CN109887553A - For the polypeptide vaccine and its design method in tumor-targeting drug drug resistance site - Google Patents

Abstract

The invention discloses a kind of polypeptide vaccine and its design method for tumor-targeting drug drug resistance site, design method includes the following steps: to collect targeted drug medicament-resistant mutation data；Intercept the interception of medicament-resistant mutation polypeptide sequence and prediction MHC molecule affinity and immunogenicity；Targeted drug and drug resistance site correlation and drug cluster；The corresponding vaccine polypeptide sequence of engineer；The targeted drug joint polypeptide vaccine combination that design method in this way obtains can cover common target therapeutic agent, tumor drug resistance probability of happening can be effectively reduced, extend targeting medicine effective acting time, increase the disease response rate of patient, with broad spectrum activity, shorten individuation polypeptide vaccine from the time for analyzing treatment, reduces medical treatment cost.

Description

For the polypeptide vaccine and its design method in tumor-targeting drug drug resistance site

Technical field

The present invention relates to tumor vaccine field, especially a kind of polypeptide vaccine for tumor-targeting drug drug resistance site and Its design method.

Background technique

In recent years, the progress of neoplasm targeted therapy with Protocols in Molecular Biology development and to pathogenesis from cell, The further understanding of molecular level has come into a completely new epoch.These fields make much progress, so far, very much Targeted drug clinic play a part of it is of crucial importance even miraculously.Molecular targeted therapy has become surgery, putting The another important means of the oncotherapies such as treatment, plays increasingly important role in oncotherapy.Due to targeted therapy High efficiency and hypotoxicity, compared with traditional treatment and chemotherapy, its advantage is that apparent.Targeted therapy is quickly grown, some International tumour educational circles the recognized standard therapeutic scheme and specification are entered according to the principle of evidence-based medicine EBM.More, more have uncommon The drug of prestige is also speeding up in development and early studies in man.

Targeting medicine has become a big sharp weapon of oncotherapy.But as treatment time is increasingly longer, most of patients occurs resistance to Medicine.As for drug resistant specific mechanism, mainly there are 3 kinds at present.Firstly, generating drug resistance by gene mutation.The genetic test positive The gene of patient about 40% can be generated new gene by original gene, this will lead to it is insensitive to original drug, to generate Drug resistance reaction.Secondly, cunning cancer cell usually " can pretend to prepare to advance along one path and advance secretly by an unknown path ", an other road of going for a stroll away.Such case Account for about 20% or so in drug resistance patient.In addition to both the above drug resistance approach, it is left the resistance mechanism of 30% or so patient, still It is indefinite.

" variable body " (drug resistance) after targeting is inevitable, and it is existing that drug resistance can all occur in almost all of targeted drug treatment As basic reason is the heterogeneity and dynamic change due to cancer cell.A kind of targeted drug is for cancer cell at present Some albumen, some molecule work, so can only inhibit an access of tumour growth.When an access by When inhibition, tumour cell can seek new " means of livelihood " certainly, other accesses is selected to synthesize substance required for own growth, long and long It, molecular targeted agents just lose effect, to generate drug resistance, for example, EGFR mutation patient take after targeting medicine occur it is resistance to Medicine, 50%~60% is because gene mutation occurs again, and most of to sport T790M mutation, patient can be used the at this time Three generations's targeted drug such as difficult to understand this replaces Buddhist nun.8-14 of the Most patients after receiving EGFR-TKI treatment will appear secondary resistance to for a month Medicine, the resistance problems for how solving targeted drug treatment have become a hot topic of research.

In recent years, the discovery due to tumor associated antigen and tumour specific antigen and people are immune to tumor inducing Response and tumour escape the further investigation and understanding of immune surveillance mechanism, and the research of tumour polypeptide vaccine has been achieved for gratifying Achievement.Neoantigen is the paraprotein of tumor cell surface expression caused by gene mutation, different from conventional Antigenic Target It is that neoantigen only not in normal cell surface expression and can be identified and activated by immune system and exempt from tumor cell surface expression Epidemic disease system.On July 13rd, 2017, Nature magazine have issued two personalized tumor vaccines based on neoantigen on the same day The successful case for treating malignant mela noma.Germany Carmen Loquai professor andT ü reci professor, utilizes RNA corresponding to neoantigen makees vaccine, has treated 13 patients altogether.On the same day, the Catherine of Harvard University The team that professor J.Wu leads makees vaccine using Antigenic Peptide corresponding to neoantigen, and also reported success treatment is pernicious black The case of melanoma.

Drug resistance site polypeptide vaccine is typically designed process, is to find out to lead to the drug resistant mutational site of targeted drug, analysis The neoantigen that these mutation generate out, preparation can be by the polypeptide epidemic disease of patient tissue histocompatibility complex (MHC) specific recognition Seedling, polypeptide preparation can both be obtained by chemically synthesized mode, nucleic acid molecules (such as DNA and RNA) also can be used and pass through The mode of transcription and translation obtains, or can also be that carrier expresses to obtain by bacterium or virus.Polypeptide vaccine is injected and is suffered from In person's body, specific T-cells response and immune storm are activated, patient's self immune system is enable effectively to identify, kill this Class has the tumour cell of medicament-resistant mutation.

Polypeptide vaccine and targeting medicine combination one side targeted drug can play the tumour cell for carrying medication target spot Lethal effect another aspect polypeptide vaccine activation body immune system can cells of resistant tumors just on the first appearance just into Row is effectively identified and is removed, to extend the action time of targeted drug, and the immune system activated can be grown in vivo Phase keeps to the lethal effect of tumour cell, it is ensured that have lasting inhibition to drug resistance phenomenon, overcome to a certain extent due to The targeted drug drug resistance that medicament-resistant mutation generates.

Market needs a set of polypeptide vaccine screening and design method for tumor-targeting drug drug resistance site, can be effective Tumor drug resistance probability of happening is reduced, there is broad spectrum activity, shortens individuation polypeptide vaccine from the time for analyzing treatment, reduces medical treatment Cost；The present invention solves such problems.

Summary of the invention

To solve the deficiencies in the prior art, the purpose of the present invention is to provide one kind to be directed to tumor-targeting drug drug resistance site Polypeptide vaccine and its design method, provide targeted drug joint polypeptide vaccine assembled scheme cover common targeted therapy medicine Tumor drug resistance probability of happening can be effectively reduced in object, extends targeting medicine effective acting time, increases the disease response rate of patient, With broad spectrum activity, shortens individuation polypeptide vaccine from the time for analyzing treatment, reduce medical treatment cost.

In order to achieve the above objectives, the present invention adopts the following technical scheme that:

For the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that it is directed to targeted drug vismodegib, it is more Peptide vaccine combination is for following mutation: SMO-A459V, SMO-C469Y, SMO-D473G, SMO-D473H, SMO-D473Y, SMO- F460L, SMO-G497W, SMO-H231R, SMO-Q477E, SMO-T241M, SMO-V321A, SMO-V321M, SMO-W281L, SMO-W535L, SMO-W535R.

For the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that Buddhist nun is replaced according to Shandong for targeted drug, it is more Peptide vaccine combination is for following mutation: BTK-C481F, BTK-C481R, BTK-C481S, BTK-C481Y, BTK-T316A.

For the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that by targeted drug abiraterone, En Zha Shandong amine, Flutamide, Ketoconazole gather for one kind,

The polypeptide vaccine combination of abiraterone is for following mutation: AR-T878A, AR-T878S；

The polypeptide vaccine combination of En Zhalu amine is for following mutation: AR-F877L, AR-T878A；

The polypeptide vaccine combination of Flutamide is for following mutation: AR-T878A, AR-T878S, AR-V716M；

The polypeptide vaccine of ketoconazole is for following mutation: AR-T878A；

For the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that by targeted drug dabrafenib, pyridine aldoxime methyliodide (PAM) Monoclonal antibody, Wei Luofeini, department's beauty are gathered for Buddhist nun for one kind,

The polypeptide vaccine combination of dabrafenib is for following mutation: MAP2K1-P124S, MAP2K2-Q60P, NRAS- G12D, NRAS-Q61R；

The polypeptide vaccine of pyridine aldoxime methyliodide (PAM) monoclonal antibody is for following mutation: NRAS-Q61R；

The polypeptide vaccine combination of Wei Luofeini is for following mutation: MAP2K1-G128V, MAP2K1-P124L, MAP2K1- Q56P, NRAS-Q61R；

Department's beauty is directed to following mutation: MAP2K1-P124L for the polypeptide vaccine of Buddhist nun.

For the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that targeted drug A Lei is auspicious for Buddhist nun, color Gather for Buddhist nun, gram azoles for Buddhist nun for one kind,

A Lei is mutated for the polypeptide vaccine of Buddhist nun for following: ALK-G1202R；

The polypeptide vaccine combination of Ceritinib is for following mutation: ALK-D1203N, ALK-F1174V, ALK-G1123S, ALK-G1202R, ALK-G1269A, ALK-L1196M；

Gram azoles for Buddhist nun polypeptide vaccine combination for following mutation: ALK-F1174L, ALK-F1174V, ALK-G1202R, ALK-G1269A, ALK-I1171T, ALK-L1196M, MET-D1246H, MET-D1246N.

For the polypeptide vaccine in tumor-targeting drug drug resistance site, replaced by targeted drug Afatinib, bosutinib, up to sand Buddhist nun, Sorafenib, Sutent, network are replaced for Buddhist nun, Imatinib, nilotinib, difficult to understand this in Buddhist nun, Tarceva, Gefitinib, Lip river difficult to understand Histidine kinase inhibitor class drug, special watt prick for Buddhist nun, Kui and gather for Buddhist nun for one kind,

The polypeptide vaccine of Afatinib is for following mutation: EGFR-T790M；

The polypeptide vaccine combination of bosutinib is for following mutation: ABL1-F359V, ABL1-V299L；

The polypeptide vaccine combination of Dasatinib is for following mutation: ABL1-E255K, ABL1-E255K, ABL1-F317L, ABL1-F359V, ABL1-T315A, ABL1-T315I, ABL1-V299L；

The polypeptide vaccine of Tarceva is for following mutation: EGFR-T790M；

The polypeptide vaccine combination of Gefitinib is for following mutation: EGFR-D761Y, EGFR-T790M；

Ao Luo is mutated for the polypeptide vaccine of Buddhist nun for following: EGFR-C797S；

The polypeptide vaccine combination of Imatinib is for following mutation: ABL1-A397P, ABL1-A399T, ABL1-A433T, ABL1-E255K, ABL1-E255V, ABL1-E275K, ABL1-E279A, ABL1-E279K, ABL1-E279Y, ABL1-E279Z, ABL1-E282G, ABL1-E292V, ABL1-E352D, ABL1-E352G, ABL1-E355A, ABL1-E355G, ABL1-E450A, ABL1-E450G, ABL1-E450K, ABL1-E453A, ABL1-E453G, ABL1-E453K, ABL1-E453L, ABL1-F311L, ABL1-F317L, ABL1-F359A, ABL1-F359V, ABL1-F382L, ABL1-F486S, ABL1-G398R, ABL1-H396P, ABL1-H396R, ABL1-I418V, ABL1-K294 > RGG, ABL1-K378R, ABL1-K419E, ABL1-L298V, ABL1- L384M, ABL1-L387F, ABL1-L387M, ABL1-M244V, ABL1-M388L, ABL1-N374Y, ABL1-P480L, ABL1- Q252E, ABL1-Q252H, ABL1-Q252K, ABL1-Q252R, ABL1-R328M, ABL1-S417Y, ABL1-T277A, ABL1- T315I, ABL1-T315N, ABL1-V289F, ABL1-V299L, ABL1-V379I, ABL1-Y253F, ABL1-Y320C, KIT- A829P, KIT-D816A, KIT-D816E, KIT-D816H, KIT-D820G, KIT-D820V, KIT-D820Y, KIT-K642E, KIT-S709F, KIT-S821F, KIT-T670E, KIT-T670I, KIT-V654A, PDGFRA-D842V；

The polypeptide vaccine combination of nilotinib is for following mutation: ABL1-E255K, ABL1-E255V, ABL1-H396R, ABL1-T315I, ABL1-T315V, KIT-N655T；

Ao Si is mutated for the polypeptide vaccine combination of Buddhist nun for following: EGFR-C797S, EGFR-G796D, EGFR-G796R, EGFR-G796S, EGFR-L792H, KIT-T790M；

The polypeptide vaccine combination of Sorafenib is for following mutation: FLT3-D835H, FLT3-D835Y；

The polypeptide vaccine combination of Sutent is for following mutation: FLT3-D835Y, PDGFRA-D842V；

The polypeptide vaccine combination of tyrosine kinase inhibitors drug is for following mutation: ABL1-A399T, ABL1- D325G, ABL1-E255K, ABL1-E255V, ABL1-E279K, ABL1-E282K, ABL1-E355G, ABL1-E453K, ABL1- F311L, ABL1-F317L, ABL1-F359V, ABL1-H396P, ABL1-H396R, ABL1-L298V, ABL1-L384M, ABL1- L387F, ABL1-L387M, ABL1-M244V, ABL1-M388L, ABL1-P310S, ABL1-Q252H, ABL1-Q252M, ABL1- T315A, ABL1-T315I, ABL1-T495R, ABL1-V289A, ABL1-V299L, ABL1-V338F, ABL1-V379I, ABL1- Y253F, EGFR-T790M；

Te Wa is mutated for the polypeptide vaccine of Buddhist nun for following: EGFR-T790M；

Kui Zha is mutated for the polypeptide vaccine combination of Buddhist nun for following: FLT3-D835Y, FLT3-D842V, FLT3-D835Y.

For the polypeptide vaccine in tumor-targeting drug drug resistance site, targeted drug everolimus, rapamycin, MEK are pressed down Preparation PD0325901, C-MET inhibitor Savolitinib gathers for one kind,

The polypeptide vaccine of everolimus is for following mutation: MTOR-F2108L；

The polypeptide vaccine of rapamycin is for following mutation: MTOR-F2108L；

The polypeptide vaccine of mek inhibitor PD0325901 is for following mutation: MAP2K1-F129L；

The polypeptide vaccine of C-MET inhibitor Savolitinib is for following mutation: MET-D1246V；

For the design method of the polypeptide vaccine in tumor-targeting drug drug resistance site, include the following steps:

Find the data of targeted drug medicament-resistant mutation data；

Intercept medicament-resistant mutation polypeptide sequence and prediction MHC molecule affinity and immunogenicity:

The polypeptide sequence that covering 16 amino acid of mutational site upstream and downstream is intercepted for point mutation intercepts frameshift mutation Extend the amino acid of 16 length forward, extend back until the polypeptide sequence of terminator codon is as the prominent of resistant mutational site Modification polypeptide, while the corresponding wild polypeptide sequence in corresponding position is intercepted,

Use at least one database as source statistic high frequency HLA parting and frequency, by the HLA counted merge duplicate removal it Afterwards as prediction candidate HLA parting,

The corresponding polypeptide in these mutational sites and HLA molecule binding affinity are analyzed using a plurality of softwares, synthesis is a plurality of soft Affinity is divided into three classes by part: strong affinity-SB, weak affinity-WB and without affinity, and true compared with versus wild type polypeptide Fixed its affinity variation,

The variation of A class is to become having strong affinity from no affinity,

The variation of B class is to become weak affinity from no affinity,

The variation of C class is to become strong affinity from weak affinity,

D class variation be it is unchanged,

Internal sort thinks that A class is better than D class better than C class better than B class,

Its immunogenicity is predicted using immunogenicity forecasting tool, and reservation mutant polypeptides affinity is strong, affinity changes Big and strong immunogenicity epitope；

Targeted drug and drug resistance site correlation and drug cluster:

It gives a mark to drug resistance site affinity:

Comprehensively consider the epitope number and each epitope affinity variation size that there be affinity in site, not to tetra- class of A, B, C, D Corresponding weight is given with affinity variation, weight is given according to the corresponding HLA frequency size of each epitope, by each of site The cumulative summation of epitope；

AC: affinity changes size, and different weights is given in the variation of tetra- class difference affinity of A, B, C, D；

F_hla: corresponding HLA frequency；

N: each epitope number in site；

Correlation combination targeted drug mechanism of action is poly- to targeted drug between comprehensive analysis targeted drug and drug resistance site Class is to be classified as 7 classes:

The first kind generates drug resistant Hedgehog signal path antagonist vismodegib for SMO mutation,

Second class replaces Buddhist nun according to Shandong for BTK mutation generation is drug resistant,

Third class is directed to some antiandrogens such as abiraterone etc. of AR medicament-resistant mutation,

4th class is directed to some luxuriant and rich with fragrance Buddhist nun's class drugs of BRAF medicament-resistant mutation,

5th class is directed to some tyrosine kinase inhibitors of the fusions such as ALK, MET,

6th class is directed to the tyrosine kinase inhibitor of EGFR access,

7th class is for kinase inhibitors such as the everolimus of PI3K/AKT/mTOR access；

Design corresponding vaccine polypeptide sequence.

The design method of polypeptide vaccine above-mentioned for tumor-targeting drug drug resistance site,

Intercept medicament-resistant mutation polypeptide sequence and prediction MHC molecule affinity and immunogenicity:

The polypeptide sequence that covering 16 amino acid of mutational site upstream and downstream is intercepted for point mutation intercepts frameshift mutation Extend the amino acid of 16 length forward, extend back until the polypeptide sequence of terminator codon is as the prominent of resistant mutational site Modification polypeptide, while the corresponding wild polypeptide sequence in corresponding position is intercepted,

Use at least one database as source statistic high frequency HLA parting and frequency, by the HLA counted merge duplicate removal it Afterwards as the candidate HLA parting for prediction, the database includes: public database, clinical patients database,

The corresponding polypeptide in these mutational sites and HLA molecule binding affinity are analyzed using a plurality of softwares, synthesis is a plurality of soft Affinity is divided into three classes by part: strong affinity-SB, weak affinity-WB and without affinity, and true compared with versus wild type polypeptide Fixed its affinity variation, a plurality of softwares include: this three sections of softwares of netMHCpan, netMHC and Pickpocket,

The variation of A class is to become having strong affinity from no affinity,

The variation of B class is to become weak affinity from no affinity,

The variation of C class is to become strong affinity from weak affinity,

D class variation be it is unchanged,

Internal sort thinks that A class is better than D class better than C class better than B class,

Its immunogenicity is predicted using immunogenicity forecasting tool, and reservation mutant polypeptides affinity is strong, affinity changes Big and strong immunogenicity epitope.

The design method of polypeptide vaccine above-mentioned for tumor-targeting drug drug resistance site,

Network is made to all targeted drugs and drug resistance site using cytoscape, the size in drug resistance site represents it Affinity goals for, correlation combination targeted drug mechanism of action is to targeting medicine between comprehensive analysis targeted drug and drug resistance site Object cluster is to be classified as 7 classes:

The first kind generates drug resistant Hedgehog signal path antagonist vismodegib for SMO mutation,

Second class replaces Buddhist nun according to Shandong for BTK mutation generation is drug resistant,

Third class is directed to some antiandrogens such as abiraterone etc. of AR medicament-resistant mutation,

4th class is directed to some luxuriant and rich with fragrance Buddhist nun's class drugs of BRAF medicament-resistant mutation,

5th class is directed to some tyrosine kinase inhibitors of the fusions such as ALK, MET,

6th class is directed to the tyrosine kinase inhibitor of EGFR access,

7th class is for kinase inhibitors such as the everolimus of PI3K/AKT/mTOR access.

The invention has the beneficial effects that:

Targeted drug joint polypeptide vaccine assembled scheme provided by the invention covers common target therapeutic agent；Comparison is single Tumor drug resistance probability of happening can be effectively reduced in only targeted therapy, extends targeting medicine effective acting time；Comparison is individually more Peptide vaccine, the scheme that we provide increase the disease response rate of patient；

The sequencing of patient's hereditary information, specific determination patient's HLA parting are needed not move through, there is certain broad spectrum activity, contract significantly Short individuation polypeptide vaccine reduces medical treatment cost from the time for analyzing treatment；In addition polypeptide vaccine also generates toxic side effect；

Polypeptide vaccine itself can keep long-term tumor-killing effect, and vaccine peptide is digested into short polypeptide simultaneously in vivo MHC molecule submission goes out to be identified by TCR, body can be stimulated to generate specific killing T cell and memory T cell, identified simultaneously The cell for carrying mutation is removed, and this effect can be kept for a long time in vivo；

The polypeptide vaccine of 31 targeted drugs is provided, it is basic to cover kinds of tumor targeted drug；

Targeted drug is classified as 7 major class according to its mechanism and medicament-resistant mutation clustering, multiple target point joint can effectively increase Peptide vaccine is added to the fragmentation effect of tumour cell, significant extension targeted drug effective acting time.

Detailed description of the invention

Fig. 1 is targeted drug and drug resistance site interaction diagram of the invention；Origin represents targeted drug, and triangle represents Drug resistance site, triangle size indicate that the site corresponds to the affinity score of polypeptide, and network is divided into 7 parts by great circle, Represent 7 products；

Fig. 2 is the negative control of the A-D of present invention experiment 1 and the ELISPOT spot point-and-figure chart of 137 peptide fragments；

Fig. 3 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-1；

Fig. 4 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-2；

Fig. 5 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-3；

Fig. 6 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-4；

Fig. 7 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-5；

Fig. 8 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-6；

Fig. 9 is the effector T cell killing-efficiency schematic diagram of each group of present invention experiment 2-7.

Specific embodiment

Specific introduce is made to the present invention below in conjunction with the drawings and specific embodiments.

For the design method of the polypeptide vaccine in tumor-targeting drug drug resistance site, include the following steps:

1, targeted drug medicament-resistant mutation data collection: human cancer correlation somatic mutation catalogue Cosmic (database official Net https: //cancer.sanger.ac.uk/cosmic) to the report of acquired resistance and primary drug resistance in clinical research Road is arranged and has been annotated, including gene, mutation, drug, the information such as cancer kind and bibliography.We downloaded FDA or CFDA approved or in the clinical test III/IV phase all target therapeutic agent resistant mutational sites, to all candidate targets The less mutation of the sample size especially supported to drug and medicament-resistant mutation further searches for document and determines its reliability, determines Really reliable targeted drug drug resistance site is used for downstream analysis comprehensively, such as cosmic has included a determining EGFR-T790M Mutation is that difficult to understand this replaces the report in Buddhist nun's drug resistance site, but finds that T790M is not that difficult to understand this replaces the drug resistance site of Buddhist nun by searching document.

2, the interception of medicament-resistant mutation polypeptide sequence and MHC molecule affinity and immunogenicity prediction:

2.1, the polypeptide sequence that covering 16 amino acid of mutational site upstream and downstream is intercepted for point mutation, for frameshift mutation Interception extends forward the amino acid of 16 length, extends back until the polypeptide sequence of terminator codon is as resistant mutational site Mutant polypeptides, while intercepting the corresponding wild polypeptide sequence in corresponding position.

2.2, count high frequency HLA parting and frequency: mainly there are two parts in the source of high frequency HLA, first is that coming from common data It is relatively common to summarize Chinese population in conjunction with information reported in the literature in recent years for the statistics (IMGT/HLA and Chinese Marrow Donor Program data bank) in library HLA allelic gene typing；Second is that one group of relatively high HLA parting of the frequency of occurrences has been counted from the database of clinical patients, Specific database is as shown in table 2.Two groups of HLA are merged into duplicate removal later as the candidate HLA parting for being used to predict.It needs to illustrate , the selection of database is unrestricted, this is a kind of preferred embodiment, is also possible to the combination system of other several databases Meter.

2.3, we analyze these mutational sites pair using tri- sections of softwares of netMHCpan, netMHC and Pickpocket Affinity is divided into three classes (Qiang Qinhe by the comprehensive three sections of softwares of HLA molecule binding affinity obtained in the polypeptide answered and 2.2 steps Power-SB, weak affinity-WB and without affinity), and determine compared with versus wild type polypeptide the variation of its affinity (by without affine Power, which becomes having strong affinity to be defined as the variation of A class, becomes weak affinity from no affinity is defined as the variation of B class, by weak affinity Become strong affinity be defined as C class variation, it is other it is unconverted be defined as D class variation, internal sort think A class better than B class it is excellent It is better than D class in C class), its immunogenicity also in addition is predicted using the immunogenicity forecasting tool that IEDB is provided, and it is more to retain saltant type Peptide affinity is strong, affinity variation greatly and the strong epitope of immunogenicity, screen that obtain 31 kinds of targeted drugs 137 corresponding altogether Medicament-resistant mutation.

3, targeted drug and drug resistance site correlation and drug cluster:

3.1 pairs of drug resistance site affinity marking:

Comprehensively consider site have affinity epitope number and each epitope affinity variation size (a. is to tetra- class of A, B, C, D Different weights is given in the variation of different affinity, and b. gives weight according to the corresponding HLA frequency size of each epitope, and c. is by site The cumulative summation of each epitope).

AC: affinity changes size, and different weights is given in the variation of tetra- class difference affinity of A, B, C, D；

F_hla: corresponding HLA frequency；

N: each epitope number in site；

3.2 make network (such as Fig. 1) to all targeted drugs and drug resistance site using cytoscape, drug resistance site Size represents its affinity goals for, and correlation combination targeted drug acts on machine between comprehensive analysis targeted drug and drug resistance site System is as shown in Figure 1 to be classified as 7 classes to targeted drug cluster.

The first kind generates drug resistant Hedgehog signal path antagonist vismodegib for SMO mutation, and the second class is directed to BTK mutation generation is drug resistant to replace Buddhist nun according to Shandong, and third class is directed to some antiandrogens such as abiraterone of AR medicament-resistant mutation Deng, some luxuriant and rich with fragrance Buddhist nun class drugs of the 4th class for BRAF medicament-resistant mutation, some networks of the 5th class for fusions such as ALK, MET Histidine kinase inhibitor, the 6th class are directed to the tyrosine kinase inhibitor of EGFR access, and the 7th class is logical for PI3K/AKT/mTOR The kinase inhibitors such as the everolimus on road.

Since above targeted drug cluster has comprehensively considered mechanism of drug action and the correlation between drug resistance site, There to be the targeted drug in similar action mechanism and crossing drug resistant site to gather for one kind, to maximumlly cover having for targeted drug The drug resistance site of immunogenicity, and phenomenon of " missing the target " as caused by patient's self-HLA molecule difference can be significantly reduced, from And make polypeptide vaccine can be to play lethal effect to the tumour cell for having similar medicament-resistant mutation in maximum magnitude, relatively single medicine Vaccine significantly improves its validity.

4, polypeptide vaccine sequence design: as one embodiment, the corresponding vaccine polypeptide sequence of engineer can be passed through Column can also input the vaccine design program iNeo-VaDes (V1.2) of our independent researches, design polypeptide vaccine sequence.As One kind being directed to the drug resistant polypeptide vaccine therapy of targeted drug, and during vaccine polypeptide design, the present invention considers epitope Distribution, covering epitope containing mutation sites as much as possible, it is also contemplated that the length of polypeptide, hydrophobic rate etc. influence amino acid Factor and toxicity, the homology of polypeptide of combined coefficient etc. influence the factor of polypeptide safety.Vaccine polypeptide group of the invention It closes and is made of the polypeptide sequence being mutated comprising following 137, shown in table 1 specific as follows.

5, vaccine preparation: the preparation of polypeptide vaccine both can be directly using preparing in a manner of chemically synthesized, also can be used Nucleic acid molecules (such as DNA and RNA) are obtained by way of transcription and translation, or can also be carrier by bacterium or virus Expression obtains.We obtain polypeptide vaccine using chemical synthesis mode in this example.

Table 1: targeted drug medicament-resistant mutation and polypeptide vaccine sequence

Table 2: the database of clinical patients:

The present invention provides the polypeptide vaccines of 31 targeted drugs by △, and basic to cover kinds of tumor targeted drug, use is following It tests and experimental verification is carried out to the immunogenicity of each polypeptide:

Experiment 1, the immunogenicity determining of polypeptide；

Experiment purpose: being tested by ELISpot, and the corresponding polypeptide of 7 products is in humanization Mice Body in the verifying present invention It is inside responsible for being immunoreacted.

Experimental method:

Test polypeptide: 7 product polypeptides are synthesized by Nanjing Genscript Biotechnology Co., Ltd.'s generation, and Purity is all larger than 90%, endotoxin content is lower than 0.5EU/mg.

In order to detect the immune response of polypeptide, implement IFN-γ Enzyme-linked Immunosorbent Assay (ELISPOT) measuring method.Detailed reality It is as follows to test process: selecting 8 week old humanization mouse B-NSG (CD34+) 24, is randomly divided into 8 groups, every group 3.It adapts to one week Afterwards, it is divided into that polypeptide group 1 (corresponding product 1), polypeptide group 2 (corresponding product 2), polypeptide group 7 is (right for polypeptide group 3 (corresponding product 3) ... Answer product 7), amount to 7 groups and negative control group # 8.Use CpG for adjuvant (0.2 μ g/ only), 50 μ g every of polypeptide, then with not Family name's Freund's incomplete adjuvant Freund ' s adjuvant (FIA, Sigma-Aldrich) 1:1 is mixed, and is emulsified 30 minutes, PBS and Freund It is used as within Freund's incomplete adjuvant 1:1 mixing and emulsifying 30 minutes negative control, four are inferior to the right chest subcutaneous inoculation of the nape of the neck, accumulated dose 0.5mL/ Only, once totally three weeks, 10 days after third time is immune, the mouse spleen taken prepared mouse lymphocyte suspension, is used within 1 week ELISPOT detection.

In ELISPOT testing result, IFN-γ is positive the polypeptide of result, that is, is determined as positive candidate polypeptide.Experiment is pressed Group carries out single peptide respectively ELISPOT test, i.e., mouse lymphocyte is diluted to concentration is 1-2*10⁶/ mL spreads 24 holes Plate, every hole 1mL are divided into control group (with the DMSO of polypeptide same concentrations), corresponding single polypeptide group, and PHA positive controls are compiled Number (PHA group lymphocyte derive from negative control group), each processing is repeated 3 times (i.e. 3 multiple holes), is separately added into more accordingly Peptide (10 μ g/mL), centrifugal separating cell after preincubate 72h, adjustment cell concentration are 2*10⁶/ mL, upper IFN-γ Elispot plate, It develops the color according to the specification method of IFN-γ ELISPOT kit, with the enzyme-linked spot of CTL-ImmunoSpotS5 series Analyzer reads the spot number generated.IFN-γ positive findings show there is T cells with antigenic specificity generation, and being considered as polypeptide can cause The immune response of body, its immune power of how much reflections of spot number.

Experimental result:

In order to further clarify the immune response of each independent polypeptide, it is corresponding that its in each polypeptide group has been carried out respectively The ELISPOT of single peptide is tested, and the spot number that each polypeptide generates is shown in Fig. 2A-D, each to be responsible for being immunoreacted, but each polypeptide The spot diversity ratio of generation is larger, differs from 10 spots to more than 200 a spots, and the basic immaculate of control group generates.

Interpretation of result: the corresponding polypeptide of 7 products is responsible for being immunoreacted in humanization Mice Body in the present invention.

The treatment and preventive effect of the drug resistance polypeptide vaccine of 7 class targeted drugs in the present invention are verified in experiment two；

In order to verify the treatment and preventive effect of gastric cancer drug resistance polypeptide vaccine in the present invention, it is therefore desirable to construct a set of containing this The steady of specific mutation site turns cell line in invention, and the experiment for carrying out following 7 embodiments respectively for 7 class targeted drugs is quasi- It is standby；Embodiment 1, the first kind generate drug resistant Hedgehog signal path antagonist vismodegib for SMO mutation (Vismodegib) building for surely turning cell line in specific mutation site:

Vismodegib is purchased from LC Laboratories, and stomach cancer cell strain AGS is purchased from ATCC, and FITC-CD44 is purchased from BD Company (BD555478).The preparation of cell: stomach cancer cell strain AGS in containing 10%FBS, 100U/mL penicillin and The DMEM of 100 μ g/mL streptomycin, 2mmol/L l-glutamine cultivates 48h, changes liquid and adds 20ng/mL OfEGF, bFGF, N-2 (1 ×) and B27 continue to cultivate 72h, it are promoted to form spheroid (" spheroid formation media").Spheroid forms unicellular through Accutase (Innovative Cell Technologies) enzymatic hydrolysis, uses FITC- CD44 streaming antibody sub-elect positive cell as subsequent transfection cell, is denoted as CD44⁺-AGS。

Experiment purpose: there is killing activity in order to verify the T cell of gastric cancer drug resistance polypeptid induction in the present invention, it is therefore desirable to Construct it is a set of containing in the present invention specific mutation site surely turn cell line, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

The mini- that can express all 15 vaccine polypeptides being mutated in the present invention is obtained using artificial synthesized method Gene consists of the following components: signal peptide moiety (lysosome-associated membrane glycoprotein 1, LAMP1), 15 mutant polypeptides and MHC class I trafficking domain (MITD) are used between 15 mutant polypeptides Flexible peptide linker GGSGGGGSGG connection, after gene carries out codon optimization, upstream introduces GATATC (EcoR V), and downstream introduces CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), is named as smo15-pcDNA3.1 (+).Simultaneously synthesizing corresponding wild polypeptide as control (since wherein adjacent polypeptide can be grouped into the long peptide of wild type, therefore A total of 7 wild polypeptides), it is named as smow7-pcDNA3.1 (+).All genetic fragments are by Nanjing Jin Sirui biotechnology Co., Ltd's generation synthesis and building,

The amino acid sequence of smo15 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQGGSGGGGSGGMLRLGIFGFLVFGFVLITFSCKKKKKKG GSGGGGSGGAFGFVLITFSYHFYDFFNQAEKKKKKGGSGGGGSGGVLITFSCHFYGFFNQAEWERKKGGSGGGGSG GLRLGIFGFLALGFVLITFSCHKKKKKKGGSGGGGSGGYVLCQANVTIWLPTKQPIPDCKGGSGGGGSGGVLITFS CHFYHFFNQAEWERSKKKGGSGGGGSGGFTEAEHQDMRSYIAAFGAVTKKGGSGGGGSGGMFGTGIAMSTLVWTKA TLLIWKKKKKKKKGGSGGGGSGGVLITFSCHFYYFFNQAEWERSKKKGGSGGGGSGGFSCHFYDFFNEAEWERSFR DYKKGGSGGGGSGGHSYIAAFGAVMGLCTLFTLAKKKKKKKKKGGSGGGGSGGTLSCVIIFVIAYYALMAGVVWKK KKKKKKKKGGSGGGGSGGNACFFVGSIGLLAQFMDGARKGGSGGGGSGGMFGTGIAMSTRVWTKATLLIWKKKKKK GGSGGGGSGGTLSCVIIFVIMYYALMAGVVWKKKKKKKKKKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMC RRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

The amino acid sequence of Smow7 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQMLRLRLGIFGFLAFGFVLITFSCHFYDFFNQAEWERSF RDGGSGGGGSGGYVLCQANVTIGLPTKQPIPDCKGGSGGGGSGGFTEAEHQDMHSYIAAFGAVTKKGGSGGGGSGG HSYIAAFGAVMGLCTLFTLAKKKKKKKKKGGSGGGGSGGTLSCVIIFVIVYYALMAGVVWKKKKKKKKKKGGSGGG GSGGNACFFVGSIGWLAQFMDGARKGGSGGGGSGGMFGTGIAMSTLVWTKATLLIWKKKKKKKGGSLGGGGSGIVG IVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column；

B. building can stablize the cell line of expression mutant polypeptide

Stomach cancer cell strain CD44⁺- AGS is with 2*10⁵/ hole kind starts to transfect in 6 orifice plates when cell covers 70-80%. 2.5 μ g smo15-pcDNA3.1 (+) plasmids and smow7-pcDNA3.1 (+) plasmid 2 are diluted in 2 part of 100 μ l serum-free respectively In RPMI-1640 culture medium, then it is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ l Lipofectamine^TMThe 100 μ l system of serum-free RPMI-1640 of LTX mixes, and is incubated at room temperature 30min.By liposome plasmid Complex compound is added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, and front and back gently shakes up, and is stood It is changed to the RPMI-1640 culture medium containing 10% serum after 6h, continues after cultivating 48h, is changed to containing 700 μ g/mLG418 and 400 μ The culture medium of 10% serum of g/mL hygromycin B carries out cell screening.It is resistance screening 10-14 days, all dead to cellular control unit It dies, transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.Dan Ke is selected under microscope Grand cell, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue culture about After 10 days, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.PcDNA3.1 (+)-Hygro sky is transfected simultaneously Vector plasmid is made to be named as CD44 as control cell with method resistance screening⁺- AGS (contains pcDNA3.1 (+)).The above method In, smo15- is respectively designated as with the cell line of smo15-pcDNA3.1 (+) plasmid and smow7-pcDNA3.1 (+) plasmid construction ASG (containing smo15-pcDNA3.1 (+)) and smow7-AGS (containing smow7-pcDNA3.1 (+)).

Embodiment 2, the second class generate the drug resistant specific mutation site that Buddhist nun Ibrutinib is replaced according to Shandong for BTK mutation Surely turn the building of cell line:

Ibrutinib (replacing Buddhist nun according to Shandong) is purchased from Selleck Chemicals, people's multiple myeloma cell line RPMI8226 ( CCL-155^TM) it is purchased from ATCC, it is incubated at the fetal calf serum that 1640 culture medium of RPMI adds 10%.

Experiment purpose: there is killing activity in order to verify the T cell of drug resistance polypeptid induction in the present invention, it is therefore desirable to construct It is a set of to turn cell line containing the steady of specific mutation site in the present invention, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

The mini- that can express all 5 vaccine polypeptides being mutated in the present invention is obtained using artificial synthesized method Gene consists of the following components: signal peptide moiety (lysosome-associated membrane glycoprotein 1, LAMP1), 5 mutant polypeptides and MHC class I trafficking domain (MITD), with soft between 5 mutant polypeptides Property link peptide GGSGGGGSGG connection, gene carry out codon optimization after, upstream introduce GATATC (EcoR V), downstream introduce CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), is named as BTK5-pcDNA3.1 (+).Simultaneously synthesizing corresponding wild polypeptide is named as BTKw2-pcDNA3.1 (+) (since wherein 4 polypeptides are as control SNP site mutation, therefore a total of 2 wild polypeptides).All genetic fragments are by Nanjing Genscript Biotechnology Co., Ltd.'s generation Synthesis and building,

The amino acid sequence of BTK5 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQFIITEYMANGFLLNYLREMRHKKKGGSGGGGSGGIITE YMANGRLLNYLREMRHKGGSGGGGSGGVRDSSKAGKYAVSVFAKSTGDGGSGGGGSGGIITEYMANGSLLNYLREM RHKGGSGGGGSGGFIITEYMANGYLLNYLREMRHKKKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKS SGGKGGSYSQAASSDSAQGSDVSLTA；

The amino acid sequence of BTKW2 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQFIITEYMANGCLLNYLREMRHKKKGGSGGGGSGGVRDS SKTGKYAVSVFAKSTGDGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGS DVSLTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

B. building can stablize the cell line of expression mutant polypeptide

People's multiple myeloma cell line RPMI8226 is with 2*10⁵/ hole kind is opened when cell covers 70-80% in 6 orifice plates Begin to transfect.BTK5-pcDNA3.1 (+) plasmid and BTKw2-pcDNA3.1 (+) plasmid 2 are diluted in 2 part of 100 μ l serum-free respectively In RPMI-1640 culture medium, then it is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ l Lipofectamine^TMThe 100 μ l system of serum-free RPMI-1640 of LTX mixes, and is incubated at room temperature 30min.By liposome plasmid Complex compound is added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, and front and back gently shakes up, and is stood It is changed to the RPMI-1640 culture medium containing 10% serum after 6h, continues after cultivating 48h, is changed to containing 700 μ g/mLG418 and 400 μ The culture medium of 10% serum of g/mL hygromycin B carries out cell screening.It is resistance screening 10-14 days, all dead to cellular control unit It dies, transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.Dan Ke is selected under microscope Grand cell, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue culture about After 10 days, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.In the above method, BTK5-pcDNA3.1 is used The cell line of (+) plasmid and BTKw2-pcDNA3.1 (+) plasmid construction names BTK5 respectively (containing BTK5-pcDNA3.1 (+)) (contain BTKw2-pcDNA3.1 (+)) with BTKw2.,

Embodiment 3, third class is for the specific prominent of some antiandrogens such as abiraterone of AR medicament-resistant mutation etc. The building for surely turning cell line of displacement point；

Prostate cancer cell line PC-3 ( CRL-1435^TM) it is purchased from ATCC, it is incubated at F-12K culture medium and adds 10% Fetal calf serum.

Experiment purpose: there is killing activity in order to verify the T cell of drug resistance polypeptid induction in the present invention, it is therefore desirable to construct It is a set of to turn cell line containing the steady of specific mutation site in the present invention, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

The mini- that can express all 4 vaccine polypeptides being mutated in the present invention is obtained using artificial synthesized method Gene consists of the following components: signal peptide moiety (lysosome-associated membrane glycoprotein 1, LAMP1), 4 mutant polypeptides and MHC class I trafficking domain (MITD), with soft between 4 mutant polypeptides Property link peptide GGSGGGGSGG connection, gene carry out codon optimization after, upstream introduce GATATC (EcoR V), downstream introduce CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), is named as AR4-pcDNA3.1 (+).Simultaneously synthesizing corresponding wild polypeptide is named as ARw2-pcDNA3.1 (+) (since wherein 4 polypeptides are as control SNP site mutation, therefore a total of 2 wild polypeptides).All genetic fragments are by Nanjing Genscript Biotechnology Co., Ltd.'s generation Synthesis and building.

The amino acid sequence of AR4 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQPIARELHQFAFDLLIKSHMKKGGSGGGGSGGVQPIARE LHQLTFDLLIKSHMKGGSGGGGSGGNELGERQLVHMVKWAKALPGFGGSGGGGSGGPIARELHQFSFDLLIKSHMG GSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

The amino acid sequence of ARW2 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQVQPIARELHQFTFDLLIKSHMGGSGGGGSGGNELGERQ LVHMVKWAKALPGFGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVS LTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

B. building can stablize the cell line of expression mutant polypeptide

Prostate cancer cell line PC-3 is with 2*10⁵/ hole kind starts to transfect in 6 orifice plates when cell covers 70-80%.Point AR4-pcDNA3.1 (+) plasmid and ARw2-pcDNA3.1 (+) plasmid 22 part of 100 μ l serum-free RPMI-1640 training is not diluted in It supports in base, then is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ l Lipofectamine^TMThe serum-free RPMI-1640100 μ l system of LTX mixes, and is incubated at room temperature 30min.By liposome plasmid network It closes object to be added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, front and back gently shakes up, and stands 6h It is changed to the RPMI-1640 culture medium containing 10% serum afterwards, continues after cultivating 48h, is changed to containing 700 μ g/mL G418 and 400 μ g/ The culture medium of 10% serum of mL hygromycin B carries out cell screening.It is resistance screening 10-14 days, all dead to cellular control unit, Transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.It is thin that monoclonal is selected under microscope Born of the same parents, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue culture about 10 days Afterwards, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.In the above method, with AR4-pcDNA3.1 (+) matter The cell line of grain and ARw2-pcDNA3.1 (+) plasmid construction names AR4 (containing AR4-pcDNA3.1 (+)) and ARw2 (to contain respectively There is ARw2-pcDNA3.1 (+)).

Embodiment 4, the 4th class turn thin for the steady of specific mutation site of some luxuriant and rich with fragrance Buddhist nun's class drugs of BRAF medicament-resistant mutation The building of born of the same parents system；

Human melanoma cell system A378 is purchased from ATCC, is incubated at the fetal calf serum that DMEM culture medium adds 10%.

Experiment purpose: there is killing activity in order to verify the T cell of drug resistance polypeptid induction in the present invention, it is therefore desirable to construct It is a set of to turn cell line containing the steady of specific mutation site in the present invention, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

The mini- that can express all 7 vaccine polypeptides being mutated in the present invention is obtained using artificial synthesized method Gene consists of the following components: signal peptide moiety (lysosome-associated membrane glycoprotein 1, LAMP1), 7 mutant polypeptides and MHC class I trafficking domain (MITD), with soft between 7 mutant polypeptides Property link peptide GGSGGGGSGG connection, gene carry out codon optimization after, upstream introduce GATATC (EcoR V), downstream introduce CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), is named as ME7-pcDNA3.1 (+).Simultaneously synthesizing corresponding wild polypeptide is named as MEw5-pcDNA3.1 (+) (wherein 124 amino of MAP2K1 as control Sour site mutant derives from same wild polypeptide, can be merged into the long peptide of wild type).All genetic fragments are by south The generation synthesis of Jing Jinsirui Biotechnology Co., Ltd and building.

The amino acid sequence of ME7 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQLHECNSPYIVVFYGAFYSDGEKKGGSGGGGSGGYKLVV VGADGVGKSALGGSGGGGSGGCLLDILDTAGREEYSAMRDQYKKKGGSGGGGSGGRKRLEAFLTPKQKVGELKGGS GGGGSGGELQVLHECNSLYIVGFYGAFYKKKGGSGGGGSGGKKRLEAFLTPKAKVGELKGGSGGGGSGGLQVLHEC NSSYIVGFYGAFYKKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDV SLTA；

The amino acid sequence of MEw5 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQYKLVVVGAGGVGKSALGGSGGGGSGGCLLDILDTAGQE EYSAMRDQYKKKGGSGGGGSGGRKRLEAFLTQKQKVGELKGGSGGGGSGGELQVLHECNSPYIVGFYGAFYSDGEK KGGSGGGGSGGKKRLEAFLTQKAKVGELKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSY SQAASSDSAQGSDVSLTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

B. building can stablize the cell line of expression mutant polypeptide

Human melanoma cell system A378 is with 2*10⁵/ hole kind starts to transfect in 6 orifice plates when cell covers 70-80%.Point ME7-pcDNA3.1 (+) plasmid and MEw5-pcDNA3.1 (+) plasmid 22 part of 100 μ l serum-free RPMI-1640 training is not diluted in It supports in base, then is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ l Lipofectamine ^TMThe 100 μ l system of serum-free RPMI-1640 of LTX mixes, and is incubated at room temperature 30min.By liposome plasmid Complex compound is added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, and front and back gently shakes up, and is stood It is changed to the RPMI-1640 culture medium containing 10% serum after 6h, continues after cultivating 48h, is changed to containing 700 μ g/mL G418 and 400 μ The culture medium of 10% serum of g/mL hygromycin B carries out cell screening.It is resistance screening 10-14 days, all dead to cellular control unit It dies, transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.Dan Ke is selected under microscope Grand cell, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue culture about After 10 days, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.In the above method, ME7-pcDNA3.1 is used The cell line of (+) plasmid and MEw5-pcDNA3.1 (+) plasmid construction names ME7 (ME7-pcDNA3.1 (+)) and MEw5 respectively (containing MEw5-pcDNA3.1 (+)).

Embodiment 5, specific mutation position of the 5th class for some tyrosine kinase inhibitors of fusions such as ALK, MET The building for surely turning cell line of point；

Non-small cell lung carcinoma H2228 (EML4-ALK variant 3a/b E6；A20 it) is purchased from ATCC, is incubated at RPMI 1640 culture mediums add 10% fetal calf serum.

Experiment purpose: there is killing activity in order to verify the T cell of drug resistance polypeptid induction in the present invention, it is therefore desirable to construct It is a set of to turn cell line containing the steady of specific mutation site in the present invention, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

The mini- that can express all 10 vaccine polypeptides being mutated in the present invention is obtained using artificial synthesized method Gene consists of the following components: signal peptide moiety (lysosome-associated membrane glycoprotein 1, LAMP1), 10 mutant polypeptides and MHC class I trafficking domain (MITD) are used between 10 mutant polypeptides Flexible peptide linker GGSGGGGSGG connection, after gene carries out codon optimization, upstream introduces GATATC (EcoR V), and downstream introduces CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), is named as ALK10-pcDNA3.1 (+).Simultaneously synthesizing corresponding wild polypeptide is named as ALKw5-pcDNA3.1 (+) (wherein 1171 amino acid of ALK as control Nearby mutant derives from same wild polypeptide, 1196 amino acid sites of ALK and 1202 for site and 1174 amino acid sites And 1203 amino acid sites nearby mutant derive from same wild polypeptide, 1246 amino acid sites of MET are from same Wild polypeptide can be merged into the long peptide of wild type, amount to 5 long peptides of wild type).All genetic fragments are by Nanjing gold The generation synthesis of Si Rui Biotechnology Co., Ltd and building.

The amino acid sequence of ALK10 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQKVADFGLARHMYDKEYYSVHKGGSGGGGSGGNITLIRG LSHGAFGEVYEGGSGGGGSGGELDFLMEALITSKFNHQNIVRGGSGGGGSGGRFILLELMAGRDLKSFLRETRGGS GGGGSGGCPGPGRVAKIADFGMARDIYRGGSGGGGSGGFLMEALIISKLNHQNIVRCIGKGGSGGGGSGGKVADFG LARNMYDKEYYSVHGGSGGGGSGGILLELMAGGNLKSFLRETRGGSGGGGSGGSLQSLPRFILMELMAGGDLKGGS GGGGSGGFLMEALIISKVNHQNIVRCIGKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSY SQAASSDSAQGSDVSLTA；

The amino acid sequence of ALKw5 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQKVADFGLARDMYDKEYYSVHKGGSGGGGSGGELDFLME ALIISKFNHQNIVRCIGKGGSGGGGSGGNITLIRGLGHGAFGEVYEGGSGGGGSGGCPGPGRVAKIGDFGMARDIY RGGSGGGGSGGSLQSLPRFILLELMAGGDLKSFLRETRGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRK SSGGKGGSYSQAASSDSAQGSDVSLTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

B. building can stablize the cell line of expression mutant polypeptide

Non-small cell lung carcinoma H2228 is with 2*10⁵/ hole kind starts to transfect in 6 orifice plates when cell covers 70-80%.Point ALK10-pcDNA3.1 (+) plasmid and ALKw5-pcDNA3.1 (+) plasmid 22 part of 100 μ l serum-free RPMI- is not diluted in In 1640 culture mediums, then it is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ lLipofectamine ^TMThe 100 μ l system of serum-free RPMI-1640 of LTX mixes, and is incubated at room temperature 30min.By lipid constitution Grain complex compound is added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, and front and back gently shakes up, quiet Be changed to the RPMI-1640 culture medium containing 10% serum after setting 6h, continue after cultivating 48h, be changed to containing 700 μ g/mL G418 and The culture medium of 10% serum of 400 μ g/mL hygromycin Bs carries out cell screening.It is resistance screening 10-14 days, complete to cellular control unit Portion is dead, and transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.It is selected under microscope Monoclonal cell, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue to train After supporting about 10 days, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.In the above method, ALK10- is used The cell line of pcDNA3.1 (+) plasmid and ALKw5-pcDNA3.1 (+) plasmid construction names ALK10 (ALK10- respectively PcDNA3.1 (+)) and ALKw5 (containing ALKw5-pcDNA3.1 (+)).

Embodiment 6, the 6th class turn cell for the steady of specific mutation site of the tyrosine kinase inhibitor of EGFR access The building of system；

Human chronic polymorpho nuclear leukemia cells system THP-1 (ATCC TIB-202) is purchased from ATCC, is incubated at RPMI-1640 training Feeding base adds 10% fetal calf serum.

Experiment purpose: there is killing activity in order to verify the T cell of drug resistance polypeptid induction in the present invention, it is therefore desirable to construct It is a set of to turn cell line containing the steady of specific mutation site in the present invention, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

In order to realize the expression of mutant polypeptide, the present invention is quasi- to pass through each polypeptide gene (i.e. mini-gene) of contacting in people It is overexpressed in chronic myeloid leukemia cell line THP-1.But from concentration, (genetic fragment is homologous since these mutantional hotspots compare Property it is high), by corresponding 4 genes of this 92 polypeptides, be divided into 2 groups, every group of gene for containing 46 polypeptides is cloned in eukaryon respectively Expression vector pcDNA3.1-hygro (+) and pcDNA3.1-zeo (+).Each carrier can express the whole being mutated in the present invention The mini-gene of 46 vaccine polypeptides, consists of the following components: signal peptide moiety (lysosome-associated Membrane glycoprotein 1, LAMP1), 13 mutant polypeptides and MHC class I trafficking domain (MITD), after using flexible peptide linker GGSGGGGSGG connection, gene to carry out codon optimization between 46 mutant polypeptides, upstream is drawn Enter GATATC (EcoR V), downstream introduces CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro In (+), it is named as mut46-hygro (+) and mut46-zeo (+).Simultaneously synthesizing corresponding wild polypeptide is as control, name For wid8-hygro (+), (wherein ABL1 albumen directly chooses 234-505 amino acid sites, and FLT3 albumen chooses the 835th amino Sour location proximate one, nearby 2 articles of the 760th and 790 amino acid sites of EGFR albumen selection, 3, KIT albumen and PDGFRA albumen 1 wild polypeptide), it is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), all genes of wid8-hygro (+) Segment was synthesized and is constructed by Nanjing Genscript Biotechnology Co., Ltd.'s generation.

The amino acid sequence of Mut46-hy are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQTSPKANKEILYEAYVMASVDKGGSGGGGSGGICLTSTV QLIMQLMPFGCLLDKKKKKGGSGGGGSGGAPESLAYNKFYIKSDVWAFGVGGSGGGGSGGAVKTLKEDTMKVEEFL KEAAVKGGSGGGGSGGAVMKEIKHPNVVQLLGVCTRKGGSGGGGSGGCLVGENHLVKIADFGLSRLMTKGGSGGGG SGGCTREPPFYIIAEFMTYGNLLDKKGGSGGGGSGGQLITQLMPFDCLLDYVREHKKGGSGGGGSGGGENHLVKVA DLGLSRLMGGSGGGGSGGIDLSQVYELLKKDYRMERGGSGGGGSGGITMKHKLGGGEYGEVYEGVWGGSGGGGSGG ITMKHKLGGGHYGEVYEGVWKGGSGGGGSGGLTSTVQLITQHMPFGCLLDYVKKKKKGGSGGGGSGGCTREPPFYI INEFMTYGNLLDKKGGSGGGGSGGKEAAVMKEIRGGHPNLVQLLGVGGSGGGGSGGKHKLGGGQYGKVYEGVWKKY SGGSGGGGSGGKVADFGLSRMMTGDTYTAHAKKGGSGGGGSGGKWEMERTDITVKHKLGGGQYGGSGGGGSGGLAA RNCLVGEYHLVKVADFKKKGGSGGGGSGGLLGVCTREPPLYIITEFMTYGKKKGGSGGGGSGGITMKHKLGGGMYG EVYEGVWKGGSGGGGSGGIDLSQVYELLLKDYRMERKGGSGGGGSGGLMTGDTYTAHPGAKFPIKWKKGGSGGGGS GGMTYGNLLDYLMECNRQEVNAVKKGGSGGGGSGGNCLVGENHLVRVADFGLSRLMGGSGGGGSGGREPPFYIITE LMTYGNLLDYLKKGGSGGGGSGGRLMTGDTYTAPAGAKFPIKWKGGSGGGGSGGSAMEYLEKKNAIHRDLAARGGS GGGGSGGITMKHKLGGGRYGEVYEGVWKGGSGGGGSGGTLKEDTMEVEGFLKEAAVMKKKGGSGGGGSGGVEEFLK EAAFMKEIKHPNLVGGSGGGGSGGYMATQISSAMGYLEKKNFIHRGGSGGGGSGGYPGIDLSQVYALLEKDYRMER KGGSGGGGSGGVKICDFGLARFIMSDSNYVVRGGSGGGGSGGKICDFGLARAIKNDSNYVVKGGSGGGGSGGDFGL ARDIKNVSNYVVKGNARGGSGGGGSGGCTIGGPTLVIIEYCCYGDLLNKKKGGSGGGGSGGSYLGNHMNIVTLLGA CTIGGPKGGSGGGGSGGYPGIDLSQVYKLLEKDYRMERGGSGGGGSGGVKICDFGLARYIMSDSNYVVRGGSGGGG SGGTQISSAMEYLAKKNFIHRDGGSGGGGSGGWQWNPSDRPSSAEIHQAFETMKGGSGGGGSGGWAFGVLLWEITT YGMSPYPGIKKKGGSGGGGSGGVAVKTLKEDAMEVEEFLKEAKKKGGSGGGGSGGVKICDFGLARVIMHDSNYVSK GGSGGGGSGGDSNYVVKGNPRLPVKWMAGGSGGGGSGGKICDFGLARHIKNDSNYVVKGGSLGGGGSGIVGIVAGL AVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

The amino acid sequence of Mut46-zeo are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQLITQLMPFGSLLDYVREHKDGGSGGGGSGGAVKTLKED TMYVEEFLKEAAVKKGGSGGGGSGGCTREPPFYIIIEFMTYGNLLDKKGGSGGGGSGGDLSQVYELLGKDYRMERK GGSGGGGSGGDTYTAHAGTKFPIKWTAPEKKGGSGGGGSGGEFLKEAAVMKVIKHPNLVQLLKGGSGGGGSGGVQL ITQLMPFRCLLDYVREHKKGGSGGGGSGGELMRACWQWNLSDRPSFAEIHGGSGGGGSGGESLAYNKFSIESDVWA FGVLLKKGGSGGGGSGGHLVKVADFGMSRLMTGDTYTKKGGSGGGGSGGIDLSQVYELLAKDYRMERKGGSGGGGS GGITMKHKLGGGEYGEVYEGVWGGSGGGGSGGCTREPPFYIIVEFMTYGNLLDKKAVKTLKEDTMVEEFLKEAKKG GSGGGGSGGKHKLGGGQYGVVYEGVWKKYSGGSGGGGSGGKTLKEDTMAVEEFLKEAAVKKGGSGGGGSGGKVADF GLSRLLTGDTYTAHAGKGGSGGGGSGGLLGVCTREPSFYIITEFMTYKKKKGGSGGGGSGGITMKHKLGGGKYGEV YEGVWKGGSGGGGSGGMTGDTYTAHARAKFPIKWTAPKKKGGSGGGGSGGPESLAYNKFSVKSDVWAFGVGGSGGG GSGGPFYIITEFMTCGNLLDYLRKKKGGSGGGGSGGQISSAMEYLGKKNFIHRDGGSGGGGSGGRECNRQEVNAFV LLYMATQIKGGSGGGGSGGRLMTGDTYTARAGAKFPIKWTKGGSGGGGSGGSAMEYLEKKNVIHRDLAARNGGSGG GGSGGTLKEDTMEVEKFLKEAAVMKKGGSGGGGSGGVEEFLKEAAAMKEIKHPNLVGGSGGGGSGGYMATQISSAM DYLEKKNFIHRGGSGGGGSGGYPGIDLSQVYGLLEKDYRMERKGGSGGGGSGGKICDFGLARHIMSDSNYVVRGGS GGGGSGGKICDFGLAREIKNDSNYVVKGGSGGGGSGGLARDIKNGSNYVVKGNGGSGGGGSGGCTIGGPTLVIEEY CCYGDLLNKKKGGSGGGGSGGLSYLGNHMNIANLLGACTIGKKGGSGGGGSGGVKICDFGLARVIMSDSNYVVRGG SGGGGSGGSFAEIHQAFERMFQESSISDEKKKGGSGGGGSGGSLTVAVKTLKKDTMEVEEFLKGGSGGGGSGGTEF MTYGNLLGYLRECNRQEVKGGSGGGGSGGTMKHKLGGGQFGEVYEGVWKKGGSGGGGSGGVKVADFGLSRFMTGDT YTAHAKKKGGSGGGGSGGVMKEIKHPNLLQLLGVCTREPGGSGGGGSGGEREALMSELEVLSYLGNHMNKKGGSGG GGSGGEAALYKNLLHFKESSCSDSTGGSGGGGSGGDFGLARDIKNYSNYVVKGNARGGSGGGGSGGFGLARDIKND FNYVVKGNARGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

The amino acid sequence of Wid8-hy are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVK TLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQI SSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVW AFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQE SSISDGGSGGGGSGGVKICDFGLARDIMSDSNYVVRGGSGGGGSGGTSPKANKEILDEAYVMASVDKGGSGGGGSG GICLTSTVQLITQLMPFGCLLDYVREHKGGSGGGGSGGVKICDFGLARDIMHDSNYVSKGGSGGGGSGGEREALMS ELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNGGSGGGGSGGEAALYKNLLHSKESSCSDSTGGSGG GGSGGKICDFGLARDIKNDSNYVVKGNARLPVKWMAGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSS GGKGGSYSQAASSDSAQGSDVSLTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

B. building can stablize the cell line of expression mutant polypeptide

Human chronic polymorpho nuclear leukemia cells system THP-1 is with 2*10⁵/ hole kind is in 6 orifice plates, when cell covers 70-80% Start to transfect.Mut46-hygro (+) plasmid and wid8-hygro (+) plasmid 2 are diluted in 2 part of 100 μ l serum-free respectively In RPMI-1640 culture medium, then it is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ lLipofectamine ^TMThe 100 μ l system of serum-free RPMI-1640 of LTX mixes, and is incubated at room temperature 30min.By lipid constitution Grain complex compound is added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, and front and back gently shakes up, quiet Be changed to the RPMI-1640 culture medium containing 10% serum after setting 6h, continue after cultivating 48h, be changed to containing 700 μ g/mL G418 and The culture medium of 10% serum of 400 μ g/mL hygromycin Bs carries out cell screening.It is resistance screening 10-14 days, complete to cellular control unit Portion is dead, and transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.It is selected under microscope Monoclonal cell, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue to train After supporting about 10 days, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.In the above method, mut46- is used The cell line of hygro (+) plasmid and wid8-hygro (+) plasmid construction name respectively mut46 (mut46-hygro (+)) and Wid8 (contains wid8-hygro (+)).

Mut46 (mut46-hygro (+)) is reactivated, and presses above-mentioned transfection protocol, it will be in mut46-zeo (+) importing Cell line is stated, is screened with 400 μ g/mL zeo, mut92 (mut46-hygro (+), mut46-zeo (+)) is named as.

Embodiment 7, specific mutation position of the 7th class for kinase inhibitors such as the everolimus of PI3K/AKT/mTOR access The building for surely turning cell line of point；

Human embryo kidney (HEK) fibroblast strain HEK 293 ( CRL-1573^TM) it is purchased from ATCC, it is incubated at 10%FBS- DMEM culture medium.

Experiment purpose: there is killing activity in order to verify the T cell of drug resistance polypeptid induction in the present invention, it is therefore desirable to construct It is a set of to turn cell line containing the steady of specific mutation site in the present invention, these polypeptides of submission can be expressed

A. mutational site eukaryon expression plasmid is constructed

The mini- that can express all 3 vaccine polypeptides being mutated in the present invention is obtained using artificial synthesized method Gene consists of the following components: signal peptide moiety (lysosome-associated membrane glycoprotein 1, LAMP1), 3 mutant polypeptides and MHC class I trafficking domain (MITD), with soft between 3 mutant polypeptides Property link peptide GGSGGGGSGG connection, gene carry out codon optimization after, upstream introduce GATATC (EcoR V), downstream introduce CTCGAG (Xho I), is cloned into eukaryotic expression vector pcDNA3.1-hygro (+), is named as MEM3-pcDNA3.1 (+).Simultaneously synthesizing corresponding wild polypeptide is named as MEMw3-pcDNA3.1 (+) as control.All genetic fragments are by Nanjing The generation synthesis of Jin Sirui Biotechnology Co., Ltd and building.

The amino acid sequence of MEM3 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQTQAWDLYYHVLRRISKQLPQGGSGGGGSGGHECNSPYI VGLYGAFYSDGEIKKGGSGGGGSGGVKVADFGLARVMYDKEYYSVHKGGSLGGGGSGIVGIVAGLAVLAVVVIGAV VATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

The amino acid sequence of MEMw3 are as follows:

MAAPGSARRPLLLLLLLLLLGLMHCASALQTQAWDLYYHVFRRISKQLPQGGSGGGGSGGHECNSPYI VGFYGAFYSDGEIKKGGSGGGGSGGVKVADFGLARDMYDKEYYSVHKGGSLGGGGSGIVGIVAGLAVLAVVVIGAV VATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；

Wherein the 1-28 amino acid is signal peptide region, is indicated with overstriking italic；Underscore mark part is MITD sequence Column.

B. building can stablize the cell line of expression mutant polypeptide

Human embryo kidney (HEK) fibroblast strain HEK 293 is with 2*10⁵/ hole kind starts when cell covers 70-80% in 6 orifice plates Transfection.MEM3-pcDNA3.1 (+) plasmid and MEMw3-pcDNA3.1 (+) plasmid 2 are diluted in 2 part of 100 μ l serum-free respectively In RPMI-1640 culture medium, then it is separately added into 2.5 μ l PLUS^TMReagents, be incubated at room temperature 5min after, respectively with contain 5 μ l Lipofectamine ^TMThe 100 μ l system of serum-free RPMI-1640 of LTX mixes, and is incubated at room temperature 30min.By lipid constitution Grain complex compound is added dropwise respectively in 2 parts of cells to be transfected containing 1000 μ l serum-free RPMI-1640, and front and back gently shakes up, quiet It is changed to the RPMI-1640 culture medium containing 10% serum after setting 6h, continues after cultivating 48h, is changed to containing 700 μ g/mLG418 and 400 The culture medium of 10% serum of μ g/mL hygromycin B carries out cell screening.It is resistance screening 10-14 days, all dead to cellular control unit It dies, transfection group cell raised growth enters cell dissociation in 96 orifice plates using limiting dilution assay kind.Dan Ke is selected under microscope Grand cell, with 700 μ g/mL G418 are contained, the culture medium of 400 μ g/mL hygromycin Bs continues to cultivate, and changes liquid every other day.Continue culture about After 10 days, monoclonal cell grows up to larger one, and digestion is transferred to 24 orifice plate cultures.In the above method, MEM3-pcDNA3.1 is used The cell line of (+) plasmid and MEMw3-pcDNA3.1 (+) plasmid construction names MEM3 respectively (containing MEM3-pcDNA3.1 (+)) (contain MEMw3-pcDNA3.1 (+)) with MEMw3.

The lymphocyte suspension of 1 mouse of polypeptide group obtained according to above embodiments 1-7 does cell in vitro killing experiments, real It is as follows to test content:

Test 2-1: the cell in vitro killing experiments of the polypeptide vaccine group of first kind targeted drug；

Experiment purpose: the cellular level in vitro of 15 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.(contained under aseptic condition with CFSE label smo15-ASG Have smo15-pcDNA3.1 (+)) and smow7-AGS (containing smow7-pcDNA3.1 (+)) target cell, respectively as experimental group and The target cell of control group.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 1, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL uses RPMI1640+10% FBS+1×penicillin(100μg/mL)+streptomycin(100μg/mL)+1×MEM non-essential amino Acids+1mM sodium pyruvate+10mM HEPES buffer culture medium is cultivated, and the rhIL2 of 50U/mL is added. The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half amounts change liquid, and add corresponding antigen Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL.

1) smo15-ASG (is contained into smo15-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1: The cell number ratio of 10 and 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, each Three parallel control holes are arranged in experimental group.

2) smow7-AGS (is contained into smow7-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1: The cell number ratio of 10 and 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as a control group, each Three parallel control holes are arranged in control group.

3) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

4) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, is gone in streaming loading pipe, With propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out machine in streaming at once Detection.

Experimental result:

As shown in figure 3, its killing-efficiency of effector T cell of experimental group (Fig. 3) induction is in 40%-80% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitating target ratio is 20:1, to target cell killing-efficiency up to 75% or more.

Test the cell in vitro killing experiments of the polypeptide vaccine group of the 2-2: the second class targeted drug

Experiment purpose: the cellular level in vitro of 5 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.(contained under aseptic condition with CFSE label BTK5 BTK5-pcDNA3.1 (+)) and BTKw2 (contain BTKw2-pcDNA3.1 (+)) target cell, respectively as experimental group and control group Target cell.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 2, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL is used

RPMI1640+10%FBS+1 × penicillin (100 μ g/mL)+streptomycin (100 μ g/mL)+1 × MEM non-essential amino acids+1mM sodium pyruvate+10mM HEPES buffer culture medium carries out Culture, adds the rhIL2 of 50U/mL.The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half Amount changes liquid, and adds corresponding Antigenic Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL。

5) BTK5 (is contained into BTK5-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and 1: 20 cell number ratio is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, each experimental group Three parallel control holes are set.

6) by BTKw2 (contain BTKw2-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and The cell number ratio of 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as a control group, each control Three parallel control holes of group setting.

7) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

8) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, is gone in streaming loading pipe, With propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out machine in streaming at once Detection.

Experimental result:

As shown in figure 4, its killing-efficiency of effector T cell of experimental group (Fig. 4) induction is in 40%-80% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitating target ratio is 20:1, to target cell killing-efficiency up to 75% or more.

Test 2-3: the cell in vitro killing experiments of the polypeptide vaccine group of third class targeted drug

Experiment purpose: the cellular level in vitro of 4 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.(contain AR4- with CFSE label AR4 under aseptic condition PcDNA3.1 (+)) and ARw2 (contain ARw2-pcDNA3.1 (+)) target cell, it is thin respectively as the target of experimental group and control group Born of the same parents.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 3, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL is used

RPMI1640+10%FBS+1 × penicillin (100 μ g/mL)+streptomycin (100 μ g/mL)+1 × MEM non-essential amino acids+1mM sodium pyruvate+10mM HEPES buffer culture medium carries out Culture, adds the rhIL2 of 50U/mL.The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half Amount changes liquid, and adds corresponding Antigenic Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL。

9) AR4 (is contained into AR4-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and 1:20 Cell number ratio mixed, be added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, each experimental group is set Set three parallel control holes.

10) by ARw2 (contain ARw2-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and The cell number ratio of 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as a control group, each control Group three parallel control holes (can delete) of setting.

11) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

12) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, goes to streaming loading pipe In, with propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out streaming at once Upper machine testing.

Experimental result:

As shown in figure 5, its killing-efficiency of effector T cell of experimental group (Fig. 5) induction is in 40%-90% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitate target ratio be 20:1 when, to target cell killing-efficiency up to 80% with

Test the polypeptide vaccine group cell in vitro killing experiments of the 2-4: the four class targeted drug

Experiment purpose: the cellular level in vitro of 7 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.ME7 (ME7- is marked with CFSE under aseptic condition PcDNA3.1 (+)) and MEw5 (contain MEw5-pcDNA3.1 (+)) target cell, it is thin respectively as the target of experimental group and control group Born of the same parents.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 4, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL uses RPMI1640+10% FBS+1×penicillin(100μg/mL)+streptomycin(100μg/mL)+1×MEM non-essential amino Acids+1mM sodium pyruvate+10mM HEPES buffer culture medium is cultivated, and the rhIL2 of 50U/mL is added. The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half amounts change liquid, and add corresponding antigen Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL.

13) by ME7 (ME7-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and 1:20 Cell number ratio is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, each experimental group setting Three parallel control holes.

14) by MEw5 (contain MEw5-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and The cell number ratio of 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as a control group, each control Group three parallel control holes (can delete) of setting.

15) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

16) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, goes to streaming loading pipe In, with propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out streaming at once Upper machine testing.

Experimental result:

As shown in fig. 6, its killing-efficiency of effector T cell of experimental group (Fig. 6) induction is in 45%-90% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitating target ratio is 20:1, to target cell killing-efficiency up to 85% or more.

Test the polypeptide vaccine group cell in vitro killing experiments of the 2-5: the five class targeted drug；

Experiment purpose: the cellular level in vitro of 10 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.ALK10 (ALK10- is marked with CFSE under aseptic condition PcDNA3.1 (+)) and ALKw5 (contain ALKw5-pcDNA3.1 (+)) target cell, respectively as the target of experimental group and control group Cell.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 5, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL uses RPMI1640+10% FBS+1×penicillin(100μg/mL)+streptomycin(100μg/mL)+1×MEM non-essential amino Acids+1mM sodium pyruvate+10mM HEPES buffer culture medium is cultivated, and the rhIL2 of 50U/mL is added. The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half amounts change liquid, and add corresponding antigen Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL.

17) by ALK10 (ALK10-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and 1: 20 cell number ratio is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, each experimental group Three parallel control holes are set.

18) ALKw5 (is contained into ALKw5-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 It is mixed, is added in U-shaped 96 orifice plate with the cell number ratio of 1:20, every 200 μ L of pore volume is as a control group, each right According to group three parallel control holes (can delete) of setting.

19) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

20) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, goes to streaming loading pipe In, with propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out streaming at once Upper machine testing.

Experimental result:

As shown in fig. 7, its killing-efficiency of effector T cell of experimental group (Fig. 7) induction is in 40%-90% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitating target ratio is 20:1, to target cell killing-efficiency up to 80% or more.

Test the polypeptide vaccine group cell in vitro killing experiments of the 2-6: the six class targeted drug；

Experiment purpose: the cellular level in vitro of 92 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.Mut92 (mut46- is marked with CFSE under aseptic condition Hygro (+), mut46-zeo (+)) and wid8 (contain wid8-hygro (+)) target cell, respectively as experimental group and control group Target cell.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 6, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL uses RPMI1640+10% FBS+1×penicillin(100μg/mL)+streptomycin(100μg/mL)+1×MEM non-essential amino Acids+1mM sodium pyruvate+10mM HEPES buffer culture medium is cultivated, and the rhIL2 of 50U/mL is added. The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half amounts change liquid, and add corresponding antigen Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL.

21) by mut92 (mut46-hygro (+), mut46-zeo (+)) target cell respectively with effector cell CTL according to 1: 5, the cell number ratio of 1:10 and 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, Three parallel control holes are arranged in each experimental group.

22) wid8 (is contained into wid8-hygro (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and 1:20 Cell number ratio mixed, be added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as a control group, each control group is set Set three parallel control holes.

23) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

24) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, goes to streaming loading pipe In, with propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out streaming at once Upper machine testing.

Experimental result:

As shown in figure 8, its killing-efficiency of effector T cell of experimental group (Fig. 8) induction is in 40%-90% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitating target ratio is 20:1, to target cell killing-efficiency up to 84% or more.

Test the polypeptide vaccine group cell in vitro killing experiments of the 2-7: the seven class targeted drug；

Experiment purpose: the cellular level in vitro of 3 polypeptides of verifying can cause the fragmentation effect of tumour cell.

Experimental method:

(1) 5- (6)-Carboxy-fluorescein succinimidyl ester (CFSE) dyestuff is purchased from Invitrogen company.Operating procedure is carried out according to kit specification.(contained under aseptic condition with CFSE label MEM3 MEM3-pcDNA3.1(+))

(contain MEMw3-pcDNA3.1 (+)) target cell with MEMw3, it is thin respectively as the target of experimental group and control group Born of the same parents.

(2) killing experiments

A. priming effect cell:

It by the lymphocyte suspension for 1 mouse of polypeptide group left and taken in embodiment 7, is resuspended with 1640 culture medium of RPMI, platform is expected Blue dyeing counting.

B. the Fiber differentiation of effector cell CTL:

It is (1-2) * 10 by 2 lymphocyte diluted concentrations of group⁶/ mL, spreads 6 orifice plates, and every hole 3mL uses RPMI1640+10% FBS+1×penicillin(100μg/mL)+streptomycin(100μg/mL)+1×MEM non-essential amino Acids+1mM sodium pyruvate+10mM HEPES buffer culture medium is cultivated, and the rhIL2 of 50U/mL is added. The corresponding Antigenic Peptide pool group of 10 μ g/mL is added into every hole, cultivates 7 days, every 3 days half amounts change liquid, and add corresponding antigen Peptide and rhIL2；Cell is resuspended after one week, is washed 2 times with PBS, is prepared into effector cell CTL.

25) by MEM3 (contain MEM3-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 and The cell number ratio of 1:20 is mixed, and is added in U-shaped 96 orifice plate, every 200 μ L of pore volume, as experimental group, each experiment Three parallel control holes of group setting.

26) MEMw3 (is contained into MEMw3-pcDNA3.1 (+)) target cell respectively with effector cell CTL according to 1:5,1:10 It is mixed, is added in U-shaped 96 orifice plate with the cell number ratio of 1:20, every 200 μ L of pore volume is as a control group, each right According to group three parallel control holes of setting.

27) 96 orifice plates are placed on 37 DEG C of incubator culture 4h.

28) supernatant is removed into the centrifugation of 96 orifice plates, cell precipitation is resuspended with the PBS that 200 μ L are pre-chilled, goes to streaming loading pipe In, with propidium iodide (Prodium Iodide, PI) dye marker, concentration is 1 μ g/m L, dyes 3min, carries out streaming at once Upper machine testing.

Experimental result:

As shown in figure 9, its killing-efficiency of effector T cell of experimental group (Fig. 9) induction is in 40%-80% etc., killing effect Rate is apparently higher than control group, illustrates that mutant polypeptide group plays lethal effect to target cell.In mutant polypeptide group, with effect target ratio Raising, the lethal effect of T cell is more and more stronger, when imitating target ratio is 20:1, to target cell killing-efficiency up to 70% or more.

It is tested according to above 7, can learn design method through the invention by targeted drug according to its mechanism and resistance to Medicine is mutated clustering 7 major class of playback, and multiple target point joint can effectively increase polypeptide vaccine to the fragmentation effect of tumour cell, show It writes and extends targeted drug effective acting time.

The present invention provides a kind of polypeptide vaccine and its design method for tumor-targeting drug drug resistance site, the target provided Common target therapeutic agent is covered to medication combined polypeptide vaccine assembled scheme, tumor drug resistance can be effectively reduced and occur generally Rate extends targeting medicine effective acting time, increases the disease response rate of patient, has broad spectrum activity, shortens individuation polypeptide vaccine From the time for analyzing treatment, medical treatment cost is reduced.

The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should Understand, the above embodiments do not limit the invention in any form, all obtained by the way of equivalent substitution or equivalent transformation Technical solution is fallen within the scope of protection of the present invention.

Sequence table

<110>Hangzhou Niu Anjin Biotechnology Co., Ltd

<120>polypeptide vaccine and its design method in tumor-targeting drug drug resistance site are directed to

<141> 2019-01-29

<160> 208

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> PRT

<213> Artificial Sequence

<400> 1

Met Leu Arg Leu Gly Ile Phe Gly Phe Leu Val Phe Gly Phe Val Leu

1 5 10 15

Ile Thr Phe Ser Cys Lys Lys Lys Lys Lys Lys

20 25

<210> 2

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 2

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

1 5 10 15

Phe Asn Gln Ala Glu Lys Lys Lys Lys Lys

20 25

<210> 3

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 3

Val Leu Ile Thr Phe Ser Cys His Phe Tyr Gly Phe Phe Asn Gln Ala

1 5 10 15

Glu Trp Glu Arg Lys Lys

20

<210> 4

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 4

Val Leu Ile Thr Phe Ser Cys His Phe Tyr His Phe Phe Asn Gln Ala

1 5 10 15

Glu Trp Glu Arg Ser Lys Lys Lys

20

<210> 5

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 5

Val Leu Ile Thr Phe Ser Cys His Phe Tyr Tyr Phe Phe Asn Gln Ala

1 5 10 15

Glu Trp Glu Arg Ser Lys Lys Lys

20

<210> 6

<211> 27

<212> PRT

<213> Artificial Sequence

<400> 6

Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Leu Gly Phe Val Leu Ile

1 5 10 15

Thr Phe Ser Cys His Lys Lys Lys Lys Lys Lys

20 25

<210> 7

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 7

Tyr Val Leu Cys Gln Ala Asn Val Thr Ile Trp Leu Pro Thr Lys Gln

1 5 10 15

Pro Ile Pro Asp Cys Lys

20

<210> 8

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 8

Phe Thr Glu Ala Glu His Gln Asp Met Arg Ser Tyr Ile Ala Ala Phe

1 5 10 15

Gly Ala Val Thr Lys Lys

20

<210> 9

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 9

Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Glu Ala Glu Trp Glu Arg

1 5 10 15

Ser Phe Arg Asp Tyr Lys Lys

20

<210> 10

<211> 29

<212> PRT

<213> Artificial Sequence

<400> 10

His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys Thr Leu

1 5 10 15

Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys

20 25

<210> 11

<211> 31

<212> PRT

<213> Artificial Sequence

<400> 11

Thr Leu Ser Cys Val Ile Ile Phe Val Ile Ala Tyr Tyr Ala Leu Met

1 5 10 15

Ala Gly Val Val Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30

<210> 12

<211> 31

<212> PRT

<213> Artificial Sequence

<400> 12

Thr Leu Ser Cys Val Ile Ile Phe Val Ile Met Tyr Tyr Ala Leu Met

1 5 10 15

Ala Gly Val Val Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

20 25 30

<210> 13

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 13

Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Leu Leu Ala Gln Phe Met

1 5 10 15

Asp Gly Ala Arg Lys

20

<210> 14

<211> 29

<212> PRT

<213> Artificial Sequence

<400> 14

Met Phe Gly Thr Gly Ile Ala Met Ser Thr Leu Val Trp Thr Lys Ala

1 5 10 15

Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Lys Lys

20 25

<210> 15

<211> 27

<212> PRT

<213> Artificial Sequence

<400> 15

Met Phe Gly Thr Gly Ile Ala Met Ser Thr Arg Val Trp Thr Lys Ala

1 5 10 15

Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys

20 25

<210> 16

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 16

Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Phe Leu Leu Asn Tyr Leu

1 5 10 15

Arg Glu Met Arg His Lys Lys Lys

20

<210> 17

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 17

Ile Ile Thr Glu Tyr Met Ala Asn Gly Arg Leu Leu Asn Tyr Leu Arg

1 5 10 15

Glu Met Arg His Lys

20

<210> 18

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 18

Ile Ile Thr Glu Tyr Met Ala Asn Gly Ser Leu Leu Asn Tyr Leu Arg

1 5 10 15

Glu Met Arg His Lys

20

<210> 19

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 19

Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Tyr Leu Leu Asn Tyr Leu

1 5 10 15

Arg Glu Met Arg His Lys Lys Lys

20

<210> 20

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 20

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

1 5 10 15

Lys Ser Thr Gly Asp

20

<210> 21

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 21

Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys

1 5 10 15

Ser His Met Lys Lys

20

<210> 22

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 22

Pro Ile Ala Arg Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys

1 5 10 15

Ser His Met

<210> 23

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 23

Val Gln Pro Ile Ala Arg Glu Leu His Gln Leu Thr Phe Asp Leu Leu

1 5 10 15

Ile Lys Ser His Met Lys

20

<210> 24

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 24

Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys

1 5 10 15

Ser His Met Lys Lys

20

<210> 25

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 25

Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys

1 5 10 15

Ser His Met Lys Lys

20

<210> 26

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 26

Pro Ile Ala Arg Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys

1 5 10 15

Ser His Met

<210> 27

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 27

Asn Glu Leu Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys

1 5 10 15

Ala Leu Pro Gly Phe

20

<210> 28

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 28

Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys

1 5 10 15

Ser His Met Lys Lys

20

<210> 29

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 29

Leu Gln Val Leu His Glu Cys Asn Ser Ser Tyr Ile Val Gly Phe Tyr

1 5 10 15

Gly Ala Phe Tyr Lys Lys

20

<210> 30

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 30

Lys Lys Arg Leu Glu Ala Phe Leu Thr Pro Lys Ala Lys Val Gly Glu

1 5 10 15

Leu Lys

<210> 31

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 31

Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu

1 5 10 15

<210> 32

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 32

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr Lys Lys Lys

20

<210> 33

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 33

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr Lys Lys Lys

20

<210> 34

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 34

Leu His Glu Cys Asn Ser Pro Tyr Ile Val Val Phe Tyr Gly Ala Phe

1 5 10 15

Tyr Ser Asp Gly Glu Lys Lys

20

<210> 35

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 35

Glu Leu Gln Val Leu His Glu Cys Asn Ser Leu Tyr Ile Val Gly Phe

1 5 10 15

Tyr Gly Ala Phe Tyr Lys Lys Lys

20

<210> 36

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 36

Arg Lys Arg Leu Glu Ala Phe Leu Thr Pro Lys Gln Lys Val Gly Glu

1 5 10 15

Leu Lys

<210> 37

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 37

Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala

1 5 10 15

Met Arg Asp Gln Tyr Lys Lys Lys

20

<210> 38

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 38

Glu Leu Gln Val Leu His Glu Cys Asn Ser Leu Tyr Ile Val Gly Phe

1 5 10 15

Tyr Gly Ala Phe Tyr Lys Lys Lys

20

<210> 39

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 39

Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Arg Asp Leu Lys Ser Phe

1 5 10 15

Leu Arg Glu Thr Arg

20

<210> 40

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 40

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

1 5 10 15

Glu Thr Arg

<210> 41

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 41

Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn Ile

1 5 10 15

Val Arg Cys Ile Gly Lys

20

<210> 42

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 42

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

1 5 10 15

Tyr Glu

<210> 43

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 43

Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Arg Asp Leu Lys Ser Phe

1 5 10 15

Leu Arg Glu Thr Arg

20

<210> 44

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 44

Cys Pro Gly Pro Gly Arg Val Ala Lys Ile Ala Asp Phe Gly Met Ala

1 5 10 15

Arg Asp Ile Tyr Arg

20

<210> 45

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 45

Ser Leu Gln Ser Leu Pro Arg Phe Ile Leu Met Glu Leu Met Ala Gly

1 5 10 15

Gly Asp Leu Lys

20

<210> 46

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 46

Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Leu Asn His Gln Asn Ile

1 5 10 15

Val Arg Cys Ile Gly Lys

20

<210> 47

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 47

Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn Ile

1 5 10 15

Val Arg Cys Ile Gly Lys

20

<210> 48

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 48

Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Arg Asp Leu Lys Ser Phe

1 5 10 15

Leu Arg Glu Thr Arg

20

<210> 49

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 49

Cys Pro Gly Pro Gly Arg Val Ala Lys Ile Ala Asp Phe Gly Met Ala

1 5 10 15

Arg Asp Ile Tyr Arg

20

<210> 50

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 50

Glu Leu Asp Phe Leu Met Glu Ala Leu Ile Thr Ser Lys Phe Asn His

1 5 10 15

Gln Asn Ile Val Arg

20

<210> 51

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 51

Ser Leu Gln Ser Leu Pro Arg Phe Ile Leu Met Glu Leu Met Ala Gly

1 5 10 15

Gly Asp Leu Lys

20

<210> 52

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 52

Lys Val Ala Asp Phe Gly Leu Ala Arg His Met Tyr Asp Lys Glu Tyr

1 5 10 15

Tyr Ser Val His Lys

20

<210> 53

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 53

Lys Val Ala Asp Phe Gly Leu Ala Arg Asn Met Tyr Asp Lys Glu Tyr

1 5 10 15

Tyr Ser Val His

20

<210> 54

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 54

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe

1 5 10 15

Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys

20 25

<210> 55

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 55

Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu

1 5 10 15

Ala Ala Arg Asn

20

<210> 56

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 56

Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val

1 5 10 15

Cys Thr Arg Glu Pro

20

<210> 57

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 57

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 58

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 58

Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr Gly Asn

1 5 10 15

Leu Leu Asp Tyr Leu Lys Lys

20

<210> 59

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 59

Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu

1 5 10 15

Ala Ala Arg Asn

20

<210> 60

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 60

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 61

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 61

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 62

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 62

Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val

1 5 10 15

Cys Thr Arg Glu Pro

20

<210> 63

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 63

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe

1 5 10 15

Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys

20 25

<210> 64

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 64

Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Tyr Glu Ala Tyr Val Met

1 5 10 15

Ala Ser Val Asp Lys

20

<210> 65

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 65

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe

1 5 10 15

Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys

20 25

<210> 66

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 66

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

1 5 10 15

Glu His Lys Asp

20

<210> 67

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 67

Leu Met Thr Gly Asp Thr Tyr Thr Ala His Pro Gly Ala Lys Phe Pro

1 5 10 15

Ile Lys Trp Lys Lys

20

<210> 68

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 68

Asp Thr Tyr Thr Ala His Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr

1 5 10 15

Ala Pro Glu Lys Lys

20

<210> 69

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 69

Trp Ala Phe Gly Val Leu Leu Trp Glu Ile Thr Thr Tyr Gly Met Ser

1 5 10 15

Pro Tyr Pro Gly Ile Lys Lys Lys

20

<210> 70

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 70

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 71

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 71

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 72

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 72

Ser Leu Thr Val Ala Val Lys Thr Leu Lys Lys Asp Thr Met Glu Val

1 5 10 15

Glu Glu Phe Leu Lys

20

<210> 73

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 73

Lys Thr Leu Lys Glu Asp Thr Met Ala Val Glu Glu Phe Leu Lys Glu

1 5 10 15

Ala Ala Val Lys Lys

20

<210> 74

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 74

Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu

1 5 10 15

Lys Glu Ala Ala Val Lys

20

<210> 75

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 75

Ala Val Lys Thr Leu Lys Glu Asp Thr Met Tyr Val Glu Glu Phe Leu

1 5 10 15

Lys Glu Ala Ala Val Lys Lys

20

<210> 76

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 76

Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glx Val Glu Glu Phe Leu

1 5 10 15

Lys Glu Ala Lys Lys

20

<210> 77

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 77

Thr Leu Lys Glu Asp Thr Met Glu Val Glu Gly Phe Leu Lys Glu Ala

1 5 10 15

Ala Val Met Lys Lys Lys

20

<210> 78

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 78

Glu Phe Leu Lys Glu Ala Ala Val Met Lys Val Ile Lys His Pro Asn

1 5 10 15

Leu Val Gln Leu Leu Lys

20

<210> 79

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 79

Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Asp Tyr Leu Glu Lys Lys

1 5 10 15

Asn Phe Ile His Arg

20

<210> 80

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 80

Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Gly Tyr Leu Glu Lys Lys

1 5 10 15

Asn Phe Ile His Arg

20

<210> 81

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 81

Thr Gln Ile Ser Ser Ala Met Glu Tyr Leu Ala Lys Lys Asn Phe Ile

1 5 10 15

His Arg Asp

<210> 82

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 82

Gln Ile Ser Ser Ala Met Glu Tyr Leu Gly Lys Lys Asn Phe Ile His

1 5 10 15

Arg Asp

<210> 83

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 83

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

1 5 10 15

Tyr Arg Met Glu Arg Lys

20

<210> 84

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 84

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

1 5 10 15

Tyr Arg Met Glu Arg Lys

20

<210> 85

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 85

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

1 5 10 15

Tyr Arg Met Glu Arg

20

<210> 86

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 86

Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Ala Lys Asp Tyr Arg Met

1 5 10 15

Glu Arg Lys

<210> 87

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 87

Asp Leu Ser Gln Val Tyr Glu Leu Leu Gly Lys Asp Tyr Arg Met Glu

1 5 10 15

Arg Lys

<210> 88

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 88

Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met

1 5 10 15

Glu Arg

<210> 89

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 89

Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Leu Lys Asp Tyr Arg Met

1 5 10 15

Glu Arg Lys

<210> 90

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 90

Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu Tyr Ile Ile Thr Glu

1 5 10 15

Phe Met Thr Tyr Gly Lys Lys Lys

20

<210> 91

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 91

Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr Gly Asn

1 5 10 15

Leu Leu Asp Tyr Leu Lys Lys

20

<210> 92

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 92

Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Ala Ile His Arg Asp Leu

1 5 10 15

Ala Ala Arg

<210> 93

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 93

Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu

1 5 10 15

Ala Ala Arg Asn

20

<210> 94

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 94

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

1 5 10 15

Met

<210> 95

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 95

Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Ser Ala Glu Ile His Gln

1 5 10 15

Ala Phe Glu Thr Met Lys

20

<210> 96

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 96

Met Thr Gly Asp Thr Tyr Thr Ala His Ala Arg Ala Lys Phe Pro Ile

1 5 10 15

Lys Trp Thr Ala Pro Lys Lys Lys

20

<210> 97

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 97

Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly Ala Lys Phe

1 5 10 15

Pro Ile Lys Trp Lys

20

<210> 98

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 98

Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly Ala Lys Phe

1 5 10 15

Pro Ile Lys Trp Thr Lys

20

<210> 99

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 99

Pro Glu Ser Leu Ala Tyr Asn Lys Phe Ser Val Lys Ser Asp Val Trp

1 5 10 15

Ala Phe Gly Val

20

<210> 100

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 100

Lys Glu Ala Ala Val Met Lys Glu Ile Arg Gly Gly His Pro Asn Leu

1 5 10 15

Val Gln Leu Leu Gly Val

20

<210> 101

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 101

Asn Cys Leu Val Gly Glu Asn His Leu Val Arg Val Ala Asp Phe Gly

1 5 10 15

Leu Ser Arg Leu Met

20

<210> 102

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 102

Glu Ser Leu Ala Tyr Asn Lys Phe Ser Ile Glu Ser Asp Val Trp Ala

1 5 10 15

Phe Gly Val Leu Leu Lys Lys

20

<210> 103

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 103

Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly

1 5 10 15

Val Cys Thr Arg Lys

20

<210> 104

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 104

His Leu Val Lys Val Ala Asp Phe Gly Met Ser Arg Leu Met Thr Gly

1 5 10 15

Asp Thr Tyr Thr Lys Lys

20

<210> 105

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 105

Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met Thr Gly Asp Thr

1 5 10 15

Tyr Thr Ala His Ala Lys Lys Lys

20

<210> 106

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 106

Lys Val Ala Asp Phe Gly Leu Ser Arg Met Met Thr Gly Asp Thr Tyr

1 5 10 15

Thr Ala His Ala Lys Lys

20

<210> 107

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 107

Lys Trp Glu Met Glu Arg Thr Asp Ile Thr Val Lys His Lys Leu Gly

1 5 10 15

Gly Gly Gln Tyr

20

<210> 108

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 108

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

1 5 10 15

Thr Ala His Ala Gly Lys

20

<210> 109

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 109

Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Tyr His Leu Val Lys Val

1 5 10 15

Ala Asp Phe Lys Lys Lys

20

<210> 110

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 110

Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Leu Ser Asp Arg Pro Ser

1 5 10 15

Phe Ala Glu Ile His

20

<210> 111

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 111

Ile Thr Met Lys His Lys Leu Gly Gly Gly Glu Tyr Gly Glu Val Tyr

1 5 10 15

Glu Gly Val Trp

20

<210> 112

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 112

Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr Gly Glu Val Tyr

1 5 10 15

Glu Gly Val Trp Lys

20

<210> 113

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 113

Ile Thr Met Lys His Lys Leu Gly Gly Gly Lys Tyr Gly Glu Val Tyr

1 5 10 15

Glu Gly Val Trp Lys

20

<210> 114

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 114

Ile Thr Met Lys His Lys Leu Gly Gly Gly Arg Tyr Gly Glu Val Tyr

1 5 10 15

Glu Gly Val Trp Lys

20

<210> 115

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 115

Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Met Glu Cys Asn Arg Gln

1 5 10 15

Glu Val Asn Ala Val Lys Lys

20

<210> 116

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 116

Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe Tyr Ile Lys Ser Asp Val

1 5 10 15

Trp Ala Phe Gly Val

20

<210> 117

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 117

Val Ala Val Lys Thr Leu Lys Glu Asp Ala Met Glu Val Glu Glu Phe

1 5 10 15

Leu Lys Glu Ala Lys Lys Lys

20

<210> 118

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 118

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 119

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 119

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Asn Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 120

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 120

Val Glu Glu Phe Leu Lys Glu Ala Ala Phe Met Lys Glu Ile Lys His

1 5 10 15

Pro Asn Leu Val

20

<210> 121

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 121

Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val

1 5 10 15

Cys Thr Arg Glu Pro

20

<210> 122

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 122

Cys Leu Val Gly Glu Asn His Leu Val Lys Ile Ala Asp Phe Gly Leu

1 5 10 15

Ser Arg Leu Met Thr Lys

20

<210> 123

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 123

Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe Gly Glu Val Tyr Glu

1 5 10 15

Gly Val Trp Lys Lys

20

<210> 124

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 124

Pro Phe Tyr Ile Ile Thr Glu Phe Met Thr Cys Gly Asn Leu Leu Asp

1 5 10 15

Tyr Leu Arg Lys Lys Lys

20

<210> 125

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 125

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

1 5 10 15

Met Ala

<210> 126

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 126

Lys Ile Cys Asp Phe Gly Leu Ala Arg Ala Ile Lys Asn Asp Ser Asn

1 5 10 15

Tyr Val Val Lys

20

<210> 127

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 127

Lys Ile Cys Asp Phe Gly Leu Ala Arg Glu Ile Lys Asn Asp Ser Asn

1 5 10 15

Tyr Val Val Lys

20

<210> 128

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 128

Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Lys Asn Asp Ser Asn

1 5 10 15

Tyr Val Val Lys

20

<210> 129

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 129

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

1 5 10 15

<210> 130

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 130

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

1 5 10 15

Lys Gly Asn Ala Arg

20

<210> 131

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 131

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

1 5 10 15

Lys Gly Asn Ala Arg

20

<210> 132

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 132

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

1 5 10 15

Asn His Met Asn Lys Lys

20

<210> 133

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 133

Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Phe Lys Glu Ser Ser Cys

1 5 10 15

Ser Asp Ser Thr

20

<210> 134

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 134

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

1 5 10 15

Gly Asn Ala Arg

20

<210> 135

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 135

Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Glu Glu Tyr Cys Cys Tyr

1 5 10 15

Gly Asp Leu Leu Asn Lys Lys Lys

20

<210> 136

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 136

Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Ile Glu Tyr Cys Cys Tyr

1 5 10 15

Gly Asp Leu Leu Asn Lys Lys Lys

20

<210> 137

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 137

Leu Ser Tyr Leu Gly Asn His Met Asn Ile Ala Asn Leu Leu Gly Ala

1 5 10 15

Cys Thr Ile Gly Lys Lys

20

<210> 138

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 138

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ile Met His Asp Ser

1 5 10 15

Asn Tyr Val Ser Lys

20

<210> 139

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 139

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 140

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 140

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 141

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 141

Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly Ala Lys Phe

1 5 10 15

Pro Ile Lys Trp Thr Lys

20

<210> 142

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 142

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 143

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 143

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Val Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 144

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 144

Ser Tyr Leu Gly Asn His Met Asn Ile Val Thr Leu Leu Gly Ala Cys

1 5 10 15

Thr Ile Gly Gly Pro Lys

20

<210> 145

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 145

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

1 5 10 15

Glu His Lys Asp

20

<210> 146

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 146

Gln Leu Ile Thr Gln Leu Met Pro Phe Asp Cys Leu Leu Asp Tyr Val

1 5 10 15

Arg Glu His Lys Lys

20

<210> 147

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 147

Val Gln Leu Ile Thr Gln Leu Met Pro Phe Arg Cys Leu Leu Asp Tyr

1 5 10 15

Val Arg Glu His Lys Lys

20

<210> 148

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 148

Gln Leu Ile Thr Gln Leu Met Pro Phe Ser Cys Leu Leu Asp Tyr Val

1 5 10 15

Arg Glu His Lys Lys

20

<210> 149

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 149

Leu Thr Ser Thr Val Gln Leu Ile Thr Gln His Met Pro Phe Gly Cys

1 5 10 15

Leu Leu Asp Tyr Val Lys Lys Lys Lys Lys

20 25

<210> 150

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 150

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe

1 5 10 15

Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys

20 25

<210> 151

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 151

Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Met Ser Asp Ser Asn

1 5 10 15

Tyr Val Val Arg

20

<210> 152

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 152

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Tyr Ile Met Ser Asp Ser

1 5 10 15

Asn Tyr Val Val Arg

20

<210> 153

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 153

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Tyr Ile Met Ser Asp Ser

1 5 10 15

Asn Tyr Val Val Arg

20

<210> 154

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 154

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ile Met His Asp Ser

1 5 10 15

Asn Tyr Val Ser Lys

20

<210> 155

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 155

Asp Thr Tyr Thr Ala His Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr

1 5 10 15

Ala Pro Glu Lys Lys

20

<210> 156

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 156

Thr Glu Phe Met Thr Tyr Gly Asn Leu Leu Gly Tyr Leu Arg Glu Cys

1 5 10 15

Asn Arg Gln Glu Val Lys

20

<210> 157

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 157

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 158

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 158

Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val

1 5 10 15

Trp Lys Lys Tyr Ser

20

<210> 159

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 159

Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu

1 5 10 15

Lys Glu Ala Ala Val Lys

20

<210> 160

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 160

Thr Leu Lys Glu Asp Thr Met Glu Val Glu Lys Phe Leu Lys Glu Ala

1 5 10 15

Ala Val Met Lys Lys

20

<210> 161

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 161

Gln Ile Ser Ser Ala Met Glu Tyr Leu Gly Lys Lys Asn Phe Ile His

1 5 10 15

Arg Asp

<210> 162

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 162

Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met

1 5 10 15

Glu Arg

<210> 163

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 163

Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu Tyr Ile Ile Thr Glu

1 5 10 15

Phe Met Thr Tyr Gly Lys Lys Lys

20

<210> 164

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 164

Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr Gly Asn

1 5 10 15

Leu Leu Asp Tyr Leu Lys Lys

20

<210> 165

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 165

Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu

1 5 10 15

Ala Ala Arg Asn

20

<210> 166

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 166

Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly Ala Lys Phe

1 5 10 15

Pro Ile Lys Trp Lys

20

<210> 167

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 167

Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly Ala Lys Phe

1 5 10 15

Pro Ile Lys Trp Thr Lys

20

<210> 168

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 168

Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly

1 5 10 15

Val Cys Thr Arg Lys

20

<210> 169

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 169

His Leu Val Lys Val Ala Asp Phe Gly Met Ser Arg Leu Met Thr Gly

1 5 10 15

Asp Thr Tyr Thr Lys Lys

20

<210> 170

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 170

Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met Thr Gly Asp Thr

1 5 10 15

Tyr Thr Ala His Ala Lys Lys Lys

20

<210> 171

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 171

Lys Val Ala Asp Phe Gly Leu Ser Arg Met Met Thr Gly Asp Thr Tyr

1 5 10 15

Thr Ala His Ala Lys Lys

20

<210> 172

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 172

Lys Trp Glu Met Glu Arg Thr Asp Ile Thr Val Lys His Lys Leu Gly

1 5 10 15

Gly Gly Gln Tyr

20

<210> 173

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 173

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

1 5 10 15

Thr Ala His Ala Gly Lys

20

<210> 174

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 174

Leu Leu Gly Val Cys Thr Arg Glu Pro Ser Phe Tyr Ile Ile Thr Glu

1 5 10 15

Phe Met Thr Tyr Lys Lys Lys Lys

20

<210> 175

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 175

Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr Gly Glu Val Tyr

1 5 10 15

Glu Gly Val Trp Lys

20

<210> 176

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 176

Ile Thr Met Lys His Lys Leu Gly Gly Gly Met Tyr Gly Glu Val Tyr

1 5 10 15

Glu Gly Val Trp Lys

20

<210> 177

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 177

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 178

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 178

Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr

1 5 10 15

Gly Asn Leu Leu Asp Lys Lys

20

<210> 179

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 179

Ser Phe Ala Glu Ile His Gln Ala Phe Glu Arg Met Phe Gln Glu Ser

1 5 10 15

Ser Ile Ser Asp Glu Lys Lys Lys

20

<210> 180

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 180

Val Glu Glu Phe Leu Lys Glu Ala Ala Ala Met Lys Glu Ile Lys His

1 5 10 15

Pro Asn Leu Val

20

<210> 181

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 181

Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val

1 5 10 15

Cys Thr Arg Glu Pro

20

<210> 182

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 182

Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Phe Val Leu Leu Tyr Met

1 5 10 15

Ala Thr Gln Ile Lys

20

<210> 183

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 183

Cys Leu Val Gly Glu Asn His Leu Val Lys Ile Ala Asp Phe Gly Leu

1 5 10 15

Ser Arg Leu Met Thr Lys

20

<210> 184

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 184

Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe Gly Glu Val Tyr Glu

1 5 10 15

Gly Val Trp Lys Lys

20

<210> 185

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 185

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe

1 5 10 15

Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys

20 25

<210> 186

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 186

Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe

1 5 10 15

Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys

20 25

<210> 187

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 187

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Phe Ile Met Ser Asp Ser

1 5 10 15

Asn Tyr Val Val Arg

20

<210> 188

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 188

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ile Met Ser Asp Ser

1 5 10 15

Asn Tyr Val Val Arg

20

<210> 189

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 189

Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Tyr Ile Met Ser Asp Ser

1 5 10 15

Asn Tyr Val Val Arg

20

<210> 190

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 190

Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys

1 5 10 15

Gln Leu Pro Gln

20

<210> 191

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 191

Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys

1 5 10 15

Gln Leu Pro Gln

20

<210> 192

<211> 23

<212> PRT

<213> Artificial Sequence

<400> 192

His Glu Cys Asn Ser Pro Tyr Ile Val Gly Leu Tyr Gly Ala Phe Tyr

1 5 10 15

Ser Asp Gly Glu Ile Lys Lys

20

<210> 193

<211> 22

<212> PRT

<213> Artificial Sequence

<400> 193

Val Lys Val Ala Asp Phe Gly Leu Ala Arg Val Met Tyr Asp Lys Glu

1 5 10 15

Tyr Tyr Ser Val His Lys

20

<210> 194

<211> 630

<212> PRT

<213> Artificial Sequence

<400> 194

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Gly Gly

20 25 30

Ser Gly Gly Gly Gly Ser Gly Gly Met Leu Arg Leu Gly Ile Phe Gly

35 40 45

Phe Leu Val Phe Gly Phe Val Leu Ile Thr Phe Ser Cys Lys Lys Lys

50 55 60

Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Phe Gly

65 70 75 80

Phe Val Leu Ile Thr Phe Ser Tyr His Phe Tyr Asp Phe Phe Asn Gln

85 90 95

Ala Glu Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

100 105 110

Gly Val Leu Ile Thr Phe Ser Cys His Phe Tyr Gly Phe Phe Asn Gln

115 120 125

Ala Glu Trp Glu Arg Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Leu Gly Phe Val Leu

145 150 155 160

Ile Thr Phe Ser Cys His Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Gly Gly Tyr Val Leu Cys Gln Ala Asn Val Thr Ile

180 185 190

Trp Leu Pro Thr Lys Gln Pro Ile Pro Asp Cys Lys Gly Gly Ser Gly

195 200 205

Gly Gly Gly Ser Gly Gly Val Leu Ile Thr Phe Ser Cys His Phe Tyr

210 215 220

His Phe Phe Asn Gln Ala Glu Trp Glu Arg Ser Lys Lys Lys Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Phe Thr Glu Ala Glu His Gln Asp

245 250 255

Met Arg Ser Tyr Ile Ala Ala Phe Gly Ala Val Thr Lys Lys Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Met Phe Gly Thr Gly Ile Ala Met

275 280 285

Ser Thr Leu Val Trp Thr Lys Ala Thr Leu Leu Ile Trp Lys Lys Lys

290 295 300

Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val

305 310 315 320

Leu Ile Thr Phe Ser Cys His Phe Tyr Tyr Phe Phe Asn Gln Ala Glu

325 330 335

Trp Glu Arg Ser Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

340 345 350

Gly Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Glu Ala Glu Trp Glu

355 360 365

Arg Ser Phe Arg Asp Tyr Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys

385 390 395 400

Thr Leu Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly

405 410 415

Gly Ser Gly Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile

420 425 430

Phe Val Ile Ala Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys

435 440 445

Lys Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Gly Gly Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Leu Leu Ala Gln

465 470 475 480

Phe Met Asp Gly Ala Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

485 490 495

Gly Met Phe Gly Thr Gly Ile Ala Met Ser Thr Arg Val Trp Thr Lys

500 505 510

Ala Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly

515 520 525

Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile Phe Val Ile

530 535 540

Met Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys Lys Lys Lys

545 550 555 560

Lys Lys Lys Lys Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

565 570 575

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

580 585 590

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

595 600 605

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

610 615 620

Asp Val Ser Leu Thr Ala

625 630

<210> 195

<211> 348

<212> PRT

<213> Artificial Sequence

<400> 195

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Met Leu

20 25 30

Arg Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Phe Gly Phe Val Leu

35 40 45

Ile Thr Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Gln Ala Glu Trp

50 55 60

Glu Arg Ser Phe Arg Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

65 70 75 80

Tyr Val Leu Cys Gln Ala Asn Val Thr Ile Gly Leu Pro Thr Lys Gln

85 90 95

Pro Ile Pro Asp Cys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

100 105 110

Phe Thr Glu Ala Glu His Gln Asp Met His Ser Tyr Ile Ala Ala Phe

115 120 125

Gly Ala Val Thr Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys Thr Leu

145 150 155 160

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

165 170 175

Gly Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile Phe Val

180 185 190

Ile Val Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys Lys Lys

195 200 205

Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

210 215 220

Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Trp Leu Ala Gln Phe Met

225 230 235 240

Asp Gly Ala Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Met

245 250 255

Phe Gly Thr Gly Ile Ala Met Ser Thr Leu Val Trp Thr Lys Ala Thr

260 265 270

Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser Leu Gly

275 280 285

Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu

290 295 300

Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg

305 310 315 320

Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser

325 330 335

Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

340 345

<210> 196

<211> 246

<212> PRT

<213> Artificial Sequence

<400> 196

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Phe Ile

20 25 30

Ile Thr Glu Tyr Met Ala Asn Gly Phe Leu Leu Asn Tyr Leu Arg Glu

35 40 45

Met Arg His Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

50 55 60

Ile Ile Thr Glu Tyr Met Ala Asn Gly Arg Leu Leu Asn Tyr Leu Arg

65 70 75 80

Glu Met Arg His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val

85 90 95

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

100 105 110

Ser Thr Gly Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Ile

115 120 125

Thr Glu Tyr Met Ala Asn Gly Ser Leu Leu Asn Tyr Leu Arg Glu Met

130 135 140

Arg His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Phe Ile Ile

145 150 155 160

Thr Glu Tyr Met Ala Asn Gly Tyr Leu Leu Asn Tyr Leu Arg Glu Met

165 170 175

Arg His Lys Lys Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

180 185 190

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

195 200 205

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

210 215 220

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

225 230 235 240

Asp Val Ser Leu Thr Ala

245

<210> 197

<211> 150

<212> PRT

<213> Artificial Sequence

<400> 197

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Phe Ile

20 25 30

Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu Leu Asn Tyr Leu Arg Glu

35 40 45

Met Arg His Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

50 55 60

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

65 70 75 80

Lys Ser Thr Gly Asp Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

85 90 95

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

100 105 110

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

115 120 125

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

130 135 140

Asp Val Ser Leu Thr Ala

145 150

<210> 198

<211> 208

<212> PRT

<213> Artificial Sequence

<400> 198

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Pro Ile

20 25 30

Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys Ser His

35 40 45

Met Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Gln Pro

50 55 60

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

65 70 75 80

His Met Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Glu Leu

85 90 95

Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys Ala Leu Pro

100 105 110

Gly Phe Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Ile Ala Arg

115 120 125

Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys Ser His Met Gly

130 135 140

Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly

145 150 155 160

Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val

165 170 175

Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln

180 185 190

Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

195 200 205

<210> 199

<211> 147

<212> PRT

<213> Artificial Sequence

<400> 199

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Val Gln

20 25 30

Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys

35 40 45

Ser His Met Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Glu Leu

50 55 60

Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys Ala Leu Pro

65 70 75 80

Gly Phe Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile

85 90 95

Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val

100 105 110

Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser

115 120 125

Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser

130 135 140

Leu Thr Ala

145

<210> 200

<211> 340

<212> PRT

<213> Artificial Sequence

<400> 200

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Leu His

20 25 30

Glu Cys Asn Ser Pro Tyr Ile Val Val Phe Tyr Gly Ala Phe Tyr Ser

35 40 45

Asp Gly Glu Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr

50 55 60

Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu Gly

65 70 75 80

Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Leu Leu Asp Ile Leu Asp

85 90 95

Thr Ala Gly Arg Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Lys Lys

100 105 110

Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys Arg Leu Glu

115 120 125

Ala Phe Leu Thr Pro Lys Gln Lys Val Gly Glu Leu Lys Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Glu Leu Gln Val Leu His Glu Cys Asn

145 150 155 160

Ser Leu Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Lys Lys Lys Gly

165 170 175

Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Arg Leu Glu Ala Phe

180 185 190

Leu Thr Pro Lys Ala Lys Val Gly Glu Leu Lys Gly Gly Ser Gly Gly

195 200 205

Gly Gly Ser Gly Gly Leu Gln Val Leu His Glu Cys Asn Ser Ser Tyr

210 215 220

Ile Val Gly Phe Tyr Gly Ala Phe Tyr Lys Lys Gly Gly Ser Leu Gly

225 230 235 240

Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu

245 250 255

Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg

260 265 270

Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser

275 280 285

Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Met Leu Arg Leu

290 295 300

Arg Leu Gly Ile Phe Gly Phe Leu Ala Phe Gly Phe Val Leu Ile Thr

305 310 315 320

Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Gln Ala Glu Trp Glu Arg

325 330 335

Ser Phe Arg Asp

340

<210> 201

<211> 238

<212> PRT

<213> Artificial Sequence

<400> 201

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Tyr Lys

20 25 30

Leu Val Val Val Gly Ala Gly Gly Val Gly Lys Ser Ala Leu Gly Gly

35 40 45

Ser Gly Gly Gly Gly Ser Gly Gly Cys Leu Leu Asp Ile Leu Asp Thr

50 55 60

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

65 70 75 80

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys Arg Leu Glu Ala

85 90 95

Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Glu Leu Gln Val Leu His Glu Cys Asn Ser

115 120 125

Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Lys

130 135 140

Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Arg Leu Glu

145 150 155 160

Ala Phe Leu Thr Gln Lys Ala Lys Val Gly Glu Leu Lys Gly Gly Ser

165 170 175

Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly Leu Ala

180 185 190

Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys

195 200 205

Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala

210 215 220

Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala

225 230 235

<210> 202

<211> 360

<212> PRT

<213> Artificial Sequence

<400> 202

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Val

20 25 30

Ala Asp Phe Gly Leu Ala Arg His Met Tyr Asp Lys Glu Tyr Tyr Ser

35 40 45

Val His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Ile Thr

50 55 60

Leu Ile Arg Gly Leu Ser His Gly Ala Phe Gly Glu Val Tyr Glu Gly

65 70 75 80

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Asp Phe Leu Met Glu

85 90 95

Ala Leu Ile Thr Ser Lys Phe Asn His Gln Asn Ile Val Arg Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Arg Phe Ile Leu Leu Glu Leu Met

115 120 125

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

130 135 140

Gly Gly Gly Gly Ser Gly Gly Cys Pro Gly Pro Gly Arg Val Ala Lys

145 150 155 160

Ile Ala Asp Phe Gly Met Ala Arg Asp Ile Tyr Arg Gly Gly Ser Gly

165 170 175

Gly Gly Gly Ser Gly Gly Phe Leu Met Glu Ala Leu Ile Ile Ser Lys

180 185 190

Leu Asn His Gln Asn Ile Val Arg Cys Ile Gly Lys Gly Gly Ser Gly

195 200 205

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

210 215 220

Met Tyr Asp Lys Glu Tyr Tyr Ser Val His Gly Gly Ser Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Ile Leu Leu Glu Leu Met Ala Gly Gly Asn Leu Lys

245 250 255

Ser Phe Leu Arg Glu Thr Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly

260 265 270

Gly Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn

275 280 285

Ile Val Arg Cys Ile Gly Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser

290 295 300

Gly Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val

305 310 315 320

Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly

325 330 335

Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln

340 345 350

Gly Ser Asp Val Ser Leu Thr Ala

355 360

<210> 203

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 203

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Val

20 25 30

Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr Tyr Ser

35 40 45

Val His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Asp

50 55 60

Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Phe Asn His Gln Asn Ile

65 70 75 80

Val Arg Cys Ile Gly Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

85 90 95

Ile Leu Leu Glu Leu Met Ala Gly Gly Asp Leu Lys Ser Phe Leu Arg

100 105 110

Glu Thr Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly

115 120 125

Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val

130 135 140

Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly

145 150 155 160

Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val

165 170 175

Ser Leu Thr Ala

180

<210> 204

<211> 1559

<212> PRT

<213> Artificial Sequence

<400> 204

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Ser

20 25 30

Pro Lys Ala Asn Lys Glu Ile Leu Tyr Glu Ala Tyr Val Met Ala Ser

35 40 45

Val Asp Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Cys Leu

50 55 60

Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe Gly Cys Leu

65 70 75 80

Leu Asp Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly

85 90 95

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

100 105 110

Val Trp Ala Phe Gly Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu

130 135 140

Lys Glu Ala Ala Val Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly

165 170 175

Val Cys Thr Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys

180 185 190

Leu Val Gly Glu Asn His Leu Val Lys Ile Ala Asp Phe Gly Leu Ser

195 200 205

Arg Leu Met Thr Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys

210 215 220

Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr Gly

225 230 235 240

Asn Leu Leu Asp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gln Leu Ile Thr Gln Leu Met Pro Phe Asp Cys Leu Leu Asp Tyr Val

260 265 270

Arg Glu His Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

275 280 285

Glu Asn His Leu Val Lys Val Ala Asp Leu Gly Leu Ser Arg Leu Met

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val

305 310 315 320

Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met Glu Arg Gly Gly Ser Gly

325 330 335

Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly

340 345 350

Glu Tyr Gly Glu Val Tyr Glu Gly Val Trp Gly Gly Ser Gly Gly Gly

355 360 365

Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr

370 375 380

Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly Ser Gly Gly Gly Gly

385 390 395 400

Ser Gly Gly Leu Thr Ser Thr Val Gln Leu Ile Thr Gln His Met Pro

405 410 415

Phe Gly Cys Leu Leu Asp Tyr Val Lys Lys Lys Lys Lys Gly Gly Ser

420 425 430

Gly Gly Gly Gly Ser Gly Gly Cys Thr Arg Glu Pro Pro Phe Tyr Ile

435 440 445

Ile Asn Glu Phe Met Thr Tyr Gly Asn Leu Leu Asp Lys Lys Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Lys Glu Ala Ala Val Met Lys Glu

465 470 475 480

Ile Arg Gly Gly His Pro Asn Leu Val Gln Leu Leu Gly Val Gly Gly

485 490 495

Ser Gly Gly Gly Gly Ser Gly Gly Lys His Lys Leu Gly Gly Gly Gln

500 505 510

Tyr Gly Lys Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Gly Gly Ser

515 520 525

Gly Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ser Arg

530 535 540

Met Met Thr Gly Asp Thr Tyr Thr Ala His Ala Lys Lys Gly Gly Ser

545 550 555 560

Gly Gly Gly Gly Ser Gly Gly Lys Trp Glu Met Glu Arg Thr Asp Ile

565 570 575

Thr Val Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Gly Ser Gly Gly

580 585 590

Gly Gly Ser Gly Gly Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Tyr

595 600 605

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

610 615 620

Gly Gly Ser Gly Gly Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu

625 630 635 640

Tyr Ile Ile Thr Glu Phe Met Thr Tyr Gly Lys Lys Lys Gly Gly Ser

645 650 655

Gly Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly

660 665 670

Gly Met Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly Ser Gly

675 680 685

Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu

690 695 700

Leu Lys Asp Tyr Arg Met Glu Arg Lys Gly Gly Ser Gly Gly Gly Gly

705 710 715 720

Ser Gly Gly Leu Met Thr Gly Asp Thr Tyr Thr Ala His Pro Gly Ala

725 730 735

Lys Phe Pro Ile Lys Trp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser

740 745 750

Gly Gly Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Met Glu Cys Asn

755 760 765

Arg Gln Glu Val Asn Ala Val Lys Lys Gly Gly Ser Gly Gly Gly Gly

770 775 780

Ser Gly Gly Asn Cys Leu Val Gly Glu Asn His Leu Val Arg Val Ala

785 790 795 800

Asp Phe Gly Leu Ser Arg Leu Met Gly Gly Ser Gly Gly Gly Gly Ser

805 810 815

Gly Gly Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr

820 825 830

Gly Asn Leu Leu Asp Tyr Leu Lys Lys Gly Gly Ser Gly Gly Gly Gly

835 840 845

Ser Gly Gly Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly

850 855 860

Ala Lys Phe Pro Ile Lys Trp Lys Gly Gly Ser Gly Gly Gly Gly Ser

865 870 875 880

Gly Gly Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Ala Ile His Arg

885 890 895

Asp Leu Ala Ala Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile

900 905 910

Thr Met Lys His Lys Leu Gly Gly Gly Arg Tyr Gly Glu Val Tyr Glu

915 920 925

Gly Val Trp Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Thr Leu

930 935 940

Lys Glu Asp Thr Met Glu Val Glu Gly Phe Leu Lys Glu Ala Ala Val

945 950 955 960

Met Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Glu

965 970 975

Glu Phe Leu Lys Glu Ala Ala Phe Met Lys Glu Ile Lys His Pro Asn

980 985 990

Leu Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Met Ala Thr

995 1000 1005

Gln Ile Ser Ser Ala Met Gly Tyr Leu Glu Lys Lys Asn Phe Ile His

1010 1015 1020

Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly Ile Asp

1025 1030 1035 1040

Leu Ser Gln Val Tyr Ala Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg

1045 1050 1055

Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp

1060 1065 1070

Phe Gly Leu Ala Arg Phe Ile Met Ser Asp Ser Asn Tyr Val Val Arg

1075 1080 1085

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys Asp Phe Gly

1090 1095 1100

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

1105 1110 1115 1120

Ser Gly Gly Gly Gly Ser Gly Gly Asp Phe Gly Leu Ala Arg Asp Ile

1125 1130 1135

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

1140 1145 1150

Gly Gly Gly Gly Ser Gly Gly Cys Thr Ile Gly Gly Pro Thr Leu Val

1155 1160 1165

Ile Ile Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Lys Lys Lys Gly

1170 1175 1180

Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Tyr Leu Gly Asn His Met

1185 1190 1195 1200

Asn Ile Val Thr Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro Lys Gly

1205 1210 1215

Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly Ile Asp Leu Ser

1220 1225 1230

Gln Val Tyr Lys Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Gly Gly

1235 1240 1245

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

1250 1255 1260

Ala Arg Tyr Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser

1265 1270 1275 1280

Gly Gly Gly Gly Ser Gly Gly Thr Gln Ile Ser Ser Ala Met Glu Tyr

1285 1290 1295

Leu Ala Lys Lys Asn Phe Ile His Arg Asp Gly Gly Ser Gly Gly Gly

1300 1305 1310

Gly Ser Gly Gly Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Ser Ala

1315 1320 1325

Glu Ile His Gln Ala Phe Glu Thr Met Lys Gly Gly Ser Gly Gly Gly

1330 1335 1340

Gly Ser Gly Gly Trp Ala Phe Gly Val Leu Leu Trp Glu Ile Thr Thr

1345 1350 1355 1360

Tyr Gly Met Ser Pro Tyr Pro Gly Ile Lys Lys Lys Gly Gly Ser Gly

1365 1370 1375

Gly Gly Gly Ser Gly Gly Val Ala Val Lys Thr Leu Lys Glu Asp Ala

1380 1385 1390

Met Glu Val Glu Glu Phe Leu Lys Glu Ala Lys Lys Lys Gly Gly Ser

1395 1400 1405

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

1410 1415 1420

Arg Val Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly Gly Ser Gly

1425 1430 1435 1440

Gly Gly Gly Ser Gly Gly Asp Ser Asn Tyr Val Val Lys Gly Asn Pro

1445 1450 1455

Arg Leu Pro Val Lys Trp Met Ala Gly Gly Ser Gly Gly Gly Gly Ser

1460 1465 1470

Gly Gly Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Lys Asn Asp

1475 1480 1485

Ser Asn Tyr Val Val Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly

1490 1495 1500

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

1505 1510 1515 1520

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

1525 1530 1535

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

1540 1545 1550

Ser Asp Val Ser Leu Thr Ala

1555

<210> 205

<211> 1511

<212> PRT

<213> Artificial Sequence

<400> 205

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Leu Ile

20 25 30

Thr Gln Leu Met Pro Phe Gly Ser Leu Leu Asp Tyr Val Arg Glu His

35 40 45

Lys Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Val Lys Thr

50 55 60

Leu Lys Glu Asp Thr Met Tyr Val Glu Glu Phe Leu Lys Glu Ala Ala

65 70 75 80

Val Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Thr Arg

85 90 95

Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr Gly Asn Leu

100 105 110

Leu Asp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Leu

115 120 125

Ser Gln Val Tyr Glu Leu Leu Gly Lys Asp Tyr Arg Met Glu Arg Lys

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Thr Tyr Thr Ala His

145 150 155 160

Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Lys Lys Gly

165 170 175

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Phe Leu Lys Glu Ala Ala

180 185 190

Val Met Lys Val Ile Lys His Pro Asn Leu Val Gln Leu Leu Lys Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Gln Leu Ile Thr Gln Leu

210 215 220

Met Pro Phe Arg Cys Leu Leu Asp Tyr Val Arg Glu His Lys Lys Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Met Arg Ala Cys Trp

245 250 255

Gln Trp Asn Leu Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gly Gly

260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Glu Ser Leu Ala Tyr Asn Lys Phe

275 280 285

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

290 295 300

Gly Ser Gly Gly Gly Gly Ser Gly Gly His Leu Val Lys Val Ala Asp

305 310 315 320

Phe Gly Met Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Lys Lys Gly

325 330 335

Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val Tyr

340 345 350

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

355 360 365

Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly

370 375 380

Glu Tyr Gly Glu Val Tyr Glu Gly Val Trp Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Val Glu

405 410 415

Phe Met Thr Tyr Gly Asn Leu Leu Asp Lys Lys Ala Val Lys Thr Leu

420 425 430

Lys Glu Asp Thr Met Val Glu Glu Phe Leu Lys Glu Ala Lys Lys Gly

435 440 445

Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys His Lys Leu Gly Gly Gly

450 455 460

Gln Tyr Gly Val Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Gly Gly

465 470 475 480

Ser Gly Gly Gly Gly Ser Gly Gly Lys Thr Leu Lys Glu Asp Thr Met

485 490 495

Ala Val Glu Glu Phe Leu Lys Glu Ala Ala Val Lys Lys Gly Gly Ser

500 505 510

Gly Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ser Arg

515 520 525

Leu Leu Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Lys Gly Gly Ser

530 535 540

Gly Gly Gly Gly Ser Gly Gly Leu Leu Gly Val Cys Thr Arg Glu Pro

545 550 555 560

Ser Phe Tyr Ile Ile Thr Glu Phe Met Thr Tyr Lys Lys Lys Lys Gly

565 570 575

Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu

580 585 590

Gly Gly Gly Lys Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly

595 600 605

Ser Gly Gly Gly Gly Ser Gly Gly Met Thr Gly Asp Thr Tyr Thr Ala

610 615 620

His Ala Arg Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Lys Lys Lys

625 630 635 640

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Glu Ser Leu Ala Tyr

645 650 655

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

660 665 670

Ser Gly Gly Gly Gly Ser Gly Gly Pro Phe Tyr Ile Ile Thr Glu Phe

675 680 685

Met Thr Cys Gly Asn Leu Leu Asp Tyr Leu Arg Lys Lys Lys Gly Gly

690 695 700

Ser Gly Gly Gly Gly Ser Gly Gly Gln Ile Ser Ser Ala Met Glu Tyr

705 710 715 720

Leu Gly Lys Lys Asn Phe Ile His Arg Asp Gly Gly Ser Gly Gly Gly

725 730 735

Gly Ser Gly Gly Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Phe Val

740 745 750

Leu Leu Tyr Met Ala Thr Gln Ile Lys Gly Gly Ser Gly Gly Gly Gly

755 760 765

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

770 775 780

Ala Lys Phe Pro Ile Lys Trp Thr Lys Gly Gly Ser Gly Gly Gly Gly

785 790 795 800

Ser Gly Gly Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His

805 810 815

Arg Asp Leu Ala Ala Arg Asn Gly Gly Ser Gly Gly Gly Gly Ser Gly

820 825 830

Gly Thr Leu Lys Glu Asp Thr Met Glu Val Glu Lys Phe Leu Lys Glu

835 840 845

Ala Ala Val Met Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

850 855 860

Val Glu Glu Phe Leu Lys Glu Ala Ala Ala Met Lys Glu Ile Lys His

865 870 875 880

Pro Asn Leu Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Met

885 890 895

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

900 905 910

Ile His Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly

915 920 925

Ile Asp Leu Ser Gln Val Tyr Gly Leu Leu Glu Lys Asp Tyr Arg Met

930 935 940

Glu Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys

945 950 955 960

Asp Phe Gly Leu Ala Arg His Ile Met Ser Asp Ser Asn Tyr Val Val

965 970 975

Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys Asp Phe

980 985 990

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

995 1000 1005

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

1010 1015 1020

Gly Ser Asn Tyr Val Val Lys Gly Asn Gly Gly Ser Gly Gly Gly Gly

1025 1030 1035 1040

Ser Gly Gly Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Glu Glu Tyr

1045 1050 1055

Cys Cys Tyr Gly Asp Leu Leu Asn Lys Lys Lys Gly Gly Ser Gly Gly

1060 1065 1070

Gly Gly Ser Gly Gly Leu Ser Tyr Leu Gly Asn His Met Asn Ile Ala

1075 1080 1085

Asn Leu Leu Gly Ala Cys Thr Ile Gly Lys Lys Gly Gly Ser Gly Gly

1090 1095 1100

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

1105 1110 1115 1120

Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser Gly Gly Gly

1125 1130 1135

Gly Ser Gly Gly Ser Phe Ala Glu Ile His Gln Ala Phe Glu Arg Met

1140 1145 1150

Phe Gln Glu Ser Ser Ile Ser Asp Glu Lys Lys Lys Gly Gly Ser Gly

1155 1160 1165

Gly Gly Gly Ser Gly Gly Ser Leu Thr Val Ala Val Lys Thr Leu Lys

1170 1175 1180

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

1185 1190 1195 1200

Gly Gly Ser Gly Gly Thr Glu Phe Met Thr Tyr Gly Asn Leu Leu Gly

1205 1210 1215

Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val Lys Gly Gly Ser Gly Gly

1220 1225 1230

Gly Gly Ser Gly Gly Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe

1235 1240 1245

Gly Glu Val Tyr Glu Gly Val Trp Lys Lys Gly Gly Ser Gly Gly Gly

1250 1255 1260

Gly Ser Gly Gly Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met

1265 1270 1275 1280

Thr Gly Asp Thr Tyr Thr Ala His Ala Lys Lys Lys Gly Gly Ser Gly

1285 1290 1295

Gly Gly Gly Ser Gly Gly Val Met Lys Glu Ile Lys His Pro Asn Leu

1300 1305 1310

Leu Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Gly Gly Ser Gly Gly

1315 1320 1325

Gly Gly Ser Gly Gly Glu Arg Glu Ala Leu Met Ser Glu Leu Glu Val

1330 1335 1340

Leu Ser Tyr Leu Gly Asn His Met Asn Lys Lys Gly Gly Ser Gly Gly

1345 1350 1355 1360

Gly Gly Ser Gly Gly Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Phe

1365 1370 1375

Lys Glu Ser Ser Cys Ser Asp Ser Thr Gly Gly Ser Gly Gly Gly Gly

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

Val Lys Gly Asn Ala Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly

1445 1450 1455

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

1460 1465 1470

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

1475 1480 1485

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

1490 1495 1500

Ser Asp Val Ser Leu Thr Ala

1505 1510

<210> 206

<211> 625

<212> PRT

<213> Artificial Sequence

<400> 206

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Trp

20 25 30

Glu Met Glu Arg Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly

35 40 45

Gln Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Leu Thr

50 55 60

Val Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe

65 70 75 80

Leu Lys Glu Ala Ala Val Met Lys Glu Ile Lys His Pro Asn Leu Val

85 90 95

Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr

100 105 110

Glu Phe Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn

115 120 125

Arg Gln Glu Val Asn Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile

130 135 140

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

145 150 155 160

Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Asn His Leu Val Lys Val

165 170 175

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

180 185 190

His Ala Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu

195 200 205

Ala Tyr Asn Lys Phe Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val

210 215 220

Leu Leu Trp Glu Ile Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile

225 230 235 240

Asp Leu Ser Gln Val Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu

245 250 255

Arg Pro Glu Gly Cys Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys

260 265 270

Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gln

275 280 285

Ala Phe Glu Thr Met Phe Gln Glu Ser Ser Ile Ser Asp Gly Gly Ser

290 295 300

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

305 310 315 320

Arg Asp Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser Gly

325 330 335

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

340 345 350

Asp Glu Ala Tyr Val Met Ala Ser Val Asp Lys Gly Gly Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr

370 375 380

Gln Leu Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys

385 390 395 400

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe

405 410 415

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

420 425 430

Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Arg Glu Ala Leu Met Ser

435 440 445

Glu Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn

450 455 460

Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu

465 470 475 480

Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Gly Gly Ser Gly Gly Gly Gly

485 490 495

Ser Gly Gly Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Ser Lys Glu

500 505 510

Ser Ser Cys Ser Asp Ser Thr Gly Gly Ser Gly Gly Gly Gly Ser Gly

515 520 525

Gly Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Asp Ser

530 535 540

Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala

545 550 555 560

Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala

565 570 575

Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr

580 585 590

Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser

595 600 605

Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr

610 615 620

Ala

625

<210> 207

<211> 244

<212> PRT

<213> Artificial Sequence

<400> 207

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Gln

20 25 30

Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys Gln Leu

35 40 45

Pro Gln Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Met Lys Cys Lys

50 55 60

Asn Val Val Pro Leu Tyr Gly Leu Leu Leu Glu Met Leu Asp Ala His

65 70 75 80

Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly His Glu Cys Asn

85 90 95

Ser Pro Tyr Ile Val Gly Leu Tyr Gly Ala Phe Tyr Ser Asp Gly Glu

100 105 110

Ile Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Ser Met

115 120 125

Lys Cys Lys Asn Val Val Pro His Tyr Asp Leu Leu Leu Glu Met Leu

130 135 140

Asp Ala Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Val

145 150 155 160

Ala Asp Phe Gly Leu Ala Arg Val Met Tyr Asp Lys Glu Tyr Tyr Ser

165 170 175

Val His Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly

180 185 190

Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val

195 200 205

Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly

210 215 220

Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val

225 230 235 240

Ser Leu Thr Ala

<210> 208

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 208

Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Gln

20 25 30

Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu

35 40 45

Pro Gln Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Ser Met Lys

50 55 60

Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp

65 70 75 80

Ala His Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly His Glu

85 90 95

Cys Asn Ser Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp

100 105 110

Gly Glu Ile Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val

115 120 125

Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr

130 135 140

Tyr Ser Val His Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

145 150 155 160

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

165 170 175

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

180 185 190

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

195 200 205

Asp Val Ser Leu Thr Ala

210

Claims (10)

1. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that be directed to targeted drug vismodegib, polypeptide Vaccine combination is for following mutation: SMO-A459V, SMO-C469Y, SMO-D473G, SMO-D473H, SMO-D473Y, SMO- F460L, SMO-G497W, SMO-H231R, SMO-Q477E, SMO-T241M, SMO-V321A, SMO-V321M, SMO-W281L, SMO-W535L, SMO-W535R.

2. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that replace Buddhist nun, polypeptide according to Shandong for targeted drug Vaccine combination is for following mutation: BTK-C481F, BTK-C481R, BTK-C481S, BTK-C481Y, BTK-T316A.

3. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that by targeted drug abiraterone, En Zhalu Amine, Flutamide, ketoconazole gather for one kind,

The polypeptide vaccine combination of abiraterone is for following mutation: AR-T878A, AR-T878S；

The polypeptide vaccine combination of En Zhalu amine is for following mutation: AR-F877L, AR-T878A；

The polypeptide vaccine combination of Flutamide is for following mutation: AR-T878A, AR-T878S, AR-V716M；

The polypeptide vaccine of ketoconazole is for following mutation: AR-T878A.

4. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that by targeted drug dabrafenib, pyridine aldoxime methyliodide (PAM) list Anti-, Wei Luofeini, department's beauty are gathered for Buddhist nun for one kind,

The polypeptide vaccine combination of dabrafenib is for following mutation: MAP2K1-P124S, MAP2K2-Q60P, NRAS-G12D, NRAS-Q61R；

The polypeptide vaccine of pyridine aldoxime methyliodide (PAM) monoclonal antibody is for following mutation: NRAS-Q61R；

The polypeptide vaccine combination of Wei Luofeini is for following mutation: MAP2K1-G128V, MAP2K1-P124L, MAP2K1-Q56P, NRAS-Q61R；

Department's beauty is directed to following mutation: MAP2K1-P124L for the polypeptide vaccine of Buddhist nun.

5. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that by targeted drug A Lei for Buddhist nun, color is auspicious replaces Buddhist nun, gram azoles gather for Buddhist nun for one kind,

A Lei is mutated for the polypeptide vaccine of Buddhist nun for following: ALK-G1202R；

The polypeptide vaccine combination of Ceritinib is for following mutation: ALK-D1203N, ALK-F1174V, ALK-G1123S, ALK- G1202R, ALK-G1269A, ALK-L1196M；

Gram azoles for Buddhist nun polypeptide vaccine combination for following mutation: ALK-F1174L, ALK-F1174V, ALK-G1202R, ALK- G1269A, ALK-I1171T, ALK-L1196M, MET-D1246H, MET-D1246N.

6. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that replace targeted drug Afatinib, rich relax Buddhist nun, Dasatinib, Tarceva, Gefitinib, Lip river difficult to understand replace Buddhist nun for Buddhist nun, Imatinib, nilotinib, difficult to understand this, Sorafenib, relax Buddhist nun, which pricks for Buddhist nun, tyrosine kinase inhibitors drug, special watt for Buddhist nun, Kui, to be gathered for Buddhist nun for one kind,

The polypeptide vaccine of Afatinib is for following mutation: EGFR-T790M；

The polypeptide vaccine combination of bosutinib is for following mutation: ABL1-F359V, ABL1-V299L；

The polypeptide vaccine combination of Dasatinib is for following mutation: ABL1-E255K, ABL1-E255K, ABL1-F317L, ABL1- F359V, ABL1-T315A, ABL1-T315I, ABL1-V299L；

The polypeptide vaccine of Tarceva is for following mutation: EGFR-T790M；

The polypeptide vaccine combination of Gefitinib is for following mutation: EGFR-D761Y, EGFR-T790M；

Ao Luo is mutated for the polypeptide vaccine of Buddhist nun for following: EGFR-C797S；

The polypeptide vaccine combination of Imatinib is for following mutation: ABL1-A397P, ABL1-A399T, ABL1-A433T, ABL1- E255K, ABL1-E255V, ABL1-E275K, ABL1-E279A, ABL1-E279K, ABL1-E279Y, ABL1-E279Z, ABL1- E282G, ABL1-E292V, ABL1-E352D, ABL1-E352G, ABL1-E355A, ABL1-E355G, ABL1-E450A, ABL1- E450G, ABL1-E450K, ABL1-E453A, ABL1-E453G, ABL1-E453K, ABL1-E453L, ABL1-F311L, ABL1- F317L, ABL1-F359A, ABL1-F359V, ABL1-F382L, ABL1-F486S, ABL1-G398R, ABL1-H396P, ABL1- H396R, ABL1-I418V, ABL1-K294 > RGG, ABL1-K378R, ABL1-K419E, ABL1-L298V, ABL1-L384M, ABL1-L387F, ABL1-L387M, ABL1-M244V, ABL1-M388L, ABL1-N374Y, ABL1-P480L, ABL1-Q252E, ABL1-Q252H, ABL1-Q252K, ABL1-Q252R, ABL1-R328M, ABL1-S417Y, ABL1-T277A, ABL1-T315I, ABL1-T315N, ABL1-V289F, ABL1-V299L, ABL1-V379I, ABL1-Y253F, ABL1-Y320C, KIT-A829P, KIT-D816A, KIT-D816E, KIT-D816H, KIT-D820G, KIT-D820V, KIT-D820Y, KIT-K642E, KIT- S709F, KIT-S821F, KIT-T670E, KIT-T670I, KIT-V654A, PDGFRA-D842V；

The polypeptide vaccine combination of nilotinib is for following mutation: ABL1-E255K, ABL1-E255V, ABL1-H396R, ABL1- T315I, ABL1-T315V, KIT-N655T；

Ao Si is mutated for the polypeptide vaccine combination of Buddhist nun for following: EGFR-C797S, EGFR-G796D, EGFR-G796R, EGFR- G796S, EGFR-L792H, KIT-T790M；

The polypeptide vaccine combination of Sorafenib is for following mutation: FLT3-D835H, FLT3-D835Y；

The polypeptide vaccine combination of Sutent is for following mutation: FLT3-D835Y, PDGFRA-D842V；

The polypeptide vaccine combination of tyrosine kinase inhibitors drug is for following mutation: ABL1-A399T, ABL1-D325G, ABL1-E255K, ABL1-E255V, ABL1-E279K, ABL1-E282K, ABL1-E355G, ABL1-E453K, ABL1-F311L, ABL1-F317L, ABL1-F359V, ABL1-H396P, ABL1-H396R, ABL1-L298V, ABL1-L384M, ABL1-L387F, ABL1-L387M, ABL1-M244V, ABL1-M388L, ABL1-P310S, ABL1-Q252H, ABL1-Q252M, ABL1-T315A, ABL1-T315I, ABL1-T495R, ABL1-V289A, ABL1-V299L, ABL1-V338F, ABL1-V379I, ABL1-Y253F, EGFR-T790M；

Te Wa is mutated for the polypeptide vaccine of Buddhist nun for following: EGFR-T790M；

Kui Zha is mutated for the polypeptide vaccine combination of Buddhist nun for following: FLT3-D835Y, FLT3-D842V, FLT3-D835Y.

7. being directed to the polypeptide vaccine in tumor-targeting drug drug resistance site, which is characterized in that targeted drug everolimus, thunder pa is mould Element, mek inhibitor PD0325901, C-MET inhibitor Savolitinib gather for one kind,

The polypeptide vaccine of everolimus is for following mutation: MTOR-F2108L；

The polypeptide vaccine of rapamycin is for following mutation: MTOR-F2108L；

The polypeptide vaccine of mek inhibitor PD0325901 is for following mutation: MAP2K1-F129L；

The polypeptide vaccine of C-MET inhibitor Savolitinib is for following mutation: MET-D1246V.

8. the design method of the polypeptide vaccine for tumor-targeting drug drug resistance site, which comprises the steps of:

Find the data of targeted drug medicament-resistant mutation data；

Intercept medicament-resistant mutation polypeptide sequence and prediction MHC molecule affinity and immunogenicity:

The polypeptide sequence that covering 16 amino acid of mutational site upstream and downstream is intercepted for point mutation, forward for frameshift mutation interception Extend the amino acid of 16 length, the saltant type of the polypeptide sequence that extends back until terminator codon as resistant mutational site Polypeptide, while the corresponding wild polypeptide sequence in corresponding position is intercepted,

It uses at least one database as source statistic high frequency HLA parting and frequency, makees after the HLA counted is merged duplicate removal For the candidate HLA parting for prediction,

The corresponding polypeptide in these mutational sites and HLA molecule binding affinity are analyzed using a plurality of softwares, comprehensive a plurality of softwares will Affinity is divided into three classes: strong affinity-SB, weak affinity-WB and determining it without affinity, and compared with versus wild type polypeptide Affinity variation,

The variation of A class is to become having strong affinity from no affinity,

The variation of B class is to become weak affinity from no affinity,

The variation of C class is to become strong affinity from weak affinity,

D class variation be it is unchanged,

Internal sort thinks that A class is better than D class better than C class better than B class,

Its immunogenicity is predicted using immunogenicity forecasting tool, and reservation mutant polypeptides affinity is strong, affinity changes greatly simultaneously And the epitope that immunogenicity is strong；

Targeted drug and drug resistance site correlation and drug cluster:

It gives a mark to drug resistance site affinity:

Comprehensively consider the epitope number and each epitope affinity variation size that there be affinity in site, parents different to tetra- class of A, B, C, D Corresponding weight is given with power variation, weight is given according to the corresponding HLA frequency size of each epitope, by each epitope in site Cumulative summation；

AC: affinity changes size, and different weights is given in the variation of tetra- class difference affinity of A, B, C, D；

F_hla: corresponding HLA frequency；

N: each epitope number in site；

Between comprehensive analysis targeted drug and drug resistance site correlation combination targeted drug mechanism of action to targeted drug cluster from And it is classified as 7 classes:

The first kind generates drug resistant Hedgehog signal path antagonist vismodegib for SMO mutation,

Second class replaces Buddhist nun according to Shandong for BTK mutation generation is drug resistant,

Third class is directed to some antiandrogens such as abiraterone etc. of AR medicament-resistant mutation,

4th class is directed to some luxuriant and rich with fragrance Buddhist nun's class drugs of BRAF medicament-resistant mutation,

5th class is directed to some tyrosine kinase inhibitors of the fusions such as ALK, MET,

6th class is directed to the tyrosine kinase inhibitor of EGFR access,

7th class is for kinase inhibitors such as the everolimus of PI3K/AKT/mTOR access；

Design corresponding vaccine polypeptide sequence.

9. the design method of the polypeptide vaccine according to claim 8 for tumor-targeting drug drug resistance site, feature It is, intercepts medicament-resistant mutation polypeptide sequence and prediction MHC molecule affinity and immunogenicity:

The polypeptide sequence that covering 16 amino acid of mutational site upstream and downstream is intercepted for point mutation, forward for frameshift mutation interception Extend the amino acid of 16 length, the saltant type of the polypeptide sequence that extends back until terminator codon as resistant mutational site Polypeptide, while the corresponding wild polypeptide sequence in corresponding position is intercepted,

It uses at least one database as source statistic high frequency HLA parting and frequency, makees after the HLA counted is merged duplicate removal For the candidate HLA parting for prediction, the database includes: public database, clinical patients database,

The corresponding polypeptide in these mutational sites and HLA molecule binding affinity are analyzed using a plurality of softwares, comprehensive a plurality of softwares will Affinity is divided into three classes: strong affinity-SB, weak affinity-WB and determining it without affinity, and compared with versus wild type polypeptide Affinity variation, a plurality of softwares include: this three sections of softwares of netMHCpan, netMHC and Pickpocket,

The variation of A class is to become having strong affinity from no affinity,

The variation of B class is to become weak affinity from no affinity,

The variation of C class is to become strong affinity from weak affinity,

D class variation be it is unchanged,

Internal sort thinks that A class is better than D class better than C class better than B class,

Its immunogenicity is predicted using immunogenicity forecasting tool, and reservation mutant polypeptides affinity is strong, affinity changes greatly simultaneously And the epitope that immunogenicity is strong.

10. the design method of the polypeptide vaccine according to claim 8 for tumor-targeting drug drug resistance site, feature It is, network is made to all targeted drugs and drug resistance site using cytoscape, it is affine that the size in drug resistance site represents its Power goals for, correlation combination targeted drug mechanism of action is poly- to targeted drug between comprehensive analysis targeted drug and drug resistance site Class is to be classified as 7 classes:

The first kind generates drug resistant Hedgehog signal path antagonist vismodegib for SMO mutation,

Second class replaces Buddhist nun according to Shandong for BTK mutation generation is drug resistant,

Third class is directed to some antiandrogens such as abiraterone etc. of AR medicament-resistant mutation,

4th class is directed to some luxuriant and rich with fragrance Buddhist nun's class drugs of BRAF medicament-resistant mutation,

5th class is directed to some tyrosine kinase inhibitors of the fusions such as ALK, MET,

6th class is directed to the tyrosine kinase inhibitor of EGFR access,

7th class is for kinase inhibitors such as the everolimus of PI3K/AKT/mTOR access.