阅读说明：本技术 一种融合蛋白、融合蛋白疫苗及其制备方法、应用 (Fusion protein, fusion protein vaccine, and preparation method and application thereof ) 是由 董彦鹏 李玉峰 张健淞 肖澄 周琳 车巧林 胡静雅 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种融合蛋白、融合蛋白疫苗及其制备方法、应用,将TbpB蛋白的氨基酸残基序列中的至少一部分抗原表位区序列替换为TbpA蛋白的氨基酸残基序列的至少一部分抗原表位区序列,所得的序列为所述融合蛋白的氨基酸残基序列。本发明为了使TbpA的抗原性得到稳定形式的表达,利用TbpA和TbpB所共有的β-桶结构特征,设计了以TbpB为支架,将TbpA的若干个表面区域的抗原决定簇替换TbpB的若干个较大的可变环区抗原决定簇,得到了TbpA+B融合蛋白,这种融合蛋白同时具有两种蛋白的抗原性,并且可以通过原核细胞表达系统得到大量的可溶形式的表达,适合于疫苗的商业化生产。(The invention discloses a fusion protein, a fusion protein vaccine, a preparation method and an application thereof, wherein at least a part of antigen epitope region sequences in an amino acid residue sequence of TbpB protein are replaced by at least a part of antigen epitope region sequences of the amino acid residue sequence of the TbpA protein, and the obtained sequences are the amino acid residue sequences of the fusion protein. In order to make the antigenicity of TbpA obtain stable expression, the invention designs that TbpB is used as a bracket and a plurality of antigenic determinants of a plurality of surface regions of TbpA replace a plurality of larger variable loop region antigenic determinants of TbpB by utilizing the common beta-barrel structure characteristics of TbpA and TbpB, so as to obtain TbpA + B fusion protein.)

1. A fusion protein is characterized in that at least a part of the epitope region sequence in the amino acid residue sequence of the TbpB protein is replaced by at least a part of the subsequence of the amino acid residue sequence of the TbpA protein, and the obtained sequence is the amino acid residue sequence of the fusion protein;

the length of the subsequence is not less than the length of the antigen epitope region sequence to be replaced;

the coding amino acid sequence of the TbpA protein is shown as SEQ ID NO. 1;

the coding amino acid sequence of the TbpB protein is shown as SEQ ID NO. 2.

2. The fusion protein of claim 1, wherein the subsequence includes at least one variable loop region sequence of amino acid residue sequences of the TbpA protein.

3. The fusion protein of claim 1, wherein the subsequence includes at least one alpha helical region sequence of amino acid residue sequences of the TbpA protein.

4. The fusion protein of claim 1, wherein the first epitope region substituted in the amino acid residue sequence of the TbpB protein is amino acid residues 402 to 409, the corresponding amino acid residue sequence of the first epitope region before substitution is SEQ ID NO. 3, and the corresponding amino acid residue sequence after substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

5. The fusion protein of claim 1, wherein the substituted second epitope region of the amino acid residue sequence of the TbpB protein is from 433 th to 440 th amino acid residues, the corresponding amino acid residue sequence of the second epitope region before the substitution is SEQ ID NO. 8, and the corresponding amino acid residue sequence after the substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

6. The fusion protein of claim 1, wherein the substituted third epitope region of the amino acid residue sequence of the TbpB protein is from 459 to 466 amino acid residues, the corresponding amino acid residue sequence of the third epitope region before substitution at least comprises SEQ ID NO. 9, and the corresponding amino acid residue sequence after substitution is one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

7. The fusion protein of claim 1, wherein the fourth epitope region substituted in the amino acid residue sequence of the TbpB protein is 484 to 504 amino acid residues, the corresponding amino acid residue sequence of the fourth epitope region before substitution is SEQ ID NO. 10, and the corresponding amino acid residue sequence after substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

8. A fusion protein vaccine, wherein the antigen of the fusion protein vaccine comprises the fusion protein of any one of claims 1-5.

9. The fusion protein vaccine of claim 8, wherein the fusion protein content of the fusion protein vaccine is at least 200ug/0.5 ml.

10. A method of preparing the fusion protein vaccine of claim 8 or 9, comprising the steps of:

preparing the fusion protein as an antigen;

and (3) compounding the fusion protein and an adjuvant according to the ratio of 9: 1.

11. Use of the fusion protein according to any one of claims 1 to 7 for the preparation of a medicament for the prevention of haemophilus parasuis.

Technical Field

The invention relates to the field of transferrin receptor protein, and particularly relates to a fusion protein, and a preparation method, a vaccine and application thereof.

Background

The transferrin receptor protein is an important vaccine target of pathogenic bacteria-haemophilus parasuis in a pig body, the transferrin receptor protein comprises transferrin receptor protein A (TbpA) and transferrin receptor protein B (TbpB), the TbpB protein is a surface membrane protein, and the TbpB protein can be expressed in a large amount through a prokaryotic cell expression system, is easy to produce and is suitable for commercial production of vaccines; the TbpA protein is a complete outer membrane protein, and a large amount of soluble expression is not easy to obtain through a prokaryotic cell expression system, so that the production cost is high, and the TbpA protein is not suitable for commercial production of vaccines.

The existing vaccine prepared by singly adopting the TbpA protein and the vaccine prepared by singly adopting the TbpB protein have the unsatisfactory protection effect on preventing the haemophilus parasuis, which is lower than the effect of simultaneously using the two proteins, but the TbpA protein is not easy to produce and has higher cost, so that a vaccine which has the similar effect to the effect of simultaneously using the two proteins, is easy to produce and has lower cost is urgently needed.

Disclosure of Invention

Therefore, the invention provides a fusion protein, a fusion protein vaccine, a preparation method and an application thereof, in order to ensure that the antigenicity of TbpA is expressed in a stable form, the TbpB is used as a bracket by utilizing the common beta-barrel structure characteristics of the TbpA and the TbpB, a plurality of antigenic determinants of a plurality of surface regions of the TbpA are used for replacing a plurality of larger variable loop region antigenic determinants of the TbpB, and the TbpA + B fusion protein is obtained.

In order to achieve the above objects, according to one aspect of the present invention, there is provided a fusion protein, wherein at least a part of an epitope region sequence of an amino acid residue sequence of a TbpB protein is replaced with at least a part of a subsequence of the amino acid residue sequence of the TbpA protein, and the resulting sequence is the amino acid residue sequence of the fusion protein;

the length of the subsequence is not less than the length of the antigen epitope region sequence to be replaced;

the coding amino acid sequence of the TbpA protein is shown as SEQ ID NO. 1;

the coding amino acid sequence of the TbpB protein is shown as SEQ ID NO. 2.

The hydrophilicity of the epitope region can be analyzed by DNAStar Protean software, and online website ABCcred can be applied

(website: https:// webs. iiiitd. edu.in/raghava/abcpred/index. html). Prediction of the epitope region of a particular protein by hydrophilicity analysis is a common technique used by those skilled in the art.

The epitope region of the TbpA protein comprises a variable loop region and a part of an alpha spiral region.

Preferably, the subsequence includes at least one variable loop region sequence of the sequence of amino acid residues of the TbpA protein.

Preferably, the subsequence includes at least one alpha helical region sequence of the sequence of amino acid residues of the protein TbpA.

Furthermore, a first epitope region which is replaced in the amino acid residue sequence of the TbpB protein is amino acid residues from 402 th position to 409 th position, the corresponding amino acid residue sequence of the first epitope region before replacement is SEQ ID NO. 3, and the corresponding amino acid residue sequence after replacement at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

Furthermore, the substituted second antigenic epitope region position in the amino acid residue sequence of the TbpB protein is from 433 th to 440 th amino acid residues, the corresponding amino acid residue position of the second antigenic epitope region position before the substitution is SEQ ID NO. 8, and the corresponding amino acid residue sequence after the substitution at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

Furthermore, a third epitope region which is replaced in the amino acid residue sequence of the TbpB protein is from 459 th to 466 th amino acid residues, the corresponding amino acid residue sequence of the third epitope region before replacement at least comprises SEQ ID NO 9, and the corresponding amino acid residue sequence after replacement is one of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.

Furthermore, a fourth epitope region which is replaced in the amino acid residue sequence of the TbpB protein is 484 th to 504 th amino acid residues, the corresponding amino acid residue sequence of the fourth epitope region before replacement is SEQ ID NO. 10, and the corresponding amino acid residue sequence after replacement at least comprises one of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

Among them, the amino acid residue sequences of the TbpA protein and the TbpB protein are the disclosures which are easily obtained by those skilled in the art, and the fusion protein of the present invention has all the amino acid residue sequences disclosed, and those skilled in the art can easily prepare the above-mentioned fusion protein, and the preparation method thereof will not be described in detail herein.

In another aspect, the invention provides a fusion protein vaccine, and the antigen of the fusion protein vaccine comprises the fusion protein.

Preferably, the fusion protein content of the fusion protein vaccine is at least 200ug/0.5 ml.

In another aspect, the present invention provides a method for preparing the fusion protein vaccine, comprising the following steps:

preparing the fusion protein as an antigen;

and (3) compounding the fusion protein and an adjuvant according to the ratio of 9: 1.

Preferably, the adjuvant is a gel adjuvant.

The invention also provides application of the fusion protein in preparation of a medicament for preventing haemophilus parasuis.

The invention has the beneficial effects that:

in order to make TbpA antigenicity obtain stable expression, the common beta-barrel structure characteristics of TbpA and TbpB are utilized, TbpB is designed as a bracket, antigenic determinants of a plurality of surface regions of TbpA are substituted for a plurality of larger variable loop region antigenic determinants of TbpB, and TbpA + B fusion protein is obtained.

Biological preservation Instructions

The Haemophilus parasuis 5-SQ strain is Haemophilus parasuis (Haemophilus parasuis)5-SQ, CCTCC NO: m2021877, deposited by the depository: china center for type culture Collection, Address: wuhan university, preservation date: 13/7/2021, accession number: CCTCC NO: m2021877, classification name: haemophilus parasuis 5-SQ Haemophilus parauis 5-SQ.

Drawings

FIG. 1 is a diagram of a TbpB space structure provided by the present invention;

FIG. 2 is a diagram of the TbpA space structure provided by the present invention.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

The spatial structure of TbpB is shown in FIG. 1, and it has two β -barrel structures (two dashed boxes in the figure), each of which is composed of 8 β -sheets and 6 variable loop regions.

The spatial structure of TbpA is shown in FIG. 2, and it has 1 β -barrel structure, which is also composed of β -sheet and variable loop regions.

In the structures of the two proteins, the variable loop region in the beta-barrel structure is used for connecting two antiparallel beta-sheets, and the variable loop regions are also important antigenic determinants of the protein, so that the variable loop region of the beta-barrel structure on the TbpA is replaced by the variable loop region of the beta-barrel structure on the TbpB, so as to realize the TbpA + B fusion protein, and the antigenicity of the two proteins can be simultaneously realized.

Hydrophilicity analysis was performed by DNAStar Protean software, and epitope prediction was performed by using the online site ABCPred (website: https:// webs. iiitd. edu.in/raghava/ABCpred/index. html), and the epitope region (epitope) of TbpA protein was found to include the variable loop region and a portion of the alpha-helical region.

Taking Haemophilus parasuis 5-SQ strain as an example, selecting TbpB protein of 5-SQ strain and TbpA protein of 5-SQ strain, replacing four fragments of the TbpA protein of 5-SQ strain with four fragments of the TbpB protein of 5-SQ strain, wherein the specific positions of replacement are shown in the following table.

An escherichia coli expression system is adopted, pET32a is used as an expression vector, a T7 promoter is used, BL21 is used as a host bacterium, IPTG is added into a bacterial culture medium to start expression, and the TbpA + B fusion protein is prepared.

50 female guinea pigs weighing 220g-250g were selected and divided into 5 groups, and the TbpA protein, TbpB protein and TbpA + B fusion protein were mixed with gel02 adjuvant at a ratio of 9:1, the final protein content of the vaccine is 200ug/0.5ml, the immunization dose is 0.5 ml/vaccine, and the negative control group is prepared by mixing sterile normal saline and gel02 adjuvant in a ratio of 9:1, performing the proportional preparation, injecting leg muscles in different points, performing immunization twice, performing second immunization 14 days after the first immunization, performing toxicity attack 28 days after the second immunization, wherein the toxicity attack dose is the lowest total lethal dose of 5-SQ haemophilus parasuis, the method is that guinea pigs are injected in abdominal cavities, the death condition of each group of guinea pigs is observed after the toxicity attack, and the survival rate is calculated 7 days after the toxicity attack;

animal experiments prove that the TbpA + B fusion protein has similar immune effect to the simultaneous use of the TbpA and the TbpB proteins. The TbpA + B fusion protein can be expressed in a large amount of soluble forms through a prokaryotic cell expression system, is suitable for commercial production of vaccines, and the cost of the TbpA + B fusion protein is inevitably lower than the cost of the simultaneous use of the TbpA protein and the TbpB protein.

As the sequences of the Haemophilus parasuis 5-SQ strain and all the Haemophilus parasuis are universal, the TbpA + B fusion protein prepared by the preparation method disclosed by the embodiment of the invention is suitable for all the Haemophilus parasuis and has universality.

The above description is only a preferred embodiment of the present invention, and any person skilled in the art may modify the present invention or modify it into an equivalent technical solution by using the technical solution described above. Therefore, any simple modifications or equivalent substitutions made in accordance with the technical solution of the present invention are within the scope of the claims of the present invention.

Sequence listing

<110> Jiangsu south agriculture high technology corporation

<120> fusion protein, fusion protein vaccine and preparation method thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 898

<212> PRT

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 1

Met Lys Asn Lys Leu Asn Leu Ile Ser Leu Ala Ile Leu Ser Leu Phe

1 5 10 15

Ala Ala Gln Ser Tyr Ala Glu Gln Ala Val Gln Leu Asn Asp Val Tyr

20 25 30

Val Thr Gly Thr Lys Lys Lys Val His Lys Lys Glu Asn Glu Val Thr

35 40 45

Gly Leu Gly Lys Val Val Lys Thr Pro Asp Ser Leu Ser Lys Asp Gln

50 55 60

Val Leu Gly Met Arg Asp Leu Thr Arg Tyr Asp Pro Gly Ile Ser Val

65 70 75 80

Val Glu Gln Gly Arg Gly Ala Thr Thr Gly Tyr Ser Ile Arg Gly Val

85 90 95

Asp Arg Asn Arg Val Gly Leu Ala Leu Asp Gly Leu Pro Gln Ile Gln

100 105 110

Ser Tyr Val Ser Gln Tyr Ser Arg Ser Ser Ser Gly Ala Ile Asn Glu

115 120 125

Ile Glu Tyr Glu Asn Leu Arg Ser Ile Gln Ile Ser Lys Gly Ala Ser

130 135 140

Ser Ser Glu Phe Gly Ser Gly Ser Leu Gly Gly Ser Val Gln Phe Arg

145 150 155 160

Thr Lys Glu Val Ser Asp Ile Ile Lys Pro Gly Gln Ser Trp Gly Leu

165 170 175

Asp Thr Lys Ser Ala Tyr Ser Ser Lys Asn Gln Gln Trp Leu Asn Ser

180 185 190

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

195 200 205

Thr His Arg Asp Gly Lys Glu Thr Lys Ala His Lys Asp Ala Glu Ser

210 215 220

Arg Ser Lys Arg Ile Gln Arg Val Asp Leu Ala Asp Asn Asn Pro Gln

225 230 235 240

Gly Ser Asn Trp Phe Lys Val Lys Asn Asp Cys Pro Ser Leu Lys Cys

245 250 255

Glu Pro Arg Gln Gln Val Gly Val Ser Tyr Asp Gly Gln Lys Tyr Ile

260 265 270

Thr Glu Gln Leu Ser Ala Lys Glu Tyr Thr Gly Glu Glu Arg Ala Leu

275 280 285

Pro Asp Pro Val Lys Tyr Lys Ser Asp Ser Trp Leu Val Lys Leu Gly

290 295 300

Tyr Ser Leu Ser Pro Lys His Tyr Val Ala Gly Val Tyr Glu His Ser

305 310 315 320

Lys Gln Arg Tyr Asp Thr Arg Asp Met Thr Tyr Pro Ala Tyr Trp Gln

325 330 335

Leu Ser Asp Leu Lys Asn Gly Asn Thr Trp Tyr Pro Met Asn Asn Ala

340 345 350

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

355 360 365

Phe Thr Glu Asp Gly Val Lys Ser Ser Lys Gly Leu Arg Trp Ala Lys

370 375 380

Ala Arg Phe Ile Asp Glu Trp His Thr Arg Asp Arg Leu Gly Ala Leu

385 390 395 400

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

405 410 415

Leu Ser Phe Asp Gln Gln Lys Ile Asp Leu Ser Thr Arg Leu Arg Glu

420 425 430

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

435 440 445

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

450 455 460

Glu Lys His Asp Thr Ile Gln Leu Ser Leu Asp Lys Thr Val Gln Thr

465 470 475 480

Gly Leu Gly Lys His Gln Leu Asn Met Leu Leu Gly Ser Asp Arg Phe

485 490 495

Asn Ser Thr Leu Lys Arg His Glu Ile Leu Ser Lys Phe Ser Val Gly

500 505 510

Thr Trp His Arg Ile Asn Gly Asn Gly Tyr Gln Asn Asn Pro Tyr Ile

515 520 525

Tyr Glu Leu Lys Asp Gln Ala Ile Tyr Ser Lys Asn Glu Cys Asn Tyr

530 535 540

Ser Gly Thr Ile Ala Gly Arg Ala Asp Cys Ala Thr Ser Lys Ile Lys

545 550 555 560

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

565 570 575

Tyr Leu Asp Ile Gly Leu Gly Tyr Arg Phe Asp Lys His Lys Phe Arg

580 585 590

Ser Thr His Arg Trp Ala Asn Gln Gly Asp Tyr Lys Asn Ser Ala Trp

595 600 605

Asn Val Gly Val Val Ala Lys Pro Thr Ser Phe Leu Ser Leu Ser Tyr

610 615 620

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

625 630 635 640

Leu Arg Tyr Asp Gly Ala Met Ala Gly Ser Ser Asp Ala Tyr Gln Lys

645 650 655

Thr Glu Lys Leu Ser Pro Glu Lys Ser Leu Asn Gln Glu Ile Ala Ala

660 665 670

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

675 680 685

Asp Tyr Lys Gln Leu Ile Ala Pro Ala Glu Arg Met His Gln Thr Gln

690 695 700

Ser Met Ile Asn Tyr Phe Asn Val Gln Asp Ile Lys Leu Asp Gly Ile

705 710 715 720

Asn Leu Ile Gly Lys Leu Asp Trp Asn Gly Val Phe Asp Lys Ile Pro

725 730 735

Glu Gly Ile Tyr Thr Thr Leu Ala Tyr Ser Lys Met Arg Val Lys Glu

740 745 750

Val Lys Asn Tyr Pro Gly Tyr Met Asn Ile Arg Thr Pro Leu Leu Asp

755 760 765

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

770 775 780

Asp Glu Lys Trp Gly Val Asn Leu Thr Met Thr His Ser Ser Gly Lys

785 790 795 800

Asn Pro Asp Glu Leu Arg Gly Asn Glu Gln Val Gly Phe Ala Asn Tyr

805 810 815

Glu Arg Thr Ala Thr Lys Lys Arg Thr Arg Ser Trp His Thr Phe Asp

820 825 830

Leu Thr Gly Tyr Ile Thr Pro Trp Lys His Ile Thr Val Arg Ala Gly

835 840 845

Val Tyr Asn Leu Met Asn Tyr Arg Tyr Thr Thr Trp Glu Ser Val Arg

850 855 860

Gln Ser Ser Leu Asn Ala Ile His Gln His Thr Asn Val Lys Asp Tyr

865 870 875 880

Val Arg Tyr Ala Ala Pro Gly Arg Asn Tyr Val Leu Ser Val Glu Met

885 890 895

Lys Phe

<210> 2

<211> 545

<212> PRT

<213> Haemophilus parasuis (Haemophilus parasuis)

<400> 2

Met His Phe Lys Leu Asn Pro Tyr Ala Leu Ala Phe Thr Ser Leu Phe

1 5 10 15

Ile Val Ala Cys Ser Gly Gly Lys Gly Ser Phe Asp Leu Glu Asp Val

20 25 30

Gln Pro Asn Lys Thr Thr Gly Val Ser Lys Glu Glu Tyr Gln Asp Val

35 40 45

Glu Thr Asp Lys Lys Val Lys Glu Gln Leu Gly Glu Leu Met Glu Pro

50 55 60

Ala Leu Gly Tyr Val Val Lys Val Pro Val Ser Gln Ser Gly Ala Arg

65 70 75 80

Arg Thr Glu Ile Ser Asn Pro Glu Ala Ile Thr Asp Glu Asp Leu Lys

85 90 95

Lys Ile Pro Asn His Glu Thr Ile Lys Ser Val Ala Glu Gln Asn Tyr

100 105 110

Gly Trp Val Val Asp Lys Thr Ala Asp Lys Thr Met Lys Tyr Val Arg

115 120 125

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

130 135 140

Tyr Val Phe Tyr Lys Gly Ile Asn Pro Ser Lys Glu Leu Pro Gln Gly

145 150 155 160

Leu Val Leu Thr Tyr Gln Gly Glu Trp Asp Phe Thr Ser Asp Ala Asn

165 170 175

Leu Asp Ala Lys Arg Pro Asn Ser Asn Pro Glu Phe Asn Gly Tyr Gly

180 185 190

Pro Gly Gln Arg Ile Ala Thr Thr Ser Ala Asn Ala Lys Gly Arg Thr

195 200 205

Tyr Thr Ser Glu Phe Glu Val Asp Phe Ser Thr Lys Lys Leu Lys Gly

210 215 220

Lys Leu Asn Ser Lys Thr Ala Asn Thr Glu Ser Leu Glu Arg Tyr Ser

225 230 235 240

Ile Glu Ala Glu Leu Lys Gly Asn Arg Phe Arg Gly Lys Ala Ile Ala

245 250 255

Lys Asp Thr Ser Asp Ala Ile Leu Gly Lys Ser Ser Asp Tyr Leu Glu

260 265 270

Gly Gly Phe Tyr Gly Pro Lys Ala Glu Glu Leu Ala Gly Lys Phe Ile

275 280 285

Thr Asn Asp Lys Ser Leu Phe Ala Val Phe Ser Ala Lys His Asn Gly

290 295 300

Ser Asn Val Asp Thr Val Arg Ile Ile Asp Ala Ser Lys Ile Asp Leu

305 310 315 320

Thr His Phe Asn Ser Ser Glu Leu Thr Asn Phe Gly Asp Ala Ser Val

325 330 335

Leu Ile Ile Asp Gly Lys Lys Met Asp Leu Ala Gly Thr Asp Phe Lys

340 345 350

Asn Ser Lys Thr Ile Glu Ile Asn Gly Lys Thr Met Val Ala Val Ala

355 360 365

Cys Cys Ser Asn Leu Glu Tyr Met Lys Phe Gly Gln Leu Trp Gln Lys

370 375 380

Glu Gly Glu Thr Ala Lys Asp Asn Ser Leu Phe Leu Gln Gly Glu Arg

385 390 395 400

Thr Thr Thr Asp Lys Met Pro Lys Gly Gly Asn Tyr Lys Tyr Ile Gly

405 410 415

Thr Trp Asp Ala Gln Val Ser Lys Glu Asn Asn Trp Val Ala Thr Ala

420 425 430

Asp Asp Asp Arg Lys Ala Gly Tyr Arg Thr Glu Phe Asp Val Asp Phe

435 440 445

Gly Asn Lys Asn Leu Ser Gly Lys Leu Phe Asp Lys Asn Gly Val Asn

450 455 460

Pro Val Phe Thr Val Asp Ala Lys Ile Asn Gly Asn Gly Phe Thr Gly

465 470 475 480

Lys Ala Lys Thr Ser Asp Ala Gly Phe Ala Leu Asp Ser Gly Ser Ser

485 490 495

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

500 505 510

Tyr Gly Pro Thr Ala Ala Glu Leu Gly Gly Gln Phe Phe His Lys Ser

515 520 525

Asp Asn Gly Ser Val Gly Ala Val Phe Gly Ala Lys Gln Gln Val Lys

530 535 540

Lys

545

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Thr Thr Asp Lys Met Pro Lys Gly

1 5

<210> 4

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Ser Tyr Val Ser Gln Tyr Ser Arg Ser Ser Ser Gly Ala Ile Asn

1 5 10 15

Glu Ile Glu Tyr Glu

20

<210> 5

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Gln Leu Ser Asp Leu Lys Asn Gly Asn Thr Trp Tyr Pro Met

1 5 10 15

<210> 6

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ser Gly Lys Asn Pro Asp Glu Leu Arg Gly Asn Glu Gln Val Gly Phe

1 5 10 15

Ala Asn Tyr Glu Arg Thr Ala Thr Lys Lys Arg Thr Arg

20 25

<210> 7

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Tyr Thr Thr Trp Glu Ser Val Arg Gln Ser Ser Leu Asn Ala Ile His

1 5 10 15

Gln His Thr Asn Val Lys Asp Tyr Val Arg Tyr Ala

20 25

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Asp Asp Arg Lys Ala Gly Tyr

1 5

<210> 9

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Asp Lys Asn Gly Val Asn Pro Val

1 5

<210> 10

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

Asn Val Lys Phe Ser

20