEV virus possesses the non-structural proteins of the SINV Girdwood strain and the structural proteins of the VEEVTC-83 strain. The green fluorescent protein is inserted into nsP3.

[0093] 1. Mouse neuroma cells Neuro-2a (ATCC CCL-131) were proliferated in MEM medium (C11095500BT, Gibco) containing 10% FBS (Gibco), 1% Sodium Pyruvate (11360-070, Gibco), and 1% MEM NEAA (11140-050, Gibco). 1×10⁻⁶ 5 The cells were seeded into each well of a 24-well plate and incubated for 18 hours in a constant temperature incubator at 37 degrees Celsius and 5% CO2.

[0094] 2. Dilute the mutant fusion protein and the control protein (WT) starting at 2 μg / ml with the culture medium from step 1, using a 2-fold serial dilution. Add an equal volume of chimeric virus (virus titer 2 × 10⁻⁶) to the diluted protein. 5 (pfu / ml), incubate at 37 degrees for 1 hour.

[0095] 3. Discard the culture medium from the 24 wells in step 1, add 400 μL of the virus-protein complex from step 2 to each well, and perform three replicates. Incubate at 37°C with 5% CO2 for 16-18 hours.

[0096] 4. Remove the 24-well plate from step 3, discard the supernatant, and wash each well with 200 μL of PBS. After removing the PBS, add 50 μL of trypsin to each well and digest for 5 min. Transfer to a 1.5 ml tube and centrifuge at 1000 g for 5 min. After discarding the supernatant, add 200 μL of 4% paraformaldehyde (P1110, Solarbio) to each tube and fix at room temperature for 15 min.

[0097] 5. Centrifuge the cells from step 4 at 1000g for 5 minutes, discard 4% paraformaldehyde, and resuspend in 180ul PBS.

[0098] 6. Using a BD Fortessa flow cytometer, read the green fluorescence signal from the cell samples in step 5. Plot the results using Prism 9 software and calculate the IC50 value. The results are shown in Table 2 and... Figure 2 .

[0099] Table 2: Inhibitory activity of LDLRAD3-D1 mutant fusion protein against VEEV infection

[0100]

[0101]

[0102] Table 2 and Figure 2In the figure, wt represents protein. The fusion protein of the present invention can effectively inhibit the activity of VEEV virus. As can be seen from Table 2, proteins 41E and 39D have the best activity against VEEV virus, with IC50 values ​​of 19 ng / ml and 15 ng / ml, respectively. Compared with the IC50 of WT, the inhibitory activity is increased by about 6 times.

[0103] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Tsinghua University Institute of Biophysics, Chinese Academy of Sciences <120> Proteins for inhibiting the biological activity of Venezuelan equine encephalitis virus and their applications <160> 52 <170> SIPOSequenceListing 1.0 <210> 1 <211> 918 <212> DNA <213> Artificial Sequence <400> 1 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcgcc atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 2 <211> 918 <212> DNA <213> Artificial Sequence <400> 2 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcgac atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 3 <211> 918 <212> DNA <213> Artificial Sequence <400> 3 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcgaa atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 4 <211> 918 <212> DNA <213> Artificial Sequence <400> 4 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgccgc atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 5 <211> 918 <212> DNA <213> Artificial Sequence <400> 5 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgctcc atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 6 <211> 918 <212> DNA <213> Artificial Sequence <400> 6 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcacc atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gaagagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg caaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 7 <211> 918 <212> DNA <213> Artificial Sequence <400> 7 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggcg cgttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 8 <211> 918 <212> DNA <213> Artificial Sequence <400> 8 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggcc tcttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 9 <211> 918 <212> DNA <213> Artificial Sequence <400> 9 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcgatggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 10 <211> 918 <212> DNA <213> Artificial Sequence <400> 10 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcgaaggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 11 <211> 918 <212> DNA <213> Artificial Sequence <400> 11 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 gcctgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gaagagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 12 <211> 918 <212> DNA <213> Artificial Sequence <400> 12 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 gaatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 13 <211> 918 <212> DNA <213> Artificial Sequence <400> 13 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 ggatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 14 <211> 918 <212> DNA <213> Artificial Sequence <400> 14 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 aagtgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 15 <211> 918 <212> DNA <213> Artificial Sequence <400> 15 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 ctgtgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 16 <211> 918 <212> DNA <213> Artificial Sequence <400> 16 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 atgtgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 17 <211> 918 <212> DNA <213> Artificial Sequence <400> 17 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 aactgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 18 <211> 918 <212> DNA <213> Artificial Sequence <400> 18 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 caatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gaagagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg caaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 19 <211> 918 <212> DNA <213> Artificial Sequence <400> 19 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 tggtgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 20 <211> 918 <212> DNA <213> Artificial Sequence <400> 20 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 tactgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 21 <211> 918 <212> DNA <213> Artificial Sequence <400> 21 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcgatggc 120 gaatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 22 <211> 918 <212> DNA <213> Artificial Sequence <400> 22 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcgatggc 120 atgtgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 23 <211> 918 <212> DNA <213> Artificial Sequence <400> 23 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcgaa atccccggca acttcatgtg cagcgatggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgctacga caagagcgac 180 gagaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttctc 300 ttccccccaa aacccaagga caccctcatg atctccccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt aaacagcac gtaccgtgtg 480 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gaagagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 24 <211> 918 <212> DNA <213> Artificial Sequence <400> 24 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcgatggc 120 gaatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgctacga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 25 <211> 918 <212> DNA <213> Artificial Sequence <400> 25 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcgatggc 120 atgtgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgctacga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918 <210> 26 <211> 305 <212> PRT <213> Artificial Sequence <400> 26 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Ala Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 27 <211> 305 <212> PRT <213> Artificial Sequence <400> 27 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asp Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 28 <211> 305 <212> PRT Artificial Sequence <400> 28 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Glu Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 29 <211> 305 <212> PRT <213> Artificial Sequence <400> 29 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Arg Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 30 <211> 305 <212> PRT <213> Artificial Sequence <400> 30 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Ser Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 31 <211> 305 <212> PRT <213> Artificial Sequence <400> 31 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Thr Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 32 <211> 305 <212> PRT <213> Artificial Sequence <400> 32 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Ala Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 33 <211> 305 <212> PRT <213> Artificial Sequence <400> 33 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Leu Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 34 <211> 305 <212> PRT <213> Artificial Sequence <400> 34 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asp Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 35 <211> 305 <212> PRT <213> Artificial Sequence <400> 35 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Glu Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 36 <211> 305 <212> PRT <213> Artificial Sequence <400> 36 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Ala Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 37 <211> 305 <212> PRT <213> Artificial Sequence <400> 37 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Glu Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 38 <211> 305 <212> PRT Artificial Sequence <400> 38 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Gly Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 39 <211> 305 <212> PRT <213> Artificial Sequence <400> 39 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Lys Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 40 <211> 305 <212> PRT <213> Artificial Sequence <400> 40 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Leu Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 41 <211> 305 <212> PRT <213> Artificial Sequence <400> 41 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Met Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 42 <211> 305 <212> PRT <213> Artificial Sequence <400> 42 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Asn Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 43 <211> 305 <212> PRT <213> Artificial Sequence <400> 43 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Gln Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 44 <211> 305 <212> PRT <213> Artificial Sequence <400> 44 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Trp Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 45 <211> 305 <212> PRT <213> Artificial Sequence <400> 45 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Tyr Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 46 <211> 305 <212> PRT <213> Artificial Sequence <400> 46 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asp Gly Glu Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 47 <211> 305 <212> PRT <213> Artificial Sequence <400> 47 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asp Gly Met Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 48 <211> 305 <212> PRT <213> Artificial Sequence <400> 48 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Glu Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asp Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Tyr Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 49 <211> 305 <212> PRT <213> Artificial Sequence <400> 49 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asp Gly Glu Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Tyr Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 50 <211> 305 <212> PRT <213> Artificial Sequence <400> 50 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asp Gly Met Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Tyr Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 51 <211> 305 <212> PRT <213> Artificial Sequence <400> 51 Met Trp Leu Leu Gly Pro Leu Cys Leu Leu Leu Ser Ser Ala Ala Glu 1 5 10 15 Ser Gln Leu Leu Pro Gly Asn Asn Phe Thr Asn Glu Cys Asn Ile Pro 20 25 30 Gly Asn Phe Met Cys Ser Asn Gly Arg Cys Ile Pro Gly Ala Trp Gln 35 40 45 Cys Asp Gly Leu Pro Asp Cys Phe Asp Lys Ser Asp Glu Lys Glu Cys 50 55 60 Pro Lys Ala Lys Ser Lys Gly Pro Leu Glu Val Leu Phe Gln Gly Pro 65 70 75 80 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 210 215 220 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 52 <211> 918 <212> DNA <213> Artificial Sequence <400> 52 atgtggctgc tgggacctct gtgtctgctg ctgtctagcg ccgctgaatc tcagctgctg 60 cccggcaaca acttcaccaa cgagtgcaac atccccggca acttcatgtg cagcaacggc 120 agatgtatcc ctggcgcctg gcagtgtgat ggcctgcctg attgcttcga caagagcgac 180 gagaaagagt gccccaaggc caagagcaag gggcccctcg aagtgctatt ccaaggtcct 240 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 300 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 360 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 420 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 480 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 540 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 600 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 660 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 720 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 780 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 840 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 900 ctgtctccgg gtaaatga 918

Claims

1. A protein, said protein being an LDLRAD3 mutant, said LDLRAD3 mutant being a protein obtained by mutating at least one of the following positions: 30, 34, 39, 41, and 56 of the amino acid described in SEQ ID No. 51 of the sequence listing; said protein being any one of the following A1) to A23): A1) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 26 of the sequence listing; A2) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 27 of the sequence listing; A3) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 28 of the sequence listing; A4) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 29 of the sequence listing; A5) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 30 of the sequence listing; A6) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 31 of the sequence listing; A7) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 32 of the sequence listing; A8) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 33 of the sequence listing; A9) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 34 of the sequence listing; A10) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 36 of the sequence listing; A11) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 37 of the sequence listing; A12) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 39 of the sequence listing; A13) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 40 of the sequence listing; A14) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 41 of the sequence listing; A15) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 42 of the sequence listing; A16) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 43 of the sequence listing; A17) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 44 of the sequence listing; A18) A protein with the amino acid sequence described in SEQ ID No. 45 of the sequence listing; A19) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 46 of the sequence listing; A20) is a protein with the amino acid sequence described in SEQ ID No. 47 of the sequence listing; A21) A protein whose amino acid sequence is the amino acid described in SEQ ID No. 48 of the sequence listing; A22) A protein with the amino acid sequence described in SEQ ID No. 49 of the sequence listing; A23) is a protein with the amino acid sequence described in SEQ ID No. 50 of the sequence listing.

2. A biomaterial, wherein the biomaterial is: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic animal cell line containing the nucleic acid molecule described in B1), or a transgenic animal cell line containing the expression cassette described in B2).

3. The biomaterial according to claim 2, characterized in that, B1) The nucleic acid molecule described below is the coding gene shown in b1)-b2): b1) The coding sequence is a cDNA molecule or DNA molecule whose nucleotides 52-915 are any of the sequences described in SEQ ID No. 1-9, 11-12 or 14-25 in the sequence listing; b2) The nucleotide is a cDNA molecule or DNA molecule of any of the nucleotides described in SEQ ID No. 1-9, 11-12 or 14-25 in the sequence listing.

4. The biomaterial according to claim 2, characterized in that, B3) The recombinant vector containing the nucleic acid molecule described in B1) is a recombinant expression vector obtained by cloning the nucleic acid molecule described in B1) into a eukaryotic expression vector or a prokaryotic expression vector.

5. The biomaterial according to claim 4, characterized in that, B3) The recombinant vector containing the nucleic acid molecule described in B1) is a recombinant expression vector obtained by cloning the nucleic acid molecule described in B1) into the expression vector pCMV.

6. A method for preparing a fusion protein, characterized in that, The procedure includes the following steps: introducing the recombinant vector described in claim 2 (B3) into mammalian cells, followed by cell culture, to obtain the LDLRAD3 mutant fusion protein.

7. Any of the following applications: Q1. The use of any of the biomaterials described in claims 2-5 in the preparation of the protein described in claim 1; Q2. The use of the protein of claim 1 or any of the biomaterials of claims 2-5 in the preparation of products for the prevention or treatment of diseases caused by VEEV infection.

8. A VEEV protein inhibitor comprising the protein of claim 1.

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