阅读说明：本技术 一种人***瘤病毒56型l1蛋白的突变体 (mutant of human papilloma virus 56 type L1 protein ) 是由 俞海 王大宁 魏双萍 郑清炳 李少伟 夏宁邵 于 2019-06-04 设计创作，主要内容包括：本发明涉及一种突变的HPV56L1蛋白(或其变体),其编码序列和制备方法,以及包含其的病毒样颗粒,所述蛋白(或其变体)和病毒样颗粒能够诱发抗至少两个型别的HPV(例如,HPV56和HPV66,或者HPV56、HPV53和HPV66)的中和抗体,从而可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。本发明还涉及上述蛋白和病毒样颗粒用于制备药物组合物或疫苗的用途,所述药物组合物或疫苗可用于预防所述至少两个型别的HPV感染以及由所述感染所导致的疾病例如宫颈癌和尖锐湿疣。(The present invention relates to a mutated HPV56L1 protein (or a variant thereof), a coding sequence and a preparation method thereof, and virus-like particles comprising the same, which are capable of inducing neutralizing antibodies against at least two types of HPV (e.g., HPV56 and HPV66, or HPV56, HPV53 and HPV66), thereby being useful for preventing infection by the at least two types of HPV and diseases caused by the infection, such as cervical cancer and condyloma acuminata. The invention also relates to the use of the above proteins and virus-like particles for the preparation of a pharmaceutical composition or vaccine useful for the prevention of HPV infections of said at least two types and diseases caused by said infections, such as cervical cancer and condyloma acuminata.)

1. A mutant HPV56L1 protein or variant thereof, wherein the mutant HPV56L1 protein has the following mutations compared to the wild-type HPV56L1 protein:

(1) The amino acid residues at the 130-150 th positions of the wild-type HPV56L1 protein are replaced by the amino acid residues at the corresponding positions of the L1 protein of the wild-type HPV of the second type; or

(2) The amino acid residue at position 347-357 in the wild-type HPV56L1 protein was replaced with the amino acid residue at the corresponding position in the L1 protein of wild-type HPV of the second type;

Alternatively, the mutated HPV56L1 protein has the mutation as defined in (2) compared to the wild-type HPV56L1 protein, and further has the following mutations:

(3) amino acid residues at positions 53-56 of the wild-type HPV56L1 protein are replaced with amino acid residues at corresponding positions of the L1 protein of wild-type HPV of the second type, and

(4) The amino acid residues at the 172-181 position of the wild-type HPV56L1 protein are replaced by the amino acid residues at the corresponding positions of the L1 protein of the wild-type HPV of the third type,

And, the variant differs from the mutated HPV56L1 protein only by substitution (preferably conservative substitution), addition or deletion of one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids and retains the function of the mutated HPV56L1 protein, i.e., is capable of inducing neutralizing antibodies against at least two types of HPV (e.g., HPV56 and HPV66 or HPV56, HPV66 and HPV 53);

Preferably, the wild-type HPV of the second type is HPV 66; preferably, the amino acid residues at the corresponding positions in (1) are the amino acid residues at the 130-150 th position of the wild-type HPV66L1 protein; preferably, the amino acid residue at the corresponding position in (2) is the amino acid residue at position 347-357 of the wild-type HPV66L1 protein; preferably, the amino acid residues at the corresponding positions in (3) are amino acid residues 53-56 of wild-type HPV66L1 protein;

Preferably, the wild-type HPV of the third type is HPV 53; preferably, the amino acid residues at the corresponding positions in (4) are the amino acid residues at positions 171 and 180 of the wild-type HPV53L1 protein;

Preferably, the wild type HPV56L1 protein has an amino acid sequence shown as SEQ ID NO. 1;

Preferably, the wild type HPV66L1 protein has an amino acid sequence as shown in SEQ ID NO 2 or 3;

preferably, the wild-type HPV53L1 protein has an amino acid sequence as shown in SEQ ID NO 40 or 42; preferably, the mutated HPV56L1 protein has an amino acid sequence selected from the group consisting of: 5, 8 and 38 SEQ ID NOs.

2. An isolated nucleic acid encoding the mutated HPV56L1 protein of claim 1 or a variant thereof,

Preferably, the isolated nucleic acid has a nucleotide sequence selected from the group consisting of: 12, 15 and 39 SEQ ID NOs.

3. A vector comprising the isolated nucleic acid of claim 2.

4. A host cell comprising the isolated nucleic acid of claim 2 and/or the vector of claim 3.

5. an HPV virus-like particle comprising or consisting of the mutated HPV56L1 protein or variant thereof of claim 1, or the mutated HPV56L1 protein or variant thereof of claim 1.

6. A composition comprising the mutated HPV56L1 protein or variant thereof of claim 1, or the isolated nucleic acid of claim 2, or the vector of claim 3, or the host cell of claim 4, or the HPV virus-like particle of claim 5.

7. A pharmaceutical composition or vaccine comprising the HPV virus-like particle of claim 5, optionally further comprising a pharmaceutically acceptable carrier and/or excipient,

Preferably, the HPV virus-like particle is present in an effective amount to prevent HPV infection or a disease caused by HPV infection;

Preferably, the HPV infection is one or more types of HPV infection (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection);

Preferably, the disease caused by HPV infection is selected from cervical cancer and condyloma acuminatum.

8. a method of making the mutant HPV56L1 protein or variant thereof of claim 1, comprising expressing the mutant HPV56L1 protein or variant thereof in a host cell, and recovering the mutant HPV56L1 protein or variant thereof from the culture of the host cell;

preferably, the host cell is escherichia coli;

Preferably, the method comprises the steps of: expressing the mutated HPV56L1 protein or variant thereof in E.coli, and purifying the mutated HPV56L1 protein or variant thereof from the E.coli lysis supernatant; preferably, said mutated HPV56L1 protein or variant thereof is recovered from the lysis supernatant of said e.coli by chromatography (e.g. cation exchange chromatography, hydroxyapatite chromatography and/or hydrophobic interaction chromatography).

9. A method of making a vaccine comprising admixing the HPV virus-like particle of claim 5 with a pharmaceutically acceptable carrier and/or excipient.

10. A method of preventing HPV infection or a disease caused by HPV infection, comprising administering to a subject a prophylactically effective amount of the HPV virus-like particle of claim 5 or the pharmaceutical composition or vaccine of claim 7,

Preferably, the HPV infection is one or more types of HPV infection (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection);

preferably, the disease caused by HPV infection is selected from cervical cancer and condyloma acuminatum.

11. use of the mutated HPV56L1 protein or variant thereof according to claim 1 or HPV virus-like particle according to claim 5 for the preparation of a pharmaceutical composition or a vaccine for the prevention of HPV infection or a disease resulting from HPV infection,

Preferably, the HPV infection is one or more types of HPV infection (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection);

Preferably, the disease caused by HPV infection is selected from cervical cancer and condyloma acuminatum.

Technical Field

The present invention relates to the fields of molecular virology and immunology. In particular, the present invention relates to a mutated HPV56L1 protein (or a variant thereof), a coding sequence and a preparation method thereof, and virus-like particles comprising the same, which are capable of inducing neutralizing antibodies against at least two types of HPV (e.g., HPV66 and HPV56, or HPV66, HPV56 and HPV53), thereby being useful for preventing infection by the at least two types of HPV and diseases caused by the infection, such as cervical cancer and condyloma acuminatum. The invention also relates to the use of the above proteins and virus-like particles for the preparation of a pharmaceutical composition or vaccine useful for the prevention of HPV infections of said at least two types and diseases caused by said infections, such as cervical cancer and condyloma acuminata.

Background

Human Papilloma Virus (HPV)) mainly causes warty lesions of the skin, mucosa. According to the relation between the HPV and tumorigenesis, the HPV can be divided into high-risk type and low-risk type, wherein the HPV infection of the high-risk type is proved to be the main reason for inducing genital cancer including female cervical cancer; the low risk type mainly causes condyloma acuminatum. The most effective way for preventing and controlling HPV infection is HPV vaccine, especially high-risk type HPV vaccine capable of causing cervical cancer.

The major capsid protein L1 of HPV has the property of self-assembling as hollow Virus-Like particles (VLPs). HPV VLPs are icosahedral stereosymmetric structures composed of pentameric interactions of the 72 major capsid proteins L1 (doorb, j.and p.h.Gallimore.1987.j Virol,61(9): 2793-9). HPV VLPs are structurally highly similar to native HPV, retain most of the neutralizing epitopes of the native virus, and induce high titers of neutralizing antibodies (Kirnbauer, R., F.Booy, et al.1992Proc Natl Acad Sci U S A89 (24): 12180-4).

Existing studies found that HPV VLPs induce mainly neutralizing antibodies against the same type of HPV, resulting in protective immunity against the same type of HPV, with only low cross-protection between some highly homologous types (Sara l. bissett, giaada mattiuzzo, et al.2014vacine.32: 6548-.

Therefore, there is still a need in the art to develop HPV virus-like particles capable of inducing protective neutralizing antibodies against multiple types of HPV to more economically and effectively prevent multiple types of HPV infection and diseases caused thereby, such as cervical cancer and condyloma acuminatum.

Disclosure of Invention

the present invention is based, at least in part, on the following unexpected findings of the inventors: after replacing a specific segment in the Human Papillomavirus (HPV) type 56L1 protein with a corresponding segment of the second type HPV (e.g., HPV66) L1 protein, the resulting mutated HPV56L1 protein is capable of inducing the body to produce high titer neutralizing antibodies against HPV56 and the second type HPV (e.g., HPV66) with protective effects comparable to mixed HPV56 VLPs and the second type HPV VLPs, and protective effects against HPV56 comparable to HPV56 VLPs alone, and protective effects against HPV of the second type (e.g., HPV66) comparable to HPV VLPs of the second type alone.

furthermore, on the basis of the above substitution, another specific segment in the HPV56L1 protein may be further substituted with a corresponding segment of the L1 protein of HPV of the third type (e.g., HPV53), whereby the resulting mutant HPV56L1 protein containing the double substitution is capable of inducing the body to produce high-titer neutralizing antibodies against HPV56, HPV of the second type (e.g., HPV66) and HPV of the third type (e.g., HPV53), with a protective effect comparable to that of mixed HPV56 VLPs, HPV VLPs of the second type and HPV VLPs of the third type; and, its protective effect against HPV56 is comparable to HPV56VLP alone, protective effect against HPV of the second type (e.g., HPV66) is comparable to HPV VLP of the second type alone, and protective effect against HPV of the third type (e.g., HPV53) is comparable to HPV VLP of the third type alone.

accordingly, in one aspect, the present invention provides a mutant HPV56L1 protein or variant thereof, wherein the mutant HPV56L1 protein has the following mutations compared to the wild-type HPV56L1 protein:

(1) The amino acid residues at the 130-150 position of the wild-type HPV56L1 protein are replaced by the amino acid residues at the corresponding position of the wild-type HPV L1 protein of the second type; or

(2) The amino acid residue at position 347-357 of the wild-type HPV56L1 protein was replaced with the amino acid residue at the corresponding position of the wild-type HPV L1 protein of the second type;

Alternatively, the mutated HPV56L1 protein has the mutation defined in (2) compared to the wild-type HPV56L1 protein, and further has the following mutations:

(3) The amino acid residues at positions 53-56 of the wild-type HPV56L1 protein are replaced with the amino acid residues at the corresponding positions of the wild-type HPV L1 protein of the second type, and

(4) The amino acid residues at the 172-181 position of the wild-type HPV56L1 protein are replaced by the amino acid residues at the corresponding positions of the wild-type HPV L1 protein of the third type;

and, the variant differs from the mutated HPV56L1 protein only in the substitution (preferably conservative substitution), addition or deletion of one or a few (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acids and retains the function of the mutated HPV56L1 protein, i.e., is capable of inducing neutralizing antibodies against at least two additional types of HPV (e.g., HPV56 and HPV66 or HPV56, HPV66 and HPV 53).

In certain preferred embodiments, the wild-type HPV of the second type is HPV 66. In certain preferred embodiments, the amino acid residues at the corresponding positions described in (1) are the amino acid residues at positions 130-150 of the L1 protein of wild-type HPV 66. In certain preferred embodiments, the amino acid residues at the corresponding positions described in (2) are amino acid residues 347-357 of the L1 protein of wild-type HPV 66. In certain preferred embodiments, the amino acid residues at the corresponding positions described in (3) are amino acid residues 53-56 of wild-type HPV66L1 protein.

in certain preferred embodiments, the wild-type HPV of the third type is HPV 53. In certain preferred embodiments, the amino acid residues at the corresponding positions described in (4) are the amino acid residues at positions 171 and 180 of the L1 protein of wild-type HPV 53.

In certain preferred embodiments, the wild-type HPV56L1 protein has the amino acid sequence shown as SEQ ID No. 1.

In certain preferred embodiments, the wild-type HPV66L1 protein has an amino acid sequence as set forth in SEQ ID No. 2 or 3.

In certain preferred embodiments, the wild-type HPV53L1 protein has an amino acid sequence as shown in SEQ ID No. 40 or 42.

in certain preferred embodiments, the sequence of amino acid residues at positions 130-150 of the wild-type HPV66L1 protein is shown in SEQ ID NO 16.

in certain preferred embodiments, the sequence of amino acid residues 347-357 of the L1 protein of wild-type HPV66 is depicted in SEQ ID NO 17.

In certain preferred embodiments, the sequence of amino acid residues 53-56 of the wild-type HPV66L1 protein is depicted in SEQ ID NO 20.

in certain preferred embodiments, the sequence of the amino acid residues at positions 171 and 180 of the L1 protein of wild-type HPV53 is shown in SEQ ID NO: 44.

In certain preferred embodiments, the mutated HPV56L1 protein has an amino acid sequence selected from the group consisting of seq id no:5, 8 and 38 SEQ ID NOs.

In another aspect, the invention provides an isolated nucleic acid encoding a mutant HPV56L1 protein or variant thereof as described above. In another aspect, the invention provides a vector comprising the isolated nucleic acid. In certain preferred embodiments, the isolated nucleic acid of the invention has a nucleotide sequence selected from the group consisting of: 12, 15 and 39 SEQ ID NOs.

Vectors useful for inserting a polynucleotide of interest are well known in the art and include, but are not limited to, cloning vectors and expression vectors. In one embodiment, the vector is, for example, a plasmid, cosmid, phage, or the like.

In another aspect, the invention also relates to a host cell comprising the isolated nucleic acid or vector described above. Such host cells include, but are not limited to, prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The host cell of the invention may also be a cell line, such as 293T cells.

In another aspect, the invention relates to an HPV virus-like particle, wherein the virus-like particle comprises or consists of a mutated HPV56L1 protein or a variant thereof of the invention, or a mutated HPV56L1 protein or a variant thereof of the invention.

In certain preferred embodiments, the HPV virus-like particle of the invention comprises a mutated HPV56L1 protein wherein the amino acid residue at position 130-150 of the wild-type HPV56L1 protein is replaced with the amino acid residue at position 130-150 of the wild-type HPV66L1 protein compared to the wild-type HPV56L1 protein.

In certain preferred embodiments, the HPV virus-like particle of the invention comprises a mutant HPV56L1 protein wherein the amino acid residue at position 347-357 in the wild-type HPV56L1 protein is replaced with the amino acid residue at position 347-357 in the wild-type HPV66L1 protein as compared to the wild-type HPV56L1 protein.

In certain preferred embodiments, the HPV virus-like particle of the invention comprises a mutant HPV56L1 protein in which the amino acid residues at positions 53-56 of the wild-type HPV56L1 protein are replaced with the amino acid residues 53-56 of the wild-type HPV66L1 protein, and the amino acid residues at positions 347-357 of the wild-type HPV56L1 protein are replaced with the amino acid residues at positions 172-180 of the wild-type HPV66L1 protein, and the amino acid residues at positions 172-181 of the wild-type HPV56L1 protein are replaced with the amino acid residues at positions 171-180 of the wild-type HPV53L1 protein, as compared to the wild-type HPV56L1 protein.

In a particularly preferred embodiment, the HPV virus-like particle of the invention comprises a mutated HPV56L1 protein having the sequence shown in SEQ ID NO 5, 8 or 38.

In another aspect, the invention also relates to a composition comprising the above mutated HPV56L1 protein or variant thereof, or the above isolated nucleic acid or vector or host cell or HPV virus-like particle. In certain preferred embodiments, the compositions comprise a mutated HPV56L1 protein of the invention or a variant thereof. In certain preferred embodiments, the composition comprises an HPV virus-like particle of the invention.

In another aspect, the present invention also relates to a pharmaceutical composition or vaccine comprising the HPV virus-like particle of the invention, optionally further comprising a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition or vaccine of the present invention can be used for preventing HPV infection or diseases caused by HPV infection such as cervical cancer and condyloma acuminatum.

In certain preferred embodiments, the HPV virus-like particle is present in an effective amount to prevent HPV infection or a disease caused by HPV infection. In certain preferred embodiments, the HPV infection is one or more HPV infection of a type(s) (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection). In certain preferred embodiments, the disease caused by HPV infection is selected from cervical cancer and condyloma acuminatum.

The pharmaceutical composition or vaccine of the present invention may be administered by methods well known in the art, such as, but not limited to, oral administration or injection. In the present invention, a particularly preferred mode of administration is injection.

In certain preferred embodiments, the pharmaceutical composition or vaccine of the invention is administered in a unit dosage form. For example, and without intending to limit the present invention, the amount of HPV virus-like particles contained per unit dose is 5. mu.g-80. mu.g, preferably 20. mu.g-40. mu.g.

in another aspect, the present invention relates to a method of preparing a mutant HPV56L1 protein or variant thereof as described above, comprising expressing the mutant HPV56L1 protein or variant thereof in a host cell and recovering the mutant HPV56L1 protein or variant thereof from the culture of the host cell.

in certain preferred embodiments, the host cell is E.coli.

In certain preferred embodiments, the method comprises the steps of: expressing the mutant HPV56L1 protein or variant thereof in Escherichia coli, and purifying the mutant HPV56L1 protein or variant thereof from the lysate supernatant of the Escherichia coli. In certain preferred embodiments, the mutated HPV56L1 protein or variant thereof is recovered from the e.coli lysis supernatant by chromatography (e.g., cation exchange chromatography, hydroxyapatite chromatography and/or hydrophobic interaction chromatography).

in another aspect, the invention relates to a method of preparing a vaccine comprising admixing the HPV virus-like particle of the invention with a pharmaceutically acceptable carrier and/or excipient.

In another aspect, the present invention relates to a method of preventing HPV infection or a disease caused by HPV infection, comprising administering to a subject a prophylactically effective amount of an HPV virus-like particle or a pharmaceutical composition or a vaccine according to the invention. In a preferred embodiment, the HPV infection is one or more HPV infection of a type(s) (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection). In another preferred embodiment, the diseases caused by HPV infection include, but are not limited to, cervical cancer and condyloma acuminatum. In another preferred embodiment, the subject is a mammal, such as a human.

In another aspect, it also relates to the use of a mutated HPV56L1 protein or variant thereof or HPV virus-like particle of the invention in the preparation of a pharmaceutical composition or a vaccine for the prevention of HPV infection or a disease caused by HPV infection. In a preferred embodiment, the HPV infection is one or more HPV infection of a type(s) (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection). In another preferred embodiment, the diseases caused by HPV infection include, but are not limited to, cervical cancer and condyloma acuminatum.

in another aspect, the invention also relates to a mutated HPV56L1 protein or a variant thereof or an HPV virus-like particle of the invention for use in the prevention of an HPV infection or a disease caused by an HPV infection. In a preferred embodiment, the HPV infection is one or more HPV infection of a type(s) (e.g., HPV56 infection, HPV53 infection, and/or HPV66 infection). In another preferred embodiment, the diseases caused by HPV infection include, but are not limited to, cervical cancer and condyloma acuminatum.

Description and explanation of related terms in the present 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. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

according to the present invention, the term "wild-type HPV of the second type" refers to a wild-type HPV of another type than HPV 56. In the present invention, the wild-type HPV of the second type is preferably wild-type HPV 66.

According to the present invention, the term "wild-type HPV of the third type" refers to a wild-type HPV of another type than HPV56 and different from the wild-type HPV of the second type. In the present invention, the wild-type HPV of the third type is preferably wild-type HPV 53.

according to the invention, the expression "corresponding position" refers to an equivalent position in the sequences being compared when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity. According to the present invention, the term "wild-type HPV56L1 protein" refers to the major capsid protein L1 naturally occurring in human papillomavirus type 56 (HPV 56). The sequence of the wild-type HPV56L1 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession numbers ALT54892.1, ALT54864.1, ASN77877.1, and P36743.1, etc.).

in the present invention, when referring to the amino acid sequence of wild-type HPV56L1 protein, reference is made to SEQ ID NO:1, is described. For example, the expression "amino acid residues 130-150 of wild-type HPV56L1 protein" means that the amino acid sequence shown in SEQ ID NO:1 at amino acid residue 130-150. "amino acid residue 347-357 of wild-type HPV56L1 protein" refers to the amino acid sequence of SEQ ID NO:1 at amino acid residue 347-357 of the polypeptide. However, it is understood by those skilled in the art that wild-type HPV56 may comprise a variety of isolates, and that there may be differences between the amino acid sequences of the L1 proteins of the various isolates. Further, it is understood by those skilled in the art that, despite sequence differences, the L1 proteins of different isolates of HPV56 have very high identity (typically greater than 95%, e.g., greater than 96%, greater than 97%, greater than 98%, or greater than 99%) in amino acid sequence and have essentially the same biological function. Therefore, in the present invention, the term "wild-type HPV56L1 protein" includes not only the protein shown in SEQ ID NO:1, but also L1 proteins of various HPV56 isolates (e.g., HPV56L1 proteins shown in ALT54892.1, ALT54864.1, ASN77877.1 and P36743.1). And, when describing a sequence fragment of wild-type HPV56L1 protein, it includes not only SEQ ID NO:1, and also includes the corresponding sequence fragment in the L1 protein of various HPV56 isolates. For example, the expression "amino acid residues 130-150 of the wild-type HPV56L1 protein" includes the amino acid sequences shown in SEQ ID NO:1, amino acid residues 130-150, and the corresponding fragments in the L1 protein of various HPV56 isolates.

According to the present invention, the term "wild-type HPV66L1 protein" refers to the major capsid protein L1 naturally occurring in human papillomavirus type 66 (HPV 66). The sequence of the wild-type HPV66L1 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession numbers ABO76865.1, Q80961.1, and ALT54961.1, etc.).

In the present invention, when referring to the amino acid sequence of wild-type HPV66L1 protein, reference is made to SEQ ID NO:2, to the sequence shown in figure 2. For example, the expression "amino acid residues 130-150 of wild-type HPV66L1 protein" means that the amino acid sequence shown in SEQ ID NO:2 at amino acid residue 130-150 of the polypeptide set forth in SEQ ID NO; the expression "amino acid residue 347-357 of the wild-type HPV66L1 protein" means that the amino acid sequence of SEQ ID NO:2 at amino acid residue 347-357 of the polypeptide. However, it is understood by those skilled in the art that wild-type HPV66 may comprise a variety of isolates, and that there may be differences between the amino acid sequences of the L1 proteins of the various isolates. Further, it is understood by those skilled in the art that, despite sequence differences, the L1 proteins of different isolates of HPV66 have very high identity (typically greater than 95%, e.g., greater than 96%, greater than 97%, greater than 98%, or greater than 99%) in amino acid sequence and have essentially the same biological function. Therefore, in the present invention, the term "wild-type HPV66L1 protein" shall include not only the protein shown in SEQ ID NO:2, but also the L1 proteins of various HPV66 isolates (e.g., the HPV66L1 proteins shown in ABO76865.1, Q80961.1, ALT54961.1, etc.). And, when describing a sequence fragment of wild-type HPV66L1 protein, it includes not only SEQ ID NO:2, and also includes the corresponding sequence fragment in the L1 protein of various HPV66 isolates. For example, the expression "amino acid residues 130-150 of the wild-type HPV66L1 protein" includes the amino acid sequences shown in SEQ ID NO:2, amino acid residues 130-150, and the corresponding fragment in the L1 protein of various HPV66 isolates.

According to the present invention, the term "wild-type HPV53L1 protein" refers to the major capsid protein L1 naturally occurring in human papillomavirus type 53 (HPV 53). The sequence of the wild-type HPV53L1 protein is well known in the art and can be found in various public databases (e.g., NCBI database accession numbers NP041848.1, ANY26596.1, ABU54090.1, and ALJ32506.1, etc.).

In the present invention, when referring to the amino acid sequence of wild-type HPV53L1 protein, reference is made to SEQ ID NO:40, is described. For example, the expression "amino acid residues 171 and 180 of the wild-type HPV53L1 protein" means that the amino acid sequence shown in SEQ ID NO:40 at amino acid residues 171 and 180. However, it is understood by those skilled in the art that wild-type HPV53 may comprise a variety of isolates, and that there may be differences between the amino acid sequences of the L1 proteins of the various isolates. Further, it is understood by those skilled in the art that, despite sequence differences, the L1 proteins of different isolates of HPV53 have very high identity (typically greater than 95%, e.g., greater than 96%, greater than 97%, greater than 98%, or greater than 99%) in amino acid sequence and have essentially the same biological function. Therefore, in the present invention, the term "wild-type HPV53L1 protein" shall include not only the protein shown in SEQ ID NO:40, but also the L1 proteins of various HPV53 isolates (e.g., HPV53L1 proteins shown in NP041848.1, ANY26596.1, ABU54090.1, and ALJ 32506.1). And, when describing a sequence fragment of wild-type HPV53L1 protein, it includes not only SEQ ID NO:40, and also includes the corresponding sequence fragment in the L1 protein of various HPV53 isolates. For example, the expression "amino acid residues 171 and 180 of the wild-type HPV53L1 protein" includes the amino acid sequences shown in SEQ ID NO:40 at amino acid residues 171 and 180, and the corresponding fragment in the L1 protein of various HPV53 isolates.

According to the invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to the fragments at equivalent positions in the sequences being compared when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity.

According to the present invention, the expression "truncated by X amino acids at the N-terminus" means that the amino acid residues 1 to X at the N-terminus of the protein are replaced with a methionine residue encoded by the start codon (for initiating translation of the protein). For example, the HPV66L1 protein truncated by 5 amino acids at the N-terminus refers to a protein obtained by replacing amino acid residues 1 to 5 of the N-terminus of wild-type HPV66L1 protein with a methionine residue encoded by the start codon.

according to the present invention, the term "variant" refers to a protein whose amino acid sequence has a substitution (preferably conservative substitution), addition or deletion of one or several (e.g., 1, 2, 3, 4, 5 or 6) amino acids, or has at least 90%, 95%, 96%, 97%, 98%, or 99% identity, compared to the amino acid sequence of the mutated HPV56L1 protein of the present invention (protein as shown in SEQ ID NO:5 or 8), and which retains the function of the mutated HPV56L1 protein. In the present invention, the term "function of mutated HPV56L1 protein" means: capable of inducing the body to produce neutralizing antibodies against at least two types of HPV (e.g., HPV56 and HPV 66). The term "identity" is a measure of similarity of nucleotide or amino acid sequences. Sequences are usually aligned to achieve maximum matching. "identity" itself has a meaning well known in the art and can be calculated using published algorithms (e.g., BLAST).

According to the invention, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.Applbiosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential characteristics of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, a conservative substitution typically refers to the replacement of a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA 94:412-417(1997), which are incorporated herein by reference).

according to the present invention, the term "E.coli expression system" refers to an expression system consisting of E.coli (strain) derived from a commercially available strain, such as, but not limited to: ER2566, BL21(DE3), B834(DE3), BLR (DE 3).

According to the present invention, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing 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; bacteriophage; cosmids, and the like.

According to the present invention, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's pharmaceutical sciences. edited by genomic AR,19th ed. pennsylvania: mack publishing Company,1995) and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvant (e.g., complete freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.

According to the present invention, the term "effective amount" means an amount effective to achieve the intended purpose. For example, a prophylactically effective amount (e.g., HPV infection) is an amount effective to prevent, or delay the onset of a disease (e.g., HPV infection). It is within the ability of those skilled in the art to determine such an effective amount.

According to the present invention, the term "chromatography" includes, but is not limited to: ion exchange chromatography (e.g., cation exchange chromatography), hydrophobic interaction chromatography, adsorption chromatography (e.g., hydroxyapatite chromatography), gel filtration (gel exclusion) chromatography, affinity chromatography.

according to the invention, the term "lysis supernatant" refers to the solution produced by the following steps: host cells (e.g., E.coli) are disrupted in a lysis solution, and insoluble matter is removed from the lysis solution containing the disrupted host cells. Various lysing solutions are known to those skilled in the art and include, but are not limited to, Tris buffer, phosphate buffer, HEPES buffer, MOPS buffer, and the like. In addition, the disruption of host cells can be accomplished by a variety of methods well known to those skilled in the art, including but not limited to homogenizer disruption, sonication, milling, high pressure extrusion, lysozyme treatment, and the like. Methods for removing insoluble materials from the lysate are also well known to those skilled in the art, and include, but are not limited to, filtration and centrifugation.

Advantageous effects of the invention

Studies have shown that although there is some cross-protection between HPV56 and other types of HPV (e.g. HPV66), the ability of such cross-protection is low, typically less than one hundredth or even less than one thousandth of the level of protection of their own type of VLP. Thus, the risk of infection with other types of HPV (e.g., HPV66) remains high for subjects vaccinated with HPV56 vaccine.

the invention provides a mutant HPV56L1 protein and an HPV virus-like particle formed by the same. The HPV virus-like particle of the invention is capable of providing significant cross-protection between HPV56 and other types of HPV (e.g. HPV 66). In particular, the HPV virus-like particle of the present invention is capable of inducing the body to produce high-titer neutralizing antibodies against at least two types of HPV (e.g., HPV56 and HPV66, or HPV56, HPV66, and HPV53) at equivalent immune doses, and is comparable in effect to a mixture of multiple types of HPV VLPs (e.g., a mixture of HPV56 VLPs and HPV66 VLPs, or a mixture of HPV56 VLPs, HPV66 VLPs, and HPV53 VLPs). Therefore, the HPV virus-like particle of the present invention can be used for simultaneously preventing infection of two or three types of HPV (e.g., HPV56 and HPV66, or HPV56, HPV66 and HPV53) and diseases associated therewith, with significant advantageous technical effects. The method has particularly remarkable advantages in the aspects of expanding the protection range of the HPV vaccine, reducing the production cost of the HPV vaccine and the like.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows the results of SDS polyacrylamide gel electrophoresis of the purified mutein of example 1. Lane 1: protein molecular weight markers; lane 2 HPV56N0 (full-length HPV56L1 protein); lane 3 HPV66N5 (HPV 66L1 protein truncated by 5 amino acids at the N-terminus); lane 4H 56N0-66T 1; lane 5, H56N0-66T 2; lane 6H 56N0-66T 3; lane 7, H56N0-66T 4; lane 8, H56N0-66T 5; lane 9: protein molecular weight markers; lane 8, H56N0-66T1-66T5-53S 3. The results show that after chromatographic purification, the purities of the mutant proteins H56N0-66T1, H56N0-66T2, H56N0-66T3, H56N0-66T4 and H56N0-66T5 reach about 90 percent, and the purities of the mutant proteins H56N0-66T1-66T5-53S3 reach about 80 percent.

FIG. 2 shows the results of Western blot detection of H56N0-66T1, H56N0-66T2, H56N0-66T3, H56N0-66T4, H56N0-66T5 prepared in example 1 using the HPV L1 broad-spectrum antibody 4B 3. Lane 1 HPV56N0 (full-length HPV56L1 protein); lane 2 HPV66N5 (HPV 66L1 protein truncated by 5 amino acids at the N-terminus); lane 3, H56N0-66T 1; lane 4H 56N0-66T 2; lane 5, H56N0-66T 3; lane 6H 56N0-66T 4; lane 7H 56N0-66T 5. The results show that the mutant proteins H56N0-66T1, H56N0-66T2, H56N0-66T3, H56N0-66T4 and H56N0-66T5 can be specifically recognized by the broad-spectrum antibody 4B 3.

FIG. 3 shows the results of a molecular sieve chromatographic analysis of samples containing proteins H56N0-66T1, H56N0-66T2, H56N0-66T3, H56N0-66T4, H56N0-66T5, H56N0-66T1-66T5-53S 3. The results show that the first protein peaks of the samples containing the proteins H56N0-66T1, H56N0-66T2, H56N0-66T4, H56N0-66T5 and H56N0-66T1-66T5-53S3 are all around 13-14min and are equivalent to HPV56N 0; the main peak time of H56N0-66T3 was about 15min, and the shoulder seam time was 14min, indicating that the VLP formed by H56N0-66T3 had non-uniform particle size. The results show that except for the mutein H56N0-66T3, the remaining muteins can form uniform VLPs.

FIG. 4 shows the results of sedimentation rate analysis of mutant H56N0-66T1 VLPs, H56N0-66T2 VLPs, H56N0-66T3 VLPs, H56N0-66T4 VLPs, H56N0-66T5 VLPs, H56N0-66T1-66T5-53S3 VLPs. The results showed that the sedimentation coefficients of H56N0-66T1VLP, H56N0-66T2VLP, H56N0-66T3VLP, H56N0-66T4VLP, H56N0-66T5VLP, H56N0-66T1-66T5-53S3VLP were 135S, 139S, 78S, 136S, 139S and 135S, respectively. This indicates that the 6 mutated HPV56L1 proteins prepared above, except H56N0-66T3, were all able to assemble into virus-like particles of similar size and morphology to wild-type VLPs (HPV56N0 VLPs, 134S).

fig. 5A to 5H show transmission electron microscopy observations of various VLP samples (magnification 100,000 times, Bar 0.1 μm). FIG. 5A: HPV56N0 VLP; FIG. 5B: H56N0-66T1 VLP; FIG. 5C: HPV56N0-66T2 VLP; FIG. 5D: H56N0-66T3 VLP; FIG. 5E: H56N0-66T4 VLP; FIG. 5F: H56N0-66T5 VLP; FIG. 5G: H56N0-66T1-66T5-53S3 VLP; FIG. 5H: HPV66N5 VLPs. The results show that H56N0-66T1VLP, H56N0-66T2VLP, H56N0-66T4VLP, H56N0-66T5VLP, H56N0-66T1-66T5-53S3VLP, like HPV56N0, are all capable of assembling into VLP with a radius of around 25 nm; the H56N0-66T3VLP assembled particles were heterogeneous with radii between 15-25 nm.

FIGS. 6A-6C show the results of evaluation of neutralizing antibody titers in mouse sera after immunization of mice with H56N0-66T1VLP, H56N0-66T2VLP, H56N0-66T3VLP, H56N0-66T4VLP, and H56N0-66T5 VLP. FIG. 6A: aluminum adjuvant group 1 (immunization dose 5.0 μ g, using aluminum adjuvant); FIG. 6B: aluminum adjuvant group 2 (immunization dose 1 μ g, using aluminum adjuvant); FIG. 6C: aluminum adjuvant group 3 (immunization dose 0.2 μ g, using aluminum adjuvant). The results show that H56N0-66T2 VLPs and H56N0-66T5 VLPs can induce mice to produce high titers of neutralizing antibodies against HPV56, with protective effects comparable to the same dose of HPV56N0 VLPs alone, mixed HPV56/HPV66 VLPs, and significantly better than the same dose of HPV66N5 VLPs alone; the H56N0-66T2VLP and H56N0-66T5VLP can also induce mice to generate higher-titer neutralizing antibodies against HPV66, and the protective effect of the H56N0-66T2VLP and H56N0-66T5VLP is slightly weaker than that of single HPV66N5VLP and mixed HPV56/HPV66VLP at the same dose, but is obviously better than that of single HPV56N0VLP at the same dose. This indicates that the H56N0-66T2VLP and H56N0-66T5VLP have good cross-immunogenicity and cross-protection against HPV56 and HPV 66.

FIG. 7 shows the results of an assessment of the immunoprotection in mice of experimental groups H56N0-66T1-66T5-53S3 VLPs and control groups HPV56N0 VLPs, HPV66N5 VLPs, HPV53N5 VLPs and mixed HPV66/56/53 VLPs. The results show that H56N0-66T1-66T5-53S3 VLPs can induce high titers of neutralizing antibodies against HPV56, HPV66 and HPV53 in mice; and its protective effect against HPV56 is slightly weaker than HPV56N0VLP alone and mixed HPV56/66/53VLP, and significantly higher than HPV66N5VLP alone and HPV53N5VLP alone; its protective effect against HPV66 is slightly weaker than HPV66N0VLP alone and mixed HPV66/56/53VLP, but significantly higher than HPV56N0VLP alone and HPV53N5VLP alone; its protective effect against HPV53 is slightly weaker than HPV53N5VLP alone, mixed HPV66/56/53VLP, but significantly higher than HPV66N5VLP alone and HPV56N0VLP alone. This indicates that the H56N0-66T1-66T5-53S3VLP can be used as an effective vaccine for preventing HPV56 infection, HPV66 infection and HPV53 infection, and can be used to replace a mixed vaccine containing HPV56VLP, HPV66VLP and HPV53 VLP.

Sequence information

information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Sequence 1(SEQ ID NO: 1):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR.

Sequence 2(SEQ ID NO: 2):

MAMWRPSDNKVYLPTPVSKVDTYVKRRTSIFYHAGSSRLLAVGHPYYSVSKSGTKTNIPKVSAYQYLVRFRVRVRFVNPPNKFGLPDPSFYNPQERLVWAVGLEVGRGQPLGLGLGGLLGGLSGHPLDLDDTEVLNLNLNLNGVGDNEDVLDNISVDCKQLCGCAPALGEGGATCGTPGNTGDCPLALVNTPDGDMVDFGKLLQQEVQEPQVPDIVQSKYPDLKDDAMSATGDMSYGFYLRLFQARQLFRAVGGEVGGGTGPHTHVPGVGGGTGVDPHTHMGTGEVDGEGTGATCGATCGATCAQVPAKLTGENYLSYVPAKVFQVPAKVFKSQVPAKFLDGEGQVPAKVFTGAVFLDGEGFPAKKFQVPAKKFQVPAK:

MPSDNKVYLPPTPVSKVVATDTYVKRTS IFYHAGSSRLLAVGHPYYSVSKSGTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDPSFYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTEVSNLAGNNVIEDSRDNISVDCKQTQLCIVGCAPALGEHWTKGAVCKSTPGNTGDCPPLALVNTPIEDGDMVDTGFGAMDFKLLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGNVGEAIPTDLYWKGGNGRDPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQVFVTVVDTTRSTNMTINAAKSTLTKYDAREINQYLRHVEEYELQFVFQLCKITLTAEVMAYLHNMNNTLLDDWNIGLSPPVATSLEDKYRYIKSTAITCQREQPPAEKQDPLAKYKFWEVNLQDSFSADLDQFPLGRKFLMQLGPRPPRPKASVSASKRRAAPTSSSSSPAKRKK.

Sequence 4(SEQ ID NO: 4):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVSKSGTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR.

Sequence 5(SEQ ID NO: 5):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTEVSNLAGNNVIEDSRDNISVDCKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR.

Sequence 6(SEQ ID NO: 6):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTPGNTGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR.

Sequence 7(SEQ ID NO: 7):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGNVGEAIPTDLYWKGGNGRDPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR.

Sequence 8(SEQ ID NO: 8):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTAKSTLTKYDAREINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR.

Sequence 9(SEQ ID NO: 9):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGACCAAGGACAACACCAAGACCAACATCCCCAAGGTGTCCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGAGGCCAGCCTCTGGGAGCTGGCCTGAGCGGCCACCCCCTGTTCAACCGGCTGGACGACACCGAGAGCAGCAACCTGGCCAACAACAACGTGATCGAGGACAGCCGGGACAACATCAGCGTGGACGGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCAGGTCACCACCGGCGACTGCCCCCCTCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGAGCCGGCAAAGTGGGCGAGACAATCCCCGCCGAGCTGTACCTGAAGGGCAGCAACGGACGGGAGCCTCCTCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCACCGAGCAGCTGTCCAAGTACGACGCCCGGAAGATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA

sequence 10(SEQ ID NO: 10):

ATGCCCAGCGACAACAAGGTGTACCTGCCCCCCACCCCCGTGAGCAAGGTGGTGGCCACCGACACCTACGTGAAGAGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCCACCCCTACTACAGCGTGAGCAAGAGCGGCACCAAGACCAACATCCCCAAGGTGAGCGCCTACCAGTACAGGGTGTTCAGGGTGAGGCTCCCCGACCCCAACAAGTTCGGCCTGCCCGACCCCAGCTTCTACAACCCCGACCAGGAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGGTGGGCAGGGGCCAGCCCCTGGGCGCCGGCCTGAGCGGCCACCCCCTGTTCAACAGGCTGGACGACACCGAGGTGAGCAACCTGGCCGGCAACAACGTGATCGAGGACAGCAGGGACAACATCAGCGTGGACTGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCGCCCCCGCCCTGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCCCGGCAACACCGGCGACTGCCCCCCCCTGGCCCTGGTGAACACCCCCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCGCCATGGACTTCAAGCTGCTGCAGGAGAGCAAGGCCGAGGTGCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGAGGAGGGAGCAGCTGTTCGCCAGGCACTACTTCAACAGGGCCGGCAACGTGGGCGAGGCCATCCCCACCGACCTGTACTGGAAGGGCGGCAACGGCAGGGACCCCCCCCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGGTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGACCATCAACGCCGCCAAGAGCACCCTGACCAAGTACGACGCCAGGGAGATCAACCAGTACCTGAGGCACGTGGAGGAGTACGAGCTGCAGTTCGTGTTCCAGCTGTGCAAGATCACCCTGACCGCCGAGGTGATGGCCTACCTGCACAACATGAACAACACCCTGCTGGACGACTGGAACATCGGCCTGAGCCCCCCCGTGGCCACCAGCCTGGAGGACAAGTACAGGTACATCAAGAGCACCGCCATCACCTGCCAGAGGGAGCAGCCCCCCGCCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGAGGTGAACCTGCAGGACAGCTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGATGCAGCTGGGCCCCAGGCCCCCCAGGCCCAAGGCCAGCGTGAGCGCCAGCAAGAGGAGGGCCGCCCCCACCAGCAGCAGCAGCAGCCCCGCCAAGAGGAAGAAGTAA

Sequence 11(SEQ ID NO: 11):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGAGCAAGAGCGGCACCAAGACCAACATCCCCAAGGTGAGCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGAGGCCAGCCTCTGGGAGCTGGCCTGAGCGGCCACCCCCTGTTCAACCGGCTGGACGACACCGAGAGCAGCAACCTGGCCAACAACAACGTGATCGAGGACAGCCGGGACAACATCAGCGTGGACGGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCAGGTCACCACCGGCGACTGCCCCCCTCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGAGCCGGCAAAGTGGGCGAGACAATCCCCGCCGAGCTGTACCTGAAGGGCAGCAACGGACGGGAGCCTCCTCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCACCGAGCAGCTGTCCAAGTACGACGCCCGGAAGATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA

sequence 12(SEQ ID NO: 12):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGACCAAGGACAACACCAAGACCAACATCCCCAAGGTGTCCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGGGGCCAGCCCCTGGGCGCCGGCCTGAGCGGCCACCCCCTGTTCAACAGGCTGGACGACACCGAGGTGAGCAACCTGGCCGGCAACAACGTGATCGAGGACAGCAGGGACAACATCAGCGTGGACTGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCAGGTCACCACCGGCGACTGCCCCCCTCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGAGCCGGCAAAGTGGGCGAGACAATCCCCGCCGAGCTGTACCTGAAGGGCAGCAACGGACGGGAGCCTCCTCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCACCGAGCAGCTGTCCAAGTACGACGCCCGGAAGATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA

sequence 13(SEQ ID NO: 13):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGACCAAGGACAACACCAAGACCAACATCCCCAAGGTGTCCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGAGGCCAGCCTCTGGGAGCTGGCCTGAGCGGCCACCCCCTGTTCAACCGGCTGGACGACACCGAGAGCAGCAACCTGGCCAACAACAACGTGATCGAGGACAGCCGGGACAACATCAGCGTGGACGGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCCCGGCAACACCGGCGACTGCCCCCCCCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGAGCCGGCAAAGTGGGCGAGACAATCCCCGCCGAGCTGTACCTGAAGGGCAGCAACGGACGGGAGCCTCCTCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCACCGAGCAGCTGTCCAAGTACGACGCCCGGAAGATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA

Sequence 14(SEQ ID NO: 14):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGACCAAGGACAACACCAAGACCAACATCCCCAAGGTGTCCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGAGGCCAGCCTCTGGGAGCTGGCCTGAGCGGCCACCCCCTGTTCAACCGGCTGGACGACACCGAGAGCAGCAACCTGGCCAACAACAACGTGATCGAGGACAGCCGGGACAACATCAGCGTGGACGGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCAGGTCACCACCGGCGACTGCCCCCCTCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGGGCCGGCAACGTGGGCGAGGCCATCCCCACCGACCTGTACTGGAAGGGCGGCAACGGCAGGGACCCCCCCCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCACCGAGCAGCTGTCCAAGTACGACGCCCGGAAGATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA

Sequence 15(SEQ ID NO: 15):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGACCAAGGACAACACCAAGACCAACATCCCCAAGGTGTCCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGAGGCCAGCCTCTGGGAGCTGGCCTGAGCGGCCACCCCCTGTTCAACCGGCTGGACGACACCGAGAGCAGCAACCTGGCCAACAACAACGTGATCGAGGACAGCCGGGACAACATCAGCGTGGACGGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCAGGTCACCACCGGCGACTGCCCCCCTCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGAGCCGGCAAAGTGGGCGAGACAATCCCCGCCGAGCTGTACCTGAAGGGCAGCAACGGACGGGAGCCTCCTCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCAAGAGCACCCTGACCAAGTACGACGCCAGGGAGATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA.

sequence 16(SEQ ID NO: 16):

VSNLAGNNVIEDSRDNISVDC

Sequence 17(SEQ ID NO: 17):

KSTLTKYDARE

Sequence 18(SEQ ID NO: 18):

PGN

Sequence 19(SEQ ID NO: 19):

NVGEAIPTDLYWKGGNGRD

Sequence 20(SEQ ID NO: 20):

SKSG

Sequence 37(SEQ ID NO: 37):

ATGGCCATGTGGAGGCCCAGCGACAACAAGGTGTACCTGCCCCCCACCCCCGTGAGCAAGGTGGTGGCCACCGACACCTACGTGAAGAGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGGCTGCTGGCCGTGGGCCACCCCTACTACAGCGTGAGCAAGAGCGGCACCAAGACCAACATCCCCAAGGTGAGCGCCTACCAGTACAGGGTGTTCAGGGTGAGGCTCCCCGACCCCAACAAGTTCGGCCTGCCCGACCCCAGCTTCTACAACCCCGACCAGGAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGGTGGGCAGGGGCCAGCCCCTGGGCGCCGGCCTGAGCGGCCACCCCCTGTTCAACAGGCTGGACGACACCGAGGTGAGCAACCTGGCCGGCAACAACGTGATCGAGGACAGCAGGGACAACATCAGCGTGGACTGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCGCCCCCGCCCTGGGCGAGCACTGGACCAAGGGCGCCGTGTGCAAGAGCACCCCCGGCAACACCGGCGACTGCCCCCCCCTGGCCCTGGTGAACACCCCCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCGCCATGGACTTCAAGCTGCTGCAGGAGAGCAAGGCCGAGGTGCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGAGGAGGGAGCAGCTGTTCGCCAGGCACTACTTCAACAGGGCCGGCAACGTGGGCGAGGCCATCCCCACCGACCTGTACTGGAAGGGCGGCAACGGCAGGGACCCCCCCCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGGTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGACCATCAACGCCGCCAAGAGCACCCTGACCAAGTACGACGCCAGGGAGATCAACCAGTACCTGAGGCACGTGGAGGAGTACGAGCTGCAGTTCGTGTTCCAGCTGTGCAAGATCACCCTGACCGCCGAGGTGATGGCCTACCTGCACAACATGAACAACACCCTGCTGGACGACTGGAACATCGGCCTGAGCCCCCCCGTGGCCACCAGCCTGGAGGACAAGTACAGGTACATCAAGAGCACCGCCATCACCTGCCAGAGGGAGCAGCCCCCCGCCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGAGGTGAACCTGCAGGACAGCTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGATGCAGCTGGGCCCCAGGCCCCCCAGGCCCAAGGCCAGCGTGAGCGCCAGCAAGAGGAGGGCCGCCCCCACCAGCAGCAGCAGCAGCCCCGCCAAGAGGAAGAAGTAA.

Sequence 38(SEQ ID NO: 38):

MATWRPSENKVYLPPTPVSKVVATDSYVKRTSIFYHAGSSRLLAVGHPYYSVSKSGTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDNISVDGKQTQLCIVGCTPAMGEHWTKGTACRSTPTTAGDCPPLALINTPIEDGDMIDTGFGAMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGKVGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMTISTAKSTLTKYDAREINQYLRHVEEYELQFVFQLCKITLSAEVMAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFWDVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR

Sequence 39(SEQ ID NO: 39):

ATGGCCACCTGGCGGCCCAGCGAGAACAAGGTGTACCTGCCCCCCACCCCCGTGTCCAAGGTGGTGGCCACCGACAGCTACGTGAAGCGGACCAGCATCTTCTACCACGCCGGCAGCAGCAGACTGCTGGCCGTGGGCCACCCCTACTACAGCGTGAGCAAGAGCGGCACCAAGACCAACATCCCCAAGGTGAGCGCCTACCAGTACCGGGTGTTCAGAGTGCGGCTGCCTGACCCTAACAAGTTCGGCCTGCCCGACACCAATATCTACAACCCCGACCAGGAACGGCTGGTCTGGGCCTGCGTGGGCCTGGAAGTGGGCAGAGGCCAGCCTCTGGGAGCTGGCCTGAGCGGCCACCCCCTGTTCAACCGGCTGGACGACACCGAGAGCAGCAACCTGGCCAACAACAACGTGATCGAGGACAGCCGGGACAACATCAGCGTGGACGGCAAGCAGACCCAGCTGTGCATCGTGGGCTGCACACCCGCCATGGGCGAGCACTGGACCAAGGGCACCGCTTGCCGTTCTACCCCGACCACCGCTGGCGACTGCCCCCCTCTGGCCCTGATCAACACCCCCATCGAGGACGGCGACATGATCGACACCGGCTTCGGCGCCATGGACTTCAAGGTGCTGCAGGAAAGCAAGGCCGAGGTCCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGCGGCGGGAGCAGCTGTTCGCCCGGCACTACTTCAACAGAGCCGGCAAAGTGGGCGAGACAATCCCCGCCGAGCTGTACCTGAAGGGCAGCAACGGACGGGAGCCTCCTCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGCGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAACCAGCTGTTCGTGACCGTCGTGGACACTACCCGGTCCACCAACATGACCATCAGCACCGCCAAATCTACCCTGACCAAATACGACGCTCGTGAAATCAACCAGTACCTGCGGCACGTGGAGGAATATGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATCACCCTGAGCGCCGAAGTGATGGCCTACCTGCACAACATGAACGCCAACCTGCTGGAAGATTGGAACATCGGCCTGAGCCCCCCTGTGGCTACCTCTCTGGAAGATAAGTACAGATACGTGCGGAGCACCGCCATCACCTGCCAGAGAGAGCAGCCCCCCACCGAGAAGCAGGACCCCCTGGCCAAGTACAAGTTCTGGGACGTGAACCTGCAGGACAGCTTCAGCACCGACCTGGACCAGTTCCCCCTGGGCCGGAAGTTCCTGATGCAGCTGGGCACCAGATCCAAGCCCGCCGTGGCCACAAGCAAGAAGCGGAGCGCCCCCACAAGCACCAGCACCCCCGCCAAGCGGAAGCGGAGATGA

Sequence 40(SEQ ID NO: 40):

MAVWRPSDSKVYLPPTPVSKVITTDAYVKRTTIFYHAGSSRLLTVGHPYYPISKSGKADIPKVSAFQYRVFRVRLPDPNKFGLPDTNIFNPDQERLVWACVGLEIGRGQPLGVGVSGHPLFNRLDDTESSSIAIQDTAPDSRDNVSVDPKQTQLCI IGCAPAIGEHWTKGTACRSTPTTAGDCPPLELINSPIEDGDMVDTGFGALNFKALQESKSDVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFTRHFFNRAGVIGEEIPNDLYIKGSNGRDPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWNNQLFVTVVDTTRNTNMTLSATTQSMSTYNSKQIKQYVRHAEEYELQFVFQLCKISLSAEVMAYLHTMNSTLLEDWNIGLSPPVATSLEDKYRYVKSAAITCQKDQPPPEKQDPLSKYKFWEVNLQNSFSADLDQFPLGRKFLMQVGVRTKPPVSSKKRSASTTSTSAPSSKRKRK.

Sequence 41(SEQ ID NO: 41):

ATGGCCGTGTGGAGGCCCAGCGACAGCAAGGTGTACCTGCCCCCCACCCCCGTGAGCAAGGTGATCACCACCGACGCCTACGTGAAGAGGACCACCATCTTCTACCACGCCGGCAGCAGCAGGCTGCTGACCGTGGGCCACCCCTACTACCCCATCAGCAAGAGCGGCAAGGCCGACATCCCCAAGGTGAGCGCCTTCCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTCGGCCTGCCCGACACCAACATCTTCAACCCCGACCAGGAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGCCACCCCCTGTTCAACAGGCTGGACGACACCGAGAGCAGCAGCATCGCCATCCAGGACACCGCCCCCGACAGCAGGGACAACGTGAGCGTGGACCCCAAGCAGACCCAGCTGTGCATCATCGGCTGCGCCCCCGCCATCGGCGAGCACTGGACCAAGGGCACCGCCTGCAGGAGCACCCCCACCACCGCCGGCGACTGCCCCCCCCTGGAGCTGATCAACAGCCCCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCGCCCTGAACTTCAAGGCCCTGCAGGAGAGCAAGAGCGACGTGCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGAGGAGGGAGCAGCTGTTCACCAGGCACTTCTTCAACAGGGCCGGCGTGATCGGCGAGGAGATCCCCAACGACCTGTACATCAAGGGCAGCAACGGCAGGGACCCCCCCCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGAACAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAACACCAACATGACCCTGAGCGCCACCACCCAGAGCATGAGCACCTACAACAGCAAGCAGATCAAGCAGTACGTGAGGCACGCCGAGGAGTACGAGCTGCAGTTCGTGTTCCAGCTGTGCAAGATCAGCCTGAGCGCCGAGGTGATGGCCTACCTGCACACCATGAACAGCACCCTGCTGGAGGACTGGAACATCGGCCTGAGCCCCCCCGTGGCCACCAGCCTGGAGGACAAGTACAGGTACGTGAAGAGCGCCGCCATCACCTGCCAGAAGGACCAGCCCCCCCCCGAGAAGCAGGACCCCCTGAGCAAGTACAAGTTCTGGGAGGTGAACCTGCAGAACAGCTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGATGCAGGTGGGCGTGAGGACCAAGCCCCCCGTGAGCAGCAAGAAGAGGAGCGCCAGCACCACCAGCACCAGCGCCCCCAGCAGCAAGAGGAAGAGGAAGTAA

Sequence 42(SEQ ID NO: 42):

MPSDSKVYLPPTPVSKVITTDAYVKRTTIFYHAGSSRLLTVGHPYYPISKSGKADIPKVSAFQYRVFRVRLPDPNKFGLPDTNIFNPDQERLVWACVGLEIGRGQPLGVGVSGHPLFNRLDDTESSSIAIQDTAPDSRDNVSVDPKQTQLCIIGCAPAIGEHWTKGTACRSTPTTAGDCPPLELINSPIEDGDMVDTGFGALNFKALQESKSDVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFTRHFFNRAGVIGEEIPNDLYIKGSNGRDPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWNNQLFVTVVDTTRNTNMTLSATTQSMSTYNSKQIKQYVRHAEEYELQFVFQLCKISLSAEVMAYLHTMNSTLLEDWNIGLSPPVATSLEDKYRYVKSAAITCQKDQPPPEKQDPLSKYKFWEVNLQNSFSADLDQFPLGRKFLMQVGVRTKPPVSSKKRSASTTSTSAPSSKRKRK

Sequence 43(SEQ ID NO: 43):

ATGCCCAGCGACAGCAAGGTGTACCTGCCCCCCACCCCCGTGAGCAAGGTGATCACCACCGACGCCTACGTGAAGAGGACCACCATCTTCTACCACGCCGGCAGCAGCAGGCTGCTGACCGTGGGCCACCCCTACTACCCCATCAGCAAGAGCGGCAAGGCCGACATCCCCAAGGTGAGCGCCTTCCAGTACAGGGTGTTCAGGGTGAGGCTGCCCGACCCCAACAAGTTCGGCCTGCCCGACACCAACATCTTCAACCCCGACCAGGAGAGGCTGGTGTGGGCCTGCGTGGGCCTGGAGATCGGCAGGGGCCAGCCCCTGGGCGTGGGCGTGAGCGGCCACCCCCTGTTCAACAGGCTGGACGACACCGAGAGCAGCAGCATCGCCATCCAGGACACCGCCCCCGACAGCAGGGACAACGTGAGCGTGGACCCCAAGCAGACCCAGCTGTGCATCATCGGCTGCGCCCCCGCCATCGGCGAGCACTGGACCAAGGGCACCGCCTGCAGGAGCACCCCCACCACCGCCGGCGACTGCCCCCCCCTGGAGCTGATCAACAGCCCCATCGAGGACGGCGACATGGTGGACACCGGCTTCGGCGCCCTGAACTTCAAGGCCCTGCAGGAGAGCAAGAGCGACGTGCCCCTGGACATCGTGCAGAGCACCTGCAAGTACCCCGACTACCTGAAGATGAGCGCCGACGCCTACGGCGACAGCATGTGGTTCTACCTGAGGAGGGAGCAGCTGTTCACCAGGCACTTCTTCAACAGGGCCGGCGTGATCGGCGAGGAGATCCCCAACGACCTGTACATCAAGGGCAGCAACGGCAGGGACCCCCCCCCCAGCAGCGTGTACGTGGCCACCCCCAGCGGCAGCATGATCACCAGCGAGGCCCAGCTGTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGAACAACCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAACACCAACATGACCCTGAGCGCCACCACCCAGAGCATGAGCACCTACAACAGCAAGCAGATCAAGCAGTACGTGAGGCACGCCGAGGAGTACGAGCTGCAGTTCGTGTTCCAGCTGTGCAAGATCAGCCTGAGCGCCGAGGTGATGGCCTACCTGCACACCATGAACAGCACCCTGCTGGAGGACTGGAACATCGGCCTGAGCCCCCCCGTGGCCACCAGCCTGGAGGACAAGTACAGGTACGTGAAGAGCGCCGCCATCACCTGCCAGAAGGACCAGCCCCCCCCCGAGAAGCAGGACCCCCTGAGCAAGTACAAGTTCTGGGAGGTGAACCTGCAGAACAGCTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAAGTTCCTGATGCAGGTGGGCGTGAGGACCAAGCCCCCCGTGAGCAGCAAGAAGAGGAGCGCCAGCACCACCAGCACCAGCGCCCCCAGCAGCAAGAGGAAGAGGAAGTAA

Sequence 44(SEQ ID NO: 44):

TACRSTPTTA

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.