CN114478718A - Recombinant novel coronavirus protein vaccine, preparation method and application thereof - Google Patents

Recombinant novel coronavirus protein vaccine, preparation method and application thereof Download PDF

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CN114478718A
CN114478718A CN202210083654.XA CN202210083654A CN114478718A CN 114478718 A CN114478718 A CN 114478718A CN 202210083654 A CN202210083654 A CN 202210083654A CN 114478718 A CN114478718 A CN 114478718A
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CN114478718B (en
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李启明
张靖
梁宇
苏计国
杜丽芳
唐芳
邵帅
张学峰
雷泽华
刘兆明
韩子泊
刘宁
靳玉琴
张�浩
侯俊伟
侯亚楠
马智静
陈实
郑凡
沈福杰
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China National Biotec Research Institute Co ltd
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Abstract

The invention discloses a recombinant novel coronavirus protein, which comprises at least two artificially constructed non-natural RBD fragments, and a recombinant vaccine taking the fragment as a target antigen has the broad-spectrum protection capability of a cross-epidemic strain.

Description

Recombinant novel coronavirus protein vaccine, preparation method and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a recombinant novel coronavirus protein vaccine, a preparation method and application thereof.
Background
Novel coronaviruses (SARS-CoV-2), belonging to the order Nidovirales (Nidovirales), the family Coronaviridae (Coronaviridae), the subfamily orthocoronaviruses, the genus Betaconoviridus, the subfamily Sarbecovirus, the SARS-like virus species, single-stranded positive-strand RNA viruses, have an envelope with a genome of about 29.9kb in overall length, encode mostly non-structural proteins, are involved in functions such as viral replication and translation, and encode a small portion of the sequence of structural proteins, such as: s protein (spike protein), M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein). In addition, there are several accessory proteins: 3a,3b, p6,7a,7b,8b,9b and orf14, all of which are involved in viral assembly. S, M and the E protein constitute the viral envelope and are the major surface antigens of the virus to elicit an immune response. Wherein the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, and forms prominent homotrimers on the surface of the virus. S consists of two functional subunits, cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage site), which remain non-covalently associated in the prefusion conformation. The S2 subunit is also composed of multiple domains, whose function is primarily to mediate fusion of the virus with the host cell. The distal S1 subunit is structurally divided into four distinct domains: the vaccine comprises an N-terminal domain (NTD), a Receptor Binding Domain (RBD), a C-terminal domain 1(CTD1) and a C-terminal domain 2(CTD2), wherein the RBD is mainly responsible for binding with a receptor angiotensin converting enzyme 2 (ACE 2) on the surface of a host cell so as to mediate virus infection on the host cell, so that the S protein and the RBD are main targets for current genetic engineering vaccine development.
Up to now, there are 8 vaccines approved worldwide for marketing, BNT162b2 and mRNA-1273 for the united states authorization for Emergency Use (EUA), AZD1222 for the british authorization for Emergency Use (EUA), 3 new crown inactivated vaccines for chinese medicine chinese (beijing and wuhan) and beijing kexing, conxino biological adenovirus vector vaccines, zhifei biological recombinant protein vaccines, and russian approved "satellite V" for marketing. In addition, there are several hundred vaccines in multiple routes in different stages of clinical research. No matter which technical route is adopted, the marketed vaccine makes different degrees of contribution to epidemic prevention and control. The new coronavirus belongs to RNA virus, is easy to mutate, and has found more than seven million new coronavirus mutant strains globally, wherein the main mutant strains comprise: alpha (B.1.1.7) mutant, Beta (B.1.351) mutant, Gamma (P1) mutant, Epsilon (B.1.429) mutant, Delta (B.1.617.2) mutant, Kappa (B.1.617.1) mutant and Omicron (B.1.1.529) mutant, the occurrence of variant strains affects the protective effect of existing vaccines to different extents.
The world health organization classifies the above 5 variants as Variants (VOCs) that need attention based on the differences in the transmissibility, pathogenicity, or immune escape capabilities of the SARS-CoV-2 variants. Among these 5 VOCs, numerous studies have demonstrated that the Beta variant has a strong immune escape capacity and the Delta variant has a strong transmission capacity. Preliminary evidence shows that the Omicron variant has strong transmission capability and strong immune escape capability, and the immune escape capability of the Omicron variant is even far higher than that of a Beta strain.
The Omicron mutant was first detected in south africa 11, 9 days earlier than 2021. 26.11.2021, the fifth "Guanjie variant" was defined by the world health organization and named the Greek letter Omicron variant. The mutant contains 36 amino acid mutation sites in total, wherein the mutation sites comprise K417N/T, E484K/Q/A, N501Y, D614G and P681H/R mutation sites which appear at high frequency in an Alpha (B.1.1.7) mutant, a Beta (B.1.351) mutant, a Gamma (P1) mutant, a Delta (B.1.617.2) mutant and a Kappa (B.1.617.1) mutant of a dominant epidemic strain. The Omicron strain has gradually become the dominant epidemic strain in most regions of the world, and the cases of Omicron infection in countries such as the United kingdom, the United states, Denmark, Australia, Israel, south Africa, Spain and Canada account for more than half of the total cases. A large number of data show that the ormikrong mutant strain can improve the affinity of the virus to an ACE2 receptor, weaken the effect of a neutralizing antibody, thereby enhancing the toxicity and infectivity of the virus, accelerating the escape of the virus and reducing the protective effect of the vaccine.
The emergence of these strains with strong immune escape capacity raises a great concern for the effectiveness of the existing new corona vaccines. The development of a new generation vaccine with cross-protection capability is an effective means for dealing with these variants. Integrating multiple variants into the same immunogen to construct mosaic antigen molecules, which is a feasible strategy for realizing single-component cross-protection vaccines. Similar strategies have been applied in the design of broad-spectrum influenza vaccines. However, since there are many mutation sites in different mutants, it is urgent to find a broad-spectrum vaccine with excellent immunogenicity against many new strains of coronavirus epidemic.
Disclosure of Invention
One aspect of the present invention is to provide a single-component broad-spectrum recombinant protein vaccine capable of simultaneously generating good cross-neutralization activity against a plurality of novel coronavirus epidemic strains, particularly against a plurality of variant strains including Omicron, Beta, Delta, Alpha, Gamma, etc., against the lack of a broad-spectrum novel coronavirus (SARS-CoV-2) vaccine in the prior art, particularly against a vaccine simultaneously having good neutralization activity against 5 VOCs variant strains, particularly against Omicron strains.
The technical scheme provided by the invention is as follows:
a recombinant novel coronavirus protein which is in a trimer form and comprises three subunits consisting of 319 th to 537 th amino acid fragments of an RBD region of a novel coronavirus S protein,
the recombinant novel coronavirus protein comprises:
one subunit containing the K417N, L452R, T478K, F490S, and N501Y mutation sites; and/or
Another subunit containing the K417T, S477N, and E484K mutation sites. The subunit of the recombinant novel coronavirus protein constructed in the invention comprises at least two artificially constructed non-natural RBD fragments, and the recombinant vaccine taking the RBD fragments as the target antigen has broad-spectrum protection capability across epidemic strains.
The receptor-binding domain (RBD) of the new coronavirus spike (S) protein is directly involved in the binding of host cell receptors and plays a key role in the process of virus invasion into host cells. Meanwhile, a great deal of research indicates that RBD contains a major neutralizing epitope, and thus, RBD is one of the major target antigens for development of a neocoronary vaccine. However, RBD monomers are not highly immunogenic due to their small molecular size. The natural S protein is of a trimeric structure, and the RBD also exists in a trimerized form. The trimerization RBD is constructed, the natural structural form of the RBD is simulated to the maximum extent, meanwhile, the molecular size of the antigen is increased through trimerization, the repetitive regular arrangement of the antigen is realized, the B cell receptor crosslinking is enhanced, and the immunogenicity of the RBD can be obviously improved. Trimerization of antigen is usually achieved by introducing exogenous linker arms or trimerization motifs, but the introduction of exogenous sequences may bring unexpected immune response with certain safety risk. We achieved trimerization of RBD without introducing a trimerization motif by computational analysis of RBD structure. The RBD has the following structural characteristics: (1) the N-end and the C-end of the RBD are provided with longer flexible loop structures, and the loop structures of the RBD can be used as connecting arms among different RBDs in the trimerization process, so that the introduction of an external connecting arm is avoided, and the trimerization space barrier is reduced; (2) in the natural structure of the S protein, RBDs are relatively independent, strong interaction does not exist between the RBDs and other structural domains, and the folding of the RBDs does not need the assistance of the other structural domains; (3) the RBD has a compact space structure, the core structure of the RBD is composed of a plurality of beta sheet layers, 4 disulfide bonds are contained in the structure domain, the stability of the structure domain is further enhanced, meanwhile, the N-end and the C-end of the RBD are close, a large space barrier does not exist in the trimerization process, and the core structure of the RBD cannot be damaged. Based on the structural characteristics of the RBD, an interception scheme of the RBD is designed, namely an amino acid fragment at 319-537 sites is intercepted, loop structures at two ends of the RBD are kept as much as possible by the interception scheme, and meanwhile, the N-end and C-end interfaces are ensured to be closer. And then, connecting the three intercepted RBD zone segments in series end to form a new RBD trimerization fusion protein.
In order to realize cross protection of different variants of the new coronavirus and construct mosaic type RBD trimerization antigen, the inventor selects residue mutations with stronger immune escape capability from 20 mutation sites with the highest occurrence frequency by analyzing mutation sites, simultaneously considers the distribution of the mutations on a spatial structure to avoid mutual influence on the spatial structure, and artificially designs 2 non-natural RBDs, wherein one artificial RBD comprises 5 residue mutations in total of K417N, L452R, T478K, F490S and N501Y (namely a first subunit and can be taken as an artificial RBD-1), the other artificially constructed RBD comprises 3 residue mutations in total of K417T, S477N and E484K (namely a second subunit and can be taken as an artificial RBD-2), and in addition, the Omicron strain RBD with the strongest immune escape capability at present can be selected as a third subunit (namely a third subunit, which may be referred to as Omicron subunit), the Omicron variants contain 15 mutated RBD regions in total of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H. These mutations occur frequently in a number of different viral lineages, suggesting that these mutations have significant selective advantages in viral evolution, and also suggesting that these mutations may occur independently or in combination again in future mutants.
Based on the structural characteristics of the RBD region of the S protein of the novel coronavirus, the invention designs a brand-new fusion protein by utilizing a computational biology method, wherein the protein comprises three RBD structural domains, and forms a trimer form with stable antigen conformation under the condition that no exogenous connecting arm or other unrelated components can be introduced, so that the trimerization of the RBD protein is realized. The RBD tripolymer protein is recombined, expressed and purified by utilizing the genetic engineering technology, and then is mixed with an adjuvant to prepare the vaccine. After a certain dosage and dose immunization, protective neutralizing antibodies against a plurality of novel coronavirus epidemic strains can be generated, and the protective neutralizing antibodies can be used for treating and/or preventing SARS-CoV-2 infection and/or novel coronavirus diseases (COVID-19). Because the RBD region has definite function and clear structure, the gene is responsible for identifying the ACE2 receptor of host cells, and simultaneously has definite function and specific target point aiming at the Antibody generated by RBD, thereby avoiding inducing organisms to generate Antibody-Dependent Enhancement (ADE) to the maximum extent.
In the present invention, the three subunits of the recombinant novel coronavirus protein may further comprise RBD fragments of other novel coronavirus variants in addition to the two artificially constructed non-natural RBD fragments. Preferably, in one embodiment of the present invention, the recombinant novel coronavirus protein further comprises a subunit consisting of a 319-537 rd amino acid fragment of the RBD region of the novel coronavirus Omicron mutant S protein.
More preferably, in certain embodiments of the invention, the subunit contains five or more mutation sites selected from the group consisting of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H mutation sites. The partial combinations of the above mutation sites are shown in Table 1.
TABLE 1
Figure BDA0003482973430000051
Preferably, in some embodiments of the present invention, the primary structure of the three subunits of the above recombinant novel coronavirus protein is such that the three subunits are connected in series in the order of N-terminus to C-terminus.
In the present invention, the first subunit (first subunit/RBD-1), the second subunit (second subunit/RBD-2), and the third subunit (third subunit) may be arranged in any suitable order. For example, as shown in table 2.
TABLE 2
Serial number Order of arrangement Sequence of
1 First subunit + second subunit + third subunit SEQ ID No.1
2 First subunit + third subunit + second subunit SEQ ID No.2
3 Second subunit + first subunit + third subunit SEQ ID No.3
4 Second subunit + third subunit + first subunit SEQ ID No.4
5 Third subunit + first subunit + second subunit SEQ ID No.5
6 Third subunit + second subunit + first subunit SEQ ID No.6
7 First subunit + third subunit SEQ ID No.7
8 Second subunit + third subunit SEQ ID No.8
9 First subunit + first subunit SEQ ID No.9
10 Second subunit + second subunit SEQ ID No.10
11 Third subunit + third subunit SEQ ID No.11
Preferably, in some embodiments of the present invention, the three subunits of the recombinant novel coronavirus protein are connected in series in the order of the first subunit (RBD-1), the second subunit (RBD-2), and the third subunit.
Preferably, in an embodiment of the present invention, the amino acid sequence of the above recombinant novel coronavirus protein is represented by SEQ ID nos. 1 to 11 or a sequence having 95% or more homology with the amino acid sequence thereof except the mutation site. More preferably, the amino acid sequence of the recombinant novel coronavirus protein is the amino acid sequence shown as SEQ ID No.1 or a sequence with more than 95% homology with the amino acid sequence except the mutation site.
In the above embodiments, the amino acid sequence of SEQ ID Nos. 1 to 11 excluding the mutation site may be substituted, deleted, inserted with 1 or more amino acids to obtain a new amino acid sequence, and the new protein consisting of the amino acid sequence has the same or substantially the same immunological activity as the protein consisting of the amino acid sequence of SEQ ID Nos. 1 to 11, and the new amino acid sequence is also considered to be included in the scope of the present invention.
The sequence having 95% or more homology thereto is an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the recombinant novel coronavirus protein or the fusion protein except for the mutation site. The amino acid sequence of the fusion protein described in the present specification can be subjected to random or engineered point mutation in a suitable manner by those skilled in the art, for example, to obtain better affinity and/or dissociation properties, to improve expression performance, etc., and these sequences can have the same or substantially the same immunological activity as the recombinant novel coronavirus RBD trimer protein or the fusion protein, and these mutated amino acid sequences are included in the protection scope of the present invention.
The trimeric forms of the above recombinant novel coronavirus proteins may also incorporate certain exogenous trimerization motifs, e.g., T4 bacteriophage fibritin, which are also considered to be within the scope of the present invention. However, the safety of the recombinant coronavirus protein is weaker than that of the recombinant coronavirus protein of the present invention, and an unexpected immune response may occur.
In another aspect of the present invention, there is provided a fusion protein comprising the above recombinant novel coronavirus protein.
Preferably, in certain embodiments of the present invention, the fusion protein further comprises one or more selected from a signal peptide, a tag, or an immune enhancing peptide. The function of the signal peptide can be more favorable for the expression of the protein; the tag may be, for example, a Flag tag, enhanced green fluorescent protein (eGFP), glutathione mercaptotransferase (GST), etc., and the role thereof may be for detection, purification, isolation, etc. The above functional sequences may be used in any combination.
In another aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the above recombinant novel coronavirus protein, or encoding the above fusion protein.
Preferably, in one embodiment of the present invention, the codon of the trimeric protein is optimized by the inventors, and the obtained nucleotide sequence is shown in SEQ ID Nos. 12-22 or a sequence having more than 95% homology thereto.
The above-mentioned sequence having 95% or more homology thereto refers to a nucleotide sequence having 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence.
The nucleic acid molecule can be prepared by a known technique such as chemical synthesis or PCR amplification based on the nucleotide sequence. In general, the codons encoding the amino acids of the domains described above can be optimized to optimize their expression in a host cell. The information on the above-mentioned nucleotide sequence can be obtained by searching a database such as a known literature or NCBI (https:// www.ncbi.nlm.nih.gov /).
In another aspect of the present invention, there is provided a vector comprising the nucleic acid molecule described above.
In the present invention, the vector may be a linear vector or a cyclic vector. The vector may be a non-viral vector such as a plasmid, a viral vector (for example, an adenovirus vector, a measles virus vector, a mumps virus vector, a rubella virus vector, a variella virus vector, a poliovirus vector, or a yellow fever virus vector), or a vector using a transposon. The vector can contain regulatory sequences such as a promoter, a terminator and the like, and marker sequences such as a drug resistance gene, a reporter gene and the like.
Preferably, in one embodiment of the present invention, the vector is an expression vector of the nucleic acid molecule of the present invention for expressing the recombinant novel coronavirus protein of the present invention.
In another aspect of the present invention, there is provided a host cell comprising the above-described nucleic acid molecule or the above-described vector.
Preferably, in an embodiment of the present invention, the host cell is escherichia coli, a yeast cell, an insect cell, or a mammalian cell;
more preferably, in one embodiment of the present invention, the host cell is a CHO cell.
In another aspect of the present invention, there is provided a method for preparing the above recombinant novel coronavirus protein or the above fusion protein, comprising the steps of:
step A) preparing the nucleic acid molecule, constructing the expression vector, and transforming or transfecting the expression vector into the host cell;
step B) protein expression using the product of step A);
step C) purifying the expression product obtained in step B) to obtain the recombinant novel coronavirus protein or the recombinant novel coronavirus fusion protein.
Wherein, the nucleic acid molecule of the step A) comprises a nucleotide sequence which codes for the recombinant novel coronavirus protein or codes for the fusion protein.
Preferably, in one embodiment of the present invention, the above nucleotide sequence is represented by SEQ ID Nos. 12 to 24 or a sequence having 95% or more homology thereto.
The nucleic acid molecules may be prepared according to the nucleotide sequences described in the present specification using any suitable molecular biological method.
Wherein, the nucleotide sequence can be constructed in the corresponding expression vector of the host cell by any suitable method for constructing the expression vector in step A).
The expression vector is then transformed or transfected into the host cell. Preferably, in one embodiment of the present invention, the inventors constructed recombinant cell lines by transfecting a CHO cell expression vector into HEK293FT cells or CHO cells after constructing the vector.
Wherein, the protein expression in the step B) can express the recombinant protein according to different expression systems. Further, in one embodiment of the present invention, the inventors screened a cell line capable of stably secreting and expressing the recombinant novel coronavirus protein or fusion protein by limiting dilution.
Wherein the purification in step C) may be by any suitable method. For example, salting out, precipitation, dialysis or ultrafiltration, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and the like. Preferably, in one embodiment of the present invention, the recombinant novel coronavirus protein or fusion protein is purified by ion exchange and hydrophobic chromatography.
Of course, according to the prior art, a collection process of the target protein should also be included before the purification step is performed, for example. Collecting cell culture solution supernatant rich in target protein; the host cell after the target protein has been expressed is disrupted by any suitable disruption method such as ultrasonic disruption, repeated freeze-thaw disruption, and chemical treatment. The above collection of host cells is also understood to be within the scope of such purification.
In another aspect of the present invention, there is provided a use of the above recombinant novel coronavirus protein, the above fusion protein, the above nucleic acid molecule, the above vector or the host cell for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus.
The disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).
In another aspect of the present invention, there is provided a vaccine comprising the above recombinant novel coronavirus protein or the above fusion protein, and an adjuvant.
In an embodiment of the present invention, the vaccine is a recombinant protein vaccine (or a genetically engineered subunit vaccine). Further, in other embodiments of the present invention, the vaccine may be a genetically engineered vector vaccine, or may be a nucleic acid vaccine comprising a nucleotide sequence described in the present specification or encoding an amino acid sequence described in the present specification.
Any suitable adjuvant may be included in the vaccines of the present invention. However, in a preferred embodiment of the present invention, the adjuvant is aluminum hydroxide, aluminum phosphate, MF59, or CpG. More preferably, the adjuvant is aluminum hydroxide.
In another aspect of the present invention, there is provided a method for preparing the vaccine, wherein the purified recombinant coronavirus protein or the fusion protein is mixed with the adjuvant.
In another aspect of the invention there is provided the use of a vaccine as described above in the treatment and/or prevention of a novel coronavirus infection and/or a disease caused by a novel coronavirus.
In another aspect, the present invention provides the use of the above recombinant novel coronavirus protein, the above fusion protein, the above nucleic acid molecule, the above vector or the above host cell for preparing a medicament for enhancing immunity of a human who has been vaccinated with the novel coronavirus vaccine; the novel coronavirus vaccine is preferably a novel coronavirus vaccine inactivated vaccine.
The disease caused by the novel coronavirus is preferably novel coronavirus pneumonia (COVID-19).
In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the vaccine, and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier may be any pharmaceutically acceptable additive, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and their compositions, stabilizers, surfactants, preservatives, isotonic agents, and the like.
The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating and/or preventing a novel coronavirus infection and/or a disease caused by a novel coronavirus at an effective and safe dose.
In another aspect of the invention, there is provided a method of eliciting an immune response against a novel coronavirus or treating a novel coronavirus infection in a subject by administering an effective dose of said vaccine or said pharmaceutical composition to said subject.
The subject may be a human or other animal.
The administration may be intramuscular, intraperitoneal or subcutaneous.
The invention has the beneficial effects that:
the recombinant novel coronavirus protein comprises at least two artificially constructed non-natural RBD fragments, and the recombinant vaccine taking the fragments as the target antigen has the broad-spectrum protection capability of crossing epidemic strains.
Drawings
FIG. 1 is a schematic diagram showing the structure of C05G12 protein in example 1 of the present invention;
FIG. 2 is a SDS-PAGE result in example 2 of the present invention, wherein lane 1 is C05G12 protein, and M is protein marker (molecular weight: kDa: 250, 130, 100, 70, 55, 35, 25, 15, 10);
FIG. 3 is a Western-blot identification result chart of the protein purified in example 2 of the present invention, wherein lanes 4-6 are C05G12 protein, and M is protein marker (molecular weight standard: kDa: 250, 130, 100, 70, 55, 35, 25, 15, 10);
FIG. 4 is a graph showing the binding of the recombinant expressed protein to the MM43 neutralizing monoclonal antibody in example 3 of the present invention;
FIG. 5 is a graph showing the binding of the recombinant expressed protein to the MM57 neutralizing monoclonal antibody in example 3 of the present invention;
FIG. 6 is a graph showing the binding of the recombinant expressed protein to MM 117-neutralizing monoclonal antibody in example 3 of the present invention;
FIG. 7 is a graph showing the binding profile of the recombinant expression protein and the R001 neutralizing monoclonal antibody in example 3 of the present invention;
FIG. 8 is a graph showing the binding of the recombinant expression protein to the R117 neutralizing monoclonal antibody in example 3 of the present invention;
FIG. 9 is a graph showing the binding of the recombinant expression protein to the R118 neutralizing monoclonal antibody in example 3 of the present invention;
FIG. 10 is a graph showing the results of measuring the neutralizing antibody titer of mouse immune sera using the wild virus microneutralization assay in example 5 of the present invention;
FIG. 11 is a graph showing the results of measuring the neutralizing antibody titer of mouse immune sera using the pseudovirus microneutralization assay in example 5 of the present invention;
FIG. 12 is a graph showing the results of measuring the neutralizing antibody titer of rat immune serum using the wild virus microneutralization assay in example 6 of the present invention.
DESCRIPTION OF THE SEQUENCES
SEQ ID Nos. 1 to 11 are amino acid sequences of the recombinant novel coronavirus proteins according to the present invention;
SEQ ID No.12 is a nucleotide sequence which codes for the amino acid sequence shown in SEQ ID No.1, namely the nucleotide sequence which codes for the C05G12 protein;
SEQ ID Nos. 13 to 22 are nucleotide sequences encoding amino acid sequences shown in SEQ ID Nos. 2 to 11, respectively;
SEQ ID No.23 is the amino acid sequence of the C05 protein in example 3 of the present invention;
SEQ ID No.24 shows the amino acid sequence of C05C protein in example 3 of the present invention.
Detailed Description
The invention discloses a recombinant novel coronavirus protein vaccine, a preparation method and application thereof, and can be realized by appropriately improving process parameters by referring to the contents in the text by a person skilled in the art. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component. Throughout the specification and claims, unless explicitly indicated otherwise, the term "RBD" refers to the RBD domain of the spike protein of the novel coronavirus, and is understood to be interchangeable with "RBD" or the "novel coronavirus RBD domain".
Some terms appearing in the present invention are explained below.
The term "novel coronavirus", SARS-CoV-2, belonging to the order Nedovirales (Nidovirales), Coronaviridae (Coronaviridae), the subfamily orthocoronaviruses, the genus Betaconoviridae, the subgenus Sarbecovirus, the SARS-like virus species, a single-stranded positive-strand RNA virus, having an envelope with a genome of about 29.9kb in length, encoding for the most part non-structural proteins, involved in functions such as viral replication and translation, and a small portion of the sequence encoding a structural protein, such as: s protein (spike protein), M protein (membrane protein), E protein (envelope protein) and N protein (nucleo protein), in addition to several accessory proteins: 3a,3b, p6,7a,7b,8b,9b and orf14, all of which are involved in viral assembly. S, M and the E protein constitute the viral envelope and are the major surface antigens of the virus to elicit an immune response. Wherein the S protein is a transmembrane glycoprotein with a molecular weight of about 150kDa, and forms prominent homotrimers on the surface of the virus. S consists of two functional subunits, cleaved at the boundary between the S1 and S2 subunits (S1/S2 cleavage site), which remain non-covalently associated in the prefusion conformation. The S2 subunit is also composed of multiple domains, whose function is primarily to mediate fusion of the virus with the host cell. The distal S1 subunit is structurally divided into four distinct domains: the vaccine comprises an N-terminal domain (NTD), a Receptor Binding Domain (RBD), a C-terminal domain 1(CTD1) and a C-terminal domain 2(CTD2), wherein the RBD is mainly responsible for binding with a receptor angiotensin converting enzyme 2 (ACE 2) on the surface of a host cell so as to mediate virus infection on the host cell, so that the S protein and the RBD are main targets for current genetic engineering vaccine development.
The term "trimeric form" is a type of higher structure of proteins. Three protein subunits are contained in the protein, namely the trimer form.
The term "at least one" is understood to mean that two of the three amino acid sequences are identical or that the three amino acid sequences differ from one another.
The term "primary structure" is a linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is from the amino-terminus (N) to the carboxy-terminus (C).
The term "fusion protein" refers to an expression product of one, two or more genes obtained by recombinant DNA techniques. The fusion protein technology is a purposeful gene fusion and protein expression method for obtaining a large number of standard fusion proteins, and a novel target protein with multiple functions can be constructed and expressed by utilizing the fusion protein technology.
The term "vector" is a nucleic acid delivery vehicle into which a polynucleotide may be inserted. When a vector is capable of providing for expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.
The term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biological techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
The term "treating" refers to reducing the likelihood of disease pathology, reducing the occurrence of disease symptoms, e.g., to the extent that a subject has a longer survival period or reduced discomfort. Treatment can refer to the ability of a therapy to reduce the symptoms, signs, or causes of a disease when administered to a subject. Treating also refers to alleviating or reducing at least one clinical symptom and/or inhibiting or delaying the progression of a disorder and/or preventing or delaying the onset of a disease or disorder.
The term "subject" refers to any human or other animal, particularly other mammals, that is being prevented, treated, diagnosed. Other mammals may include, for example, dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, guinea pigs, mice, and the like. In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1: novel coronavirus RBD trimer protein based on protein structure and computational biological design
The new coronavirus is continuously mutated, multiple rounds of outbreaks of epidemic situations are caused by the emergence of multiple variant strains, and a large number of researches prove that the variant strains have stronger immune escape capacity. Especially, the Omicron variant strains which are exploded recently have preliminary research evidence that the variant strains have very strong transmission capability and strong immune escape capability. The emergence of strains with strong immune escape capacity, especially the Omicron variant strains, has caused great worry of people about the effectiveness of the existing new crown vaccines. The development of a new generation vaccine with cross-protection capability is an effective means for dealing with these variants. Integrating multiple variants into the same immunogen to construct mosaic antigen molecules, which is a feasible strategy for realizing single-component cross-protection vaccines.
The receptor-binding domain (RBD) of the new coronavirus spike (S) protein is directly involved in the binding of host cell receptors and plays a key role in the process of virus invasion into host cells. Meanwhile, a great deal of research indicates that RBD contains a major neutralizing epitope, and thus, RBD is one of the major target antigens for development of a neocoronary vaccine. However, RBD monomers are not highly immunogenic due to their small molecular size. The natural S protein is of a trimeric structure, and the RBD also exists in a trimerized form. The trimerization RBD is constructed, the natural structural form of the RBD is simulated to the maximum extent, meanwhile, the molecular size of the antigen is increased through trimerization, the repetitive regular arrangement of the antigen is realized, the B cell receptor crosslinking is enhanced, and the immunogenicity of the RBD can be obviously improved. Trimerization of antigen is usually achieved by introducing exogenous linker arms or trimerization motifs, but the introduction of exogenous sequences may bring unexpected immune response with certain safety risk. We achieved trimerization of RBD without introducing a trimerization motif by computational analysis of RBD structure. The RBD has the following structural characteristics: (1) the N-end and the C-end of the RBD are provided with longer flexible loop structures, and the loop structures of the RBD can be used as connecting arms among different RBDs in the trimerization process, so that the introduction of an external connecting arm is avoided, and the trimerization space barrier is reduced; (2) in the natural structure of the S protein, RBDs are relatively independent, strong interaction does not exist between the RBDs and other structural domains, and the folding of the RBDs does not need the assistance of the other structural domains; (3) the RBD has a compact space structure, the core structure of the RBD is composed of a plurality of beta sheet layers, 4 disulfide bonds are contained in the structure domain, the stability of the structure domain is further enhanced, meanwhile, the N-end and the C-end of the RBD are close, a large space barrier does not exist in the trimerization process, and the core structure of the RBD cannot be damaged. Based on the structural characteristics of the RBD, an interception scheme of the RBD is designed, namely an amino acid fragment at 319-537 sites is intercepted, loop structures at two ends of the RBD are kept as much as possible by the interception scheme, and meanwhile, the N-end and C-end interfaces are ensured to be closer. And then, connecting the three intercepted RBD zone segments in series end to form a new RBD trimerization fusion protein.
In order to realize cross protection of different variants of the new coronavirus, a mosaic RBD trimerization antigen is constructed, and key mutation sites of various variants are integrated into three RBDs in the antigen. One of the RBDs was derived from an Omicron variant and contained 15 mutations in total, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N63 501Y and Y505H. The other two RBDs are artificially constructed non-natural RBDs, and key mutation sites with strong immune escape capacity, which frequently appear in different variants, are integrated. In order to realize the screening and integration of key mutation sites, the mutation sites of various variant strains of the new coronavirus are analyzed, residue mutations with strong immune escape capacity are selected from 20 mutation sites with the highest occurrence frequency, and meanwhile, the distribution of the mutations on a spatial structure is considered, so that the mutual influence on the spatial structure is avoided. Based on the above analysis, two artificially constructed RBDs incorporated 8 key site mutations in total, one of which contained 5 residue mutations of K417N, L452R, T478K, F490S and N501Y, and the other of which contained 3 residue mutations of K417T, S477N and E484K. Numerous studies have demonstrated that these residue mutations can cause strong immune escape and occur frequently in a number of different viral lineages, suggesting that these mutations have significant selective advantages in viral evolution and also suggest that these mutations may occur independently or in combination again in future mutants. The three RBDs are sequentially connected in series to construct mosaic type trimerization RBD fusion protein, and the specific series connection mode is as follows: the C-terminus of RBD containing the "K417N, L452R, T478K, F490S and N501Y" mutation was ligated to the N-terminus of RBD containing the "K417T, S477N and E484K" mutations, and the C-terminus of RBD containing the "K417T, S477N and E484K" mutations was ligated to the N-terminus of the Omicron variant RBD. Through the series connection mode, the fused sequence is shown as SEQ ID No.1, namely the C05G12 protein.
By utilizing a homologous modeling method, a possible spatial structure of mosaic-type trimerized RBD fusion protein is built, and the result is shown in figure 1. The fusion protein is shown to contain three independent RBD domains, and can form an antigen conformation stable trimer form, and the protein contains 23 mutation sites, and the mutation sites are shown in a ball and stick model in figure 1. Theoretically, the recombinant vaccine taking the antigen as the target antigen covers the frequently-occurring key mutation sites in a large number of variant strains and has broad-spectrum protection capability across epidemic strains. In addition, compared with the traditional strategy of preparing a plurality of monovalent vaccines and realizing broad-spectrum protection by multivalent combination, the method realizes the broad-spectrum protection effect of the cross-epidemic strains on one antigen molecule, and has obvious advantages in the time cost, the economic cost and the vaccine productivity of vaccine preparation.
Example 2: recombinant protein expression, purification and identification
According to the codon preference of a CHO cell expression system, the nucleotide sequence of the coded recombinant protein C05G12 (the amino acid sequence is shown as SEQ ID NO. 1) is subjected to codon optimization, and the optimized nucleotide sequence is shown as SEQ ID NO. 12. The CHO cell expression vector is constructed and then transfected into 293FT cells or CHO cells to construct recombinant cell strains, the cell strains which can stably secrete and express the recombinant protein C05G12 are obtained by screening through a limiting dilution method, the supernatant is harvested after cell culture, and the recombinant protein C05G12 with the purity of more than or equal to 95 percent is obtained after serial chromatography purification. The SDS-PAGE detection result is shown in figure 2, the molecular weight of the protein is 70-100 kD, and meanwhile, part of product related substances such as dimeric protein, monomeric protein and the like can be seen.
The purified C05G12 protein is electrophoresed through SDS-PAGE and then electrically transferred to a PVDF membrane, and Western-blot identification is carried out by using a RBD specific antibody (manufacturer: Beijing Yiqian Shenzhou science and technology Co., Ltd.; product number: 40591-T62; dilution: 2000 times) (the result is shown in figure 3), so that the C05G12 protein can be combined with the RBD specific antibody, and has good biological activity. The purified C05G12 protein is subjected to molecular exclusion chromatography by using a TSKgel G2500PW gel chromatographic column, and the protein purity is more than 90%.
Example 3: detection of physicochemical properties and biological activity of C05G12 protein
The purified C05G12 protein, C05 protein (amino acid sequence is shown as SEQ ID No.23, which is obtained by 293FT cell or CHO cell recombinant expression and chromatography purification) and C05C protein (amino acid sequence is shown as SEQ ID No.24, which is obtained by 293FT cell or CHO cell recombinant expression and chromatography purification), prototype strain RBD protein (manufacturer: Beijing-Yi-Qian-Shenzhou technology Co., Ltd.; No. 371: 40592-V08B), RBD protein consistent with Delta strain virus mutation site (K417N, E484K, N501Y; manufacturer: Beijing-Yi-Qian-Shenzhou technology Co., Ltd.; No. 40592-V08H85), RBD protein consistent with Delta strain virus mutation site (L452R, T K; manufacturer: Beijing-Shenzhou technology Co., No. 373: 40592-V02H3), RBD protein consistent with Omicron strain virus mutation site (G417, S6346, S8646, S8653, S86478, S8653, Q498R, N501Y, Y505H; the manufacturer: beijing Yiqiao Shenzhou science and technology Co., Ltd; the goods number is: 40592-V08H121), respectively diluting to 1.0000 μ g/ml, 0.3333 μ g/ml, 0.1111 μ g/ml, 0.0370 μ g/ml, 0.0123 μ g/ml, 0.0041 μ g/ml, 0.0013 μ g/ml, 0.0004 μ g/ml and 100 μ l/well by using a coating solution, coating the coating solution on a 96-well enzyme label plate, carrying out 4 ℃, 8-12H and taking a blank well as a negative control; adding a sealing liquid after washing the plate by using the PBST solution, and sealing for 2h at 37 ℃; after the PBST solution is washed, the PBST solution is respectively added with and diluted to 1 mu g/ml of antibody, and the antibody specifically comprises MM43 monoclonal antibody (manufacturer: Beijing Yi Qiao Shen technology Limited company; manufacturer: 40591-MM43), MM57 monoclonal antibody (manufacturer: Beijing Yi Qiao Shen technology Limited company; manufacturer: 40592-MM57), MM117 monoclonal antibody (manufacturer: Beijing Yi Qiao Shen technology Limited company; manufacturer: 592-MM117), R001 monoclonal antibody (manufacturer: Beijing Yi Qiao Shen technology Limited company; manufacturer: 40592-R001), R117 monoclonal antibody (manufacturer: Beijing Yi Qiao Shen technology Limited company; manufacturer: 40592-R117), R118 monoclonal antibody (manufacturer: Beijing Yi Qiao Shen technology Limited company; manufacturer: 40592-R118), 100 mu l/well, 37 ℃ for 1 h; after washing the plate with the PBST solution, adding a diluted horseradish peroxidase-labeled goat anti-mouse or goat anti-rabbit IgG antibody, 100 mu l/hole, and incubating for 1h at 37 ℃; after the PBST solution is washed, adding the developing solutions A and B in sequence, developing at room temperature for 5-10 min, and adding the stop solution C; the readings of the enzyme-linked immunosorbent assay were carried out at two wavelengths (OD450nm and 630nm), cut-off values were determined, and protein concentration-absorbance value curves were plotted.
The results of the activity of binding to MM43 monoclonal antibody are shown in FIG. 4, the results of the activity of binding to MM57 monoclonal antibody are shown in FIG. 5, the results of the activity of binding to MM117 monoclonal antibody are shown in FIG. 6, the results of the activity of binding to R001 monoclonal antibody are shown in FIG. 7, the results of the activity of binding to R117 monoclonal antibody are shown in FIG. 8, and the results of the activity of binding to R118 monoclonal antibody are shown in FIG. 9, and it was found that C05G12 protein has the protein biological activities of a plurality of variants such as Beta strain, Delta strain, and Omicron strain.
Example 4: preparation of recombinant coronavirus vaccine
Diluting the purified recombinant protein C05G12 to 2 times of target antigen concentration, mixing and adsorbing the purified recombinant protein C05G12 with 1.2mg/ml aluminum hydroxide adjuvant according to a ratio of 1:1 (w/w), stirring the mixture on a magnetic stirrer for 40-120 min at a rotating speed of 200-300 rpm to obtain a semi-finished vaccine product, wherein the content of residual protein in supernatant is less than 10% of the content of total protein, and each bottle of the semi-finished vaccine product is aseptically subpackaged according to the loading of 0.5ml to obtain the finished vaccine product.
Example 5: immunological effect evaluation of recombinant novel coronavirus vaccine in mice
The prepared recombinant novel coronavirus vaccine (wherein, the C05G12 protein vaccine is the vaccine prepared in the embodiment 4 of the invention, the C05 protein in the C05 protein vaccine is the recombinant protein in the form of homotrimer formed by 319-537 amino acid fragments of RBD regions of three novel coronavirus original strain S proteins, the C05 protein vaccine is prepared by the same method as the embodiment 4 of the invention), BALB/C mice (purchased from Beijing Wintolite laboratory animal technology Limited company, SPF grade, female, 6-8 weeks old) which are inoculated with 1-time inactivated vaccine (0.5 mu G/dose) are respectively immunized by intraperitoneal injection, 0.5 mu G/dose is obtained, specifically after 1-time immunization of the 0w inactivated vaccine, then immunizing 1 needle of recombined novel coronavirus vaccine or inactivated vaccine at the 3w, and collecting blood and separating serum at the 4 w. The test scheme is adopted for the purpose of investigating and simulating the neutralizing capacity of people who are inoculated with the inactivated vaccine and then boosted to a plurality of variant strains.
The neutralizing activity of the mouse serum after immunization against the prototype strain, the Beta strain, the Delta strain and the Omicron strain viruses is detected by using a wild virus micro-neutralization test, the result is shown in figure 10, the GMT value of a serum neutralizing antibody is shown in table 3, the C05G12 protein can generate wide neutralizing activity against various viruses, the neutralizing capacity against the prototype strain, the Beta strain and the Delta strain viruses is equivalent to that of the C05 protein, the neutralizing capacity against the Omicron virus is obviously superior to that of the C05 protein, the neutralizing activity against the prototype strain, the Beta strain, the Delta strain and the Omicron strain viruses is obviously superior to that of an inactivated vaccine, and the broad-spectrum protective capacity is expected to be generated, so that the inactivated vaccine is an ideal enhanced immunity candidate vaccine.
TABLE 3 mouse immune serum neutralizing antibody GMT values (wild virus microneutralization test)
Figure BDA0003482973430000171
The neutralizing activity of the sera of the immunized mice against the prototype strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Lambda strain, Mu strain and the Omicron strain pseudoviruses was examined by using a pseudovirus micro-neutralization test, and as a result, as shown in FIG. 11, the values of serum neutralizing antibodies GMT were as shown in Table 4, and it was found that the C05G12 protein could produce a broad neutralizing activity against a variety of pseudoviruses, the neutralizing activity against the prototype strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Lambda strain and Mu strain pseudoviruses was comparable to that of C05 protein, the neutralizing activity against the Omicron virus was significantly superior to that of C05 protein, and the neutralizing activity against the prototype strain, Alpha strain, Beta strain, Delta strain, Gamma strain, Lambda strain, Mu strain and Omicron strain pseudoviruses was significantly superior to that of C3556 protein, and a broad-spectrum vaccine was expected to be an ideal vaccine candidate for boosting immunity.
TABLE 4 mouse immune serum neutralizing antibody GMT values (pseudovirus microneutralization test)
Figure BDA0003482973430000181
Example 6: immunological effect evaluation of recombinant novel coronavirus vaccine in rat body
The recombinant new coronavirus vaccine prepared (wherein, the C05G12 protein vaccine is the vaccine prepared in the embodiment 4 of the invention, the C05 protein in the C05 protein vaccine is the recombinant protein in the form of homotrimer composed of 319 th to 537 th amino acid fragments of RBD region of S protein of three new original strains of coronavirus, the C05 protein vaccine is prepared by the same method as the embodiment 4 of the invention, the C05C protein in the C05C protein vaccine is the recombinant protein in the form of heterotrimer composed of 319 th to 537 th amino acid fragments of RBD region of three new strains of coronavirus, wherein two subunits are respectively from Beta strain and Kappa strain, the C05C protein vaccine is prepared by the same method as the embodiment 4 of the invention), Wistar rats which have been inoculated with 1-needle inactivated vaccine (human dose) are respectively immunized by intramuscular injection (purchased from Beijing Wistar Tuoli animal technology, SPF grade, female and male, 6-8 weeks old), 10 mu g/dose, specifically 1 needle after 0w of immune inactivated vaccine, 1 needle of 3w of immune inactivated vaccine, 1 needle of recombinant novel coronavirus vaccine or inactivated vaccine, and 4w of blood collection and serum separation. The test scheme is adopted for the purpose of investigating and simulating the neutralizing capacity of people who are inoculated with the inactivated vaccine and then boosted to a plurality of variant strains.
The neutralizing activity of the immunized rat serum against the prototype strain, the Beta strain, the Delta strain and the Omicron strain viruses is detected by using a wild virus micro-neutralization test, and the result is shown in figure 12, the GMT value of a serum neutralizing antibody is shown in table 5, so that the C05G12 protein can generate wide neutralizing activity against various viruses, the neutralizing capacity against the prototype strain, the Beta strain and the Delta strain viruses is equivalent to that of the C05 protein and the C05C protein, the neutralizing capacity against the Omicron virus is obviously superior to that of the C05 protein and that of the C05C protein, and the neutralizing activity against the prototype strain, the Beta strain, the Delta strain and the Omicron strain viruses is obviously superior to that of an inactivated vaccine, and the broad-spectrum protective capacity is expected to be generated, so that the immune enhancing candidate vaccine is an ideal type.
TABLE 5 neutralizing antibody GMT values in rat immune sera
Figure BDA0003482973430000191
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> a recombinant novel coronavirus protein vaccine, preparation method and application thereof
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Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
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Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
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Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
545 550 555 560
Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly
610 615 620
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 2
<211> 657
<212> PRT
<213> Artificial
<400> 2
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
260 265 270
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
370 375 380
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
385 390 395 400
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
545 550 555 560
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly
610 615 620
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 3
<211> 657
<212> PRT
<213> Artificial
<400> 3
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val
370 375 380
Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
545 550 555 560
Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly
610 615 620
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 4
<211> 657
<212> PRT
<213> Artificial
<400> 4
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
260 265 270
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
370 375 380
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
385 390 395 400
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
545 550 555 560
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys
595 600 605
Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly
610 615 620
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 5
<211> 657
<212> PRT
<213> Artificial
<400> 5
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys
145 150 155 160
Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser
165 170 175
Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val
370 375 380
Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
545 550 555 560
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly
610 615 620
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 6
<211> 657
<212> PRT
<213> Artificial
<400> 6
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys
145 150 155 160
Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser
165 170 175
Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
545 550 555 560
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys
595 600 605
Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly
610 615 620
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 7
<211> 657
<212> PRT
<213> Artificial
<400> 7
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val
370 375 380
Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
545 550 555 560
Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly
610 615 620
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 8
<211> 657
<212> PRT
<213> Artificial
<400> 8
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
545 550 555 560
Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly
610 615 620
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 9
<211> 657
<212> PRT
<213> Artificial
<400> 9
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val
370 375 380
Glu Gly Phe Asn Cys Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
545 550 555 560
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys
595 600 605
Tyr Ser Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly
610 615 620
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 10
<211> 657
<212> PRT
<213> Artificial
<400> 10
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr
145 150 155 160
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
260 265 270
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Thr Pro Cys Asn Gly Val
370 375 380
Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
385 390 395 400
Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
545 550 555 560
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly
610 615 620
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 11
<211> 657
<212> PRT
<213> Artificial
<400> 11
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys
145 150 155 160
Pro Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser
165 170 175
Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr
210 215 220
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
225 230 235 240
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
245 250 255
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
260 265 270
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
275 280 285
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
290 295 300
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
305 310 315 320
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
325 330 335
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
340 345 350
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
355 360 365
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
370 375 380
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
385 390 395 400
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
405 410 415
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
420 425 430
Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val Arg
435 440 445
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala
450 455 460
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
465 470 475 480
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr
485 490 495
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
500 505 510
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
515 520 525
Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys
530 535 540
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys
545 550 555 560
Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
565 570 575
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
580 585 590
Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys
595 600 605
Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly
610 615 620
His Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
625 630 635 640
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
645 650 655
Lys
<210> 12
<211> 1971
<212> DNA
<213> Artificial
<400> 12
agggtgcagc ccaccgagag catcgtgagg ttccctaaca tcaccaacct gtgccctttt 60
ggcgaggtgt tcaacgctac aagattcgct agcgtgtacg cttggaacag aaagagaatt 120
tccaactgtg tggccgatta cagcgtcctg tataattccg cctcctttag cacctttaaa 180
tgctacggcg tgagccctac caagctgaac gacctgtgct tcaccaacgt ctacgccgat 240
agcttcgtga ttagaggcga tgaggtcaga cagatcgccc ctggacagac cggcaacatc 300
gccgattaca attataagct gcctgacgac ttcaccggat gtgtcattgc ttggaacagc 360
aataacctcg atagcaaagt gggaggcaac tacaattaca gatacagact gttcagaaag 420
tccaacctga aacctttcga gagggatatc agcaccgaga tttaccaagc cggcagcaag 480
ccttgcaacg gcgtcgaggg cttcaattgc tacagccctc tgcagagcta cggattccaa 540
cccacatacg gcgtgggcta tcagccttac agagtggtcg tgctcagctt tgagctgctg 600
cacgcccctg ccaccgtgtg cggccccaag aagtccacca atctggtcaa gaacaaaaga 660
gtgcagccta ccgaatccat cgtcagattt cccaatatca caaacctctg tcctttcgga 720
gaagtgttca atgccacaag attcgcctcc gtctacgcct ggaacagaaa aagaatcagc 780
aactgcgtgg ctgactacag cgtgctgtac aacagcgcta gcttcagcac cttcaaatgt 840
tacggcgtgt cccctaccaa gctgaacgat ctctgcttca ccaacgtgta cgctgattcc 900
ttcgtgatca gaggcgatga ggtgagacag atcgcccccg gacagaccgg caccattgcc 960
gactacaact ataaactgcc tgatgacttt accggatgcg tcattgcttg gaacagcaac 1020
aatctggaca gcaaggtggg cggcaattac aattacctgt atagactctt tagaaagagc 1080
aacctcaagc ctttcgagag agatatcagc acagagattt accaagccgg caatacccct 1140
tgcaacggag tcaagggctt caactgttac ttccctctgc agagctacgg atttcagcct 1200
accaatggcg tgggatatca gccctacaga gtggtcgtgc tgagcttcga gctgctgcat 1260
gctcccgcca ccgtgtgcgg ccctaaaaag agcaccaacc tggtcaagaa caagagagtc 1320
caacctaccg agtccatcgt gagattcccc aacatcacaa atctgtgccc tttcgacgag 1380
gtcttcaatg ccacaagatt cgctagcgtg tacgcctgga acagaaagag aatcagcaat 1440
tgcgtcgccg actacagcgt cctctacaac ctggcccctt tcttcacatt caagtgttat 1500
ggagtgtccc ccaccaaact gaacgatctg tgtttcacaa atgtgtacgc tgactccttt 1560
gtgatcagag gagatgaggt gaggcagatc gcccctggac agaccggaaa tattgccgat 1620
tacaactaca aactgcctga cgatttcacc ggctgtgtca tcgcctggaa ctccaacaag 1680
ctggatagca aggtgtccgg caactacaac tatctctaca gactgtttag gaagtccaat 1740
ctgaagcctt ttgagaggga tattagcacc gaaatttacc aagccggaaa taagccttgt 1800
aacggcgtgg ctggattcaa ctgctacttt cctctgagaa gctacagctt cagacccacc 1860
tatggcgtgg gacaccaacc ttacagagtc gtggtgctct ccttcgaact gctccacgct 1920
cctgccacag tgtgtggccc caagaaatcc accaatctcg tgaagaacaa g 1971
<210> 13
<211> 1971
<212> DNA
<213> Artificial
<400> 13
agggtgcagc ccaccgagag catcgtgagg ttccctaaca tcaccaacct gtgccctttt 60
ggcgaggtgt tcaacgctac aagattcgct agcgtgtacg cttggaacag aaagagaatt 120
tccaactgtg tggccgatta cagcgtcctg tataattccg cctcctttag cacctttaaa 180
tgctacggcg tgagccctac caagctgaac gacctgtgct tcaccaacgt ctacgccgat 240
agcttcgtga ttagaggcga tgaggtcaga cagatcgccc ctggacagac cggcaacatc 300
gccgattaca attataagct gcctgacgac ttcaccggat gtgtcattgc ttggaacagc 360
aataacctcg atagcaaagt gggaggcaac tacaattaca gatacagact gttcagaaag 420
tccaacctga aacctttcga gagggatatc agcaccgaga tttaccaagc cggcagcaag 480
ccttgcaacg gcgtcgaggg cttcaattgc tacagccctc tgcagagcta cggattccaa 540
cccacatacg gcgtgggcta tcagccttac agagtggtcg tgctcagctt tgagctgctg 600
cacgcccctg ccaccgtgtg cggccccaag aagtccacca atctggtcaa gaacaaaaga 660
gtccaaccta ccgagtccat cgtgagattc cccaacatca caaatctgtg ccctttcgac 720
gaggtcttca atgccacaag attcgctagc gtgtacgcct ggaacagaaa gagaatcagc 780
aattgcgtcg ccgactacag cgtcctctac aacctggccc ctttcttcac attcaagtgt 840
tatggagtgt cccccaccaa actgaacgat ctgtgtttca caaatgtgta cgctgactcc 900
tttgtgatca gaggagatga ggtgaggcag atcgcccctg gacagaccgg aaatattgcc 960
gattacaact acaaactgcc tgacgatttc accggctgtg tcatcgcctg gaactccaac 1020
aagctggata gcaaggtgtc cggcaactac aactatctct acagactgtt taggaagtcc 1080
aatctgaagc cttttgagag ggatattagc accgaaattt accaagccgg aaataagcct 1140
tgtaacggcg tggctggatt caactgctac tttcctctga gaagctacag cttcagaccc 1200
acctatggcg tgggacacca accttacaga gtcgtggtgc tctccttcga actgctccac 1260
gctcctgcca cagtgtgtgg ccccaagaaa tccaccaatc tcgtgaagaa caagagagtg 1320
cagcctaccg aatccatcgt cagatttccc aatatcacaa acctctgtcc tttcggagaa 1380
gtgttcaatg ccacaagatt cgcctccgtc tacgcctgga acagaaaaag aatcagcaac 1440
tgcgtggctg actacagcgt gctgtacaac agcgctagct tcagcacctt caaatgttac 1500
ggcgtgtccc ctaccaagct gaacgatctc tgcttcacca acgtgtacgc tgattccttc 1560
gtgatcagag gcgatgaggt gagacagatc gcccccggac agaccggcac cattgccgac 1620
tacaactata aactgcctga tgactttacc ggatgcgtca ttgcttggaa cagcaacaat 1680
ctggacagca aggtgggcgg caattacaat tacctgtata gactctttag aaagagcaac 1740
ctcaagcctt tcgagagaga tatcagcaca gagatttacc aagccggcaa taccccttgc 1800
aacggagtca agggcttcaa ctgttacttc cctctgcaga gctacggatt tcagcctacc 1860
aatggcgtgg gatatcagcc ctacagagtg gtcgtgctga gcttcgagct gctgcatgct 1920
cccgccaccg tgtgcggccc taaaaagagc accaacctgg tcaagaacaa g 1971
<210> 14
<211> 1971
<212> DNA
<213> Artificial
<400> 14
agagtgcagc ctaccgaatc catcgtcaga tttcccaata tcacaaacct ctgtcctttc 60
ggagaagtgt tcaatgccac aagattcgcc tccgtctacg cctggaacag aaaaagaatc 120
agcaactgcg tggctgacta cagcgtgctg tacaacagcg ctagcttcag caccttcaaa 180
tgttacggcg tgtcccctac caagctgaac gatctctgct tcaccaacgt gtacgctgat 240
tccttcgtga tcagaggcga tgaggtgaga cagatcgccc ccggacagac cggcaccatt 300
gccgactaca actataaact gcctgatgac tttaccggat gcgtcattgc ttggaacagc 360
aacaatctgg acagcaaggt gggcggcaat tacaattacc tgtatagact ctttagaaag 420
agcaacctca agcctttcga gagagatatc agcacagaga tttaccaagc cggcaatacc 480
ccttgcaacg gagtcaaggg cttcaactgt tacttccctc tgcagagcta cggatttcag 540
cctaccaatg gcgtgggata tcagccctac agagtggtcg tgctgagctt cgagctgctg 600
catgctcccg ccaccgtgtg cggccctaaa aagagcacca acctggtcaa gaacaagagg 660
gtgcagccca ccgagagcat cgtgaggttc cctaacatca ccaacctgtg cccttttggc 720
gaggtgttca acgctacaag attcgctagc gtgtacgctt ggaacagaaa gagaatttcc 780
aactgtgtgg ccgattacag cgtcctgtat aattccgcct cctttagcac ctttaaatgc 840
tacggcgtga gccctaccaa gctgaacgac ctgtgcttca ccaacgtcta cgccgatagc 900
ttcgtgatta gaggcgatga ggtcagacag atcgcccctg gacagaccgg caacatcgcc 960
gattacaatt ataagctgcc tgacgacttc accggatgtg tcattgcttg gaacagcaat 1020
aacctcgata gcaaagtggg aggcaactac aattacagat acagactgtt cagaaagtcc 1080
aacctgaaac ctttcgagag ggatatcagc accgagattt accaagccgg cagcaagcct 1140
tgcaacggcg tcgagggctt caattgctac agccctctgc agagctacgg attccaaccc 1200
acatacggcg tgggctatca gccttacaga gtggtcgtgc tcagctttga gctgctgcac 1260
gcccctgcca ccgtgtgcgg ccccaagaag tccaccaatc tggtcaagaa caaaagagtc 1320
caacctaccg agtccatcgt gagattcccc aacatcacaa atctgtgccc tttcgacgag 1380
gtcttcaatg ccacaagatt cgctagcgtg tacgcctgga acagaaagag aatcagcaat 1440
tgcgtcgccg actacagcgt cctctacaac ctggcccctt tcttcacatt caagtgttat 1500
ggagtgtccc ccaccaaact gaacgatctg tgtttcacaa atgtgtacgc tgactccttt 1560
gtgatcagag gagatgaggt gaggcagatc gcccctggac agaccggaaa tattgccgat 1620
tacaactaca aactgcctga cgatttcacc ggctgtgtca tcgcctggaa ctccaacaag 1680
ctggatagca aggtgtccgg caactacaac tatctctaca gactgtttag gaagtccaat 1740
ctgaagcctt ttgagaggga tattagcacc gaaatttacc aagccggaaa taagccttgt 1800
aacggcgtgg ctggattcaa ctgctacttt cctctgagaa gctacagctt cagacccacc 1860
tatggcgtgg gacaccaacc ttacagagtc gtggtgctct ccttcgaact gctccacgct 1920
cctgccacag tgtgtggccc caagaaatcc accaatctcg tgaagaacaa g 1971
<210> 15
<211> 1971
<212> DNA
<213> Artificial
<400> 15
agagtgcagc ctaccgaatc catcgtcaga tttcccaata tcacaaacct ctgtcctttc 60
ggagaagtgt tcaatgccac aagattcgcc tccgtctacg cctggaacag aaaaagaatc 120
agcaactgcg tggctgacta cagcgtgctg tacaacagcg ctagcttcag caccttcaaa 180
tgttacggcg tgtcccctac caagctgaac gatctctgct tcaccaacgt gtacgctgat 240
tccttcgtga tcagaggcga tgaggtgaga cagatcgccc ccggacagac cggcaccatt 300
gccgactaca actataaact gcctgatgac tttaccggat gcgtcattgc ttggaacagc 360
aacaatctgg acagcaaggt gggcggcaat tacaattacc tgtatagact ctttagaaag 420
agcaacctca agcctttcga gagagatatc agcacagaga tttaccaagc cggcaatacc 480
ccttgcaacg gagtcaaggg cttcaactgt tacttccctc tgcagagcta cggatttcag 540
cctaccaatg gcgtgggata tcagccctac agagtggtcg tgctgagctt cgagctgctg 600
catgctcccg ccaccgtgtg cggccctaaa aagagcacca acctggtcaa gaacaagaga 660
gtccaaccta ccgagtccat cgtgagattc cccaacatca caaatctgtg ccctttcgac 720
gaggtcttca atgccacaag attcgctagc gtgtacgcct ggaacagaaa gagaatcagc 780
aattgcgtcg ccgactacag cgtcctctac aacctggccc ctttcttcac attcaagtgt 840
tatggagtgt cccccaccaa actgaacgat ctgtgtttca caaatgtgta cgctgactcc 900
tttgtgatca gaggagatga ggtgaggcag atcgcccctg gacagaccgg aaatattgcc 960
gattacaact acaaactgcc tgacgatttc accggctgtg tcatcgcctg gaactccaac 1020
aagctggata gcaaggtgtc cggcaactac aactatctct acagactgtt taggaagtcc 1080
aatctgaagc cttttgagag ggatattagc accgaaattt accaagccgg aaataagcct 1140
tgtaacggcg tggctggatt caactgctac tttcctctga gaagctacag cttcagaccc 1200
acctatggcg tgggacacca accttacaga gtcgtggtgc tctccttcga actgctccac 1260
gctcctgcca cagtgtgtgg ccccaagaaa tccaccaatc tcgtgaagaa caagagggtg 1320
cagcccaccg agagcatcgt gaggttccct aacatcacca acctgtgccc ttttggcgag 1380
gtgttcaacg ctacaagatt cgctagcgtg tacgcttgga acagaaagag aatttccaac 1440
tgtgtggccg attacagcgt cctgtataat tccgcctcct ttagcacctt taaatgctac 1500
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtctacgc cgatagcttc 1560
gtgattagag gcgatgaggt cagacagatc gcccctggac agaccggcaa catcgccgat 1620
tacaattata agctgcctga cgacttcacc ggatgtgtca ttgcttggaa cagcaataac 1680
ctcgatagca aagtgggagg caactacaat tacagataca gactgttcag aaagtccaac 1740
ctgaaacctt tcgagaggga tatcagcacc gagatttacc aagccggcag caagccttgc 1800
aacggcgtcg agggcttcaa ttgctacagc cctctgcaga gctacggatt ccaacccaca 1860
tacggcgtgg gctatcagcc ttacagagtg gtcgtgctca gctttgagct gctgcacgcc 1920
cctgccaccg tgtgcggccc caagaagtcc accaatctgg tcaagaacaa a 1971
<210> 16
<211> 1971
<212> DNA
<213> Artificial
<400> 16
agagtccaac ctaccgagtc catcgtgaga ttccccaaca tcacaaatct gtgccctttc 60
gacgaggtct tcaatgccac aagattcgct agcgtgtacg cctggaacag aaagagaatc 120
agcaattgcg tcgccgacta cagcgtcctc tacaacctgg cccctttctt cacattcaag 180
tgttatggag tgtcccccac caaactgaac gatctgtgtt tcacaaatgt gtacgctgac 240
tcctttgtga tcagaggaga tgaggtgagg cagatcgccc ctggacagac cggaaatatt 300
gccgattaca actacaaact gcctgacgat ttcaccggct gtgtcatcgc ctggaactcc 360
aacaagctgg atagcaaggt gtccggcaac tacaactatc tctacagact gtttaggaag 420
tccaatctga agccttttga gagggatatt agcaccgaaa tttaccaagc cggaaataag 480
ccttgtaacg gcgtggctgg attcaactgc tactttcctc tgagaagcta cagcttcaga 540
cccacctatg gcgtgggaca ccaaccttac agagtcgtgg tgctctcctt cgaactgctc 600
cacgctcctg ccacagtgtg tggccccaag aaatccacca atctcgtgaa gaacaagagg 660
gtgcagccca ccgagagcat cgtgaggttc cctaacatca ccaacctgtg cccttttggc 720
gaggtgttca acgctacaag attcgctagc gtgtacgctt ggaacagaaa gagaatttcc 780
aactgtgtgg ccgattacag cgtcctgtat aattccgcct cctttagcac ctttaaatgc 840
tacggcgtga gccctaccaa gctgaacgac ctgtgcttca ccaacgtcta cgccgatagc 900
ttcgtgatta gaggcgatga ggtcagacag atcgcccctg gacagaccgg caacatcgcc 960
gattacaatt ataagctgcc tgacgacttc accggatgtg tcattgcttg gaacagcaat 1020
aacctcgata gcaaagtggg aggcaactac aattacagat acagactgtt cagaaagtcc 1080
aacctgaaac ctttcgagag ggatatcagc accgagattt accaagccgg cagcaagcct 1140
tgcaacggcg tcgagggctt caattgctac agccctctgc agagctacgg attccaaccc 1200
acatacggcg tgggctatca gccttacaga gtggtcgtgc tcagctttga gctgctgcac 1260
gcccctgcca ccgtgtgcgg ccccaagaag tccaccaatc tggtcaagaa caaaagagtg 1320
cagcctaccg aatccatcgt cagatttccc aatatcacaa acctctgtcc tttcggagaa 1380
gtgttcaatg ccacaagatt cgcctccgtc tacgcctgga acagaaaaag aatcagcaac 1440
tgcgtggctg actacagcgt gctgtacaac agcgctagct tcagcacctt caaatgttac 1500
ggcgtgtccc ctaccaagct gaacgatctc tgcttcacca acgtgtacgc tgattccttc 1560
gtgatcagag gcgatgaggt gagacagatc gcccccggac agaccggcac cattgccgac 1620
tacaactata aactgcctga tgactttacc ggatgcgtca ttgcttggaa cagcaacaat 1680
ctggacagca aggtgggcgg caattacaat tacctgtata gactctttag aaagagcaac 1740
ctcaagcctt tcgagagaga tatcagcaca gagatttacc aagccggcaa taccccttgc 1800
aacggagtca agggcttcaa ctgttacttc cctctgcaga gctacggatt tcagcctacc 1860
aatggcgtgg gatatcagcc ctacagagtg gtcgtgctga gcttcgagct gctgcatgct 1920
cccgccaccg tgtgcggccc taaaaagagc accaacctgg tcaagaacaa g 1971
<210> 17
<211> 1971
<212> DNA
<213> Artificial
<400> 17
agagtccaac ctaccgagtc catcgtgaga ttccccaaca tcacaaatct gtgccctttc 60
gacgaggtct tcaatgccac aagattcgct agcgtgtacg cctggaacag aaagagaatc 120
agcaattgcg tcgccgacta cagcgtcctc tacaacctgg cccctttctt cacattcaag 180
tgttatggag tgtcccccac caaactgaac gatctgtgtt tcacaaatgt gtacgctgac 240
tcctttgtga tcagaggaga tgaggtgagg cagatcgccc ctggacagac cggaaatatt 300
gccgattaca actacaaact gcctgacgat ttcaccggct gtgtcatcgc ctggaactcc 360
aacaagctgg atagcaaggt gtccggcaac tacaactatc tctacagact gtttaggaag 420
tccaatctga agccttttga gagggatatt agcaccgaaa tttaccaagc cggaaataag 480
ccttgtaacg gcgtggctgg attcaactgc tactttcctc tgagaagcta cagcttcaga 540
cccacctatg gcgtgggaca ccaaccttac agagtcgtgg tgctctcctt cgaactgctc 600
cacgctcctg ccacagtgtg tggccccaag aaatccacca atctcgtgaa gaacaagaga 660
gtgcagccta ccgaatccat cgtcagattt cccaatatca caaacctctg tcctttcgga 720
gaagtgttca atgccacaag attcgcctcc gtctacgcct ggaacagaaa aagaatcagc 780
aactgcgtgg ctgactacag cgtgctgtac aacagcgcta gcttcagcac cttcaaatgt 840
tacggcgtgt cccctaccaa gctgaacgat ctctgcttca ccaacgtgta cgctgattcc 900
ttcgtgatca gaggcgatga ggtgagacag atcgcccccg gacagaccgg caccattgcc 960
gactacaact ataaactgcc tgatgacttt accggatgcg tcattgcttg gaacagcaac 1020
aatctggaca gcaaggtggg cggcaattac aattacctgt atagactctt tagaaagagc 1080
aacctcaagc ctttcgagag agatatcagc acagagattt accaagccgg caatacccct 1140
tgcaacggag tcaagggctt caactgttac ttccctctgc agagctacgg atttcagcct 1200
accaatggcg tgggatatca gccctacaga gtggtcgtgc tgagcttcga gctgctgcat 1260
gctcccgcca ccgtgtgcgg ccctaaaaag agcaccaacc tggtcaagaa caagagggtg 1320
cagcccaccg agagcatcgt gaggttccct aacatcacca acctgtgccc ttttggcgag 1380
gtgttcaacg ctacaagatt cgctagcgtg tacgcttgga acagaaagag aatttccaac 1440
tgtgtggccg attacagcgt cctgtataat tccgcctcct ttagcacctt taaatgctac 1500
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtctacgc cgatagcttc 1560
gtgattagag gcgatgaggt cagacagatc gcccctggac agaccggcaa catcgccgat 1620
tacaattata agctgcctga cgacttcacc ggatgtgtca ttgcttggaa cagcaataac 1680
ctcgatagca aagtgggagg caactacaat tacagataca gactgttcag aaagtccaac 1740
ctgaaacctt tcgagaggga tatcagcacc gagatttacc aagccggcag caagccttgc 1800
aacggcgtcg agggcttcaa ttgctacagc cctctgcaga gctacggatt ccaacccaca 1860
tacggcgtgg gctatcagcc ttacagagtg gtcgtgctca gctttgagct gctgcacgcc 1920
cctgccaccg tgtgcggccc caagaagtcc accaatctgg tcaagaacaa a 1971
<210> 18
<211> 1971
<212> DNA
<213> Artificial
<400> 18
agggtgcagc ccaccgagag catcgtgagg ttccctaaca tcaccaacct gtgccctttt 60
ggcgaggtgt tcaacgctac aagattcgct agcgtgtacg cttggaacag aaagagaatt 120
tccaactgtg tggccgatta cagcgtcctg tataattccg cctcctttag cacctttaaa 180
tgctacggcg tgagccctac caagctgaac gacctgtgct tcaccaacgt ctacgccgat 240
agcttcgtga ttagaggcga tgaggtcaga cagatcgccc ctggacagac cggcaacatc 300
gccgattaca attataagct gcctgacgac ttcaccggat gtgtcattgc ttggaacagc 360
aataacctcg atagcaaagt gggaggcaac tacaattaca gatacagact gttcagaaag 420
tccaacctga aacctttcga gagggatatc agcaccgaga tttaccaagc cggcagcaag 480
ccttgcaacg gcgtcgaggg cttcaattgc tacagccctc tgcagagcta cggattccaa 540
cccacatacg gcgtgggcta tcagccttac agagtggtcg tgctcagctt tgagctgctg 600
cacgcccctg ccaccgtgtg cggccccaag aagtccacca atctggtcaa gaacaaaagg 660
gtgcagccca ccgagagcat cgtgaggttc cctaacatca ccaacctgtg cccttttggc 720
gaggtgttca acgctacaag attcgctagc gtgtacgctt ggaacagaaa gagaatttcc 780
aactgtgtgg ccgattacag cgtcctgtat aattccgcct cctttagcac ctttaaatgc 840
tacggcgtga gccctaccaa gctgaacgac ctgtgcttca ccaacgtcta cgccgatagc 900
ttcgtgatta gaggcgatga ggtcagacag atcgcccctg gacagaccgg caacatcgcc 960
gattacaatt ataagctgcc tgacgacttc accggatgtg tcattgcttg gaacagcaat 1020
aacctcgata gcaaagtggg aggcaactac aattacagat acagactgtt cagaaagtcc 1080
aacctgaaac ctttcgagag ggatatcagc accgagattt accaagccgg cagcaagcct 1140
tgcaacggcg tcgagggctt caattgctac agccctctgc agagctacgg attccaaccc 1200
acatacggcg tgggctatca gccttacaga gtggtcgtgc tcagctttga gctgctgcac 1260
gcccctgcca ccgtgtgcgg ccccaagaag tccaccaatc tggtcaagaa caaaagagtc 1320
caacctaccg agtccatcgt gagattcccc aacatcacaa atctgtgccc tttcgacgag 1380
gtcttcaatg ccacaagatt cgctagcgtg tacgcctgga acagaaagag aatcagcaat 1440
tgcgtcgccg actacagcgt cctctacaac ctggcccctt tcttcacatt caagtgttat 1500
ggagtgtccc ccaccaaact gaacgatctg tgtttcacaa atgtgtacgc tgactccttt 1560
gtgatcagag gagatgaggt gaggcagatc gcccctggac agaccggaaa tattgccgat 1620
tacaactaca aactgcctga cgatttcacc ggctgtgtca tcgcctggaa ctccaacaag 1680
ctggatagca aggtgtccgg caactacaac tatctctaca gactgtttag gaagtccaat 1740
ctgaagcctt ttgagaggga tattagcacc gaaatttacc aagccggaaa taagccttgt 1800
aacggcgtgg ctggattcaa ctgctacttt cctctgagaa gctacagctt cagacccacc 1860
tatggcgtgg gacaccaacc ttacagagtc gtggtgctct ccttcgaact gctccacgct 1920
cctgccacag tgtgtggccc caagaaatcc accaatctcg tgaagaacaa g 1971
<210> 19
<211> 1971
<212> DNA
<213> Artificial
<400> 19
agagtgcagc ctaccgaatc catcgtcaga tttcccaata tcacaaacct ctgtcctttc 60
ggagaagtgt tcaatgccac aagattcgcc tccgtctacg cctggaacag aaaaagaatc 120
agcaactgcg tggctgacta cagcgtgctg tacaacagcg ctagcttcag caccttcaaa 180
tgttacggcg tgtcccctac caagctgaac gatctctgct tcaccaacgt gtacgctgat 240
tccttcgtga tcagaggcga tgaggtgaga cagatcgccc ccggacagac cggcaccatt 300
gccgactaca actataaact gcctgatgac tttaccggat gcgtcattgc ttggaacagc 360
aacaatctgg acagcaaggt gggcggcaat tacaattacc tgtatagact ctttagaaag 420
agcaacctca agcctttcga gagagatatc agcacagaga tttaccaagc cggcaatacc 480
ccttgcaacg gagtcaaggg cttcaactgt tacttccctc tgcagagcta cggatttcag 540
cctaccaatg gcgtgggata tcagccctac agagtggtcg tgctgagctt cgagctgctg 600
catgctcccg ccaccgtgtg cggccctaaa aagagcacca acctggtcaa gaacaagaga 660
gtgcagccta ccgaatccat cgtcagattt cccaatatca caaacctctg tcctttcgga 720
gaagtgttca atgccacaag attcgcctcc gtctacgcct ggaacagaaa aagaatcagc 780
aactgcgtgg ctgactacag cgtgctgtac aacagcgcta gcttcagcac cttcaaatgt 840
tacggcgtgt cccctaccaa gctgaacgat ctctgcttca ccaacgtgta cgctgattcc 900
ttcgtgatca gaggcgatga ggtgagacag atcgcccccg gacagaccgg caccattgcc 960
gactacaact ataaactgcc tgatgacttt accggatgcg tcattgcttg gaacagcaac 1020
aatctggaca gcaaggtggg cggcaattac aattacctgt atagactctt tagaaagagc 1080
aacctcaagc ctttcgagag agatatcagc acagagattt accaagccgg caatacccct 1140
tgcaacggag tcaagggctt caactgttac ttccctctgc agagctacgg atttcagcct 1200
accaatggcg tgggatatca gccctacaga gtggtcgtgc tgagcttcga gctgctgcat 1260
gctcccgcca ccgtgtgcgg ccctaaaaag agcaccaacc tggtcaagaa caagagagtc 1320
caacctaccg agtccatcgt gagattcccc aacatcacaa atctgtgccc tttcgacgag 1380
gtcttcaatg ccacaagatt cgctagcgtg tacgcctgga acagaaagag aatcagcaat 1440
tgcgtcgccg actacagcgt cctctacaac ctggcccctt tcttcacatt caagtgttat 1500
ggagtgtccc ccaccaaact gaacgatctg tgtttcacaa atgtgtacgc tgactccttt 1560
gtgatcagag gagatgaggt gaggcagatc gcccctggac agaccggaaa tattgccgat 1620
tacaactaca aactgcctga cgatttcacc ggctgtgtca tcgcctggaa ctccaacaag 1680
ctggatagca aggtgtccgg caactacaac tatctctaca gactgtttag gaagtccaat 1740
ctgaagcctt ttgagaggga tattagcacc gaaatttacc aagccggaaa taagccttgt 1800
aacggcgtgg ctggattcaa ctgctacttt cctctgagaa gctacagctt cagacccacc 1860
tatggcgtgg gacaccaacc ttacagagtc gtggtgctct ccttcgaact gctccacgct 1920
cctgccacag tgtgtggccc caagaaatcc accaatctcg tgaagaacaa g 1971
<210> 20
<211> 1971
<212> DNA
<213> Artificial
<400> 20
agggtgcagc ccaccgagag catcgtgagg ttccctaaca tcaccaacct gtgccctttt 60
ggcgaggtgt tcaacgctac aagattcgct agcgtgtacg cttggaacag aaagagaatt 120
tccaactgtg tggccgatta cagcgtcctg tataattccg cctcctttag cacctttaaa 180
tgctacggcg tgagccctac caagctgaac gacctgtgct tcaccaacgt ctacgccgat 240
agcttcgtga ttagaggcga tgaggtcaga cagatcgccc ctggacagac cggcaacatc 300
gccgattaca attataagct gcctgacgac ttcaccggat gtgtcattgc ttggaacagc 360
aataacctcg atagcaaagt gggaggcaac tacaattaca gatacagact gttcagaaag 420
tccaacctga aacctttcga gagggatatc agcaccgaga tttaccaagc cggcagcaag 480
ccttgcaacg gcgtcgaggg cttcaattgc tacagccctc tgcagagcta cggattccaa 540
cccacatacg gcgtgggcta tcagccttac agagtggtcg tgctcagctt tgagctgctg 600
cacgcccctg ccaccgtgtg cggccccaag aagtccacca atctggtcaa gaacaaaagg 660
gtgcagccca ccgagagcat cgtgaggttc cctaacatca ccaacctgtg cccttttggc 720
gaggtgttca acgctacaag attcgctagc gtgtacgctt ggaacagaaa gagaatttcc 780
aactgtgtgg ccgattacag cgtcctgtat aattccgcct cctttagcac ctttaaatgc 840
tacggcgtga gccctaccaa gctgaacgac ctgtgcttca ccaacgtcta cgccgatagc 900
ttcgtgatta gaggcgatga ggtcagacag atcgcccctg gacagaccgg caacatcgcc 960
gattacaatt ataagctgcc tgacgacttc accggatgtg tcattgcttg gaacagcaat 1020
aacctcgata gcaaagtggg aggcaactac aattacagat acagactgtt cagaaagtcc 1080
aacctgaaac ctttcgagag ggatatcagc accgagattt accaagccgg cagcaagcct 1140
tgcaacggcg tcgagggctt caattgctac agccctctgc agagctacgg attccaaccc 1200
acatacggcg tgggctatca gccttacaga gtggtcgtgc tcagctttga gctgctgcac 1260
gcccctgcca ccgtgtgcgg ccccaagaag tccaccaatc tggtcaagaa caaaagggtg 1320
cagcccaccg agagcatcgt gaggttccct aacatcacca acctgtgccc ttttggcgag 1380
gtgttcaacg ctacaagatt cgctagcgtg tacgcttgga acagaaagag aatttccaac 1440
tgtgtggccg attacagcgt cctgtataat tccgcctcct ttagcacctt taaatgctac 1500
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtctacgc cgatagcttc 1560
gtgattagag gcgatgaggt cagacagatc gcccctggac agaccggcaa catcgccgat 1620
tacaattata agctgcctga cgacttcacc ggatgtgtca ttgcttggaa cagcaataac 1680
ctcgatagca aagtgggagg caactacaat tacagataca gactgttcag aaagtccaac 1740
ctgaaacctt tcgagaggga tatcagcacc gagatttacc aagccggcag caagccttgc 1800
aacggcgtcg agggcttcaa ttgctacagc cctctgcaga gctacggatt ccaacccaca 1860
tacggcgtgg gctatcagcc ttacagagtg gtcgtgctca gctttgagct gctgcacgcc 1920
cctgccaccg tgtgcggccc caagaagtcc accaatctgg tcaagaacaa a 1971
<210> 21
<211> 1971
<212> DNA
<213> Artificial
<400> 21
agagtgcagc ctaccgaatc catcgtcaga tttcccaata tcacaaacct ctgtcctttc 60
ggagaagtgt tcaatgccac aagattcgcc tccgtctacg cctggaacag aaaaagaatc 120
agcaactgcg tggctgacta cagcgtgctg tacaacagcg ctagcttcag caccttcaaa 180
tgttacggcg tgtcccctac caagctgaac gatctctgct tcaccaacgt gtacgctgat 240
tccttcgtga tcagaggcga tgaggtgaga cagatcgccc ccggacagac cggcaccatt 300
gccgactaca actataaact gcctgatgac tttaccggat gcgtcattgc ttggaacagc 360
aacaatctgg acagcaaggt gggcggcaat tacaattacc tgtatagact ctttagaaag 420
agcaacctca agcctttcga gagagatatc agcacagaga tttaccaagc cggcaatacc 480
ccttgcaacg gagtcaaggg cttcaactgt tacttccctc tgcagagcta cggatttcag 540
cctaccaatg gcgtgggata tcagccctac agagtggtcg tgctgagctt cgagctgctg 600
catgctcccg ccaccgtgtg cggccctaaa aagagcacca acctggtcaa gaacaagaga 660
gtgcagccta ccgaatccat cgtcagattt cccaatatca caaacctctg tcctttcgga 720
gaagtgttca atgccacaag attcgcctcc gtctacgcct ggaacagaaa aagaatcagc 780
aactgcgtgg ctgactacag cgtgctgtac aacagcgcta gcttcagcac cttcaaatgt 840
tacggcgtgt cccctaccaa gctgaacgat ctctgcttca ccaacgtgta cgctgattcc 900
ttcgtgatca gaggcgatga ggtgagacag atcgcccccg gacagaccgg caccattgcc 960
gactacaact ataaactgcc tgatgacttt accggatgcg tcattgcttg gaacagcaac 1020
aatctggaca gcaaggtggg cggcaattac aattacctgt atagactctt tagaaagagc 1080
aacctcaagc ctttcgagag agatatcagc acagagattt accaagccgg caatacccct 1140
tgcaacggag tcaagggctt caactgttac ttccctctgc agagctacgg atttcagcct 1200
accaatggcg tgggatatca gccctacaga gtggtcgtgc tgagcttcga gctgctgcat 1260
gctcccgcca ccgtgtgcgg ccctaaaaag agcaccaacc tggtcaagaa caagagagtg 1320
cagcctaccg aatccatcgt cagatttccc aatatcacaa acctctgtcc tttcggagaa 1380
gtgttcaatg ccacaagatt cgcctccgtc tacgcctgga acagaaaaag aatcagcaac 1440
tgcgtggctg actacagcgt gctgtacaac agcgctagct tcagcacctt caaatgttac 1500
ggcgtgtccc ctaccaagct gaacgatctc tgcttcacca acgtgtacgc tgattccttc 1560
gtgatcagag gcgatgaggt gagacagatc gcccccggac agaccggcac cattgccgac 1620
tacaactata aactgcctga tgactttacc ggatgcgtca ttgcttggaa cagcaacaat 1680
ctggacagca aggtgggcgg caattacaat tacctgtata gactctttag aaagagcaac 1740
ctcaagcctt tcgagagaga tatcagcaca gagatttacc aagccggcaa taccccttgc 1800
aacggagtca agggcttcaa ctgttacttc cctctgcaga gctacggatt tcagcctacc 1860
aatggcgtgg gatatcagcc ctacagagtg gtcgtgctga gcttcgagct gctgcatgct 1920
cccgccaccg tgtgcggccc taaaaagagc accaacctgg tcaagaacaa g 1971
<210> 22
<211> 1971
<212> DNA
<213> Artificial
<400> 22
agagtccaac ctaccgagtc catcgtgaga ttccccaaca tcacaaatct gtgccctttc 60
gacgaggtct tcaatgccac aagattcgct agcgtgtacg cctggaacag aaagagaatc 120
agcaattgcg tcgccgacta cagcgtcctc tacaacctgg cccctttctt cacattcaag 180
tgttatggag tgtcccccac caaactgaac gatctgtgtt tcacaaatgt gtacgctgac 240
tcctttgtga tcagaggaga tgaggtgagg cagatcgccc ctggacagac cggaaatatt 300
gccgattaca actacaaact gcctgacgat ttcaccggct gtgtcatcgc ctggaactcc 360
aacaagctgg atagcaaggt gtccggcaac tacaactatc tctacagact gtttaggaag 420
tccaatctga agccttttga gagggatatt agcaccgaaa tttaccaagc cggaaataag 480
ccttgtaacg gcgtggctgg attcaactgc tactttcctc tgagaagcta cagcttcaga 540
cccacctatg gcgtgggaca ccaaccttac agagtcgtgg tgctctcctt cgaactgctc 600
cacgctcctg ccacagtgtg tggccccaag aaatccacca atctcgtgaa gaacaagaga 660
gtccaaccta ccgagtccat cgtgagattc cccaacatca caaatctgtg ccctttcgac 720
gaggtcttca atgccacaag attcgctagc gtgtacgcct ggaacagaaa gagaatcagc 780
aattgcgtcg ccgactacag cgtcctctac aacctggccc ctttcttcac attcaagtgt 840
tatggagtgt cccccaccaa actgaacgat ctgtgtttca caaatgtgta cgctgactcc 900
tttgtgatca gaggagatga ggtgaggcag atcgcccctg gacagaccgg aaatattgcc 960
gattacaact acaaactgcc tgacgatttc accggctgtg tcatcgcctg gaactccaac 1020
aagctggata gcaaggtgtc cggcaactac aactatctct acagactgtt taggaagtcc 1080
aatctgaagc cttttgagag ggatattagc accgaaattt accaagccgg aaataagcct 1140
tgtaacggcg tggctggatt caactgctac tttcctctga gaagctacag cttcagaccc 1200
acctatggcg tgggacacca accttacaga gtcgtggtgc tctccttcga actgctccac 1260
gctcctgcca cagtgtgtgg ccccaagaaa tccaccaatc tcgtgaagaa caagagagtc 1320
caacctaccg agtccatcgt gagattcccc aacatcacaa atctgtgccc tttcgacgag 1380
gtcttcaatg ccacaagatt cgctagcgtg tacgcctgga acagaaagag aatcagcaat 1440
tgcgtcgccg actacagcgt cctctacaac ctggcccctt tcttcacatt caagtgttat 1500
ggagtgtccc ccaccaaact gaacgatctg tgtttcacaa atgtgtacgc tgactccttt 1560
gtgatcagag gagatgaggt gaggcagatc gcccctggac agaccggaaa tattgccgat 1620
tacaactaca aactgcctga cgatttcacc ggctgtgtca tcgcctggaa ctccaacaag 1680
ctggatagca aggtgtccgg caactacaac tatctctaca gactgtttag gaagtccaat 1740
ctgaagcctt ttgagaggga tattagcacc gaaatttacc aagccggaaa taagccttgt 1800
aacggcgtgg ctggattcaa ctgctacttt cctctgagaa gctacagctt cagacccacc 1860
tatggcgtgg gacaccaacc ttacagagtc gtggtgctct ccttcgaact gctccacgct 1920
cctgccacag tgtgtggccc caagaaatcc accaatctcg tgaagaacaa g 1971
<210> 23
<211> 658
<212> PRT
<213> Artificial
<400> 23
Met Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
1 5 10 15
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
20 25 30
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
35 40 45
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
50 55 60
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
65 70 75 80
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
85 90 95
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
100 105 110
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
115 120 125
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
130 135 140
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
145 150 155 160
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
165 170 175
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
180 185 190
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
195 200 205
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro
210 215 220
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
225 230 235 240
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
245 250 255
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
260 265 270
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
275 280 285
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
290 295 300
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
305 310 315 320
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
325 330 335
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
340 345 350
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
355 360 365
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
370 375 380
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
385 390 395 400
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
405 410 415
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
420 425 430
Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val
435 440 445
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
450 455 460
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
465 470 475 480
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
485 490 495
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
500 505 510
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
515 520 525
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
530 535 540
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
545 550 555 560
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
565 570 575
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
580 585 590
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
595 600 605
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
610 615 620
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
625 630 635 640
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
645 650 655
Asn Lys
<210> 24
<211> 658
<212> PRT
<213> Artificial
<400> 24
Met Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
1 5 10 15
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
20 25 30
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
35 40 45
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
50 55 60
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
65 70 75 80
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
85 90 95
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
100 105 110
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
115 120 125
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
130 135 140
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
145 150 155 160
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
165 170 175
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
180 185 190
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
195 200 205
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro
210 215 220
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
225 230 235 240
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
245 250 255
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
260 265 270
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
275 280 285
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
290 295 300
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile
305 310 315 320
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
325 330 335
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
340 345 350
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
355 360 365
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
370 375 380
Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
385 390 395 400
Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
405 410 415
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
420 425 430
Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr Glu Ser Ile Val
435 440 445
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
450 455 460
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
465 470 475 480
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
485 490 495
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
500 505 510
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
515 520 525
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
530 535 540
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
545 550 555 560
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu
565 570 575
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
580 585 590
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Gln Gly Phe Asn
595 600 605
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
610 615 620
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
625 630 635 640
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
645 650 655
Asn Lys

Claims (24)

1. A recombinant novel coronavirus protein which is in a trimer form and comprises three subunits consisting of 319 th to 537 th amino acid fragments of an RBD region of a novel coronavirus S protein,
the recombinant novel coronavirus protein comprises:
one subunit containing the K417N, L452R, T478K, F490S, and N501Y mutation sites; and/or
Another subunit containing the K417T, S477N, and E484K mutation sites.
2. The recombinant novel coronavirus protein according to claim 1, wherein said recombinant novel coronavirus protein further comprises a subunit consisting of a 319-537 amino acid fragment of the RBD region of the novel coronavirus Omicron mutant S protein.
3. The recombinant novel coronavirus protein as claimed in claim 2, wherein the subunit comprises five or more mutation sites selected from the group consisting of G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H mutation sites.
4. The recombinant novel coronavirus protein according to any one of claims 1 to 3, wherein the three subunits of the recombinant novel coronavirus protein have a primary structure in which the three subunits are connected in series in the order of N-terminus to C-terminus.
5. The recombinant novel coronavirus protein according to claim 4, wherein the amino acid sequence of the recombinant novel coronavirus protein is represented by SEQ ID Nos. 1-11 or a sequence having 95% or more homology with the amino acid sequence thereof except the mutation site, preferably the amino acid sequence represented by SEQ ID No.1 or a sequence having 95% or more homology with the amino acid sequence thereof except the mutation site.
6. A fusion protein, characterized in that the amino acid sequence of the fusion protein comprises the amino acid sequence of the recombinant novel coronavirus protein as defined in any one of claims 1 to 5.
7. The fusion protein of claim 6, further comprising one or more selected from a signal peptide, a tag, and an immune enhancing peptide.
8. A nucleic acid molecule comprising a nucleotide sequence encoding a recombinant novel coronavirus protein as claimed in any one of claims 1 to 5, or encoding a fusion protein as claimed in claim 6 or 7.
9. The nucleic acid molecule of claim 8, wherein the nucleotide sequence of said nucleic acid molecule is the nucleotide sequence shown as SEQ ID nos. 12-22.
10. A vector comprising the nucleic acid molecule of claim 8.
11. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 10.
12. The host cell of claim 11, wherein the host cell is an escherichia coli, yeast cell, insect cell, or mammalian cell.
13. The host cell of claim 12, wherein the host cell is a CHO cell.
14. A method for producing a recombinant novel coronavirus protein according to any one of claims 1 to 5 or a fusion protein according to claim 6 or 7, comprising the steps of:
step A) preparing the nucleic acid molecule of claim 8, constructing an expression vector for the nucleic acid molecule, and transforming or transfecting the expression vector into a host cell;
step B) protein expression using the product of step A);
and C) purifying the expression product obtained in the step B) to obtain the recombinant novel coronavirus protein or the recombinant novel coronavirus fusion protein.
15. Use of a recombinant novel coronavirus protein as defined in any one of claims 1 to 5, a fusion protein as defined in claim 6 or 7, a nucleic acid molecule as defined in claim 8, a vector as defined in claim 10 or a host cell as defined in claim 11 for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus infection and/or a novel coronavirus-induced disease.
16. Use of a recombinant novel coronavirus protein as defined in any one of claims 1 to 5, a fusion protein as defined in claim 6 or 7, a nucleic acid molecule as defined in claim 8, a vector as defined in claim 10 or a host cell as defined in claim 11 for the preparation of a medicament for boosting the immunity of a human being vaccinated with a novel coronavirus vaccine; the novel coronavirus vaccine is preferably a novel coronavirus vaccine inactivated vaccine.
17. A recombinant protein vaccine, comprising a recombinant novel coronavirus protein according to any one of claims 1-5 or a fusion protein according to claim 6 or 7, and an adjuvant.
18. The recombinant protein vaccine of claim 17, wherein the adjuvant is aluminum hydroxide, aluminum phosphate, MF59, or CpG.
19. The recombinant protein vaccine of claim 18, wherein the adjuvant is aluminum hydroxide.
20. The method of producing a recombinant protein vaccine according to claim 17, 18 or 19, wherein the purified recombinant novel coronavirus protein or the fusion protein is mixed with the adjuvant.
21. A genetically engineered vector vaccine comprising the nucleic acid molecule of claim 8.
22. A nucleic acid vaccine comprising the nucleic acid molecule of claim 8.
23. A pharmaceutical composition comprising the vaccine of claim 17, 21 or 22, and a pharmaceutically acceptable carrier.
24. A method of eliciting an immune response against a novel coronavirus or treating a novel coronavirus infection in a subject, wherein an effective amount of the vaccine of claim 17, 21 or 22 or the pharmaceutical composition of claim 23 is administered to the subject.
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