阅读说明：本技术 小反刍兽疫诊断试剂盒 (Peste des petits ruminants diagnostic kit ) 是由 薛青红 孙淼 陈延飞 陈建 李岭 于 2018-05-18 设计创作，主要内容包括：本发明涉及小反刍兽疫诊断试剂盒。本发明的检测试剂盒包括基底,以及独立地连接于所述基底上的特定多肽或特定多肽组合。(The present invention relates to peste des petits ruminants diagnostic kits.Detection kit of the invention includes substrate, and particular polypeptide or the particular polypeptide combination being independently connected in the substrate.)

1. a kind of purposes of polypeptides in combination 1 in preparation peste des petits ruminants diagnostic kit；

The polypeptide shown in NO:1~11 SEQ ID of polypeptides in combination 1 forms,

Sequence shown in SEQ ID NO:1 is the polypeptide of N50:SAEALFRLQAMAKILEDQEE,

Sequence shown in SEQ ID NO:2 is the polypeptide of N46:TKRTRSGKPRGETPGPLLPE,

Sequence shown in SEQ ID NO:3 is the polypeptide of N47:GETPGPLLPEIMQEDELSRE,

Sequence shown in SEQ ID NO:4 is the polypeptide of N48:IMQEDELSRESSQNPREAQR,

Sequence shown in SEQ ID NO:5 is the polypeptide of H1:MSAQRERINAFYKDNLHNKT,

Sequence shown in SEQ ID NO:6 is the polypeptide of N40:AKLVSEIASQTGDERTVRGT,

Sequence shown in SEQ ID NO:7 is the polypeptide of H9:TESIDHQTKDVLTPLFKIIG,

Sequence shown in SEQ ID NO:8 is the polypeptide of H36:LSSHRGIIKDNEANWVVPST,

Sequence shown in SEQ ID NO:9 is the polypeptide of F54:LKPDLTGTSKSYVRSL,

Sequence shown in SEQ ID NO:10 is the polypeptide of F52:CCCKGRCRNKEIPASKINPG, and

Sequence shown in SEQ ID NO:11 is the polypeptide of F45:EIDLGPAISLEKLDVGTNLG.

2. purposes according to claim 1, wherein the peste des petits ruminants diagnostic kit is used for raw by test object It whether there is the IgG antibody of anti-PPR virus in the biological sample in object source, whether diagnosis object organisms are ruminated by small Epizootic disease virus infection or peste des petits ruminants vaccine immunity.

3. purposes according to claim 2, wherein the object organisms small ruminant.

4. purposes according to claim 2, wherein the object organisms are goat or sheep.

5. purposes according to claim 2, wherein the biological sample is whole blood, blood plasma or serum.

6. a kind of peste des petits ruminants diagnostic kit comprising substrate, and be individually secured to following more in the substrate Peptide combination 1；

The polypeptide shown in NO:1~11 SEQ ID of polypeptides in combination 1 forms,

Sequence shown in SEQ ID NO:1 is the polypeptide of N50:SAEALFRLQAMAKILEDQEE,

Sequence shown in SEQ ID NO:2 is the polypeptide of N46:TKRTRSGKPRGETPGPLLPE,

Sequence shown in SEQ ID NO:3 is the polypeptide of N47:GETPGPLLPEIMQEDELSRE,

Sequence shown in SEQ ID NO:4 is the polypeptide of N48:IMQEDELSRESSQNPREAQR,

Sequence shown in SEQ ID NO:5 is the polypeptide of H1:MSAQRERINAFYKDNLHNKT,

Sequence shown in SEQ ID NO:6 is the polypeptide of N40:AKLVSEIASQTGDERTVRGT,

Sequence shown in SEQ ID NO:7 is the polypeptide of H9:TESIDHQTKDVLTPLFKIIG,

Sequence shown in SEQ ID NO:8 is the polypeptide of H36:LSSHRGIIKDNEANWVVPST,

Sequence shown in SEQ ID NO:9 is the polypeptide of F54:LKPDLTGTSKSYVRSL,

Sequence shown in SEQ ID NO:10 is the polypeptide of F52:CCCKGRCRNKEIPASKINPG, and

Sequence shown in SEQ ID NO:11 is the polypeptide of F45:EIDLGPAISLEKLDVGTNLG.

7. peste des petits ruminants diagnostic kit according to claim 6, is used for the life by test object biological source Whether the IgG antibody that whether there is anti-PPR virus in object sample diagnoses object organisms by PPR virus sense Dye or peste des petits ruminants vaccine immunity.

8. peste des petits ruminants diagnostic kit according to claim 7, wherein the object organisms small ruminant.

9. peste des petits ruminants diagnostic kit according to claim 7, wherein the object organisms are goat or sheep.

10. peste des petits ruminants diagnostic kit according to claim 7, wherein the biological sample be whole blood, blood plasma or Serum.

Technical field

The invention mainly relates to diagnostic kit for animals and diagnostic methods.Specifically, the present invention relates to detections pair As biological source biological sample in the presence or absence of anti-PPR virus antibody (IgG) kit.

Background technique

Peste des petits ruminants (Peste des petits ruminants, PPR) is by PPR virus (Pestedes petits ruminants virus, PPRV) Yin Qi ー kind is acute, strong, contagious disease, because of it Higher morbidity and mortality have the animals such as goat, sheep, white-tailed deer, wild gazelle sheep and Tibetan antelope great It threatens, is the deadly infectious disease generally acknowledged in world wide, China is also by disease Lie Wei ー class zoonosis.Therefore, sensitivity The foundation of high, high specificity peste des petits ruminants diagnostic method just seems especially urgent.PPR virus (PPRV) belongs to Paramyxoviridae (Paramyxoviriade) fiber crops epidemic disease Tobamovirus (Morbollivirus), other members belonged to have rinderseuche Malicious (RPV), canine distemper virus (CDV), sea dog pestivirus (PDV), dolphin pestivirus (DDV), ox measles virus (MVKI) and People measles virus (MV) etc..The genome structure of PPRV is sub-thread minus strand, without segment RNA.Currently, the diagnosis of peste des petits ruminants Method mainly has Virus Isolation, antibody serum detection, antigen detection etc..Antigen detection method has agar gel immune Spread the methods of (AGID), counter immunoelectrophoresis (CIEP), indirect fluorescent antibody test (IFAT).Antibody serum diagnosis is normal Using the OIE virus neutralization tests (VNT) recommended and competitive ELISA (c-ELISA).It is swift and violent with Protocols in Molecular Biology Development, round pcr are developed rapidly.

World Organization for Animal Health recommends the PPR virus antibody detection method used mainly to have virus to neutralize examination Test (VNT) and enzyme-linked immunosorbent assay (ELISA).Wherein, the testing result of virus neutralization tests method is accurate, is that detection is small anti- The goldstandard of hay epizootic disease virus, but this method detection time is long and is unsuitable for detecting great amount of samples.In contrast, enzyme linked immunological The specificity and sensibility of measurement are higher, detection time is shorter than virus neutralization tests and the suitable a large amount of samples of detection, therefore, quilt It is widely used in the detection of PPR virus antibody.

Enzyme-linked immunosorbent assay method for PPR virus antibody test mainly includes competitive enzyme-linked immune measurement (c-ELISA), enzyme-linked immunosorbent assay (b-ELISA) and indirect enzyme-linked immunosorbent measurement (indirect ELISA) are blocked.C-ELISA and b- The specificity and sensibility of ELISA is relatively high, be by clinical generally accepted PPR virus antibody detection method, Such as the peste des petits ruminants diagnostic kit in the general French laboratory BIRAD just uses c-ELISA method in the world.But It is that c-ELISA and b-ELISA are both needed to using monoclonal antibody, this causes testing cost to greatly improve；Moreover, c-ELISA and The cumbersome of b-ELISA, detection time long (although having been shortened relative to VNT), criterion are complicated.On the other hand, it passes The indirect ELISA of system uses intact proteins (such as H protein, N protein, F protein etc.) or recombination from PPR virus Albumen detects the antibody in serum as envelope antigen, and cost is lower than c-ELISA and b-ELISA；But due to the party Method uses intact proteins as antigen, is easy to happen the wrong identification and non-specific identification of antibody, therefore, specificity and Sensibility is all not as good as c-ELISA and b-ELISA.

Therefore, it is necessary to develop, testing cost is low, detection time is short, easy to operate, specific and high sensibility small anti- Hay epizootic disease diagnostic kit.

Summary of the invention

In view of above-mentioned problems of the prior art, low, detection that the purpose of the present invention is to provide a kind of testing costs Time short, easy to operate, specific and high sensibility peste des petits ruminants diagnostic kit, and can be used in preparing the examination The polypeptide or polypeptides in combination of agent box.

Indirect ELISA method based on antigen epitope polypeptide uses the antigen epitope polypeptide (single of 20 amino acid or so Polypeptide usually only includes an epitope) it is used as envelope antigen.Inventor's discovery: the sensibility of this method is often relatively low, single The sensibility of polypeptide does not exceed 50% generally；And the polypeptide that sensitivity is high, false positive rate are also high, i.e., it is specific low.If The specificity and sensibility of detection can be improved simultaneously by certain mode, so that it may overcome lacking for traditional indirect ELISA Point, developing specificity and sensibility can match in excellence or beauty c-ELISA and b-ELISA, even higher peste des petits ruminants diagnostic reagent Box.

Inventor has made intensive studies to solve above-mentioned technical problem, as a result, it has been found that following polypeptides in combination 1.When with It at least any two in following polypeptides in combination 1 when having response as index to the biological sample in object organisms source, can be with It, completely can be with c-ELISA method and b- with 94.2% sensibility and 92.1% specific diagnosis peste des petits ruminants ELISA method matches in excellence or beauty.

Therefore, the present invention includes:

1. a kind of peste des petits ruminants diagnostic kit comprising substrate, and under being individually secured in the substrate State polypeptides in combination 1；

The polypeptide shown in NO:1~11 SEQ ID of polypeptides in combination 1 forms,

Sequence shown in SEQ ID NO:1 is the polypeptide of N50:SAEALFRLQAMAKILEDQEE,

Sequence shown in SEQ ID NO:2 is the polypeptide of N46:TKRTRSGKPRGETPGPLLPE,

Sequence shown in SEQ ID NO:3 is the polypeptide of N47:GETPGPLLPEIMQEDELSRE,

Sequence shown in SEQ ID NO:4 is the polypeptide of N48:IMQEDELSRESSQNPREAQR,

Sequence shown in SEQ ID NO:5 is the polypeptide of H1:MSAQRERINAFYKDNLHNKT,

Sequence shown in SEQ ID NO:6 is the polypeptide of N40:AKLVSEIASQTGDERTVRGT,

Sequence shown in SEQ ID NO:7 is the polypeptide of H9:TESIDHQTKDVLTPLFKIIG,

Sequence shown in SEQ ID NO:8 is the polypeptide of H36:LSSHRGIIKDNEANWVVPST,

Sequence shown in SEQ ID NO:9 is the polypeptide of F54:LKPDLTGTSKSYVRSL,

Sequence shown in SEQ ID NO:10 is the polypeptide of F52:CCCKGRCRNKEIPASKINPG, and

Sequence shown in SEQ ID NO:11 is the polypeptide of F45:EIDLGPAISLEKLDVGTNLG.

2. being used for the biology by test object biological source according to peste des petits ruminants diagnostic kit described in item 1 Whether the antibody (IgG) that whether there is anti-PPR virus in sample diagnoses object organisms by PPR virus sense Dye or peste des petits ruminants vaccine immunity.

3. according to peste des petits ruminants diagnostic kit described in item 2, wherein the object organisms small ruminant.

4. the peste des petits ruminants diagnostic kit according to item 2 or 3, wherein the object organisms are goat or silk floss Sheep.

5. the peste des petits ruminants diagnostic kit according to any one of item 2~4, wherein the biological sample is complete Blood, blood plasma or serum.

6. purposes of following polypeptides in combination 1 in preparation peste des petits ruminants diagnostic kit；

The polypeptide shown in NO:1~11 SEQ ID of polypeptides in combination 1 forms,

Sequence shown in SEQ ID NO:1 is the polypeptide of N50:SAEALFRLQAMAKILEDQEE,

Sequence shown in SEQ ID NO:2 is the polypeptide of N46:TKRTRSGKPRGETPGPLLPE,

Sequence shown in SEQ ID NO:3 is the polypeptide of N47:GETPGPLLPEIMQEDELSRE,

Sequence shown in SEQ ID NO:4 is the polypeptide of N48:IMQEDELSRESSQNPREAQR,

Sequence shown in SEQ ID NO:5 is the polypeptide of H1:MSAQRERINAFYKDNLHNKT,

Sequence shown in SEQ ID NO:6 is the polypeptide of N40:AKLVSEIASQTGDERTVRGT,

Sequence shown in SEQ ID NO:7 is the polypeptide of H9:TESIDHQTKDVLTPLFKIIG,

Sequence shown in SEQ ID NO:8 is the polypeptide of H36:LSSHRGIIKDNEANWVVPST,

Sequence shown in SEQ ID NO:9 is the polypeptide of F54:LKPDLTGTSKSYVRSL,

Sequence shown in SEQ ID NO:10 is the polypeptide of F52:CCCKGRCRNKEIPASKINPG, and

Sequence shown in SEQ ID NO:11 is the polypeptide of F45:EIDLGPAISLEKLDVGTNLG.

7. according to purposes described in item 6, wherein the peste des petits ruminants diagnostic kit is used for raw by test object It whether there is the antibody (IgG) of anti-PPR virus in the biological sample in object source, whether diagnosis object organisms are by small anti- Hay epizootic disease virus infection or peste des petits ruminants vaccine immunity.

8. according to purposes described in item 7, wherein the object organisms small ruminant.

9. the purposes according to item 7 or 8, wherein the object organisms are goat or sheep.

10. the purposes according to any one of item 7~9, wherein the biological sample is whole blood, blood plasma or serum.

Aforementioned polypeptides and kit, which can be used for whether there is in the biological sample of test object biological source, resists small ruminate The antibody (IgG) of epizootic disease virus.In general, the PPR virus antibody (IgG) in biological sample is due to object organisms quilt PPR virus is infected or is generated by peste des petits ruminants vaccine immunity, and therefore, aforementioned polypeptides and kit can be used In diagnosis object organisms whether by PPR virus infection or peste des petits ruminants vaccine immunity.

The specific embodiment of invention

Firstly, the present invention provides following polypeptides in combination 1:

The polypeptide shown in NO:1~11 SEQ ID of polypeptides in combination 1 forms,

Sequence shown in SEQ ID NO:1 is the polypeptide of N50:SAEALFRLQAMAKILEDQEE,

Sequence shown in SEQ ID NO:2 is the polypeptide of N46:TKRTRSGKPRGETPGPLLPE,

Sequence shown in SEQ ID NO:3 is the polypeptide of N47:GETPGPLLPEIMQEDELSRE,

Sequence shown in SEQ ID NO:4 is the polypeptide of N48:IMQEDELSRESSQNPREAQR,

Sequence shown in SEQ ID NO:5 is the polypeptide of H1:MSAQRERINAFYKDNLHNKT,

Sequence shown in SEQ ID NO:6 is the polypeptide of N40:AKLVSEIASQTGDERTVRGT,

Sequence shown in SEQ ID NO:7 is the polypeptide of H9:TESIDHQTKDVLTPLFKIIG,

Sequence shown in SEQ ID NO:8 is the polypeptide of H36:LSSHRGIIKDNEANWVVPST,

Sequence shown in SEQ ID NO:9 is the polypeptide of F54:LKPDLTGTSKSYVRSL,

Sequence shown in SEQ ID NO:10 is the polypeptide of F52:CCCKGRCRNKEIPASKINPG, and

Sequence shown in SEQ ID NO:11 is the polypeptide of F45:EIDLGPAISLEKLDVGTNLG.

In the indirect ELISA method based on antigen epitope polypeptide, the sensibility for being usually no more than 50% of single polypeptide It compares, is having response as index the biological sample in object organisms source using at least any two in the polypeptides in combination 1 In the case of, it can be with 94.2% sensibility and 92.1% specific diagnosis peste des petits ruminants.

In this specification, sensibility refers to: in the positive sample with the confirmation of " goldstandard " method, being measured by other methods For the ratio of positive sample.Specificity refers to: in the negative sample with the confirmation of " goldstandard " method, being measured as by other methods The ratio of negative sample.For the detection of PPR virus antibody, " goldstandard " of the art is in virus With test (VNT).

Aforementioned polypeptides combination can be used as detection probe be used to prepare in the biological sample of test object biological source whether There are the kits of the antibody of anti-PPR virus (IgG).In general, the PPR virus antibody in biological sample It (IgG) is to be infected due to object organisms by PPR virus or generated by peste des petits ruminants vaccine immunity, Whether aforementioned polypeptides combination and kit can be used for diagnosing object organisms by PPR virus infection or peste des petits ruminants Vaccine immunity.

Therefore, the present invention also provides a kind of peste des petits ruminants diagnostic kits comprising substrate, and be independently connected In the aforementioned polypeptides combination 1 in the substrate.

In the present specification, substrate is normally solid, can be one, be also possible to it is multiple, but preferably one, i.e., Whole polypeptides are independently connected in same substrate.In the present invention, substrate is not particularly limited, as long as solid or not Soluble materials carrier.The connection of polypeptide and substrate can be using well known to a person skilled in the art polypeptides and solid material Connection method carry out.

In the present specification, the object organisms are preferably small ruminant, more preferably goat or sheep.

In the present specification, the biological sample can be whole blood, blood plasma or serum.

In the case where stating kit diagnosis peste des petits ruminants in use, when any two in the polypeptides in combination 1 or When two or more polypeptides have response to the biological sample in object organisms source, determine the object organisms by peste des petits ruminants disease Poison infection or peste des petits ruminants vaccine immunity (i.e. positive)；Conversely, determining that the object organisms are not infected by PPR virus Or peste des petits ruminants vaccine immunity (i.e. negative).

In the present specification, " response " refers to: signal-to-noise ratio (SNR) is greater than or equal to 2, wherein signal-to-noise ratio=(polypeptide point Signal value-negative control point signal value)/negative control point signal value.

Embodiment

1. the preparation and confirmation of polypeptide

The polypeptide of NO:1~11 SEQ ID is synthesized by gill biochemistry (Shanghai) Co., Ltd., and is carried out by mass spectrum to it Confirmation.Wherein,

The sequence of polypeptide shown in SEQ ID NO:1 is N50:SAEALFRLQAMAKILEDQEE,

The sequence of polypeptide shown in SEQ ID NO:2 is N46:TKRTRSGKPRGETPGPLLPE,

The sequence of polypeptide shown in SEQ ID NO:3 is N47:GETPGPLLPEIMQEDELSRE,

The sequence of polypeptide shown in SEQ ID NO:4 is N48:IMQEDELSRESSQNPREAQR,

The sequence of polypeptide shown in SEQ ID NO:5 is H1:MSAQRERINAFYKDNLHNKT,

The sequence of polypeptide shown in SEQ ID NO:6 is N40:AKLVSEIASQTGDERTVRGT,

The sequence of polypeptide shown in SEQ ID NO:7 is H9:TESIDHQTKDVLTPLFKIIG,

The sequence of polypeptide shown in SEQ ID NO:8 is H36:LSSHRGIIKDNEANWVVPST,

The sequence of polypeptide shown in SEQ ID NO:9 is F54:LKPDLTGTSKSYVRSL,

The sequence of polypeptide shown in SEQ ID NO:10 is F52:CCCKGRCRNKEIPASKINPG,

The sequence of polypeptide shown in SEQ ID NO:11 is F45:EIDLGPAISLEKLDVGTNLG.

2. the preparation of kit (polypeptide chip) 1

Kit 1

Polypeptide chip is the solution difference point sample by the polypeptide of NO:1~11 SEQ ID in solid support material --- " 0+ Be prepared on X " film (iPDMS film) (while one goat IgG of point sample is as positive quality control point and a PB point conduct Negative Quality Control point)." 0+X " film is that the initiator with olefin-terminal, surface initiated polymerization is added to poly dimethyl silicon In oxygen alkane material, then in the three-dimensional structure in a manner of heat cross-linking (si-h bond bonding) fixed to dimethyl silicone polymer, obtain A kind of material arrived.Its manufacturing process can be found in published International patent WO2014/044184.

3. being detected with kit

Polypeptide chip is placed in room temperature 20min, visually observes chip surface, there will be the polypeptide chip of defect to eliminate, simultaneously The correct direction for determining polypeptide chip, is sandwiched in tongue, and the number of chip is carried out according to the quantity of serum, then in the more of qualification TBST (0.4M Tris-HCl, 2.74M NaCl, 2%Tween20, pH7.2 ± 0.2) is filled in peptide chips, is placed at room temperature for 2- 4min checks for that in the dead of night, the polypeptide chip of leakage being discarded.The polypeptide chip of vacancy is filled up, operation is same as above.It is closed examining The polypeptide chip of lattice is sandwiched on tongue, and every 8 chips are one group, and with TBST rinse 2 times, watering can elution a, hole Kong Jinyi goes out, Flowing is formed, is finally gently patted on gauze, another one takes lid to dry, but it is noted that keeps polypeptide chip surface wet Profit, it is spare.

Serum is diluted 50 times (+245 μ l serum dilutions of 5 μ l serum sample) in super-clean bench to number respectively, is shaken It mixes, chip number is made to number consistent with each other, the loading on the chip of rinse with serum, 200 holes μ l/ avoid generating gas Bubble, is added the 200 μ l of serum after diluting, room temperature, and shaking table 150rpm is incubated for 30min.

Liquid is abandoned, 4 times (method is same as above) is eluted, drying is added secondary antibody (rabbit-anti goat IgG), room temperature, and shaking table 150rpm is incubated Educate 30min.

Abandon liquid, abandon lid, elute 4 times, drying, every 8 chips be one group, add 20 holes μ l/ luminescent solution (Thermo, Prod#37074), 2min is exposed.Data are acquired using GenePix Pro6.0.

For every a serum, whether each polypeptide counted in the kit respectively has response (that is, signal-to-noise ratio (SNR) it is more than or equal to 2), and determined.

For mentioned reagent box 1, when detecting any two or two in polypeptide shown in NO:1~11 SEQ ID When having response above, it is determined as the peste des petits ruminants positive；Conversely, being determined as feminine gender.

Wherein, signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point Signal value refers to the chemiluminescence intensity value for the polypeptide point that software is read, and negative control point signal value refers to the yin that software is read The chemiluminescence intensity value of property control point.

For 755 parts of goats and sheep serum sample from China regions, it is respectively adopted in above-mentioned steps 2 and prepares Kit (press above-mentioned 3 the step of) and virus neutralization tests (VNT " in PPR virus and is tried by OIE (2010) Test " the method) it is detected, testing result is as follows.

Table 1

Sensibility=376/399*100%=94.2%

Specificity=328/356=92.1%

As known from the above, the sensibility and specificity of kit 1 90% or more (and be using VNT method validation, It is not to be verified using contrast agents cassette method), it is sufficient to the kit to match in excellence or beauty using c-ELISA method or b-ELISA method.

Sequence table

<110>China Veterinery Drug Inspection Office

<120>peste des petits ruminants diagnostic kit

<130> PB00109

<141> 2018-04-24

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

Ser Ala Glu Ala Leu Phe Arg Leu Gln Ala Met Ala Lys Ile Leu Glu

1 5 10 15

Asp Gln Glu Glu

20

<210> 2

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Thr Lys Arg Thr Arg Ser Gly Lys Pro Arg Gly Glu Thr Pro Gly Pro

1 5 10 15

Leu Leu Pro Glu

20

<210> 3

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

Gly Glu Thr Pro Gly Pro Leu Leu Pro Glu Ile Met Gln Glu Asp Glu

1 5 10 15

Leu Ser Arg Glu

20

<210> 4

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Ile Met Gln Glu Asp Glu Leu Ser Arg Glu Ser Ser Gln Asn Pro Arg

1 5 10 15

Glu Ala Gln Arg

20

<210> 5

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Met Ser Ala Gln Arg Glu Arg Ile Asn Ala Phe Tyr Lys Asp Asn Leu

1 5 10 15

His Asn Lys Thr

20

<210> 6

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Ala Lys Leu Val Ser Glu Ile Ala Ser Gln Thr Gly Asp Glu Arg Thr

1 5 10 15

Val Arg Gly Thr

20

<210> 7

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Lys Ile Ile Gly

20

<210> 8

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Leu Ser Ser His Arg Gly Ile Ile Lys Asp Asn Glu Ala Asn Trp Val

1 5 10 15

Val Pro Ser Thr

20

<210> 9

<211> 16

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 9

Leu Lys Pro Asp Leu Thr Gly Thr Ser Lys Ser Tyr Val Arg Ser Leu

1 5 10 15

<210> 10

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Cys Cys Cys Lys Gly Arg Cys Arg Asn Lys Glu Ile Pro Ala Ser Lys

1 5 10 15

Ile Asn Pro Gly

20

<210> 11

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

Glu Ile Asp Leu Gly Pro Ala Ile Ser Leu Glu Lys Leu Asp Val Gly

1 5 10 15

Thr Asn Leu Gly

20