CN107043422A - Anticomplement factor D humanized Fab and humanized antibody and application thereof - Google Patents

Anticomplement factor D humanized Fab and humanized antibody and application thereof Download PDF

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CN107043422A
CN107043422A CN201710041945.1A CN201710041945A CN107043422A CN 107043422 A CN107043422 A CN 107043422A CN 201710041945 A CN201710041945 A CN 201710041945A CN 107043422 A CN107043422 A CN 107043422A
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val
amino acid
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CN107043422B (en
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柯潇
雷刚
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Chengdu Kanghong Biotechnologies Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention discloses the humanized Fab of anticomplement factor D a kind of and humanized antibody and application thereof, the humanized Fab and humanized antibody contain light chain variable district and weight chain variable district, and the purposes behaviour Complement Factor D relevant disease is to raise caused disease by people's Complement Factor D.

Description

Anticomplement factor D humanized Fab and humanized antibody and application thereof
Technical field
The present invention relates to biological technical field, and in particular to a kind of anticomplement factor D humanized Fab and humanized antibody And application thereof.
Background technology
Senile macular degeneration is also known as AMD (AMD), is a kind of cell by foveal region of retina area Or disease caused by tissue deterioration and blood vessel hyperplasia, two kinds of dryness AMD and moist AMD can be divided into.Senile macular degeneration master Show as retinal pigment epithelium to decline acromere disk film phagocytosis digestion power, as a result make not digested completely Disk film residual body retention in basal part cell magma, and to extracellular discharge, be deposited on Bruch films, form glass-film Wart.Due to macula area structure and particularity functionally, such a change becomes apparent.Glass-film wart also sees the old of twenty-twenty vision After year people, but thus secondary a variety of pathological changes, then cause macular degeneration.Or cause this fracture of Bruch films, arteries and veins Network film capillary is entered under RPE and under neuroepithelium of retina by the Bruch films of rupture, forms CNV. Due to the textural anomaly of new vessels wall, cause seepage and the bleeding of blood vessel, and then trigger a series of Secondary cases pathological change. Senile macular degeneration is betided more than 45 years old mostly, and its illness rate increases and increased with the age, is current the elderly's blinding Important diseases.AMD blindings account for the 8.7% of global blinding, and the whole world there are about 30,000,000 AMD patients, there are about every year 500000 people because AMD and blinding.The AMD of the annual report of the U.S. 2004 illness rate is 1.47%, there are about 1,750,000 AMD patients, and to the year two thousand twenty, is pushed away Survey by have up to 3,000,000 AMD patient.One of the works such as China Zou Hai Dong investigates display, the crowd's more than more than 50 years old AMD illness rate is 15.5%.Wherein moist AMD accounts for 11.9%.According to the demographic data data of the United Nations, China was in 2005 Population more than more than 50 years old is close to 300,000,000.With aging population, AMD illness rate will be dramatically increased.
Complement is to be present in normal person and animal blood serum and one group of activated rear albumen with enzymatic activity in tissue fluid Matter.Its effect can aid in and supplement specific antibody, mediated immunity bacteriolyze, haemocylolysis.Complement system is to plant solvable by more than 30 Property albumen, membrane-binding albumen and complement receptors composition polymolecular system, according to its biological function, complement can be divided into intrinsic Composition, complement regulatory composition and the class of complement receptors (CR) three, the regulation and control of complement system mainly have three approach：Classical pathway (Classical pathway), alternative pathway (Alternative pathway) and agglutinin (MBL) approach (Lectin Pathway), different regulatory pathways can influence to produce different complement proteins, and play different effects.Complement system anon-normal Often activation can usually cause the overreaction of human immune system, cause the damage of human body itself normal structure.Complement system can To cause local inflammation, tissue damage and the up-regulation of angiogenesis factor, the disease related to excess complement activation has class Rheumatic arthritis, senile dementia, systemic loupus erythematosus, ischemic damage and reperfusion, acute myocardial infarction AMI, acute respiratory distress Syndrome etc..Recent studies suggest that, alternative pathway (Alternative pathway) and agglutinin (MBL) approach (Lectin Pathway) be antibody-mediated reaction discrete actuation mechanism, be topmost mechanism of causing a disease in the AMD causes of disease [Paul A.et, al.The Immune System and AMD,Curr Ophthalmol Rep(2014)2:14-19], wherein Complement Factor D (complement factor D, EC:3.4.21.46 it is) to start the important component that alternative route is activated, belongs to trypsase man Race.Complement Factor D mainly exists in serum with activated form, and the active D factors (D) act on C3bB, Ke Neng Factor B is cracked into two fragments of Ba and Bb at the arginine-lysine key of 234-235, so as to start alternative route Cascade activation reacts.
Anticomplement D antibody has species specificity, anticomplement factor antibody (AFD in the prior art；FCFD4514S； Lampalizumab) for the alternative pathway of complement (alternative complement pathway, abbreviation AP) of dog or sheep There is faint suppression, and do not suppress for the AP of rabbit, rat, mouse, cavy, hamster or pig, and have for people or monkey Corresponding stronger suppression (Kelly M, et, al.Complement Inhibition in Cynomolgus Monkeys by Anti-Factor D Antigen-Binding Fragment for the Treatment of an Advanced Form of Dry Releated Macular Degeneration,J Pharmacol Exp Ther 351:527-537, December 2014)。
The content of the invention
An object of the present invention is to provide humanized antibodies' binding fragment Fab or anti-human complements of anti-human Complement Factor D Factor D humanized antibody, the Fab or antibody have preferable affinity, with more preferable stability and biology with people's Complement Factor D Activity.
One aspect of the present invention provides a kind of anticomplement factor D humanized antibody or its antigen-binding fragment Fab, containing light Chain variable region and weight chain variable district, wherein light chain variable district contain amino acid sequence ITSTDIDDDX1N, amino acid sequence GGX2TLRP and amino acid sequence X3QSDS X4PYX5, the X1For V, M or L, X2For N or A, X3For M or L, X4For L or Y, X5For I or T；Weight chain variable district contains amino acid sequence 1, sequence 2 and amino acid sequence Y1GGY2NY3, the Y1For A or E, Y2For S, V Or P, Y3For N, Y, F, H, S or R；X1、X2、X3、X4、X5Asynchronously meet following conditions：X1For M, X2For N, X3For L, X4For L, X5 For T.
Described humanized antibody or its antigen-binding fragment Fab, preferably described X1For M, X2For A, X3For L, X3For L and X5 During for T, Y1It is not A, Y2It is not V and Y3It is not N or Y1It is not E, Y2It is not P and Y3It is not N.X1、X2、X3、X4、Y1、Y2And Y3Together When meet above-mentioned condition.
The humanized antibody or its antigen-binding fragment Fab, preferably described X1For V, X2For N, X3For L, X4For L and X5For During I, Y1It is not E, Y2It is not V and Y3It is not S.X1、X2、X3、X4、X5、Y1、Y2And Y3Meet above-mentioned condition simultaneously.
The humanized antibody or its antigen-binding fragment Fab, preferably described X1For V, X2For N, X3For L, X4For Y and X5For During T, Y1It is not E, Y2It is not V and Y3It is not N or H, or Y1It is not E, Y2It is not P and Y3It is not N.X1、X2、X3、X4、X5、Y1、Y2With Y3Meet above-mentioned condition simultaneously.
The humanized antibody or its antigen-binding fragment Fab, preferably described X1For V, X2For N, X3For M, X4For L and X5For During T, Y1It is not E, Y2It is not P and Y3It is not N.X1、X2、X3、X4、X5、Y1、Y2And Y3Meet above-mentioned condition simultaneously.
The humanized antibody or its antigen-binding fragment Fab, preferably described X1For V, X2For A, X3For L, X4For L and X5For During T, Y1It is not E, Y2It is not V and Y3It is not N.X1、X2、X3、X4、X5、Y1、Y2And Y3Meet above-mentioned condition simultaneously.
The humanized antibody or its antigen-binding fragment Fab, preferably described X1For L, X2For A, X3For L, X4For L and X5For During T, Y1It is not E, Y2It is not V and Y3It is not N.X1、X2、X3、X4、X5、Y1、Y2And Y3Meet above-mentioned condition simultaneously.
The humanized antibody or its antigen-binding fragment Fab of the anticomplement factor D, comprising following weight chain variable district and gently Chain variable region sequence：The amino acid sequence of the light chain variable district is any in sequence 3,5,8,13,15,17 in sequence table Kind；The amino acid sequence of the weight chain variable district is sequence 19,21,23,25,27,29,31, any of 33 in sequence table.
Described anticomplement factor D humanized antibody or its antigen-binding fragment Fab, it is characterised in that the antigen Binding fragment Fab is selected from following any：
KH001：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH002：The amino acid sequence of the light chain variable district is sequence 8, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH003：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 21 in sequence table；
KH004：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 31 in sequence table；
KH005：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 19 in sequence table；
KH006：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 23 in sequence table；
KH007：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 25 in sequence table；
KH008：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 27 in sequence table；
KH009：The amino acid sequence of the light chain variable district is sequence 3, the amino of the weight chain variable district in sequence table Acid sequence is sequence 29 in sequence table；
KH010：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH011：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH012：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 21 in sequence table；
KH013：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 31 in sequence table；
KH014：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 23 in sequence table；
KH015：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 25 in sequence table；
KH016(XY1252)：The amino acid sequence of the light chain variable district is sequence 13, the weight chain variable in sequence table The amino acid sequence in area is sequence 29 in sequence table；
KH017：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 31 in sequence table；
KH018：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 19 in sequence table；
KH019：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 23 in sequence table；
KH020：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 25 in sequence table；
KH021：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 27 in sequence table；
KH022：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 29 in sequence table；
KH024：The amino acid sequence of the light chain variable district is sequence 17, the amino of the weight chain variable district in sequence table Acid sequence is sequence 19 in sequence table；
KH027：The amino acid sequence of the light chain variable district is sequence 5, the amino of the weight chain variable district in sequence table Acid sequence is sequence 31 in sequence table；
KH028：The amino acid sequence of the light chain variable district is sequence 5, the amino of the weight chain variable district in sequence table Acid sequence is sequence 25 in sequence table.
The CH1 of the Fd fragments of the Fab can be any type (IgG, IgA, IgM, IgE, IgD) or subclass (IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2) people source constant region CH1；The constant region of light chain can be any type The people source constant region of (κ types or λ types), hypotype (λ 1, λ 2, λ 3, λ 4) or different in nature (κ m (1), κ m (2), κ m (3)) light chain of the same race.
The Fab is made up of heavy chain fragment and light chain, and the heavy chain fragment is made up of weight chain variable district and heavy chain constant region, The light chain is made up of light chain variable district and constant region of light chain.The amino acid sequence of wherein described heavy chain constant region is preferably anti-with people The amino acid sequence of body heavy chain constant region is identical, the amino acid sequence of the constant region of light chain preferably with human antibody light chain constant region Amino acid sequence is identical；The amino acid sequence of wherein described heavy chain constant region is sequence 39；The amino acid of the constant region of light chain Sequence is sequence 41.
In the humanized antibody of the anticomplement factor D, the heavy chain constant region can be any type (IgG, IgA, IgM, IgE, IgD) or subclass (IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2) people source constant region；It is described Constant region of light chain can be any type (κ types or λ types), hypotype (λ 1, λ 2, λ 3, λ 4) or the opposite sex (κ m (1), κ m (2), κ m of the same race (3)) the people source constant region of light chain.
The antibody is made up of heavy chain or light chain, amino acid sequence and the human antibody heavy chain's constant region of the heavy chain constant region Amino acid sequence it is identical, the amino acid sequence of the constant region of light chain is identical with human antibody light chain constant region amino acid sequence. The amino acid sequence of human antibody heavy chain's constant region is preferably sequence 43, and the human antibody light chain constant region amino acid sequence is excellent Elect sequence 41 as.
The present invention further provides and is selected from following any antibody：
KH-A：Its sequence of light chain is sequence 45, and its sequence of heavy chain is sequence 47；
KH-B：Its sequence of light chain is sequence 49, and its sequence of heavy chain is sequence 51；
KH-C：Its sequence of light chain is sequence 45, and its sequence of heavy chain is sequence 53；
KH-D：Its sequence of light chain is sequence 49, and its sequence of heavy chain is sequence 55；
KH-E：Its sequence of light chain is sequence 57, and its sequence of heavy chain is sequence 59；
KH-F：Its sequence of light chain is sequence 57, and its sequence of heavy chain is sequence 61.
The present invention also provides a kind of polynucleotides, and it encodes any of the above-described humanized antibody or antigen-binding fragment Fab。
Above-mentioned polynucleotides, the light chain variable of antibody described in preferably described humanized antibody or antigen-binding fragment Fab The coded sequence in area is any of sequence 4, sequence 6, sequence 9, sequence 14, sequence 16 and sequence 18 in sequence table, heavy chain The coded sequence of variable region is sequence 20, sequence 22, sequence 24, sequence 26, sequence 28, sequence 30, sequence 32, sequence in sequence table Any of row 34.
The coded sequence of preferred Fab heavy chain constant region is as shown in sequence 40 in sequence table in above-mentioned polynucleotides； The constant region of light chain coded sequence of the Fab is as shown in sequence 42 in sequence table；The code sequence of the heavy chain constant region of the antibody Row are as shown in sequence 44 in sequence table；The coded sequence of the constant region of light chain of the antibody is as shown in sequence 42 in sequence table.
The present invention is selected from following any polynucleotides with further providing for：
KH-A：Its sequence of light chain is sequence 46, and its sequence of heavy chain is sequence 48；
KH-B：Its sequence of light chain is sequence 50, and its sequence of heavy chain is sequence 52；
KH-C：Its sequence of light chain is sequence 46, and its sequence of heavy chain is sequence 54；
KH-D：Its sequence of light chain is sequence 50, and its sequence of heavy chain is sequence 56；
KH-E：Its sequence of light chain is sequence 58, and its sequence of heavy chain is sequence 60；
KH-F：Its sequence of light chain is sequence 58, and its sequence of heavy chain is sequence 62.
The present invention furthermore provides above-mentioned humanized antibody or antigen-binding fragment Fab, or above-mentioned polynucleotides use On the way, it is selected from following any purposes：
D1) the purposes in prevention and/or treatment people's Complement Factor D relevant disease medicine is prepared, or
D2 the purposes in neutralizing people's Complement Factor D product) is being prepared, or
D3) the purposes in the kit for preparing qualitative or quantitative detection people's Complement Factor D.
It is that caused disease is raised by people's Complement Factor D the present invention relates to people's Complement Factor D (CFD) relevant disease；It is preferred that Rheumatoid arthritis, myocardial infarction, reperfusion injury or illness in eye；Wherein illness in eye be more preferably AMD or Diabetic retinopathy.Wherein senile macular degeneration (AMD) includes nonexudativeage (such as mid-term dryness AMD or ground pattern Atrophy (GA)) and exudative (such as moist AMD (choroidal neovascular formation (CNV)) AMD, diabetic retinopathy (DR), eye Interior inflammation (endophthalmitis) and uveitis.In one example, the complement associated eye condition is mid-term dryness AMD.
The present invention still further provides a kind of pharmaceutical composition, and containing auxiliary material and active component, the active component contains There are at least one of following substances：Any of the above-described humanized antibody or antigen-binding fragment Fab, or it is any of the above-described described Polynucleotides；The auxiliary material is pharmaceutically acceptable carrier or excipient.
The present invention furthermore provides the humanized Fab of the anticomplement factor D or the production method of antibody, specific steps For：By the recombinant vector transfection containing the antibody-encoding genes or conversion host cell, expressed under optimum conditions, pure Change.Wherein, the host cell is eukaryotic or prokaryotic, and preferably mammalian cell, bacterium, yeast, insect is thin Born of the same parents etc..It is thin that wherein described mammalian cell includes but is not limited to 293 cells, Chinese hamster ovary celI, SP20 cells, NS0 cells, COS Born of the same parents, bhk cell or PerC6 cells etc., wherein the method for the transfectional cell includes but is not limited to：Electroporation, it is liposome-mediated Method, calcium mediated method etc..
A kind of preferably described antibody expression mode is that recombinant vector is carried out in the host cell of stable transfection Gene magnification, to improve the expression quantity of corresponding recombinant protein, such as with the recombinant vector containing dihyrofolate reductase (DHFR) Stable transfection lacks after DHFR host cell, and the concentration of amethopterin (MTX) can be added in cell culture fluid to expand weight Number of copies of the group carrier in host cell.
After antibody expression containing the coding gene sequence, enzyme linked immunosorbent assay (ELISA) or other method can be used Determine the protein concentration in nutrient solution.For Fab fragments, it can be purified with Protein G affinity chromatographies；IgG albumen can use Protein A affinity chromatographies are purified.
Correlated series correspondence is as follows：
KH023：The amino acid sequence of the light chain variable district is sequence 17, the amino of the weight chain variable district in sequence table Acid sequence is sequence 21 in sequence table；
KH025：The amino acid sequence of the light chain variable district is sequence 17, the amino of the weight chain variable district in sequence table Acid sequence is sequence 25 in sequence table；
KH026：The amino acid sequence of the light chain variable district is sequence 5, the amino of the weight chain variable district in sequence table Acid sequence is sequence 21 in sequence table；
KH029：The amino acid sequence of the light chain variable district is sequence 5, the amino of the weight chain variable district in sequence table Acid sequence is sequence 19 in sequence table；
KH030：The amino acid sequence of the light chain variable district is sequence 5, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH031：The amino acid sequence of the light chain variable district is sequence 7, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH032：The amino acid sequence of the light chain variable district is sequence 10, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH033：The amino acid sequence of the light chain variable district is sequence 11, the amino of the weight chain variable district in sequence table Acid sequence is sequence 35 in sequence table；
KH034：The amino acid sequence of the light chain variable district is sequence 11, the amino of the weight chain variable district in sequence table Acid sequence is sequence 36 in sequence table；
KH035：The amino acid sequence of the light chain variable district is sequence 12, the amino of the weight chain variable district in sequence table Acid sequence is sequence 35 in sequence table；
KH036：The amino acid sequence of the light chain variable district is sequence 17, the amino of the weight chain variable district in sequence table Acid sequence is sequence 33 in sequence table；
KH037：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 19 in sequence table；
KH038：The amino acid sequence of the light chain variable district is sequence 13, the amino of the weight chain variable district in sequence table Acid sequence is sequence 27 in sequence table；
KH039：The amino acid sequence of the light chain variable district is sequence 15, the amino of the weight chain variable district in sequence table Acid sequence is sequence 21 in sequence table；
KH040：The amino acid sequence of the light chain variable district is sequence 17, the amino of the weight chain variable district in sequence table Acid sequence is sequence 31 in sequence table；
KH000：The amino acid sequence of the light chain variable district is sequence 37, the amino of the weight chain variable district in sequence table Acid sequence is sequence 38 in sequence table.
KH111：The chain variable region amino acid sequence is sequence 11, and the heavy chain variable amino acid sequence is sequence 33。
The amino acid of part light chain variable district and the corresponding relation of nucleotides：The nucleotides sequence of coded sequence 3 is classified as sequence 4；The nucleotides sequence of coded sequence 5 is classified as sequence 6；The nucleotides sequence of coded sequence 8 is classified as sequence 9；The nucleosides of coded sequence 13 Acid sequence is sequence 14；The nucleotides sequence of coded sequence 15 is classified as sequence 16；The nucleotides sequence of coded sequence 17 is classified as sequence 18.
The amino acid of Partial heavy variable region and the corresponding relation of nucleotides：The nucleotides sequence of coded sequence 11 is classified as sequence 63；The nucleotides sequence of coded sequence 19 is classified as sequence 20；The nucleotides sequence of coded sequence 21 is classified as sequence 22；Coded sequence 23 Nucleotides sequence is classified as sequence 24；The nucleotides sequence of coded sequence 25 is classified as sequence 26；The nucleotides sequence of coded sequence 27 is classified as sequence Row 28；The nucleotides sequence of coded sequence 29 is classified as sequence 30；The nucleotides sequence of coded sequence 31 is classified as sequence 32；Coded sequence 33 Nucleotides sequence be classified as sequence 34；The nucleotides sequence of coded sequence 29 is classified as sequence 30.
The amino acid of Partial heavy constant region and the corresponding relation of nucleotides：The nucleotides sequence of coded sequence 39 is classified as sequence 40；The nucleotides sequence of coded sequence 43 is classified as sequence 44.
The amino acid of part constant region of light chain and the corresponding relation of nucleotides：The nucleotides sequence of coded sequence 41 is classified as sequence 42。
The amino acid sequence of KH102 light chains is sequence 106, and the nucleotides sequence of coded sequence 106 is classified as sequence 107；Its is heavy Chain amino acid sequence is sequence 108, and the nucleotides sequence of coded sequence 108 is classified as sequence 109.
The present invention also provides the antigen-binding fragment A or antigen-binding fragment B as derived from the Fab.Antigen-binding fragment A For Fab ', F (ab ') 2, Fv, weight chain variable district, light chain variable district, the polypeptide piece selected from weight chain variable district as derived from the Fab Section or the polypeptide fragment selected from light chain variable district；Antigen-binding fragment B be as derived from the antibody Fab, Fab ', F (ab ') 2, Fv (antibody variable region fragment), weight chain variable district, light chain variable district, the polypeptide fragment selected from weight chain variable district or can selected from light chain Become the polypeptide fragment in area.
Heavy chain (be referred to as Fd ') of the above-mentioned F (ab ') 2 by a pair of light chains and a pair slightly larger than Fd is constituted, pepsin hydrolysis IgG molecules produce this fragment, and it includes two Fab, can combine 2 epitopes.Fd ' is about containing 235 amino acid residues, bag Include VH, CH1 and hinge region.Fv is made up of light chain variable district (VL) and weight chain variable district (VH), and the two is combined with non-covalent bond Together, molecular weight is about the 1/6 of complete antibody molecule, with single antigen binding site.Fv include ScFv (single-chain antibody), DsFv (disulfide stabilized antibody) etc..ScFv is to connect VH and VL with one section of appropriate oligonucleotide (joint, linker) Come, be allowed to be expressed as single peptide chain.DsFv is respectively to introduce one and half Guangs in light chain variable district and weight chain variable district suitable position Propylhomoserin, forms the Fv sections that a disulfide bond is fixed, it turned out that its binding ability and stability are superior to ScFv.
The antibody fragment of the present invention can be such as Fab, Fab ', F (ab ') 2, scFv, (scFv) 2, dAb, complementary determine Area (CDR) fragment, linear antibodies, single-chain antibody molecules, miniantibody, double antibody or the polyspecific from antibody fragment formation Antibody.
Another aspect of the present invention is that these humanized antibodies or its antibody fragment (such as antigen-binding fragment) are prepared and used In prevention and/or treatment medicine or the purposes of composition with illness excessively or that uncontrolled complement activation is relevant.It Include cardiopulmonary bypass surgery during complement activation；Acute myocardial infarction AMI, aneurysm, apoplexy, hemorrhagic shock, crush injury, Complement activation caused by ischemia reperfusion injury after MOF, intestine ischemia or other events for causing ischemic.Complement is lived Change has shown that relevant with inflammatory condition, and such as severe burn, endotoxemia, infectious shock, adult respiratory distress syndrome are comprehensive Simulator sickness, haemodialysis；Anaphylactic shock, serious asthma, angioedema, Crohn's disease, sickle cell anemia, streptococcus Glomerulonephritis and pancreatitis after infection.The illness can be adverse drug reactions, drug allergy, the blood of IL-2 inductions The result of pipe leaky syndrome or radiophotography (contrast) contrast agent allergy.It also includes autoimmunity disease, such as system Property lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Alzheimer's and multiple sclerosis.More importantly mend Body was activated also with eye diseases about (its pathology, which is related to complement, includes all eye conditions and disease of classical and alternative com-plement pathway), Such as but it is not limited to the senile macular degeneration (AMD) in macular degenerative disease such as all stages and includes dryness and moist (nonexudativeage and exudative) form, diabetic retinopathy and other ischemic related retinopathies, choroidal neovascular are formed (CNV), uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau diseases, the histoplasma capsulatum of eye Disease, thrombosis of central vein of retina (CRVO), cornea neovascularization and retina neovascular are formed.Wherein Age related macular Being denatured (AMD) includes nonexudativeage (such as mid-term (intermediate) dryness AMD or geographic atrophy (geographic Atrophy, GA)) and exudative (such as moist AMD (choroidal neovascular formation (CNV)) AMD, diabetic retinopathy (DR), entophthamia and uveitis, in addition nonexudativeage AMD may include Bohemian glass wart, soft glass wart, geographic atrophy and/or The presence of pigment caking.
The present invention there may be the difference on antibody and antigen binding epitope with the antibody of prior art, the present invention have compared with High-affinity, stability and bioactivity.
A further object of the present invention is to provide following genetic stocks：Recombinant vector, recombinant bacterium containing the gene, again Group cell line, recombinant virus or expression cassette.
The recombinant vector is the expression antibody or the prokaryotic expression carrier or eukaryotic expression of Fab or antigen-binding fragment Carrier.The recombinant bacterium is the Escherichia coli for carrying the gene.The recombinant cell lines can be transgenic cell line or melt Cell line is closed, wherein transgenic cell line can be the anticomplement D humanized antibodies or Fab or antigen-binding fragment for being transferred to the present invention The mammal cell line of encoding gene, preferably Chinese hamster ovary celI system, or 293 cells and its subbreed；Fused cell system can be for secretion originally The hybridoma of the anticomplement D humanized antibodies of invention.The recombinant virus is the recombined adhenovirus for carrying the gene Or recombinant adeno-associated virus etc..The expression cassette is a DNA molecular, and following three fragment is followed successively by from upstream to downstream：Open Encoding gene and the termination of mover, the antibody or Fab by promoter startup transcription, or the antigen-binding fragment Son.
The antibody, or the Fab will be contained, or the recombinant vector of the antigen-binding fragment encoding gene is transfected or converted Host cell, you can the corresponding albumen of expression, obtains the antibody or Fab, or the antigen-binding fragment.The host cell Can be eukaryotic or prokaryotic, they are thin including but not limited to mammalian cell, bacterium, yeast, insect Born of the same parents etc..Have a variety of available for the mammalian cell that protein is expressed on a large scale, such as 293 cells, Chinese hamster ovary celI, SP20 cells, NS0 cells, COS cells, bhk cell or PerC6 cells etc..The method of transfectional cell has a variety of, including but not limited to：Electricity Perforation, liposome mediated-method, calcium mediated method etc..
A kind of preferably described antibody or Fab, or the expression way of the antigen-binding fragment is in stable transfection Host cell in gene magnification is carried out to recombinant vector, to improve the expression quantity of corresponding recombinant protein, such as with containing dihydro The recombinant vector stable transfection of folic acid reductase (DHFR) lacks after DHFR host cell, and ammonia can be added in cell culture fluid First talk endlessly pyridine (MTX) concentration to expand number of copies of the recombinant vector in host cell.
After Fab or the IgG expression combined containing the coding gene sequence, can with enzyme linked immunosorbent assay (ELISA) or Other method determines the protein concentration in nutrient solution.For Fab fragments, it can be purified with Protein G affinity chromatographies；IgG eggs Available Protein A affinity chromatographies purification in vain.
A further object of the present invention is to provide pharmaceutical composition, and the pharmaceutical composition contains auxiliary material and active component, institute State active component and contain at least one of following substances：The antibody, the Fab, the antigen-binding fragment, the base Cause, and the genetic stocks；The auxiliary material is pharmaceutically acceptable carrier or excipient.The active component of the pharmaceutical composition Also can only be any of the above-described Fab or antibody, or any of the above-described antigen-binding fragment, or any of the above-described gene, or on State any of genetic stocks.
Application of any of the following substances in treatment complement D relevant diseases falls within protection scope of the present invention： The antibody, the Fab, the antigen-binding fragment, the gene, the genetic stocks, and described pharmaceutical composition.
On the other hand, the present invention provides kit, and it includes the antibody or its fragment (such as antigen binding fragment of the present invention Section).The present invention provides kit, and it is comprising antibody or its fragment (such as antigen-binding fragment) of the invention and uses instruction. The present invention provide kit include equipped with composition the first container, said composition comprising the present invention one or more antibody or Its antigen-binding fragment；With the second container equipped with buffer.
Embodiment
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
It is to pass through that the humanization Complement Factor D antibody PCR that the present invention is described, which introduces site mutation and the screening of mutant antibodies, Conventional gene recombinant technique and immunological technique based on antigen antibody interaction are completed, and specific experiment method and step is such as<< Molecular cloning>>The third edition (Joseph Sambrook, Science Press, on August 1st, 2002) and similar laboratory manual are remembered Carry.It is that OD values (OD405) the input graphpad prism5 softwares that will be measured are obtained that the haemolysis being related in embodiment, which suppresses IC50, .
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.
Embodiment 1, antigen-binding fragment Fab expression
(1) acquisition of light chain, heavy chain
The method synthesized by artificial gene, synthesizes monoclonal antibody light chain and sequence of heavy chain (the Suzhou gold only intelligence life of humanization Thing Co., Ltd).Every sequence is constituted by variable region and constant-region sequences, wherein：The light-chain variable sequence of synthesis is sequence Row 63, weight chain variabl area sequence is sequence 34, and the constant region of light chain is sequence 42, and the constant region of heavy chain is sequence 40.
(2) structure of humanized Fab carrier
The structure of humanized Fab expression vector is carried out using the method for PCR rite-directed mutagenesises, is concretely comprised the following steps：With synthesis Weight chain-ordering (plasmid) or acquired mutation Fab plasmids are template, and the weight chain building of each Fab expression vectors is distinguished Containing 4 primers, point 3 PCR are expanded, first the complementary difference of the inner primer containing identical base mutation of application two Enter performing PCR amplification with both sides primer, then using this PCR primer as template, both sides primer enters the final people for obtaining mutation of performing PCR amplification Source Fab antibody light and weight chain fragment.
The primer of light chain amplification is as follows：
Both sides primer is respectively：Sequence 64 and sequence 65
Middle mutant primer is：Sequence 66 and sequence 67；Sequence 68 and sequence 69；Sequence 70 and sequence 71；Sequence 72 with Sequence 73；Sequence 74 and sequence 75；Sequence 76 and sequence 77.
The primer of heavy chain amplification is as follows：
Both sides primer is respectively：Sequence 78 and sequence 79
Middle mutant primer is：Sequence 80 and sequence 81；Sequence 82 and sequence 83；Sequence 84 and sequence 85；Sequence 86 with Sequence 87；Sequence 88 and sequence 89；Sequence 90 and sequence 91；Sequence 92 and sequence 93；Sequence 94 and sequence 95；Sequence 96 and sequence Row 97.
The method that the light chain and heavy chain fragment of above-mentioned acquisition are connected by digestion connects that be inserted into can be while express respectively The pTLR carriers of Fab protein light chains and heavy chain (by pET22b (+) carrier of transformation, can resist in expression in escherichia coli Fab Body) in.Concrete operations are that prepare two ends by PCR method using above-mentioned primer contains restriction enzyme site NdeI and EcoRI respectively Light chain DNA, and two ends each heavy chain fragment DNA containing restriction enzyme site NotI and XhoI respectively, by the two DNA respectively gram It is grand enter pTLR carriers (by pET22b (+) carrier of transformation) in corresponding restriction endonuclease sites between, i.e. light chain DNA point It is not inserted between restriction enzyme NdeI and EcoRI sites, each heavy chain fragment (Fd) DNA is inserted respectively into restriction enzyme Between enzyme NotI and XhoI sites, several Fab expression vectors are built.
The specific method that the above-mentioned pTLR carriers of transformation acquisition are carried out to pET22b (+) carrier is as follows：First by manually closing Into section of DNA fragment (sequence 98), the fragment contains T7 terminators (T7terminator), T7 promoters successively (T7promoter), lactose operon (lac operator), ribosome bind site (RBS) sequence, and upper and lower ends bag Containing SalI and NotI restriction enzyme sites, the DNA sequence dna of pET22b (+) carrier and synthesis uses Sal I and Not I double digestions respectively Afterwards, T4Converted after ligase (NEB) connection, conventional method chooses monoclonal sequencing and filters out the carrier correctly transformed.
(3) IgG antibody recombinant expression carrier is built
After above-mentioned obtained weight chain variable district fragment codon optimization (Suzhou Jin Weizhi Co., Ltds), with weight Heavy chain fragment is stitched together by folded PCR with constant region, obtains sequence of heavy chain.The light chain of above-mentioned acquisition and sequence of heavy chain are distinguished Recombinate successively into expression plasmid Freedom pCHO1.0 (Life technologies), by the recombinant expression plasmid built Transfected CHO-S cells, express full length antibody.
Concrete operations are as described below：(1) primer containing appropriate restriction enzyme site and signal peptide sequence is expanded light chain by light chain Codon optimization template (Suzhou Jin Weizhi Co., Ltds), amplimer is：Sequence 99 and sequence 100.The PCR primer of acquisition with After AvrII enzymes (NEB Products) and BstZ17I enzymes (NEB Products) double digestion, T4 ligases (Takara companies are used Product) be connected acquisition pCHO-L with the Freedom pCHO1.0 large fragments of same double digestion.2) heavy chain need to be with overlap-extension PCR Variable region is stitched together by PCR with artificial synthesized IgG4 constant regions (Suzhou Jin Weizhi Co., Ltds), overlapping nucleotides Quantity is 40, then is recombinated to pCHO-L.Primer amplification heavy chain template containing appropriate restriction enzyme site and signal peptide sequence, can Become area and design the complementary overlapping PCR primers in inner side with constant region boundary, enter performing PCR amplification with both sides primer respectively, then with this PCR primer is template, and both sides primer enters the final humanized heavy chain's PCR primer for obtaining splicing of performing PCR amplification.Amplimer is such as Under：
Both sides primer is：Sequence 101, sequence 102 and sequence 103
Middle primer is：Sequence 104 and sequence 105.
By the PCR primer of splicing to be weighed again after EcoRV enzymes (NEB Products) and PacI enzymes (NEB Products) digestion Group obtains pCHO-HL into the expression plasmid pCHO-L of same double digestion, and correct through mirror digestion and sequencing identification.
The expression of embodiment 2Fab periplasm proteins
The Fab expression vectors of above-mentioned structure are converted into competent escherichia coli cell BL21 (DE3) (Tiangen) respectively, Apply amicillin resistance 2-YT flat boards (peptone 1.6%, yeast extract 1%, NaCl 0.5%, agar powder 1.5%).The Select within two days the plate picking monoclonal colony inoculation 5ml amicillin resistance 2-YT fluid nutrient mediums of suitable colony density, mistake Night cultivates, and condition is：37 DEG C, 220rpm, 16h.Next day, 5ml bacterium solutions are inoculated into 500ml amicillin resistance 2-YT liquid Culture medium, 33 DEG C, 220rmp is cultivated to OD600For 0.6.IPTG to final concentration of 50 μM are added in the medium, and induction is different Fab protein expressions, induction time is 3h.Nutrient solution after induced expression is terminated is under the conditions of 4500rpm, 10 DEG C, centrifugation 15min, abandons supernatant, (Tris24.23g, sucrose 171.2g, EDTA0.186g, is dissolved in precipitation with the TES solution of 40ml precoolings 1000ml deionized waters, 8.0) adjustment pH value to being fully resuspended bacterial sediment；The 1/5TES that 66ml precoolings are added after resuspension is molten In bacterium solution after liquid to resuspension, 40min is incubated on ice；After ice bath terminates, 13000rpm, centrifuges 10min by 4 DEG C；Collected after centrifugation Supernatant, supernatant is the periplasmic extract solution containing Fab albumen.Periplasmic extract crosses G-25 (GE companies, 17-0034-01) After post desalting processing, prepare Protein G (GE company 17-0618-04) prepacked column, equilibrium liquid (20mM phosphate buffers pH 6.5) albumen loading after balancing, pillar is cleaned in continuation with equilibrium liquid after end of the sample, then with eluent (0.1M Gly-HCl, PH2.5) directly elute, collect elution samples, and compare 1 according to Tris buffer solutions and sample volume before sample is collected in advance：9 add Enter 1.0M Tris-HCl (PH9.0) in sample collection tube, fast velocity modulation PH7.0, the liquid being collected into as destination protein, egg In vain after SDS-PAGE purity assays, concentration mensuration is carried out to protein sample, -80 DEG C is stored in after finally dispensing, obtains pure The higher Fab albumen of degree.
The expression of the different full length antibodies of embodiment 3
Full length antibody protein expression process comprises the following steps：The IgG antibody recombinant expression carrier of above-mentioned structure is transfected Chinese hamster ovary celI；Utilize Puromycin (invitrogen companies) and MTX (Sigma companies) joint screening overexpression protection complement factors The engineering cell strain of D albumen, expands and cultivates the cell line, full length antibody is finally isolated and purified from cell culture fluid.Specific bag Include：
(1) transfection of expression vector
Host cell CHO-S cells are selected, Cell line are pressed using the expression vector plasmid for linearizing above-mentioned structure Nucleofector kit V transfection reagents (Lonza companies) carry out electrotransfection.6 orifice plates are placed in 37 DEG C, 8%CO after transfection2Shake Bed culture 48h.
(2) foundation of CHO-S engineering cell strains
Polyclonal cells obtained by transfection are carried out with the progressively pressurization primary dcreening operation in two stages.Two pool gradients of first stage 10P/100M, 20P/200M (P=μ g/mL Puromycin, M=nM MTX) are set to, Cell viability reaches 85% i.e. progress Second stage pressurization screening, antibiotic concentration is set to 30P/500M, 50P/1000M, and culture to motility rate is completed higher than 90% Primary dcreening operation is obtained behind resisting cell storehouse, is assessed into 14 days batches of Shaking culture, selection destination protein expression quantity is higher, active more excellent Stable expression pool carry out monoclonal screening, obtain 100~150 monoclonals, select the positive monoclonal cell of high yield Expand culture.Antibody protein is produced using these cell lines.
(3) prepared by the separation of full length antibody
Using CHO-S CD Medium (Life technologies companies) culture medium, in 37 DEG C, 8%CO2Shaking table is trained Above-mentioned CHO-S engineering cell strains 13 days are supported, after 2000rpm centrifugations, supernatant are collected, then with MabSelect SuRe affinity columns (GE companies), which isolates and purifies, obtains anticomplement factor D antibody.
The difference Fab of embodiment 4. and antibody suppress alternative pathway of complement hemolytic experiment
Alternative pathway of complement activity is determined using ACH50 methods, by family's rabbit erythrocyte (self-control) in GVB/Mg-EGTA Wash 3 times, and be resuspended to 6 × 10 in Buffer8/ml.Resisted with the GVB/Mg-EGTA Buffer anti-factor Ds for preparing various concentrations Former binding fragment Fab or antibody, concentration is respectively 44.44nM, 22.22nM, 11.11nM, 5.56nM, 2.78nM, 1.39nM.Will The μ l of antibody 50 and 20% human serum (Complement C1q-Depleted Serum, Human, 234401-1ml, Calbiochem) 50 μ l are mixed in equal volume, are incubated at room temperature 7min；30 μ l red cell suspension is added, 4 DEG C vibrate 30s, 37 DEG C It is incubated 30min；2000rpm, 4 DEG C of centrifugation 3min, takes 80 μ l supernatants to be transferred to 96 hole elisa Plates, and 405nm determines light absorption value.Data State the percentage relative to the haemolysis induced when being not added with antibody as.While setting serum dissolving control group (being not added with antibody), Serum-free control group (being not added with human serum) and Quality Control control group (human serum being replaced with deionized water, be not added with antibody).
The antigen-binding fragment Fab alternative pathway of complement Activity Results of the anti-factor D of table 1
The antibody complement alternative pathway Activity Results of the anti-factor D of table 2
Embodiment 5. is based on quantitative fluorescent PCR and analyzes different Fab albumen heat endurance experiments
Albumen (antibody) dissociates exposure fluorophor and then inspires fluorescence signal at a certain temperature, passes through fluorescent quantitation PCR instrument (MJ Research) continuously collects albumen change of fluorescence signal intensity during continuous warming and draws out albumen Melting curve, calculates the Tm values of the albumen, the size forward direction reflection of the value be albumen heat endurance ability.First by It is 0.4 μ g/uL that 1 × PBS solution, which prepares anti-factor D antigen-binding fragment Fab concentration, and lucifuge adds the SYPRO dyestuffs of 5 times of dilutions (Cat No.S6650, lift technologies), total reaction volume is 50 μ l, and insufficient section is supplied using 1 × PBS solution. Melting curve experiment is run on real-time PCR, melting curve is set to 25 DEG C and is gradually heating to 95 DEG C, every 0.5 DEG C of reading Once, software option monitor3 are carried using instrument and carries out the analysis of Tm values.
The Fab albumen heat endurances of the anti-factor D of table 3
Embodiment 6. is based on differential canning calorimetry (DSC) analysis different antibodies albumen heat endurance experiment
By differential scanning Microcalorimetric method (DSC) can with the hot melt during temperature scanning protein heat denaturation change Change, and finally fitting obtains including half denaturation temperature TmVarious thermodynamic parameters (enthalpy, entropy, free energy etc.) inside.For For full length antibody molecule, differential canning calorimetry usually can more be accurately obtained three of antibody CH2, CH3 and Fab areas Thermodynamics half denaturation temperature.In general, the Fc sections being made up of CH2, CH3 are relatively fixed, thus the stability pair in Fab areas There is important influence in the stability of antibody.Utilize Malvern company Microcal Auto VP-Capillary DSC instruments With the thermal denaturation process of antibody KH-A, KH-F and KH102 between 1 DEG C/min of 25-100 DEG C of programming rate scanning, and with The subsidiary Origin program analyze datas of instrument fit TmThe thermodynamic parameters such as value.Sample is configured in PBS, pH 7.4,0.22 μm of membrane filtrations, concentration 1mg/mL.The automatic cleaning procedures of DSC are set in software end, after being cleaned with DECON cleaning solutions With ultra-pure water thoroughly cleaning sample introduction needle, auto injection pipeline and sample cell, whole flow process about 30min is completed；Setting scanning ginseng Number：20-100 DEG C of temperature range, 60 DEG C/h of sweep speed are scanned, wherein first scanning buffer liquid balances instrument 4 times, then with 1X PBS Buffer solution scans test antibodies molecule successively for control.The sweep time averagely about 2h of each sample.After the end of scan with DSC certainly Dynamic cleaning procedure thoroughly cleaning instrument.Raw scanning data is deducted into buffer control and with the molar concentration normalizing of each sample Change, recycle Origin softwares to even up baseline, Fab correspondence area's Tm values are carried out with Non-two-state model fitting data Interpretation of result.
Monoclonal antibody correspondence area's albumen heat endurance of the anti-factor D of table 4
The antibody different genera dynamic experiment of embodiment 7.
By using OctetQKProtein interaction platform, selection Ni-NTA Biosensor are passed as detection with biological Sensor；With suitable concentration (such as：10 μ g/ml) CFD (monkey CFD or rat CFD) as detection antigen；With 200nM, The combination situation of 100nM, 50nM, 25nM, 12.25nM, 6.25nM, 0nM full length antibody Protein Detection antibody and antigen, program is set It is set to：Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s.Selection Ni-NTA Biosensor are used as detection biology sensor；With CFD (the monkey CFD or big of 10ug/ml concentration Mouse CFD) it is used as detection antigen；Using 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125nM, 0nM as The combination situation of anti-CFD Fab Protein Detection concentration gradients, detection Fab albumen and antigen.Program is set to：Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s.According to combination Curve calculating antibody affinity KD.
The difference Fab of table 5. or antibody and monkey CFD affinity result
Table 5 shows that Fab of the present invention or antibody have higher combination with monkey CFD.
The different antibodies of table 6. and rat CFD affinity result
Table 6 shows that antibody of the present invention and rat CFD have certain adhesion.
The different antibodies of embodiment 8. or Fab suppress rat blood serum and cause rabbit erythrocyte hemolytic experiment
Alternative pathway of complement activity is determined using ACH50 methods, by family's rabbit erythrocyte (self-control) in GVB/Mg-EGTA 3 times (4 DEG C, 1600rpm centrifugations) are washed in Buffer, the thin of 1 × 109cells/ml is diluted to after counting with GVB/Mg-EGTA Born of the same parents' suspension, is positioned over cooled on ice.Resisted with the GVB/Mg-EGTA Buffer anti-factor D antigen binding total lengths for preparing various concentrations Body, concentration gradient is respectively 23mg/ml, 20mg/ml, 15mg/ml, 10mg/ml, 5mg/L, 1mg/ml, and anti-factor D bonding pad Section Fab concentration is 1mg/ml, and 50 μ l sample and 50 μ l rat blood serum (self-control) are mixed, and shaken at room temperature 30s, room temperature is incubated Educate 7min；Add 30 μ l red cell suspension, 4 DEG C of vibrations 30s, 37 DEG C of incubation 40min；Take out rear refrigerated centrifuge (4 DEG C, 2000rpm, 3min)；80ul supernatants are shifted to 96 orifice plates, 405nm surveys OD values.Data are stated as to lure relative to when being not added with antibody The percentage for the haemolysis led.KH-000 is not detected by activity in 1.5mg/ml.
The full length antibody of table 7. suppresses rat blood serum and causes rabbit erythrocyte hemolytic experiment result
Sample IC50
KH-A 3.860mg/ml
KH-F 3.956mg/ml
Table 8.Fab antibody suppresses rat blood serum and causes rabbit erythrocyte hemolytic experiment result
Fab sample concentrations (mg/ml) Inhibiting rate (%)
KH021(1.5mg/ml) 3.191
KH027(1.0mg/ml) 28.210
The dynamic experiment of the different antibodies of embodiment 9. or Fab to cavy or pig
Entered using the preliminary CFD albumen to transiently transfecting the pig obtained and cavy of OctetQK protein interactions platform The preliminary combination situation of row is estimated, first by pig (GeneID：396877), cavy (GeneID：100724693) gene sequence C-terminal fusion 10 × His tag of row are optimized and synthesized by Nanjing Genscript Biotechnology Co., Ltd..Then by after synthesis optimizing Gene be inserted into expression vector pCHO1.0, plasmid press Cell line Nucleofector kit V transfection reagents (Lonza companies) carries out electrotransfection host cell DG44, and 6 orifice plates are placed in 37 DEG C, after 5%CO2 shaking table cultures 48h after transfection, 1000g, is centrifuged 3 minutes, and protein dynamics analysis is carried out using supernatant.Specific method is carried out as follows：Full length antibody KH-A, KH-F dynamic analyses selection Protein A are used as detection biology sensor；First with suitable concentration (such as：10μg/ml) In antibody binding to protein A biology sensors, the combination feelings of the transient transfection supernatant synantibody of pig and cavy CFD will be tested Condition, program is set to：Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s.Negative control is set to the CHO negative cells nutrient solutions of untransfected plasmid.Fab antibody KH027 and KH021 dynamic analyses selection Ni-NTA is used as detection biology sensor；Transfection supernatants are joined respectively to Ni- first On NTA biology sensors, the knot of the transient transfection supernatant synantibody of different genera is then tested using 10 μ g/ml Fab antibody Conjunction situation, program is set to：Baseline 120s, Loding 700s, Baseline120s, Association 700S, Dissociation 1500s。
The difference Fab of table 9. or antibody and cavy CFD dissociation constant
The difference Fab of table 10. or antibody and pig CFD dissociation constant
Table 9 and the result of table 10 show that antibody and Fab and the CFD of cavy or pig have good adhesion.
The antibody of embodiment 10. kinetic test in rabbit vitreous humor is tested
The activity of antibody can be affected according to different buffer system environment, by taking rabbit vitreous humor to replace people Vitreous humor carrys out activity of the analog antibody in people's vitreous humor, and the activity to antibody carries out preliminary assessment.In this experiment, lead to Cross using OctetQK protein interaction platforms, selection Ni-NTA Biosensor are used as detection biology sensor；With 10 μ g/ml people CFD albumen is used as detection antigen；With 125nM, 0nM is anti-CFD full length antibodies KH-A detectable concentration, with vitreum Liquid is detected as detection matrix, dynamics of the confrontation CFD full length antibodies in vitreous humor.Program is set to： Baseline 120s, Loding 700s, Baseline 120s, Association 700S, Dissociation 1500s. The Preliminary activation of antibody is judged according to dynamics binding curve.
The antibody KH-A of table 11. and dissociation constants of the people CFD in rabbit vitreous humor
Table 11 shows that antibody KH-A has good binding activity in rabbit vitreous humor with people CFD.
SEQUENCE LISTING
<110>Kanghong Biotech Co., Ltd., Chengdu
<120>Anticomplement factor D humanized Fab and humanized antibody and application thereof
<130> KH20160104
<160> 109
<170> PatentIn version 3.3
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Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Ala Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 8
<211> 107
<212> PRT
<213>Artificial sequence
<400> 8
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 9
<211> 321
<212> DNA
<213>Artificial sequence
<400> 9
gatattcagg tgacccagag tccgagcagc ctgagcgcaa gcgttggtga tcgcgtgacc 60
attacctgca tcaccagcac cgatatcgac gacgacttga actggtacca gcagaaaccg 120
ggcaaagtgc cgaaactgct gattagcggt ggtaacaccc tgcgtccggg tgtgccgagc 180
cgttttagtg gtagcggtag cggtaccgac tttaccctga ccatcagcag cctgcagccg 240
gaagatgtgg ccacctatta ttgcctgcag agcgatagcc tgccgtatac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 10
<211> 107
<212> PRT
<213>Artificial sequence
<400> 10
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Ala Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 11
<211> 107
<212> PRT
<213>Artificial sequence
<400> 11
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 12
<211> 107
<212> PRT
<213>Artificial sequence
<400> 12
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Ala Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 13
<211> 107
<212> PRT
<213>Artificial sequence
<400> 13
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Ile Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 14
<211> 321
<212> DNA
<213>Artificial sequence
<400> 14
gatattcagg tgacccagag tccgagcagc ctgagcgcaa gcgttggtga tcgcgtgacc 60
attacctgca tcaccagcac cgatatcgac gacgacgtga actggtacca gcagaaaccg 120
ggcaaagtgc cgaaactgct gattagcggt ggtaacaccc tgcgtccggg tgtgccgagc 180
cgttttagtg gtagcggtag cggtaccgac tttaccctga ccatcagcag cctgcagccg 240
gaagatgtgg ccacctatta ttgcctgcag agcgatagcc tgccgtatat ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 15
<211> 107
<212> PRT
<213>Artificial sequence
<400> 15
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Met Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 16
<211> 321
<212> DNA
<213>Artificial sequence
<400> 16
gatattcagg tgacccagag tccgagcagc ctgagcgcaa gcgttggtga tcgcgtgacc 60
attacctgca tcaccagcac cgatatcgac gacgacgtga actggtacca gcagaaaccg 120
ggcaaagtgc cgaaactgct gattagcggt ggtaacaccc tgcgtccggg tgtgccgagc 180
cgttttagtg gtagcggtag cggtaccgac tttaccctga ccatcagcag cctgcagccg 240
gaagatgtgg ccacctatta ttgcatgcag agcgatagcc tgccgtatac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 17
<211> 107
<212> PRT
<213>Artificial sequence
<400> 17
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 18
<211> 321
<212> DNA
<213>Artificial sequence
<400> 18
gatattcagg tgacccagag tccgagcagc ctgagcgcaa gcgttggtga tcgcgtgacc 60
attacctgca tcaccagcac cgatatcgac gacgacgtga actggtacca gcagaaaccg 120
ggcaaagtgc cgaaactgct gattagcggt ggtaacaccc tgcgtccggg tgtgccgagc 180
cgttttagtg gtagcggtag cggtaccgac tttaccctga ccatcagcag cctgcagccg 240
gaagatgtgg ccacctatta ttgcctgcag agcgatagct atccgtatac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 19
<211> 115
<212> PRT
<213>Artificial sequence
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Phe Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 20
<211> 345
<212> DNA
<213>Artificial sequence
<400> 20
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggcgtgaatt tttggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 21
<211> 115
<212> PRT
<213>Artificial sequence
<400> 21
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Pro Asn Asn Trp Gly Pro Gly Pro Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 22
<211> 345
<212> DNA
<213>Artificial sequence
<400> 22
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggccctaata attggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 23
<211> 115
<212> PRT
<213>Artificial sequence
<400> 23
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Ser Asn Asn Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 24
<211> 345
<212> DNA
<213>Artificial sequence
<400> 24
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggctctaata attggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 25
<211> 115
<212> PRT
<213>Artificial sequence
<400> 25
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn His Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 26
<211> 345
<212> DNA
<213>Artificial sequence
<400> 26
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggcgtgaatc attggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 27
<211> 115
<212> PRT
<213>Artificial sequence
<400> 27
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Ser Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 28
<211> 345
<212> DNA
<213>Artificial sequence
<400> 28
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggcgtgaatt cttggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 29
<211> 115
<212> PRT
<213>Artificial sequence
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Arg Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 30
<211> 345
<212> DNA
<213>Artificial sequence
<400> 30
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggcgtgaatc ggtggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 31
<211> 115
<212> PRT
<213>Artificial sequence
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 32
<211> 345
<212> DNA
<213>Artificial sequence
<400> 32
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggcgtgaatt attggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 33
<211> 115
<212> PRT
<213>Artificial sequence
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Asn Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 34
<211> 345
<212> DNA
<213>Artificial sequence
<400> 34
caggtgcagc tggttcagag cggtccggag ctgaaaaaac cgggcgccag cgttaaagtg 60
agctgcaaag ccagcggcta caccttcacc aactacggca tgaactgggt tcgtcaggcc 120
ccgggtcagg gtctggaatg gatgggctgg atcaacacct ataccggcga aaccacctac 180
gccgacgatt ttaagggccg cttcgtgttt agcctggata ccagcgtgag caccgcctat 240
ctgcagatta gcagcctgaa agccgaagac accgccgtgt actattgtga acgcgagggc 300
ggcgtgaata attggggtca gggtaccctg gtgaccgtga gcagc 345
<210> 35
<211> 115
<212> PRT
<213>Artificial sequence
<400> 35
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Ala Gly Gly Val Asn Asn Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 36
<211> 115
<212> PRT
<213>Artificial sequence
<400> 36
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Ala Gly Val Asn Asn Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 37
<211> 107
<212> PRT
<213>Artificial sequence
<400> 37
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 38
<211> 115
<212> PRT
<213>Artificial sequence
<400> 38
Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Asn Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 39
<211> 103
<212> PRT
<213>Artificial sequence
<400> 39
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys
100
<210> 40
<211> 309
<212> DNA
<213>Artificial sequence
<400> 40
gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaacctgt gacggtctcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgt 309
<210> 41
<211> 107
<212> PRT
<213>Artificial sequence
<400> 41
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 42
<211> 321
<212> DNA
<213>Artificial sequence
<400> 42
cgtacggttg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60
ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120
tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180
agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240
aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagttcacc ggtgacaaag 300
agcttcaaca ggggagagtg t 321
<210> 43
<211> 327
<212> PRT
<213>Artificial sequence
<400> 43
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 44
<211> 981
<212> DNA
<213>Artificial sequence
<400> 44
gcttccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60
agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300
aaatatggtc ccccatgccc accatgccca gcacctgagt tcgagggggg accatcagtc 360
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840
gacggctcct tcttcctcta cagcaggctc accgtggaca agagcaggtg gcaggagggg 900
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960
ctctccctgt ctctgggtaa a 981
<210> 45
<211> 214
<212> PRT
<213>Artificial sequence
<400> 45
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Ala Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 46
<211> 642
<212> DNA
<213>Artificial sequence
<400> 46
gacatccagg tgacacagtc ccctagcagc ctgtccgctt ccgtgggcga cagggtgacc 60
atcacctgca tcaccagcac cgacatcgac gacgacatga attggtacca gcagaagcct 120
ggcaaggtgc ctaagctgct gatctccggc ggcgctaccc tcaggcctgg agtgcctagc 180
cggttcagcg gttctggttc tggcaccgac ttcaccctga ccatctcctc cctgcagccc 240
gaggacgtgg ctacctacta ttgcctccag agcgactccc tgccttatac cttcggccag 300
ggcaccaagc tggaaatcaa gaggaccgtg gccgctccta gcgtgttcat cttcccccct 360
tccgacgagc agctgaagtc cggcaccgcc agcgtggtgt gtctgctgaa caacttctat 420
ccccgggagg ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg aaacagccaa 480
gagtccgtga ccgagcagga cagcaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctcagctccc ctgtgaccaa gtccttcaac aggggcgagt gt 642
<210> 47
<211> 442
<212> PRT
<213>Artificial sequence
<400> 47
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 48
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 48
caggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaact actggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326
<210> 49
<211> 214
<212> PRT
<213>Artificial sequence
<400> 49
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Ile Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 50
<211> 642
<212> DNA
<213>Artificial sequence
<400> 50
gacatccagg tgacacagtc ccctagcagc ctgtccgctt ccgtgggcga cagggtgacc 60
atcacctgca tcaccagcac cgacatcgac gacgacgtga attggtacca gcagaagcct 120
ggcaaggtgc ctaagctgct gatctccggc ggcaacaccc tcaggcctgg agtgcctagc 180
cggttcagcg gttctggttc tggcaccgac ttcaccctga ccatctcctc cctgcagccc 240
gaggacgtgg ctacctacta ttgcctccag agcgactccc tgccttatat cttcggccag 300
ggcaccaagc tggaaatcaa gaggaccgtg gccgctccta gcgtgttcat cttcccccct 360
tccgacgagc agctgaagtc cggcaccgcc agcgtggtgt gtctgctgaa caacttctat 420
ccccgggagg ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg aaacagccaa 480
gagtccgtga ccgagcagga cagcaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctcagctccc ctgtgaccaa gtccttcaac aggggcgagt gt 642
<210> 51
<211> 442
<212> PRT
<213>Artificial sequence
<400> 51
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn His Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 52
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 52
caggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaacc attggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326
<210> 53
<211> 442
<212> PRT
<213>Artificial sequence
<400> 53
Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 54
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 54
gaggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaact actggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326
<210> 55
<211> 442
<212> PRT
<213>Artificial sequence
<400> 55
Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn His Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 56
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 56
gaggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaacc attggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326
<210> 57
<211> 214
<212> PRT
<213>Artificial sequence
<400> 57
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Met Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 58
<211> 642
<212> DNA
<213>Artificial sequence
<400> 58
gacatccagg tgacacagtc ccctagcagc ctgtccgctt ccgtgggcga cagggtgacc 60
atcacctgca tcaccagcac cgacatcgac gacgacgtga attggtacca gcagaagcct 120
ggcaaggtgc ctaagctgct gatctccggc ggcaacaccc tcaggcctgg agtgcctagc 180
cggttcagcg gttctggttc tggcaccgac ttcaccctga ccatctcctc cctgcagccc 240
gaggacgtgg ctacctacta ttgcatgcag agcgactccc tgccttatac cttcggccag 300
ggcaccaagc tggaaatcaa gaggaccgtg gccgctccta gcgtgttcat cttcccccct 360
tccgacgagc agctgaagtc cggcaccgcc agcgtggtgt gtctgctgaa caacttctat 420
ccccgggagg ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg aaacagccaa 480
gagtccgtga ccgagcagga cagcaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctcagctccc ctgtgaccaa gtccttcaac aggggcgagt gt 642
<210> 59
<211> 442
<212> PRT
<213>Artificial sequence
<400> 59
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Phe Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 60
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 60
caggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaact tctggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326
<210> 61
<211> 442
<212> PRT
<213>Artificial sequence
<400> 61
Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Ser Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 62
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 62
caggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaaca gctggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326
<210> 63
<211> 321
<212> DNA
<213>Artificial sequence
<400> 63
gatattcagg tgacccagag tccgagcagc ctgagcgcaa gcgttggtga tcgcgtgacc 60
attacctgca tcaccagcac cgatatcgac gacgacatga actggtacca gcagaaaccg 120
ggcaaagtgc cgaaactgct gattagcggt ggtaacaccc tgcgtccggg tgtgccgagc 180
cgttttagtg gtagcggtag cggtaccgac tttaccctga ccatcagcag cctgcagccg 240
gaagatgtgg ccacctatta ttgcctgcag agcgatagcc tgccgtatac ctttggccag 300
ggcaccaaac tggaaattaa a 321
<210> 64
<211> 92
<212> DNA
<213>Artificial sequence
<400> 64
actcatatga aatacctatt gcctacggca gccgctggat tgttattact cgcggcacag 60
ccggctatgg ccgatattca ggtgacccag ag 92
<210> 65
<211> 32
<212> DNA
<213>Artificial sequence
<400> 65
caggaattct caacactctc ccctgttgaa gc 32
<210> 66
<211> 21
<212> DNA
<213>Artificial sequence
<400> 66
ctgctggtac cagttcacgt c 21
<210> 67
<211> 21
<212> DNA
<213>Artificial sequence
<400> 67
gacgtgaact ggtaccagca g 21
<210> 68
<211> 21
<212> DNA
<213>Artificial sequence
<400> 68
gacgcagggt ggcaccaccg c 21
<210> 69
<211> 21
<212> DNA
<213>Artificial sequence
<400> 69
gcggtggtgc caccctgcgt c 21
<210> 70
<211> 21
<212> DNA
<213>Artificial sequence
<400> 70
ctgctggtac cagttcaagt c 21
<210> 71
<211> 21
<212> DNA
<213>Artificial sequence
<400> 71
gacttgaact ggtaccagca g 21
<210> 72
<211> 21
<212> DNA
<213>Artificial sequence
<400> 72
cctggccaaa gatatacggc a 21
<210> 73
<211> 21
<212> DNA
<213>Artificial sequence
<400> 73
tgccgtatat ctttggccag g 21
<210> 74
<211> 21
<212> DNA
<213>Artificial sequence
<400> 74
atcgctctgc atgcaataat a 21
<210> 75
<211> 21
<212> DNA
<213>Artificial sequence
<400> 75
tattattgca tgcagagcga t 21
<210> 76
<211> 38
<212> DNA
<213>Artificial sequence
<400> 76
tggccaaagg tatacggata gctatcgctc tgcaggca 38
<210> 77
<211> 38
<212> DNA
<213>Artificial sequence
<400> 77
tgcctgcaga gcgatagcta tccgtatacc tttggcca 38
<210> 78
<211> 94
<212> DNA
<213>Artificial sequence
<400> 78
actgcggccg catgggtaag aaacagttgg ttgtgtttgc tctgcttttg gcttttcttt 60
ctccggccat ggcccaggtg cagctggttc agag 94
<210> 79
<211> 34
<212> DNA
<213>Artificial sequence
<400> 79
cagctcgagt caacaagatt tgggctcaac tttc 34
<210> 80
<211> 39
<212> DNA
<213>Artificial sequence
<400> 80
ccaattattc acgccgccgg cgcgttcaca atagtacac 39
<210> 81
<211> 39
<212> DNA
<213>Artificial sequence
<400> 81
gtgtactatt gtgaacgcgc cggcggcgtg aataattgg 39
<210> 82
<211> 39
<212> DNA
<213>Artificial sequence
<400> 82
accccaatta ttcacgccgg cctcgcgttc acaatagta 39
<210> 83
<211> 39
<212> DNA
<213>Artificial sequence
<400> 83
tactattgtg aacgcgaggc cggcgtgaat aattggggt 39
<210> 84
<211> 39
<212> DNA
<213>Artificial sequence
<400> 84
accctgaccc caattattag ggccgccctc gcgttcaca 39
<210> 85
<211> 39
<212> DNA
<213>Artificial sequence
<400> 85
tgtgaacgcg agggcggccc taataattgg ggtcagggt 39
<210> 86
<211> 39
<212> DNA
<213>Artificial sequence
<400> 86
cagggtaccc tgaccccaat aattcacgcc gccctcgcg 39
<210> 87
<211> 39
<212> DNA
<213>Artificial sequence
<400> 87
cgcgagggcg gcgtgaatta ttggggtcag ggtaccctg 39
<210> 88
<211> 39
<212> DNA
<213>Artificial sequence
<400> 88
ccagggtacc ctgaccccaa aaattcacgc cgccctcgc 39
<210> 89
<211> 39
<212> DNA
<213>Artificial sequence
<400> 89
gcgagggcgg cgtgaatttt tggggtcagg gtaccctgg 39
<210> 90
<211> 39
<212> DNA
<213>Artificial sequence
<400> 90
accctgaccc caattattag agccgccctc gcgttcaca 39
<210> 91
<211> 39
<212> DNA
<213>Artificial sequence
<400> 91
tgtgaacgcg agggcggctc taataattgg ggtcagggt 39
<210> 92
<211> 39
<212> DNA
<213>Artificial sequence
<400> 92
cagggtaccc tgaccccaat gattcacgcc gccctcgcg 39
<210> 93
<211> 39
<212> DNA
<213>Artificial sequence
<400> 93
cgcgagggcg gcgtgaatca ttggggtcag ggtaccctg 39
<210> 94
<211> 39
<212> DNA
<213>Artificial sequence
<400> 94
cagggtaccc tgaccccaag aattcacgcc gccctcgcg 39
<210> 95
<211> 39
<212> DNA
<213>Artificial sequence
<400> 95
cgcgagggcg gcgtgaattc ttggggtcag ggtaccctg 39
<210> 96
<211> 39
<212> DNA
<213>Artificial sequence
<400> 96
cagggtaccc tgaccccacc gattcacgcc gccctcgcg 39
<210> 97
<211> 39
<212> DNA
<213>Artificial sequence
<400> 97
cgcgagggcg gcgtgaatcg gtggggtcag ggtaccctg 39
<210> 98
<211> 231
<212> DNA
<213>Artificial sequence
<400> 98
catgtcgacg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 60
gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 120
cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc 180
tctagaaata attttgttta actttaagaa ggagatatag cggccgcttg c 231
<210> 99
<211> 101
<212> DNA
<213>Artificial sequence
<400> 99
catcctaggg ccaccatgga catgagggtg cccgcccaac tgctgggact gctgctgctg 60
tggctgaggg gcgctaggtg cgacatccag gtgacacagt c 101
<210> 100
<211> 29
<212> DNA
<213>Artificial sequence
<400> 100
caggtatact taacactcgc ccctgttga 29
<210> 101
<211> 92
<212> DNA
<213>Artificial sequence
<400> 101
actgatatcg ccaccatggc ctggatgatg ctgctgctgg gactgctggc ctatggcagc 60
ggcgtggact cccaggtgca gctggtgcag tc 92
<210> 102
<211> 92
<212> DNA
<213>Artificial sequence
<400> 102
actgatatcg ccaccatggc ctggatgatg ctgctgctgg gactgctggc ctatggcagc 60
ggcgtggact ccgaggtgca gctggtgcag tc 92
<210> 103
<211> 33
<212> DNA
<213>Artificial sequence
<400> 103
catttaatta atcatttacc cagagacagg gag 33
<210> 104
<211> 40
<212> DNA
<213>Artificial sequence
<400> 104
gatgggccct tggtggaagc ggaggacact gtcaccaggg 40
<210> 105
<211> 40
<212> DNA
<213>Artificial sequence
<400> 105
ccctggtgac agtgtcctcc gcttccacca agggcccatc 40
<210> 106
<211> 214
<212> PRT
<213>Artificial sequence
<400> 106
Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30
Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 107
<211> 642
<212> DNA
<213>Artificial sequence
<400> 107
gacatccagg tgacacagtc ccctagcagc ctgtccgctt ccgtgggcga cagggtgacc 60
atcacctgca tcaccagcac cgacatcgac gacgacatga attggtacca gcagaagcct 120
ggcaaggtgc ctaagctgct gatctccggc ggcaacaccc tcaggcctgg agtgcctagc 180
cggttcagcg gttctggttc tggcaccgac ttcaccctga ccatctcctc cctgcagccc 240
gaggacgtgg ctacctacta ttgcctccag agcgactccc tgccttatac cttcggccag 300
ggcaccaagg tggaaatcaa gaggaccgtg gccgctccta gcgtgttcat cttcccccct 360
tccgacgagc agctgaagtc cggcaccgcc agcgtggtgt gtctgctgaa caacttctat 420
ccccgggagg ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg aaacagccaa 480
gagtccgtga ccgagcagga cagcaaggac agcacctact ccctgtcctc caccctgacc 540
ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600
ctcagctccc ctgtgaccaa gtccttcaac aggggcgagt gt 642
<210> 108
<211> 442
<212> PRT
<213>Artificial sequence
<400> 108
Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Glu Arg Glu Gly Gly Val Asn Asn Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys
210 215 220
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
245 250 255
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
260 265 270
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 109
<211> 1326
<212> DNA
<213>Artificial sequence
<400> 109
gaggtgcagc tggtgcagtc cggacctgag ctgaagaagc ccggcgcctc cgtgaaggtg 60
tcctgcaagg cctccggcta caccttcacc aactacggca tgaactgggt gcggcaggct 120
cctggacagg gactggagtg gatgggctgg atcaacacct acaccggcga gaccacctac 180
gccgacgact tcaagggcag gttcgtgttc tccctggaca ccagcgtgtc caccgcctac 240
ctgcaaattt cttccctgaa ggccgaggac accgccgtgt actactgcga gagggaggga 300
ggcgtgaaca actggggcca gggcaccctg gtgacagtgt cctccgcttc caccaagggc 360
ccatcggtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 420
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480
ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540
agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaacgta 600
gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 660
tgcccaccat gcccagcacc tgagttcgag gggggaccat cagtcttcct gttcccccca 720
aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 780
gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 840
aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 900
ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 960
aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1020
ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1080
acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1140
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1200
ctctacagca ggctcaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1260
tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1320
ggtaaa 1326

Claims (10)

1. a kind of anticomplement factor D humanized antibody or its antigen-binding fragment Fab, contain light chain variable district and weight chain variable Amino acid sequence ITSTDIDDDX is contained in area, wherein light chain variable district1N, amino acid sequence GGX2TLRP and amino acid sequence X3QSDS X4PYX5, the X1For V, M or L, X2For N or A, X3For M or L, X4For L or Y, X5For I or T；Weight chain variable district contains Amino acid sequence 1, sequence 2 and amino acid sequence Y1GGY2NY3, the Y1For A or E, Y2For S, V or P, Y3For N, Y, F, H, S or R；The X1、X2、X3、X4、X5Asynchronously meet following conditions：X1For M, X2For N, X3For L, X4For L, X5For T.
2. anticomplement factor D humanized antibody or its antigen-binding fragment Fab, include following weight according to claim 1 Chain variable region and light-chain variable sequence：The amino acid sequence of the light chain variable district be sequence table in sequence 3,5,8,13,15, Any of 17；The amino acid sequence of the weight chain variable district is sequence 19,21,23,25,27,29,31,33 in sequence table Any of；It is preferred that the antigen-binding fragment Fab is selected from following any：
KH001：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 33 in sequence table；
KH002：The amino acid sequence of the light chain variable district is sequence 8, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 33 in sequence table；
KH003：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 21 in sequence table；
KH004：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 31 in sequence table；
KH005：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 19 in sequence table；
KH006：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 23 in sequence table；
KH007：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 25 in sequence table；
KH008：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 27 in sequence table；
KH009：The amino acid sequence of the light chain variable district is sequence 3, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 29 in sequence table；
KH010：The amino acid sequence of the light chain variable district is sequence 13, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 33 in sequence table；
KH011：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 33 in sequence table；
KH012：The amino acid sequence of the light chain variable district is sequence 13, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 21 in sequence table；
KH013：The amino acid sequence of the light chain variable district is sequence 13, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 31 in sequence table；
KH014：The amino acid sequence of the light chain variable district is sequence 13, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 23 in sequence table；
KH015：The amino acid sequence of the light chain variable district is sequence 13, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 25 in sequence table；
KH016：The amino acid sequence of the light chain variable district is sequence 13, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 29 in sequence table；
KH017：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 31 in sequence table；
KH018：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 19 in sequence table；
KH019：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 23 in sequence table；
KH020：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 25 in sequence table；
KH021：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 27 in sequence table；
KH022：The amino acid sequence of the light chain variable district is sequence 15, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 29 in sequence table；
KH024：The amino acid sequence of the light chain variable district is sequence 17, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 19 in sequence table；
KH027：The amino acid sequence of the light chain variable district is sequence 5, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 31 in sequence table；
KH028：The amino acid sequence of the light chain variable district is sequence 5, the amino acid sequence of the weight chain variable district in sequence table It is classified as sequence 25 in sequence table.
3. anticomplement factor D humanized antibody or its antigen-binding fragment Fab according to claim 2, it is characterised in that The Fab is made up of heavy chain fragment and light chain, and the heavy chain fragment is cut with scissors by the CH1 and part of weight chain variable district and heavy chain constant region Sequence is constituted, and the light chain is made up of light chain variable district and constant region of light chain, the CH1 amino acid sequences of preferably described heavy chain constant region Row are identical with human antibody heavy chain's constant region CH1 amino acid sequence, and the amino acid sequence and human antibody of the constant region of light chain are light Chain amino acid constant region sequence is identical, and the amino acid sequence in more preferably described heavy chain constant region CH1 and part hinge area is sequence 39, the amino acid sequence of the constant region of light chain is sequence 41；
The antibody is made up of heavy chain or light chain, the amino acid sequence of the heavy chain constant region and the ammonia of human antibody heavy chain's constant region Base acid sequence is identical, and the amino acid sequence of the constant region of light chain is identical with human antibody light chain constant region amino acid sequence, preferably The amino acid sequence of human antibody heavy chain's constant region is sequence 43, and the human antibody light chain constant region amino acid sequence is sequence 41；
More preferably described antibody is selected from following any：
KH-A：Its sequence of light chain is sequence 45, and its sequence of heavy chain is sequence 47；
KH-B：Its sequence of light chain is sequence 49, and its sequence of heavy chain is sequence 51；
KH-C：Its sequence of light chain is sequence 45, and its sequence of heavy chain is sequence 53；
KH-D：Its sequence of light chain is sequence 49, and its sequence of heavy chain is sequence 55；
KH-E：Its sequence of light chain is sequence 57, and its sequence of heavy chain is sequence 59；
KH-F：Its sequence of light chain is sequence 57, and its sequence of heavy chain is sequence 61.
4. as derived from the humanized antibody or its antigen-binding fragment Fab of any anticomplement factor D in claim 1-3 Antigen-binding fragment, it is characterised in that：The derivative antigen-binding fragment is Fab ', F (ab ')2, Fv, weight chain variable district, light chain Variable region, the polypeptide fragment selected from weight chain variable district or the polypeptide fragment selected from light chain variable district.
5. a kind of polynucleotides, it encodes any humanized antibody, antigen-binding fragment Fab or preceding in claim 1-4 State humanized antibody, antigen-binding fragment derived from antigen-binding fragment Fab；It is preferred that the humanized antibody or antigen binding fragment The coded sequence of the light chain variable district of antibody is sequence 4, sequence 6, sequence 9, sequence 14, sequence in sequence table described in section Fab Any of 16 and sequence 18, the coded sequence of weight chain variable district is sequence 20, sequence 22, sequence 24, sequence in sequence table 26th, any of sequence 28, sequence 30, sequence 32, sequence 34.
6. polynucleotides according to claim 5, it is characterised in that：The coded sequence of the heavy chain constant region of the Fab is such as In sequence table shown in sequence 40；The constant region of light chain coded sequence of the Fab is as shown in sequence 42 in sequence table；
The coded sequence of the heavy chain constant region of the antibody is as shown in sequence 44 in sequence table；The constant region of light chain of the antibody Coded sequence is as shown in sequence 42 in sequence table；
It is preferred that the antibody is selected from following any polynucleotides：
KH-A：Its sequence of light chain is sequence 46, and its sequence of heavy chain is sequence 48；
KH-B：Its sequence of light chain is sequence 50, and its sequence of heavy chain is sequence 52；
KH-C：Its sequence of light chain is sequence 46, and its sequence of heavy chain is sequence 54；
KH-D：Its sequence of light chain is sequence 50, and its sequence of heavy chain is sequence 56；
KH-E：Its sequence of light chain is sequence 58, and its sequence of heavy chain is sequence 60；
KH-F：Its sequence of light chain is sequence 58, and its sequence of heavy chain is sequence 62.
7. following genetic stocks：It is thin containing the recombinant vector of any polynucleotides, recombinant bacterium, restructuring in claim 5-6 Born of the same parents system, recombinant virus or expression cassette；Wherein described recombinant vector is preferably the expression Fab or antibody or antigen-binding fragment Prokaryotic expression carrier or carrier for expression of eukaryon；The recombinant bacterium is preferably the Escherichia coli for carrying the gene；The restructuring Cell line is preferably the mammal cell line for being transferred to the gene, more preferably Chinese hamster ovary celI system, or 293 cells and its subbreed；Institute It is preferably the recombined adhenovirus or recombinant adeno-associated virus for carrying the gene to state recombinant virus.
8. any humanized antibody or antigen-binding fragment Fab in claim 1-4, or any institute in claim 5-6 State polynucleotides, or genetic stocks described in claim purposes：
D1) the purposes in prevention and/or treatment people's Complement Factor D relevant disease medicine is prepared, or
D2 the purposes in neutralizing people's Complement Factor D product) is being prepared, or
D3) the purposes in the kit for preparing qualitative or quantitative detection people's Complement Factor D.
9. purposes according to claim 8, it is characterised in that people's Complement Factor D relevant disease is by people's complement factor Disease caused by D rises；It is preferred that illness in eye, rheumatic arthritis, myocardial infarction or reperfusion injury；The illness in eye is more preferably year Age is macular degeneration related or diabetic retinopathy；The senile macular degeneration is more preferably the change of nonexudativeage macula lutea Property.
10. a kind of pharmaceutical composition, containing auxiliary material and active component, the active component contains at least one in following substances Kind：Any humanized antibody or antigen-binding fragment Fab in claim 1-4, or in claim 5-6 it is any described many Nucleotides, or any genetic stocks of claim 7；The auxiliary material is pharmaceutically acceptable carrier or excipient.
CN201710041945.1A 2016-01-28 2017-01-20 Humanized Fab and humanized antibody against complement factor D and use thereof Active CN107043422B (en)

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