CN1451045A

CN1451045A - IFN-alpha homologues

Info

Publication number: CN1451045A
Application number: CN00816713A
Authority: CN
Inventors: 沃尔克·海因里希斯; 特迪·陈; 菲利普·A·帕滕
Original assignee: Maxygen Inc
Current assignee: Maxygen Inc
Priority date: 1999-10-07
Filing date: 2000-10-06
Publication date: 2003-10-22
Also published as: JP2003511031A; WO2001025438A2; WO2001025438A3; CA2385045A1; AR025969A1; MXPA02003492A; EP1238082A2; AU8001300A

Abstract

Alpha interferon homologues (both nucleic acids and polypeptides) are provided. Compositions including these interferon homologue polypeptides and nucleic acids, recombinant cells comprising said homologue polypeptides and nucleic acids, methods of making the new homologues, antibodies to the new homologues, and methods of using the homologues are provided. Integrated systems comprising the sequences of the nucleic acids or polypeptides are also provided.

Description

The IFN-alpha homologues

Copyright statement

According to 37C.F.R.1.71 (e), the part of this patent document comprises data protected by copyright.When it appeared in the patent file of patent and trademark office or the archives, the copyright holder did not oppose that anyone duplicates the patent document or patent disclosure text, and in other cases, the copyright holder will keep all copyrights.

Cross reference with related application

The application is the part continuation application of the U.S. Patent application serial number 09/145,483 of submission on October 7th, 1999, and requires 09/145,483 interests and right of priority, and for this reason, 09/145,483 lists this paper in as a reference in full.

Invention field

The present invention relates to produce new interferon-' alpha ' homologue.

Background of invention

Interferon-' alpha ' is the member (Sprang, S.R. etc. (1993) Curr.Opin.Struct.Biol.3:815-827) of the various helical bundle superfamily of cytokine gene.Human interferon-alpha is by the nonallelic family coding that contains more than 20 inline copy, and these genes have the sequence identity (Henco, K. etc. (1985) molecular biology magazine .185:227-260) of 85-98% on amino acid levels.

Confirmed that interferon-' alpha ' can suppress polytype cell proliferation, especially can be used for treating the various kinds of cell proliferative disease, these diseases usually with cancer, particularly leukemia, relevant as leukemia.These protein demonstrate the antiproliferative activity to following disease: multiple myeloma, chronic lymphocytic leukemia, rudimentary lymphoma, Kaposi sarcoma, chronic myelogenous leukemia, renal cell carcinoma, tumor of bladder and ovarian cancer (Bonnem, E.M. etc. (1984) J.Biol.Response Modifiers3:580; Oldham, R.K (1985) Hospital Practice 20:71).

Interferon-' alpha ' also can be used for anti-polytype virus infection (Finter, N.B etc. (1991) medicine 42 (5): 749).Interferon-' alpha ' also demonstrates anti-human papilloma virus (anti-HPV) and infects, and the activity of hepatitis B virus and hepatitis c virus infection (Finter, N.B. etc., 1991, document is the same; Kashima, H etc. (1988) laryngoscope 98:334; Dusheiko, G.M. etc. (1986) hematology magazine 3 (supplementary issue 2): S199; Davis, GL etc. (1989) New England Journal of Medicine .321:1501).In addition, Interferon, rabbit and Interferon Receptors pathogenetic effect (Benoit, P etc. (1993) Journal of Immunology .150 (3): 707) have also been studied in some autoimmunization and inflammatory diseases.

Although these protein all have therapeutic value to multiple disease, do not make their optimizations as yet to be used as medicine.For example, the toxicity of dose limitation, acceptor cross reactivity and short serum half life, significantly weakened clinical application (Dusheiko, G. (1997) the hepatology 26:112S-121S of multiple this type cytokines; Vial, T and Descotes, J. (1994) Drug Experience 10:115150; Funke, I. etc. (1994) Ann.Hematol.68:49-52; Schomburg, A. etc. (1993) J.Cancer Res.Clin.Oncol.119:745-755).With use the diversified serious side effects performance that Interferon, rabbit accompanies and comprise influenza-like symptom, fatigue, neurological disorder comprises illusion, fever, the liver enzyme raises and oligoleukocythemia (Pontzer, C.H. etc. (1991) cancer research, 51:5304; Oldham, 1985, document is the same).

A large amount of in the interferon-' alpha ' native sequences is multifarious (so recombinant chou has big sequence space), and together with the complicacy of interferon-' alpha '/acceptor interaction, and multiple treatment and prophylactic activity have been created chance for making up senior Interferon, rabbit homologue.

The invention summary

The invention provides new interferon-' alpha ' (IFN-α) homologue polypeptide, the nucleic acid of coding said polypeptide, and complementary nucleotide sequence, the segment of described polypeptide and nucleic acid, the antibody of anti-polypeptide, and uses thereof, contain the many sets of data of interferon-' alpha ' homologue sequence signature chain and the automation system of the described character chain of use.

On the one hand, the present invention includes the interferon-' alpha ' nucleic acids homologue of separation or reorganization.Comprise being selected from SEQID NO:1 to SEQ ID NO:35, or to polynucleotide sequence and the complementary polynucleotide sequence thereof of SEQ ID NO:72 to SEQ ID NO:78.Polynucleotide sequence and complementary polynucleotide sequence thereof that coding is selected from the polypeptide of SEQ ID NO:36 to SEQ ID NO:81 or SEQ ID NO:79 to SEQ ID NO:85 also are features of the present invention.Similarly, under highly tight condition, also be feature of the present invention with the polynucleotide sequence of above-mentioned arbitrary polynucleotide sequence hybridization of total length basically.In addition, the polynucleotide sequence that contains the nucleotide fragment of above-mentioned arbitrary polynucleotide sequence also is a feature of the present invention, the wherein said nucleotide fragment peptide species of encoding, this polypeptide has antiproliferative activity in the cell proliferation test based on people Daudi clone.Similarly, the polynucleotide sequence that contains the nucleotide fragment of above-mentioned and following arbitrary polynucleotide sequence of the present invention also is a feature of the present invention, the wherein said nucleotide fragment peptide species of encoding, this polypeptide has antiviral activity in the test based on mouse cell line/EMCV.

The present invention also comprises the nucleic acid of separation or reorganization, and it contains the polynucleotide sequence of coded polypeptide, and wherein said polypeptide contains following aminoacid sequence: CDLPQTHSLG-X ₁₁-X ₁₂-RA-X ₁₅-X ₁₆-LL-X ₁₉-QM-X ₂₂-R-X ₂₄-S-X ₂₆-FSCLKDR-X ₃₄-DFG-X ₃₈-P-X ₄₀-EEFD-X ₄₅-X ₄₆-X ₄₇-FQ-X ₅₀-X ₅₁-QAI-X ₅₅-X ₅₆-X ₅₇-HE-X ₆₀-X ₆₁-QTFN-X ₆₇-FSTK-X ₇₂-SS-X ₇₅-X ₇₆-W-X ₇₈-X ₇₉-X ₈₀-LL-X ₈₃-K-X ₈₅-X ₈₆-T-X ₈₈-L-X ₉₀-QQLN-X ₉₅-LEACV-X ₁₀₁-Q-X ₁₀₃-V-X ₁₀₅-X ₁₀₆-X ₁₀₇-X ₁₀₈-TPLMN-X ₁₁₄-D-X ₁₁₆-ILAV-X ₁₂₁-KY-X ₁₂₄-QRITLYL-X ₁₃₂-E-X ₁₃₄-KYSPC-X ₁₄₀-WEVVRAEIMRSFSFSTNLQKRLRRKE, or its conservative variant that replaces, wherein X ₁₁Be N or D; X ₁₂Be R, S or K; X ₁₅Be L or M; X ₁₆Be I, M or V; X ₁₉Be A or G; X ₂₂Be G or R; X ₂₄Be I or T; X ₂₆Be P or H; X ₃₄Be H, Y or Q; X ₃₈Be F or L; X ₄₀Be Q or R; X ₄₅Be G or S; X ₄₆Be N or H; X ₄₇Be Q or R; X ₅₀Be K or R; X ₅₁Be A or T; X ₅₅Be S or F; X ₅₆Be V or A; X ₅₇Be L or F; X ₆₀Be M or I; X ₆₁Be I or M; X ₆₇Be L or F; X ₇₂Be D or N; X ₇₅Be A or V; X ₇₆Be A or T; X ₇₈Be E or D; X ₇₉Be Q or E; X ₈₀Be S, R, T or N; X ₈₃Be E or D; X ₈₅Be F or L; X ₈₆Be S or Y; X ₈₈Be E or G; X ₉₀Be Y, H, N; X ₉₅Be D, E or N; X ₁₀₁Be I, M or V; X ₁₀₃Be E or G; X ₁₀₅Be G or W; X ₁₀₆Be V or M; X ₁₀₇Be E, G or K; X ₁₀₈Be E or G; X ₁₁₄Be V, E or G; X ₁₁₆Be S or P; X ₁₂₁Be K or R; X ₁₂₄Be F or L; X ₁₃₂Be T, I or M; X ₁₃₄Be K or R; And X ₁₄₀Be A or S.According to those skilled in the art's general general knowledge, each single-letter in this aminoacid sequence is represented specified amino acid residues.

The invention still further relates to and have above-mentioned arbitrary polypeptide of sequence, described sequence such as SEQ ID NO:36 to SEQ ID NO:54.

In other embodiments, encoded polypeptides contains the aminoacid sequence that is selected from SEQ ID NO:36 to SEQ IDNO:54; Nucleic acid contains the polynucleotide sequence that is selected from SEQ ID NO:1 to SEQ ID NO:19.

The present invention also provides among SEQ ID NO:36-70 and the SEQ ID NO:72-79 polypeptide fragments of any.In one aspect of the invention, described polypeptide fragments shows antiproliferative activity in the cell proliferation test based on people Daudi clone, or shows antiviral activity in the test based on mouse cell line/EMCV, perhaps shows this two kinds of activity simultaneously.Below will describe in detail based on the cell proliferation test of people Daudi clone with based on the antiviral activity in the test of mouse cell line/EMCV.On the other hand, the invention provides the polynucleotide sequence of the nucleotide fragment that contains the above-mentioned and following arbitrary nucleic acid of the present invention, wherein said nucleotide fragment coded polypeptide segment, as hereinafter described in detail, described polypeptide fragments shows antiproliferative activity in the cell proliferation test based on people Daudi clone, or in test, show antiviral activity based on mouse cell line/EMCV, perhaps show this two kinds of activity simultaneously.

The present invention also comprises nucleic acid isolating or reorganization, and it contains the polynucleotide sequence of coded polypeptide, and wherein polypeptide contains the aminoacid sequence of at least 20 continuous amino acids of any among the SEQ ID NO:36-70.In other embodiments, polypeptide of the present invention contains the aminoacid sequence that comprises following one or more amino-acid residues: (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein the numbering of amino-acid residue is numbered corresponding to the residue in the SEQ ID NO:36 aminoacid sequence.In a plurality of embodiments, the polypeptide that is encoded of the present invention contains among the SEQ ID NO:36-70 any at least 30, at least 50, at least 70, at least 75, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 155, at least 160, or at least 165 successive amino-acid residues.In other embodiments, encoded polypeptide length is at least 150, at least 155, at least 160, at least 163, or at least 165 amino acid.In another embodiment, encoded polypeptide length is about 166 amino acid.In other embodiments, encoded polypeptide contains and is selected from SEQ ID NO:36, SEQ ID N0:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, the aminoacid sequence of SEQ IDNO:45 and SEQ ID NO:46.

In other embodiments, the invention provides and contain the nucleic acid that is selected from following polynucleotide sequence: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

In other embodiments, in test, have antiproliferative activity by any nucleic acid encoded polypeptide or its segment in the invention described herein, or in test, have antiviral activity based on people WISH cell/EMCV based on people Daudi clone.In other embodiments, encoded polypeptide has in the test based on people Daudi clone and is at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/milligram (1 unit is the protein mass in milligram (mg) of inducing 50% antiproliferative activity required) perhaps has in the test based on people WISH cell/EMCV and is at least about 2.1 * 10 ⁷The antiviral activity of individual unit/milligram (1 unit is the protein mass in mg of inducing 50% antiviral activity required).In other embodiments, encoded polypeptide can be in conjunction with I type Interferon Receptors, and preferred combination people I type Interferon Receptors is more preferably in conjunction with people's (for example I type) interferon-' alpha ' acceptor.

The present invention also comprises any nucleic acid that contains in the invention described herein, perhaps expresses the cell of any polypeptide in the invention described herein.In one embodiment, described cell expressing is by the nucleic acid encoded polypeptide in the invention described herein.

The present invention also comprises the carrier that contains any nucleic acid in the above-mentioned and following invention.Described carrier comprises plasmid, clay, phage, or virus; Carrier can be an expression vector for example, cloning vector, package carrier, integrative vector etc.The present invention also comprises by the cell of carrier transduction of the present invention.The present invention also comprises composition, and it contains any nucleic acid and vehicle (being preferably pharmaceutically-acceptable excipients) in the above-mentioned and following invention.The present invention also comprises by for example transduction vector and producing, and comprises any polypeptide in the above-mentioned and following invention or the cell and the transgenic animal of nucleic acid.

The present invention also comprises by using restriction endonuclease, one or more nucleic acid in the above-mentioned or following invention of RNA enzyme or dnase digestion and the composition that produces; And by at deoxyribonucleotide triphosphoric acid and nucleic acid polymerase, under the existence as the polysaccharase of thermostability, be incubated one or more nucleic acid in the above-mentioned or following invention and the composition that produces.

The present invention also comprises the composition that contains above-mentioned or following two or more nucleic acid.Described composition can comprise nucleic acid library, and wherein said library is contained at least about 5,10,20 or 50 kind of nucleic acid.

On the other hand, the present invention includes polypeptide isolating or reorganization, described polypeptide is coded by above-mentioned or following any nucleic acid.In one embodiment, described polypeptide can contain and is selected from SEQ ID NO:36 to SEQ ID NO:70, or the sequence of SEQ ID NO:79 to SEQ ID NO:85.

The present invention also comprises polypeptide, and it contains by coded proteinic 50 the successive amino acid of polynucleotide sequence at least, and wherein polynucleotide sequence is selected from: (a) SEQ ID NO:1 to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78; (b) coding is selected from the polynucleotide sequence of the polypeptide of SEQ ID NO:36 to SEQ IDNO:70 or SEQ ID NO:79 to SEQ ID NO:85; (c) under the height stringent condition, with the polynucleotide sequence of total length (a) or (b) complementary sequence of the polynucleotide sequence of hybridization basically.In a plurality of embodiments, polypeptide contains coded proteinic at least about 70,100,120,130,140,150,155,160,165 or 166 successive amino acid.

The present invention also comprises polypeptide isolating or reorganization, and it contains aminoacid sequence, and this aminoacid sequence contains among the SEQ ID NO:36-70 at least 50 the continuous amino acid residues of any, with following one or more amino acid: Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein amino acid whose numbering is corresponding to the numbering among the SEQ ID NO:36.In a plurality of embodiments, polypeptide contain among the SEQ ID NO:36-70 any at least about 50,70,75,100,110,120,130,140,150,155,160,163,165 or 166 successive amino acid.In a more preferred embodiment, polypeptide contains SEQ ID NO:36, SEQ ID N0:37, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, any at least about 50 among SEQ ID NO:45 or the SEQ ID NO:46,70,75,100,110,120,130,140,150,155,160,163,165 or 166 successive amino-acid residues.In other embodiments, the length of polypeptide of the present invention is at least about 50,70, and 75,100,110,120,130,140,150,155,160,163,165 or 166 amino-acid residues, or preferably its length is 166 amino acid.Also can use long polypeptide, for example contain the polypeptide of purifying mark etc.Described polypeptide can demonstrate antiproliferative activity in the test based on people Daudi clone, and/or demonstrates antiviral activity in the test based on people WISH cell/EMCV.

The present invention also comprises and anti-at least a antigenic polyclonal antiserum specificity bonded polypeptide, and described at least a antigen contains and is selected from aminoacid sequence or its pulsating peptide sequence shown in SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to the SEQ ID NO:85.Especially, the invention provides and anti-at least a antigenic polyclonal antiserum bonded polypeptide, wherein said at least a antigen contains at least a aminoacid sequence shown in SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to the SEQ ID NO:85, or the segment of above-mentioned arbitrary aminoacid sequence, wherein with one or more known interferon-alpha polypeptides or protein subduction (subtract) polyclonal antiserum, described polypeptide or protein for example comprise by having or corresponding to following one or more GenBank ^TMThe nucleic acid of registration number and other similar or homologous interferon-' alpha ' nucleic acids sequence encoded polypeptide or the protein described in the GenBank, described GenBank ^TMRegistration number is: and J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, and V00549 (α-2a) and I08313 (α-Con1).

Above-mentioned or following any polypeptide is chosen wantonly in the test based on people Daudi clone has antiproliferative activity, and/or has antiviral activity in the test based on people WISH cell/EMCV.Above-mentioned or following any polypeptide has in the test based on people Daudi clone and is at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/mg perhaps has in the test based on people WISH cell/EMCV and is at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg.In other embodiments, above-mentioned or following any polypeptide can be in conjunction with I type Interferon Receptors, and preferred combination people I type Interferon Receptors is more preferably in conjunction with the human interferon-alpha acceptor.

In other embodiments, above-mentioned or following any polypeptide can further comprise secretion/positioning sequence, as signal sequence, and organoid target sequence, film positioning sequence etc.Any polypeptide as herein described can further include the sequence that is beneficial to purifying, epi-position mark (as the FLAG epi-position) for example, poly histidine mark, GST fusions etc.Randomly, the N-terminal of polypeptide can comprise methionine(Met).Any polypeptide in the invention described herein is optional to comprise one or more modified amino acid, as glycosylation amino acid, and PEGization amino acid; farnesylation amino acid, ethanoyl amino acid, biotinylation amino acid; carboxylated amino acid, phosphorylated amino acid, acylated amino etc.

The present invention also comprises composition, and it contains any polypeptide as herein described and vehicle, is preferably pharmaceutically-acceptable excipients.

The present invention also comprises antibody or the antiserum(antisera) that produces by the polypeptide of giving one or more inventions described herein of administration, wherein said antibody or antiserum(antisera) do not combine with known alpha-interferon polypeptide or protein specific, and described polypeptide or protein for example comprise by having or corresponding to following one or more GenBank ^TMThe nucleic acid of registration number and other similar or homologous interferon-' alpha ' sequence is coded any polypeptide or the protein described in the GenBank, described GenBank ^TMRegistration number is: and J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, and V00549 (α-2a) and I08313 (α-Con1).

The present invention also comprises antibody, it can with contain the polypeptide of sequence specificity that is selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85 and combine.Described antibody is polyclonal antibody for example, monoclonal antibody, chimeric antibody, humanized antibody, single-chain antibody, Fab segment, the segment that produces by the Fab expression library etc.

The present invention also comprises the method for production polypeptide of the present invention.A kind of method comprises: with any nucleic acid transfered cell as herein described colony, described nucleic acid can be operated with the adjusting sequence that can effectively produce coded polypeptide and link to each other, in substratum culturing cell producing polypeptide, and optional from cell or substratum isolated polypeptide.Nucleic acid can be the part of carrier, for example recombinant expression vector.

The present invention also comprises the method that suppresses growth of tumour cell, and described method comprises tumour cell is contacted with the polypeptide of invention described herein, thereby suppresses the growth of tumour cell.In one embodiment, the present invention includes the method that suppresses the tumour cell population growth, described method comprises: tumour cell colony is contacted with the polypeptide of the present invention of significant quantity of growth of tumour cell in being enough to suppress described tumour cell colony, thereby suppress growth of tumour cell in the described cell colony.In a plurality of embodiments, tumour cell can be a human cancer cell, human leukemia cell, people T-lymphoma cell, human melanoma cell, other human cancer cell as herein described etc.Tumour cell can be intravital, ex vivo or external (for example through cultured cells).

The present invention comprises that also inhibition is by the method for one or more intracellular virus replications of virus infection, described method comprises: the polypeptide in the above-mentioned and following invention of one or more infected cells and significant quantity is contacted, thereby suppress the virus replication in described one or more cell, wherein said amount is enough to suppress the interior virus replication of described one or more cells infected.In a plurality of embodiments, virus can be RNA viruses, and for example human immune deficiency virus or hepatitis C virus also can be dna virus, for example hepatitis B virus.Infected cells can be intravital, ex vivo or external (for example through cultured cells).

The experimenter that the present invention also is included as the treatment demand treats the method for autoimmune disease, and described method comprises: using to the experimenter is enough to treat autoimmune disease, the polypeptide of the invention described herein of significant quantity.In a plurality of embodiments, autoimmune disease can be a multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type i diabetes etc.The present invention comprises that also to by use the improvement of the treatment of diseases method that interferon-' alpha ' can treat to the experimenter, described improvement comprises: using to the experimenter is enough to treat described disease, the polypeptide of the invention described herein of significant quantity.By use the disease that interferon-' alpha ' can treat to the experimenter can be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type i diabetes, AIDS or AIDS-related syndromes etc.

In general, the present invention also comprises the sequence nucleic acid and the protein that obtain by sudden change this paper.Similarly, the present invention also comprises those nucleic acid and the protein by diversity generates or tumor-necrosis factor glycoproteins reorganization (RSR) method (for example DNA reorganization) produces.The present invention also comprises sudden change and the recombination method that uses nucleic acid described herein to carry out.For example, a kind of method of the present invention comprises one or more nucleotide sequences in the above-mentioned and following invention and one or more other nucleic acid (include but not limited to as herein described those) reorganization repeatedly, each the sequence encoding interferon-' alpha ' homologue in one or more other nucleic acid or its amino acid subsequence.Reconstitution steps is optional in vivo, ex vivo, in silico or external carrying out.Described repeat the reorganization can produce at least one recombinant interferon-alpha homologues nucleic acid library.The present invention also comprises the recombinant interferon-alpha homologues nucleic acid that produces by this method, the cell that contains described recombinant interferon-alpha homologues nucleic acid, repeat the nucleic acid library that recombination method produces by this, contain the composition of two or more described recombinant interferon-'alpha ' nucleic acids and contain described recombinant interferon-'alpha ' nucleic acids or contain the cell colony in described library.In one embodiment, at least 10 kinds of described recombinant nucleic acids are contained in described library.

The present invention also provides the method for producing interferon-' alpha ' homologue nucleic acid modified or reorganization, and this method comprises the nucleic acid of the invention described herein that suddenlys change.

The present invention also provides the nucleic acid of coded interference element-alpha homologues, respectively with respect to the growth inhibitory activity of the known interferon-' alpha ' pair cell of human interferon-alpha 2a or other colony, static (cytostatic) activity of cell or cytotoxic activity, described homologue pair cell colony (for example cancer cells) has the growth inhibitory activity of increase, static (cytostatic) activity of cell or cytotoxic activity.

With the following detailed description part, can more clearly understand these and other objects of the present invention and feature in conjunction with the accompanying drawings.

The accompanying drawing summary

Figure 1A-1E has shown the sequence contrast arrangement of the ripe Interferon, rabbit homologue peptide sequence (SEQ IDNO:36-70 and 79-85) that the present invention exemplifies.

What Fig. 2 showed is: respectively with respect to two kinds of control compounds, be human interferon-alpha-2a (" IFN-α-2a " or " 2a ") and joint owner's Interferon, rabbit (" IFN-Conl " or " Con1 ") antiproliferative activity and antiviral activity separately, the Interferon, rabbit homologue that the present invention exemplifies is based on the antiproliferative activity in the test of people Daudi clone with based on the antiviral activity in the test of people WISH cell/EMCV.

Fig. 3 A, 3B and 3C have illustrated IFN-alpha homologues 3DA11 (SEQ ID NO:40) and contrast Interferon, rabbit, and human interferon-alpha-2a (" 2a ") and joint owner's interferon alpha (" Con1 ") are to the profile of activity of a series of tumor cell lines.Fig. 3 A has shown that IFN-alpha homologues 3DA11 and every kind of contrast IFN are to the total growth inhibitory activity of the cell of each clone, described activity reflects with the GI50 value, GI50 makes the growth 50% of specific cells system be suppressed the concentration (μ g/ml) of required interferon alpha homologue or contrast IFN α, this value is by when soak finishes, and the increase degree minimizing 50% of clean protein/polypeptide is measured in interferon alpha homologue or the contrast IFN α experimental group.

Fig. 3 B has shown IFN-alpha homologues 3DA11 and every kind of static activity that contrasts IFN to each clone in a series of clones.Static activity refers to the activity of energy cell growth inhibiting and breeding.Static activity is rated as the growth of the cell of specific cells system and/or breeding is suppressed fully, so that amount (" total growth-inhibiting " or " TGI ") the required interferon alpha homologue or the reflection of contrast IFN α concentration (μ g/ml) of soak cell protein when the amount of cell protein equals soak and begins when finishing.

Fig. 3 C has illustrated IFN-alpha homologues 3DA11 and every kind of cytotoxic activity that contrasts IFN to each clone.The degree of the cytotoxicity of medicament (as IFN homologue or IFN compound) the specific destruction that to be this medicament have some cell, or refer to have described destruction.This term generally refers to the medicament that can cause necrocytosis, and is used in reference to the compound drug that lysis that immunological phenomena causes and selectivity are killed somatoblast especially.In Fig. 3 C, illustrate cytotoxicity with LC50, the clean protein mass of cell when observation begins with soak is compared, when insulation finishes, make clean protein increases degree in the control cells (contrast IFN α) reduce by the concentration (μ g/ml) of 50% required IFN-alpha homologues 3DA11, be illustrated in and add certain interference element cell net loss afterwards.Cytotoxicity can be rated as: for control cells, destroy or kill specific cells and be the concentration of 50% required IFN-alpha homologues 3DA11 of total cell count (being total group).

Fig. 4 A, 4B, what 4C and 4D showed respectively is: with respect to two kinds of contrast interferon alphas, be that human interferon-alpha 2a (" 2a ") and joint owner's interferon-' alpha ' (" Con1 ") are to leukemia clone (RPMI-8226) (Fig. 4 A), lung cancer cell line (NCI-H23) (Fig. 4 B), the static activity of cell of renal carcinoma cell line (ACHN) (Fig. 4 C) and ovarian cancer cell line (OVCAR-3) (Fig. 4 D), the static activity of corresponding cell of selected interferon-' alpha ' homologue of the present invention.The static activity of cell is reflected by the TGI value of the plain α of certain interference (making the cell growth of clone be suppressed required interferon alpha concentration, the amount of the cell protein when wherein the amount of the cell protein during the soak end equals soak and begins) comprehensively.

Fig. 5 is illustrated in and has used dosage respectively is 2 μ g, two kinds of interferon-' alpha ' homologues of the present invention that exemplify (being called as " IFN-CH2.2 " and " IFN-CH2.3 ") of 10 μ g and 50 μ g, dosage is 2 μ g, the mouse IFN-α-4 of 10 μ g and 50 μ g and dosage are 2 μ g, after humanIFN--2a of 10 μ g and 50 μ g, the comparison of the mouse number of survival (total mice is 6).Result shown in Figure 5 shows: in the mouse model system, the outer antiviral activity of the modification of these the two kinds IFN-alpha homologues that exemplify can be kept and continue in vivo.Phosphoric acid-buffer saline (PBS) is used as contrast.

Detailed Description Of The Invention Definition

Unless definition is separately arranged in the context of this specification remainder, all technology used herein are all identical with the general implication of understanding of those skilled in the art with scientific terminology.

Based on context, " polynucleotide sequence " is the character chain of nucleic acid (polymer of nucleotides (A, C, T, U, G etc., or natural or artificial nucleotide analog)) or statement nucleic acid. Can determine given nucleic acid or complementary nucleic acid by any specific polynucleotide sequence.

Similarly, based on context, " amino acid sequence " is amino acid whose polymer (protein, polypeptide etc.), or the character chain of statement amino acid polymer. Can determine given nucleic acid or complementary nucleic acid by any specific polynucleotide sequence.

Work as nucleic acid, protein, peptide, (protein (comprises compound to component associated with it under polypeptide or other component and native state for other peptide, polypeptide, the polymerase and the ribosomes that for example accompany with native sequences), nucleic acid, cell, synthetic agent, cell impurity, groups of cells is graded), when for example normal other component associated with it is partially or completely separated in its derived cell, can think that they are " separation ". Work as nucleic acid, polypeptide or other component are partially or completely separated or are therefrom partially or completely reclaimed with other component in its natural surroundings, make them at composition, exist (namely on mole foundation as the kind that has comparative advantage in mixture or component gleanings, they are higher than the content of any other various species in composition) time, can say that also they separate. In preferred embodiments, goods are by more than 70%, generally more than 80%, or preferably more than this separated species composition of 90%.

On the one hand, " basically pure " or " separation " nucleic acid (for example RNA or DNA), polypeptide, protein or component also the kind of feeling the pulse with the finger-tip (for example nucleic acid or polypeptide) account at least existing all large molecular species weight (on mole foundation) 50,60 or 70%. Basically pure or component that separate also can account at least existing all large molecular species in composition weight 80,90 or 95%. The purpose kind of separating also can be purified into and be essentially homogeneous (detection method by routine can't detect the dopant species in component), and wherein component is comprised of the derivative of single large molecular species in fact.

Term " nucleic acid of separation " can refer to the nucleic acid (as DNA or RNA) that two coded sequences its immediate in natural gene group with organism producing nucleic acid of the present invention (namely at 5 ' end, at 3 ' end) are not close to. Therefore, this term for example comprises processes by polymerase chain reaction (PCR) or restriction endonuclease cDNA or the genomic DNA segment that produces, no matter and this cDNA or genomic DNA segment are to mix carrier, be integrated in the genome with the identical or different species of its source organism (for example comprise virus), or be connected to form the heterozygous genes of coding chimeric polyeptides with other coded sequence, or do not rely on any other DNA sequence dna. DNA can be two strands or strand, sense or antisense.

, when nucleic acid or polypeptide are artificial or through the gene engineering method transformation, perhaps, derived from artificial or when the protein of transformation or the nucleic acid, can say that they are " restructuring ". When being used for referring to for example cell, nucleotides, when carrier or polypeptide, the general expression of term " restructuring " is modified described cell by importing allos (or external source) nucleic acid or changing natural acid, nucleotides or carrier, perhaps expression carrys out modified polypeptide by importing allogeneic amino acid, perhaps represents the cell that cell source is hung oneself and so modified. Recombinant cell is expressed the nucleotide sequence (for example gene) of not found in natural (non--restructuring) form of cell, and perhaps expressing may be by unconventionality expression, the natural acid sequence (for example gene) of expressing or not expressing on a small quantity. Term " recombinant nucleic acid " (for example DNA or RNA) molecule refers to for example non-natural nucleotide sequence, or by at least two, generally do not occur simultaneously, uncorrelated each other, perhaps otherwise be each other the participation (for example artificial combination) of sequence section separately and the nucleotide sequence of preparation. Recombinant nucleic acid can contain the nucleic acid molecules that links together or combine and form by with synthetic nucleic acid segment separate sources and/or artificial. It is attainable artificial combination usually by the following method that term " restructuring produces " refers to, described method comprises chemical synthesis process, the repetitive sequence restructuring of nucleic acid segment or the generation method of other diverse oligonucleotide (for example reorganization), or by for example technique for gene engineering well known by persons skilled in the art, the nucleic acid segment of separating is operated. " recombinant expressed " refers generally at external generation recombinant nucleic acid, recombinant nucleic acid is transferred in body, the cell of external or ex vivo, and express therein or the technology of propagation recombinant nucleic acid. " recombinant polypeptide " or " recombinant protein " refers to respectively the polypeptide or the protein that are produced by the gene of clone or restructuring or nucleic acid usually.

" subsequence " or " segment " is any part of complete sequence, reaches and comprise complete sequence.

When any given polymers compositions (amino acid residue, the nucleotides that mixes etc.) identical residue position in amino acid or nucleotides is selected by reference in position, rather than the physical location of the component in given polymer is while specifying, the numbering of given amino acid or nucleotide polymer " corresponding to " " numbering " of selected amino acid polymer or nucleic acid.

Carrier is to be convenient to selected nucleic acid is transduceed to cell, or at the composition of cells nucleic acid. Carrier comprises for example plasmid, clay, virus, YAC, bacterium, poly-lysine etc. " expression vector " is restructuring or the synthetic nucleic acid construct that produces, and it has the specific nucleic acid element of a series of permissions at host cell transcription specific nucleic acid. Expression vector can be the part of plasmid, virus or nucleic acid fragment. Expression vector generally comprises with promoter can operate the nucleic acid to be transcribed that is connected.

" polynucleotides of total length or amino acid sequence basically " refers to be at least about 50% of total length, be at least about 60%, generally be at least about 70%, generally be at least about 80%, or generally be at least about 90%, 95%, 96%, 97%, 98% or 99% or longer amino acid sequence or nucleotide sequence.

" humanα-interferon's acceptor " is by the acceptor of the natural activation of IFN-α in people's cell.

A certain material is that " natural " refers to and can find this fact of this material at occurring in nature. For example, be present in organism and comprise in virus, can separate and obtain from its natural origin, and be natural by polypeptide or the polynucleotide sequence modified intentionally artificially in laboratory. In one aspect, " natural " nucleic acid (for example DNA or RNA) molecule is the nucleic acid molecules that exists with the state identical with naturally occurring state; Be that nucleic acid molecules does not separate, restructuring or clone.

" antibody " used herein refers to protein, its contain one or more basically or part by the polypeptide of immunoglobulin gene or immunoglobulin gene fragment encoding. Known immunoglobulin gene comprises κ, λ, alpha, gamma, δ, ε and μ constant region gene, and various immune globulin variable region gene. Light chain is divided into κ or λ. Heavy chain is divided into γ, μ, and α, δ or ε, they also are defined as respectively IgG with the type of immunoglobulin (Ig) conversely, IgM, IgA, IgD and IgE. The tetramer is contained in typical immunoglobulin (Ig) (as antibody) structural units. Each tetramer is comprised of two pairs of identical polypeptide chains, and every pair of polypeptide chain has one " light chain " (approximately 25kD) and one " heavy chain " (approximately 50-70kD). The N-end of every chain limits approximately 100 to 110 or more amino acid whose variable region, and antigen recognizing mainly is responsible in described variable region. The variable light chain of term (VL) and variable heavy chain (VH) refer to respectively these light chains and heavy chain. The existence form of antibody can be complete immunoglobulin (Ig), or a plurality of fragments of having been identified in detail that produced by multiple peptide enzymic digestion. Therefore, for example, the disulfide bond place digestion antibody of pepsin in hinge region, dimer F (ab) ' 2 that produce Fab, Fab self is the light chain that is connected with VH-CH1 by disulfide bond. Can reduce F (ab) ' 2 under the condition of gentleness, interrupt the disulfide bond in hinge region, thereby change F (ab) ' 2 dimer into Fab ' monomer. Fab ' monomer is in fact the Fab (detailed description about other antibody fragment can be referring to " basic immunology ", and W.E.Paul compiles, Raven publishing house, New York (1993)) with part hinge region. Although by the digestion complete antibody, can obtain a plurality of antibody fragments, those skilled in the art still wish and can or utilize recombinant DNA method from the beginning to synthesize these Fab ' fragments by chemical method. Therefore, term antibody used herein also comprises the antibody fragment that produces by modifying complete antibody, or the antibody fragment that uses recombinant DNA method from the beginning to synthesize. Antibody comprises single-chain antibody, and described single-chain antibody comprises scFv (sFv) antibody, and wherein variable heavy chain and variable light chain (directly or pass through peptide linker) link together, and forms the polypeptide that extends.

" antigen-binding fragment " of antibody is peptide or the polypeptide fragment of being combined with antigen in antibody. Be of value in antibody, participate in, or those amino acid of the combination of impact and antigen formed antigen-binding site. Referring to Scott, T.A and Mercer, E.I., concise encyclopedia: biochemistry and molecular biology (de Gruyter, the 3rd edition, 1997) [hereinafter referred is " Scott, concise encyclopedia "] and Watson, J.D etc., recombinant DNA (the 2nd edition, 1992) [hereinafter referred is, and " Watson, recombinant DNA "], every piece of document is all listed this paper in as a reference in full.

" immunogene " refers to the material that can cause immune response. Immunogenic example comprises for example antigen, the autoantigen that works when inducing autoimmunity disease, and the tumor-associated antigen of expressing on cancer cell.

" antigen " is a kind of like this material, and it can cause antibody and form in the host, perhaps can produce the specificity lymphocyte population with described substance reaction. Antigen is generally for the large molecule (for example protein and polysaccharide) of host for external source.

Term " immunoassay " comprises the detection method of using antibody or immunogene combination or specific binding antigen. The characteristic feature of immunoassay is to utilize the specific binding characteristic of specific antibodies to separate, target, and/or quantitative antigen.

Term " homology " refers generally to the similitude degree between two or more structures. Term " homologous sequence " refers to have in large molecule the zone of similar monomer sequence. When being used for nucleotide sequence, term " homology " refers to the similitude degree between two or more nucleotide sequences (as gene) or its fragment. In general, the similitude degree between two or more nucleotide sequences refers to: the composition of the two or more nucleotide bases in two or more nucleotide sequences (or other yielding characteristics), order or the similitude degree of arranging. Term " homologous nucleic acid " refers generally to contain at nucleotide base and forms, and has the to a certain degree nucleic acid of the nucleotide sequence of similitude on arrangement or order. Two or more nucleic acid can be identical or different kind or groups. Term " percent homology " when being used for nucleotide sequence, generally refers to the percentage degree of similitude between the nucleotide sequence of two or more nucleic acid.

When being used for polypeptide (or protein) sequence, term " homology " refers to the similitude degree between two or more polypeptide (or protein) sequences (as gene) or its fragment. In general, the similitude degree between two or more polypeptide (or protein) sequence refers to: the two or more amino acid whose composition in two or more polypeptide (or protein), order or the similitude degree of arranging. Two or more polypeptide (or protein) can be identical or different kind or groups. Term " percent homology " when being used for polypeptide (or protein) sequence, generally refers to the percentage degree of similitude between the amino acid sequence of two or more polypeptide (or protein) sequence. Polypeptide or protein that term " homeopeptide " or " homologous protein " generally refer to respectively have similar amino acid sequence and function. The correlation of this homeopeptide or protein is embodied in has similar amino acid sequence and function, but uses the techniques described herein, can derive from similar and different species or obtain them.

Term used herein " experimenter " includes but not limited to biology; Mammal, comprise for example people, non-human primate (as monkey), mouse, pig, milk cow, goat, rabbit, rat, cavy, hamster, horse, monkey, sheep or other non-human mammal; Non--mammal, comprise for example non--mammalian vertebrates, as birds (as chicken or duck) or fish; With non--mammality invertebrate.

Term " pharmaceutical composition " refers to and is suitable for the composition as medicine to the experimenter who comprises the animal or human. Pharmaceutical composition generally comprises activating agent and the medicine acceptable carrier of effective dose.

Term " effective dose " refers to dosage or the amount that is enough to produce required effect. Required effect is included in the objective or subjective improvement that produces in the acceptor of described dosage or amount.

" prophylactic treatment " is to not demonstrating disease indication or symptom, or only demonstrates experimenter's administering therapeutic of early indication or the symptom of disease, makes the treatment of using to weaken, and prevents or reduce the risk of generation disease. Preventative-therapeutic effect is prophylactically to treat disease. " prophylactic activity " refers to such as nucleic acid, carrier, gene, polypeptide, protein, material, the pharmacy application of its composition is in not demonstrating disease indication or symptom, or after only demonstrating the experimenter of the early indication of disease or symptom, can weaken, prevent from or reduce the experimenter producing the activity of the risk of disease. " can be used for preventing " medicament or compound (as nucleic acid or polypeptide) and refer to and can be used for weakening, prevent, treatment or reduce medicament or the compound of the generation of disease.

" therapeutic treatment " is the treatment that the experimenter to the symptom that demonstrates disease or sign uses, and wherein giving the purpose of experimenter's administering therapeutic is sign or the symptom that weakens or eliminate a disease. " therapeutic activity " refers to such as nucleic acid, carrier, and gene, polypeptide, protein, material, or the activity of these signs or symptom can be eliminated or weaken to the pharmacy application of its composition after the experimenter of the sign that demonstrates disease or symptom. " can be used for treating " medicament or compound (as nucleic acid or polypeptide) and refer to and can be used for weakening, treat or the sign that eliminates a disease or medicament or the compound of symptom.

Term " gene " broadly refers to any DNA section relevant to biological function. Gene comprises coded sequence and/or the required regulating and controlling sequence of its expression. That gene also comprises is non--the DNA nucleic acid segment expressed, for example form the section of the recognition sequence of other oroteins.

in general, hereinafter term used and cell are hereinafter described cultivated, molecular genetics, and molecular biology, the laboratory method of nucleic acid chemistry and protein chemistry is all those skilled in the art's those terms and methods well-known and commonly used. as Sambrook etc., molecular cloning-laboratory manual (the 2nd edition), Vol.1-3, cold spring harbor laboratory, the cold spring port, New York, 1989 (hereinafter referred is " Sambrook ") and up-to-date molecular biology methods, the volumes such as F.M.Ausubel, fresh approach, Greene Publishing Associates, Inc and John Wiley ﹠ Sons, standard technique described in co-partnership company's (augmenting in 1999) (hereinafter referred is " Ausubel ") can be used for the recombinant nucleic acid method, nucleic acid is synthetic, cell culture processes and transgenosis are mixed, as electroporation, injection and lipofection. generally, carrying out oligonucleotides according to specification synthesizes and purification step. generally according to the conventional method of this area and many pieces of general references that provide, carry out the operation of technology and method herein. method wherein it is believed that it is that those skilled in the art are well-known,, in order to help reader, provides these methods.

This paper definition or characterized a plurality of other terms.Polynucleotides of the present invention Interferon-' alpha ' homologue sequence

The invention provides the interferon-' alpha ' homologue polypeptide of separation or reorganization, and the polynucleotides of the separation or reorganization of this polypeptide of encoding.

as hereinafter described in detail, according to the present invention, the polynucleotide sequence of the interferon-' alpha ' homologue polypeptide of encoding new, the nucleotide sequence of the nucleotide sequence (as subsequence) of coded interference element-alpha homologues polypeptide fragment and coding correlation fusion polypeptide or protein or its function equivalent, be collectively referred to as in this article " interferon-' alpha ' homologue ", " interferon homologue nucleic acid ", " IFN-alpha homologues ", " IFN homologue ", " IFN nucleic acid ", " interferon homologue ", " interferon nucleic acid ", " recombinant interferon-α ", " recombinant interferon-'alpha ' nucleic acids ", " nucleic acid of the present invention ", " polynucleotides of the present invention " or " nucleotides of the present invention ". polynucleotides of the present invention, also comprise and comprise polynucleotides, nucleotides and the nucleic acid fragment of above-mentioned each term in nucleotides and nucleic acid. term " nucleic acid " and term " nucleotides " can Alternates.

The polynucleotide of code book invention polypeptide in the library of interferon-' alpha ' correlated series, have been found through reorganizing.Screen the antiproliferative activity of library member, and analyze them in some cases having infected the antiviral activity of viral people's cell to human tumor cell line.Filter out at infected viral mouse cell have antiviral activity through reorganization the library in, found sequence subset provided herein.Press the encoding sequence of identifying the Interferon, rabbit homologue described in the embodiment.

Briefly, will import intestinal bacteria through the library of the ripe interferon-' alpha ' encoding sequence of reorganization.It is described to press embodiment 1, screens bacterium colony in the antiproliferative activity test at the height-streams flux of people Daudi tumor cell line, selects to express the bacterium colony of active polypeptide, again-screening, measure expression level.The DNA that separates selected bacterium colony carries out-reorganizes to produce the s-generation library s-generation library being imported intestinal bacteria to it, screens antiproliferative activity in the cell proliferation test based on people Daudi clone.Be selected from first and second generation library screening the DNA of bacterium colony transduce to Chinese hamster ovary (CHO) cell, produce stable clone.It is described to press embodiment 1, purifying, and quantitatively CHO expressed protein, and end user Daudi clone is analyzed its antiproliferative activity, and the optional people's WISH cell that is infected by encephalomyocarditis virus (EMCV) of using is analyzed its antiviral activity.The example through reorganization nucleic acid of coded interference element-alpha homologues polypeptide is accredited as SEQ ID NO:1 to SEQ ID NO:35 in this article, described polypeptide has antiproliferative activity in the test based on people Daudi clone, described nucleic acid is coded in the ripe interferon-' alpha ' homologue polypeptide that is accredited as SEQ ID NO:36 to SEQ ID NO:70 herein respectively.In addition, at screening in by the antiviral activity shaker test of the high material circulation of EMCV mice infected cell through the ripe interferon-' alpha ' encoding sequence library of reorganization.The example through reorganization nucleic acid that is coded in the polypeptide that has antiviral activity in the test based on mouse cell/EMCV is accredited as SEQ IDNO:72 to SEQ ID NO:78 in this article, and described nucleic acid encoding is accredited as the ripe Interferon, rabbit homologue polypeptide of SEQ ID NO:79 to SEQ ID NO:85 in this article.

On the other hand, the invention provides the nucleic acid of separation or reorganization, it contains and is selected from following polynucleotide sequence: (a) SEQ ID NO:1 to SEQ ID NO:35, or its complementary polynucleotide sequence; (b) coding is selected from the polynucleotide sequence of the polypeptide of SEQ ID NO:36 to SEQ ID NO:71, or its complementary polynucleotide sequence; (c) under tight at least or at least highly tight hybridization conditions (or super-highly tight or super-super-highly tight hybridization conditions), with the polynucleotide sequence of total length (a) basically or (b), with polynucleotide sequence (a) or (b) 50,120,130,140,145, the polynucleotide sequence of the subsequence of 150,155,160 or 165 nucleotide bases or fragment hybridization; (d) contain (a), (b) or segmental polynucleotide sequence (c), complete or the part of polypeptide of described fragment coding, described polypeptide has antiproliferative activity in the test based on people Daudi clone, or has antiviral activity in the test that is used for measuring antiviral activity known in the art.

On the other hand, the invention provides the nucleic acid of separation or reorganization, it contains and is selected from following polynucleotide sequence: (a) SEQ ID NO:72 to SEQ ID NO:78, or its complementary polynucleotide sequence; (b) coding is selected from the polynucleotide sequence of the polypeptide of SEQ ID NO:79 to SEQ ID NO:85, or its complementary polynucleotide sequence; (c) under tight at least or at least highly tight hybridization conditions (or super-highly tight or super-super-highly tight hybridization conditions), with the polynucleotide sequence of total length (a) basically or (b), with polynucleotide sequence (a) or (b) 50,120,130,140,145, the polynucleotide sequence of the subsequence of 150,155,160 or 165 nucleotide bases or fragment hybridization; (d) contain (a), (b) or segmental polynucleotide sequence (c), complete or the part of polypeptide of described fragment coding, described polypeptide has antiproliferative activity in the test based on people Daudi clone, or has antiviral activity in the test based on mouse cell line/EMCV.

The present invention also comprises sophisticated interferon-' alpha ' homologue polypeptide, it contains the amino acid that is accredited as SEQ ID NO:71 in this article, the present invention also comprises the segmental polynucleotide sequence of coding said polypeptide or described polypeptide, described polypeptide fragment has antiproliferative activity in the test based on people Daudi clone, and/or has antiviral activity in the test based on mouse cell/EMCV.

The present invention also comprises the nucleic acid of separation or reorganization, and it contains the polynucleotide sequence of coded polypeptide, and wherein polypeptide contains following aminoacid sequence: CDLPQTHSLG-X ₁₁-X ₁₂-RA-X ₁₅-X ₁₆-LL-X ₁₉-QM-X ₂₂-R-X ₂₄-S-X ₂₆-FSCLKDR-X ₃₄-DFG-X ₃₈-P-X ₄₀-EEFD-X ₄₅-X ₄₆-X ₄₇-FQ-X ₅₀-X ₅₁-QAI-X ₅₅-X ₅₆-X ₅₇-HE-X ₆₀-X ₆₁-QTFN-X ₆₇-FSTK-X ₇₂-SS-X ₇₅-X ₇₆-W-X ₇₈-X ₇₉-X ₈₀-LL-X ₈₃-K-X ₈₅-X ₈₆-T-X ₈₈-L-X ₉₀-QQLN-X ₉₅-LEACV-X ₁₀₁-Q-X ₁₀₃-V-X ₁₀₅-X ₁₀₆-X ₁₀₇-X ₁₀₈-TPLMN-X ₁₁₄-D-X ₁₁₆-ILAV-X ₁₂₁-KY-X ₁₂₄-QRITLYL-X ₁₃₂-E-X ₁₃₄-KYSPC-X ₁₄₀-WEVVRAEIMRSFSFSTNLQKRLRRKE, or its conservative variant that replaces, wherein X ₁₁Be N or D; X ₁₂Be R, S or K; X ₁₅Be L or M; X ₁₆Be I, M or V; X ₁₉Be A or G; X ₂₂Be G or R; X ₂₄Be I or T; X ₂₆Be P or H; X ₃₄Be H, Y or Q; X ₃₈Be F or L; X ₄₀Be Q or R; X ₄₅Be G or S; X ₄₆Be N or H; X ₄₇Be Q or R; X ₅₀Be K or R; X ₅₁Be A or T; X ₅₅Be S or F; X ₅₆Be V or A; X ₅₇Be L or F; X ₆₀Be M or I; X ₆₁Be I or M; X ₆₇Be L or F; X ₇₂Be D or N; X ₇₅Be A or V; X ₇₆Be A or T; X ₇₈Be E or D; X ₇₉Be Q or E; X ₈₀Be S, R, T or N; X ₈₃Be E or D; X ₈₅Be F or L; X ₈₆Be S or Y; X ₈₈Be E or G; X ₉₀Be Y, H, N; X ₉₅Be D, E or N; X ₁₀₁Be I, M or V; X ₁₀₃Be E or G; X ₁₀₅Be G or W; X ₁₀₆Be V or M; X ₁₀₇Be E, G or K; X ₁₀₈Be E or G; X ₁₁₄Be V, E or G; X ₁₁₆Be S or P; X ₁₂₁Be K or R; X ₁₂₄Be F or L; X ₁₃₂Be T, I or M; X ₁₃₄Be K or R; And X ₁₄₀Be A or S.According to those skilled in the art's general general knowledge, each single-letter in this aminoacid sequence is represented specified amino acid residues.Described polypeptide has antiproliferative activity and (for example is at least about 8.3 * 10 in the test based on people Daudi clone ⁶Individual unit/mg), and/or in the test based on people WISH cell/EMCV, have antiviral activity and (be at least about 2.1 * 10 ⁷Individual unit/mg).

Such as hereinafter detailed description, polynucleotide of the present invention can be used for many aspects, include but not limited to: be used as therapeutical agent and preventive in vivo with in the method for a plurality of experimenters' of ex vivo ground treatment multiple disease; Be used in vitro method, as diagnostic method to detect the multiple disease of diagnosing and treating a plurality of experimenters; Be used for for example gene therapy; In for example therapeutic with prophylactically treat in the method for disease and be used as therapeutical agent and preventive; As immunogen; Be used for diagnosis and shaker test; Be used as diagnostic probe to survey exist (the comprising the nucleic acid that detects coding IFN-α) of complementation or part complementary nucleic acid. Prepare polynucleotide of the present invention

Can be according to known synthetic method, the solid phase method by standard prepares polynucleotide of the present invention and oligonucleotide.In general, independent synthesizing is about 20,30,40,50,60,70,80, the fragment of 90 and/or 100 nucleotide bases, then (by for example enzymatic or chemical connection method, or polymerase-mediated recombination method) they are coupled together, to form any in fact required continuous sequence.On the other hand, separately synthetic greater than 100 nucleotide bases (as 150,180,200,210,240,270,300,330,360,390,400,420,450,465,474,470,475,489,490,495,496 bases) nucleotide fragments, then (by for example enzymatic or chemical connection method, or polymerase-mediated recombination method) they are coupled together, to form any in fact required continuous sequence.For example, can use Beaucage etc., (1981) the described classical phosphoramidite method of tetrahedron communication 22:1859-69, or Matthes etc., (1984) EMBO J, the described method of 3:801-05 (for example, these methods are generally implemented in automatic synthesis method), prepare polynucleotide of the present invention and oligonucleotide by chemosynthesis, comprise its fragment (with those fragments as herein described).According to phosphoramidite method, synthetic oligonucleotide in the dna synthesizer of for example automatization carries out purifying then, and annealing connects, and is cloned into appropriate carriers.

In addition, any in fact nucleic acid all can any family's order from a plurality of businessmans obtain, TheMidland Certified Reagent Company (mcrc@oligos.com) for example, The Great AmericanGene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and a lot of other company.Similarly, peptide and antibody also can any family's order from a plurality of businessmans obtain, PeptidoGenic (pkim@ccnet.com) for example, HTI Bio-products, Inc. (http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc. and a lot of other company.

Also can be by using oligonucleotide probe screening cDNA library (for example by resembling at typical diversity method of formation, as recombinate homologous nucleic acid reorganizing in the method and the library that produces), obtain specific polynucleotide of the present invention, wherein said probe can be hybridized or PCR-amplification coding Interferon, rabbit homologue polypeptide and segmental polynucleotide thereof.Screening is that those skilled in the art are well-known with the method for separating the cDNA clone.This technical description is in for example: Sambrook etc., molecular cloning-laboratory manual (the 2nd edition), Vol.1-3, cold spring harbor laboratory, cold spring port, New York, 1989 (hereinafter referred is " Sambrook ") and up-to-date molecular biology methods, volumes such as F.M.Ausubel, fresh approach, Greene Publishing Associates, Inc and John Wiley ﹠amp; Sons co-partnership company (augmenting in 1999) (hereinafter referred is " Ausubel ").

Describe in detail as this paper, polynucleotide of the present invention comprise the new ripe interferon-' alpha ' homologue of coding sequence and with this encoding sequence complementary sequence, and the new segment of this encoding sequence and complementary sequence thereof.Polynucleotide can be the forms of RNA, or the form of DNA, comprise mRNA, cRNA, synthetic RNA and DNA, and cDNA.Polynucleotide can be two strands or strand, if strand then can be coding strand or non--coding (antisense, complementation) chain.The optional unpack format that comprises the encoding sequence (i) of interferon-' alpha ' homologue of polynucleotide, (ii) unite with for example fusion rotein of encoding with other encoding sequence, before-protein, preceding-protein is former etc., (iii) with can be at the non--encoding sequence of effective expression encoding sequence among the suitable host, as promotor, termination element, or the regional associating of 5 ' and/or 3 ' untranslated, and/or (iv) in carrier or host environment, wherein the encoding sequence of interferon-' alpha ' homologue is heterologous nucleic acid sequence or gene.Sequence also can be united with general nucleic acid combination preparation, comprising carrier, and damping fluid, adjuvant, vehicle etc.

The term DNA or the RNA of each interferon-' alpha ' homologue polypeptide of encoding comprises any oligodeoxynucleotide or oligodeoxyribonucleotide sequence, and they cause producing interferon-' alpha ' homologue polypeptide of the present invention by suitably expressing in the host cell.DNA or RNA can be at suitable host cells, or produce in acellular (external) system, and be perhaps can (by for example amplification technique, as PCR) synthetic or produce with chemical process. Utilize polynucleotide of the present invention

Polynucleotide of the present invention have a plurality of purposes, for example: recombinant production (promptly expressing) interferon-' alpha ' homologue polypeptide of the present invention; In for example therapeutic with prophylactically treat in the method for disease and be used as therapeutical agent and preventive; Be used for gene therapy method and related application field; As immunogen; Be used for diagnosis and shaker test; Be used as diagnostic probe to survey exist (the comprising the nucleic acid that detects the natural IFN-α of coding) of complementation or part complementary nucleic acid; As other reaction, for example the substrate of reorganization reaction or jump reaction is to produce IFN-alpha homologues new and/or through improveing etc. Polypeptide expression

According to the present invention, encode new and/or sophisticated interferon-' alpha ' homologue, the fragment of interferon-alpha proteins matter, relevant fusion rotein, or the polynucleotide sequence of its function equivalent is collectively referred to as " interferon-' alpha ' homologue polypeptide " in this article, " interferon-' alpha ' homologue protein ", or " interferon-' alpha ' homologue ", " Interferon, rabbit homologue ", " IFN-alpha homologues ", " IFN homologue ", " IFN polypeptide ", " IFN protein ", " polypeptide of the present invention " or " protein of the present invention ".Polypeptide of the present invention or protein desire to comprise and comprise the polypeptide or the amino acid fragment of above-mentioned each term.Polynucleotide sequence of the present invention is used to mediate those recombinant DNAs (or RNA) molecule that interferon-' alpha ' homologue polypeptide is expressed in suitable host cell.Because genetic code intrinsic degeneracy also can use other nucleotide sequence of the aminoacid sequence that is equal on encode substantially the same or the function to clone and express the Interferon, rabbit homologue. Modified encoding sequence

Those skilled in the art should understand that modifying encoding sequence (comprising for example encode interferon-' alpha ' homologue of the present invention or its segmental nucleotide sequence) is favourable to strengthen its expression in specific host.Genetic code is Feng Yu, and it has 64 possible codons, but most of biological preference is used the part in these codons.The codon of normal use is called as the suitableeest codon in species, and those codons that seldom use are divided into rare or the codon that is of little use (example is seen (1991) gene 105:61-72 such as Zhang S.P.).Can replace the preferred codon service condition of codon with the reflection host, this method is called as " codon optimization " or " control of species codon bias ".

Can prepare the codon (Murray that contains specific protokaryon or eucaryon host institute preference, (1989) nucleic acids research 17:477-508 such as E.) optimized encoding sequence, thereby make and compare without the transcript that optimized sequence produced, can improve translation speed, or produce and to have desired characteristic, as the recombinant RNA transcript of longer half life.Also can modify translation stop codon to reflect host's preference.For example, yeast saccharomyces cerevisiae and mammiferous preferred terminator codon are respectively UAA and UGA.Monocotyledonous preferred terminator codon is UGA, and insect and intestinal bacteria preferably use UAA as terminator codon ((1996) nucleic acids research 24:216-218 such as Dalphin M.E.).

For multiple reason, can transform to change Interferon, rabbit homologue encoding sequence polynucleotide sequence of the present invention, described change includes but not limited to: the clone of modifying factor product, processing and/or the change of expressing.For example, can use technology well-known in the art, for example site-directed mutagenesis imports and changes, and to insert new restriction site, changes glycosylation pattern, changes the preference of codon, imports splice site etc. Carrier, promotor and expression system

The present invention also comprises recombinant precursor, its contain one or more this paper the nucleotide sequence (for example encode interferon-' alpha ' homologue of the present invention or its segmental nucleotide sequence) of wide in range description.Construct comprises carrier, plasmid for example, and clay, phage, virus (comprising retrovirus), bacterial artificial chromosome (BAC), yeast artificial chromosomes (YAC) etc. have wherein inserted nucleotide sequence of the present invention on forward or backwards.Aspect this embodiment preferred, construct also contains the adjusting sequence, for example comprises can operating the promotor that links to each other with sequence.A large amount of appropriate carriers and promotor are well known by persons skilled in the art, also can be commercially available.

Describe Protocols in Molecular Biology used herein, comprise carrier, the utilization of promotor comprises with the common textbook of a lot of other relevant propositions: Juo, P-S., simple and clear biomedical and molecular biology dictionary (CRC press, 1996); Singleton etc., microbiology and molecular biology dictionary (the 2nd edition, 1994); Cambridge Science and Technology dictionary (Walker compiles, 1988); Hale ﹠amp; Marham, HARPERCOLLINS biology dictionary (1991); Scott and Mercer, simple and clear biological chemistry and molecular biology encyclopedia (the 3rd edition, 1997); Berger and Kimmel, molecule clone technology guide, Enzymology method, the 152nd volume, Academic publishing company, San Diego, CA (hereinafter referred is " Berger "); Sambrook etc., molecular cloning-laboratory manual (the 2nd edition), Vol.1-3, cold spring harbor laboratory, cold spring port, New York, 1989 (" Sambrook ") and up-to-date molecular biology methods, volumes such as F.M.Ausubel, fresh approach, Greene Publishing Associates, Inc and John Wiley ﹠amp; Sons co-partnership company (augmenting in 1999) (" Ausubel ").Be enough to instruct those of skill in the art to carry out amplification in vitro method, comprise polymerase chain reaction (PCR), ligase chain reaction (LCR), the technology (for example NASBA) of Q β-replicative enzyme amplification and the mediation of other RNA polymerase can be referring to for example Berger with the technology that produces homologous nucleic acid of the present invention, Sambrook and Ausubel, and Mullis etc. (1987), United States Patent (USP) 4,683,202; On July 28th, 1997, laid-open U.S. Patents 4,683,195; PCR method: methods and applications guide (volume such as Innis) Academic publishing company, San Diego, CA (1990) is (Innis); Arnheim ﹠amp; Levinson (October 1 nineteen ninety) C﹠amp; EN36-47; NIH research magazine (1991) 3,81-94; KWoh etc., (1989) Proc.Natl Acad.Sci.USA86,1173; Guatelli etc., (1990) Proc.Natl Acad.Sci.USA87,1874; Lomell etc., (1989) clinical chemistry magazine, 35,1826; Landegren etc., (1988) science 241,1077-1080; Van Brunt (1990) biotechnology 8,291-294; Wu and Wallace (1989) gene 4,560; Barringer etc., (1990) gene 89,117 and Sooknanan and Malek (1995) biotechnology 13:563-564.

PCR generally refers to: by the specific nucleic acid of method amplification minute quantity well-known in the art, the method for RNA and/or dna fragmentation (referring to for example United States Patent (USP) 4,683,195 and above-mentioned other reference).The general sequence information of desired zone end or farther place that uses comes the design oligonucleotides primer.The sequence of described primer and the opposite strand of template to be amplified are same or similar.5 ' terminal nucleotide of opposite strand can conform to the end of amplified material.Can use specific RNA of pcr amplification or specific dna sequence dna, recombinant DNA or RNA sequence are from the DNA and the RNA sequence of total genomic dna with transcribe from total cell RNA, the cDNA of phage or plasmid sequence etc.PCR is to use another kind (as is known) nucleic acid as primer, comes an example of the nucleic acid polymerase reaction method of amplification of nucleic acid test sample, but is not unique example.The clone is described in Wallace etc. through the modification method of the nucleic acid of amplification in vitro, United States Patent (USP) 5,426,039.Sketch in Cheng etc. by the modification method of pcr amplification large nucleic acids, (1994) natural 369:684-685 and reference have wherein wherein produced pcr amplification that grows to 40kb.Those skilled in the art should understand: use reversed transcriptive enzyme and polysaccharase any RNA can be changed in fact and be suitable for restrictive diges-tion, the double-stranded DNA of PCR expansion and order-checking.Referring to Ausubel, Sambrook and Berger, document is the same.

The invention still further relates to by the host cell of carrier transduction of the present invention with by recombinant technology and produce polypeptide of the present invention (comprising its fragment).With carrier of the present invention host cell is carried out genetic engineering modified (i.e. transduction transforms or transfection), described carrier can be for example cloning vector or expression vector.Carrier can be a plasmid for example, virion, forms such as phage.Can in conventional nutritional medium, cultivate host cell, can change with the activation promotor medium component in case of necessity, select transformant or amplification Interferon, rabbit homologue gene through transforming.As temperature, the culture condition of pH etc. and the host cell of selecting to be used to express are previous used identical, they are conspicuous to those skilled in the art, the also reference that can mention referring to this paper, comprise for example Freshney (1994) animal cell culture, basic fundamental handbook, the 3rd edition, Wiley-Liss, New York and the reference of wherein mentioning.

Also can be at non--zooblast, as plant, yeast is produced Interferon, rabbit homologue polypeptide of the present invention and protein in the fungi, bacterium etc.Except Sambrook, outside Berger and the Ausubel, the detail file that cells involved is cultivated can be referring to Payne etc., the vegetable cell and the tissue culture of (1992) liquid system, John Wiley ﹠amp; Sons, Inc. New York, NY; Gamborg and Phillips (volume) (1995) vegetable cell, tissue and organ culture; Basic skills, Springer laboratory manual, Springer-Verlag (BerlinHeidelberg New York) and Atlas and Parks (volume), microbiological culture media handbook (1993), CRC press, Boca Raton, FL.

Polynucleotide of the present invention can be included in any of a plurality of expression vectors with express polypeptide.Described carrier comprises karyomit(e), and non-chromosome and synthetic dna sequence dna are as the derivative of SV40; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; Derived from plasmid and phage DNA, viral DNA, as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, adeno-associated virus, the carrier of the combination of retrovirus and a lot of other viruses.Can use any genetic material can transduction to cell, and the carrier that in the time need duplicating, can duplicate and in relevant host, can survive.

Nucleotide sequence in the expression vector can be operated to link to each other with suitable transcriptional control sequence (promotor) and synthesize with mediation mRNA.The example of described promotor comprises: LTR or SV40 promotor, intestinal bacteria lac or trp promotor, phage P _LPromotor and other the known promotor that can control the genetic expression in protokaryon or eukaryotic cell or its virus.Expression vector also contains ribosome bind site and the transcription terminator that is useful on translation initiation.Carrier is optional to be comprised and is used to the suitable sequence that increases and express.In addition, expression vector is also optional to contain one or more selectable marker genes so that phenotypic character to be provided, be used for selecting through transformed host cells, as Tetrahydrofolate dehydrogenase or the neomycin resistance of cultivating at eukaryotic cell, or at colibacillary tsiklomitsin or amicillin resistance.

Can use and contain suitable dna sequence dna as herein described, and suitably the carrier of promotor or control sequence transforms suitable host, so that host expresses protein.Suitably the example of expressive host comprises: bacterial cell, and as intestinal bacteria, streptomyces and Salmonella typhimurium; The fungal cell, as yeast saccharomyces cerevisiae, Pichia pastoris and Neurospora crassa; Insect cell is as fruit bat and fall army worm; Mammalian cell, as CHO, COS, BHK, HEK293 or Bowes melanoma; Vegetable cell etc.Should understand not all cell or clone and all need to produce full functionality Interferon, rabbit homologue; For example, can in bacterium or other expression system, produce the antigenicity fragment of Interferon, rabbit homologue.The present invention is not subjected to the restriction of used host cell.

In bacterial system, can select multiple expression vector according to the purposes of Interferon, rabbit homologue.For example, when a large amount of Interferon, rabbit homologues of needs or its fragment when inducing antibody, then need to mediate the carrier of the fusion rotein high level expression that is easy to purifying.Described carrier includes but not limited to multi-functional escherichia coli cloning and expression vector, as BLUESCRIPT (Stratagene), wherein Interferon, rabbit homologue encoding sequence is connected in the carrier, be positioned at same reading frame with 7 residues of aminoterminal Met sequence and beta-galactosidase enzymes subsequently, thereby produce hybrid protein; PIN carrier (Van Heeke ﹠amp; Schuster (1989) journal of biological chemistry 264:5503-5509); PET carrier (Novagen, Madison WI) etc.

Similarly, in the yeast yeast saccharomyces cerevisiae, can use multiple composing type or the inducible promoter of containing, as alpha factor, the carrier of alcohol oxidase and PGH is produced Interferon, rabbit homologue protein of the present invention.Relevant commentary can be referring to Ausubel etc., (document is the same) and Grant etc., (1987; Enzymology method 153:516-544).

In mammalian host cell, can utilize multiple expression system, as system based on virus.When using adenovirus as expression vector, optionally encoding sequence is connected to adenovirus transcribes/translate in the mixture, described mixture is made up of late promoter and tripartite leader[.Inserting in the viral genome nonessential E1 or E3 zone can cause depositing the poison of curing the disease can express Interferon, rabbit homologue (Logan and Shenk (1984) Proc.Natl.Acad.Sci.81:3655-3659) in infected host cell.In addition, can use transcriptional enhancer, strengthen expression in the mammalian host cell as Rous sarcoma virus (RSV) enhanser. Other Expression element

Specific start signal helps effectively to translate Interferon, rabbit homologue encoding sequence.These signals comprise for example sequence of ATG initiator codon and adjacency.When with Interferon, rabbit homologue encoding sequence, when its initiator codon and upstream sequence insert suitable expression vector, need not other translation control signal.Yet, when only inserting encoding sequence (as the encoding sequence of mature protein) or its part, must provide external source to transcribe control signal, comprise the ATG initiator codon.In addition, initiator codon must be positioned at correct reading frame to guarantee to transcribe complete inset.It can be different sources with initiator codon that external source is transcribed element, can be natural and synthetic.By comprising the enhanser that is suitable for used cell system, can strengthen to express and render a service (Schaf, D. etc. (1994) Results Probl.Cell Differ.20:125-62; Bittner etc. (1987) Enzymology method, 153:516-544). Secretion/positioning sequence

Polynucleotide of the present invention also can merge with the nucleic acid of coding secretion/positioning sequence, frame endomixis for example, thus with expression of polypeptides target required cellular compartment extremely, film or organoid perhaps mediate polypeptide and secrete to periplasmic space or cell culture medium.Described sequence is well known by persons skilled in the art, and it comprises the secretion leading peptide, organoid target sequence (nuclear localization sequence for example, ER is detained signal, plastosome transposition sequence, chloroplast transgenic bit sequence), film location/anchor sequence (as stop-transfer sequence, the GPI anchor sequence) etc.Comprise corresponding to the secretion and/or the aminoacid sequence of positioning sequence by polynucleotide polypeptide expressed of the present invention. Expressive host

In other embodiments, the present invention relates to contain the host cell of above-mentioned construct.Host cell can be an eukaryotic cell, as mammalian cell, and yeast cell or vegetable cell, or host cell can be prokaryotic cell prokaryocyte, as bacterial cell.By calcium phosphate transfection, the transfection of DEAE-Dextran mediation, electroporation, or other ordinary skill (Davis, L, Dibner, M and Battey, the basic molecular biology method of I. (1986)) can import host cell with construct.Can comprise nucleic acid of the present invention in the cell, described nucleic acid encoding polypeptide, wherein said cell expressing polypeptide (, having the interferon-' alpha ' homologue polypeptide of antiviral or antiproliferative activity) for example by test determination as herein described.The present invention also comprises the carrier that contains any nucleic acid of the present invention, and comprises by the cell of described carrier transduction.In addition, the present invention also comprises cell and the transgenic animal that contain described any polypeptide of above-mentioned or whole specification sheets or nucleic acid, for example cell and the transgenic animal that produce by the carrier of the present invention of transduceing.

Optional processing through the ability of expressed protein according to the expression of cell adjusting insertion sequence or by required mode selected host cell strain.Protein modification includes but not limited to: ethanoylization, and carboxylated, glycosylation, phosphorylation, lipidization and acylations.The translation post-treatment of " preceding " of crack protein matter or " preceding former " form is for correct insertion, and folding and/or functionating is vital.For the activity after this translation, such as CHO, HeLa, BHK, MDCK, 293, the different host cells of WI38 etc. have specific cell machine and distinctive mechanism, can select in them to guarantee the correct exogenous protein that imports of modifying and process.

For for a long time, the high place of production produces recombinant protein, can use stable expression.For example, can use the clone of expression vector transduction energy stably express polypeptide of the present invention, described expression vector contains virus replication starting point or endogenous expression element and selectable marker gene.Import after the carrier, cell was cultivated in enriched medium 1 to 2 day, be transferred to then and select in the substratum.The purpose of selective marker is to give resistance to select the cell of its existence permission cultivation and recovery successful expression importing sequence.For example, use the tissue culture technique that is suitable for cell type can breed the resistance group of stable transformed cells.

Choose wantonly being suitable for expressing and from cell culture, reclaiming under the condition of coded protein, cultivate the nucleotide sequence transformed host cells of the polypeptide of the present invention that is encoded.According to the difference of used sequence and/or carrier, can be secreted by protein or its fragment that reconstitution cell produces, combine or be included in the cell with film.Those skilled in the art should understand: can invent modelled signal sequence in the expression vector of polynucleotide of ripe Interferon, rabbit homologue containing code book, described signal sequence can mediate that sophisticated polypeptide passes protokaryon or eukaryotic cell membrane is secreted out. Other peptide sequence

Polynucleotide of the present invention also can comprise and the encoding sequence of flag sequence (for example being convenient to the sequence of purifying coded polypeptide of the present invention) fusion in a frame.This structural domain of being convenient to purifying includes but not limited to: can be on immobilized metal the metal chelating peptide of purifying, as Histidine-tryptophane assembly, with gsh bonded sequence (as GST), hemagglutinin (HA) mark is (corresponding to the proteic epi-position of influenza virus hemagglutinin; Wilson, I. etc. (1984) cell 37:767), maltose binding protein sequence, FLAGS extension/affinity purification system (Immunex Corp., Seattle, used FLAG epi-position etc. in WA).Between purification structure territory and Interferon, rabbit homologue sequence, comprise and to be helped purifying by the peptide linker sequence of protease cracking.An expression vector that is expected to be used for composition described herein and method provides Expression of Fusion Protein, and the polypeptide of the present invention that described fusion rotein contains and merge in poly Histidine zone is separated by the enteropeptidase cracking site between them.Histidine residues is convenient to go up purifying at IMIAC (immobilized metal ion affinity chromatography is as (1992) protein expression and purifying 3:263-281 such as Porath), and the enteropeptidase cracking site can make Interferon, rabbit homologue polypeptide separate from fusion rotein.Also can use pGEX carrier (Promega; Madison WI) is expressed as allogenic polypeptide fusion rotein with glutathione S-transferase (GST).Usually, this fusion rotein is a solubility, by (for example being adsorbed to part-sepharose 4B, when merging with GST, be gsh-agarose), then in the presence of free ligand, carry out wash-out, can easily from lysing cell, be purified into fusion rotein. The production of polypeptide and recovery

Host's strain (being) of transduceing suitable also is cultured to host's strain (being) after the suitable cell density, induces selected promotor by suitable mode (for example temperature transition or chemical reagent are induced), and cell is cultivated for some time again.General by centrifugal collecting cell, with physics or chemical process smudge cells, keep the gained crude extract to be further purified.Can comprise multigelation, supersound process, Mechanical Crushing or use lysis agent or other method by the used microorganism cells of the broken marking protein of any method easily well-known in the art.

As mentioned above, a lot of piece of writing reference can be used for cultivating and producing a variety of cells, comprise being derived from bacterium plant, the cell of animal (especially Mammals) and archeobacteria.Referring to for example Sambrook, Ausubel and Berger (document is the same), and Freshney (1994) animal cell culture, the basic fundamental handbook, the 3rd edition, Wiley-Liss, New York and the reference of wherein mentioning, Doyle and Griffiths (1997) mammalian cell is cultivated: basic fundamental, John Wiley and Sons, New York; Humason (1979) animal tissues technology, the 4th edition, W.H.Freeman and Company; With (1989) cell in vitro developmental biology such as Ricciardelli, 25:1016-1024.About culture plant cell and regeneration, can be referring to vegetable cell and the tissue culture in (1992) liquid systems such as Payne, JohnWiley﹠amp; Sons, Inc., New York, NY; Gamborg and Phillips (volume) (1995) vegetable cell, tissue and organ culture; Basic skills, the Springer laboratory manual, Springer-Verlag (Berlin HeidelbergNew York) and molecular biology of plants (1993) R.R.D.Croy compile, Bios ScientificPublishers, Oxford, U.K.ISBN 0 12 1983706.Cell culture medium usually can be referring to Atlas and Parks (volume) microbiological culture media handbook (1993) CRC press, Boca Raton, FL.The document that other data that cells involved is cultivated can provide referring to businessman, (the St.Louis of Sigma-Aldrich company for example, MO) life science that provides, cell cultures catalogue (1998) (" Sigma-LSRCCC "), Sigma-Aldrich company (St.Louis, plant culturing catalogue that MO) provides and enlarged edition (1997) (" Sigma-PCCS ") for example.

Also can be by in the several different methods well-known in the art any, from the reconstitution cell culture, reclaim and purifying polypeptide of the present invention, described method comprises: ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography (as using any Mk system as herein described), hydroxyapatite chromatography and lectin chromatography.In case of necessity, when finishing the configuration of mature protein, can use proteinic folding step again.Finally, in last purification step, can use high performance liquid chromatography (HPLC).Except reference mentioned above, multiple purification process is well-known in the art, comprises for example proteinic bioseparation of Sandana (1997), Academic publishing company; With (1996) protein methods such as Bollag, the 2nd edition, Wiley-Liss, New York; Walker (1996) protein method handbook, Humana press, NJ, Hahis and Angal (1990) protein purification is used: working method, Oxford IRL press, Oxford, Britain; Harris and Angal, method of purifying protein: working method, Oxford IRL press, Oxford, Britain; Scopes (1993) protein purification: principle and practice, the 3rd edition, Springer Verlag, New York; Janson and Ryden (1998) protein purification: principle, high-resolution methods and applications, the 2nd edition, Wiley-VCH, New York; And the protein method of Walker (1998) CD-ROM version, Humana press, NJ. The vivoexpression system

Also can use DNA of the present invention or RNA, utilize cell-free transcription/translation system to produce polypeptide.Several such systems can be commercially available.The general guide of in-vitro transcription and interpretation method can be referring to Tymms (1995) in-vitro transcription and interpretation method: molecular biology method, the 37th volume, GarlandPublishing, New York. Modified amino acid

Polypeptide of the present invention can contain one or more modified amino acid.Modified occurrence of amino acid prolongs the serum half life of polypeptide to for example (a), (b) reduces the antigenicity of polypeptide, and the stability in storage that (c) increases polypeptide is favourable.For example, when recombinant production, can be total to-translate or posttranslational modification amino acid (when for example in mammalian cell, expressing, the N-linked glycosylation of N-X-S/T primitive), or by synthetic mode modified amino acid.

Modified amino acid whose non-limitative example comprises: glycosylation amino acid; sulfated amino acids; isoprenylation is (as farnesylation; the geranyl geranylization) amino acid, ethanoyl amino acid, acylated amino acid; PEGization amino acid; biotinylation amino acid, carboxylated amino acid, phosphorylated amino acid etc.The reference of the detailed those skilled in the art's of the guidance modified amino acid of energy has a lot, and the method that exemplifies can be referring to the protein method of Walker (1998) CD-ROM version, Human press, Towata, NJ.

Polynucleotide of the present invention and polypeptide have a plurality of purposes, for example include but not limited to: recombinant production (promptly expressing) recombinant interferon-alpha homologues of the present invention; Be used as therapeutical agent and preventive in vivo with in the method for a plurality of experimenters' of ex vivo ground treatment multiple disease; Be used in vitro method, with detection, diagnose and treat a plurality of experimenters' (as Mammals) multiple disease (for example cancer, based on the disease of virus, based on the disease of vasculogenesis) as diagnosis and screening method; As immunogen; In gene therapy method and transmission method, be used for in the biologically active polypeptides body of the present invention ex vivo or the external tissue that is passed to or is applied to the experimenter based on DNA or RNA, colony or cell, organ, graft, body system (as tract, lymphsystem, blood system etc.); As dna vaccination, the vaccine of many-composition is with being used for the treatment of property with the multiple disease (for example cancer, based on the disease of virus, based on the disease of vasculogenesis) of prophylactically treating a plurality of experimenters (as Mammals); As the immunne response of adjuvant with enhancing or expansion experimenter; As the multistep booster immunization method composition of (for example comprise by transmitting DNA or RNA Nucleotide (for example Nucleotide of the code book invention polypeptide or the another kind of polypeptide of encoding) and cause immunity, then use the form of polypeptide (polypeptide for example of the present invention or other polypeptide) booster immunization once more); Be used as diagnostic probe to survey exist (the comprising the nucleic acid that detects coding natural interferon-α) of complementation or part complementary nucleic acid; As other reaction, for example reorganization reaction, the substrate of jump reaction or other diversity formation reaction to produce the new interferon-' alpha ' nucleic acids of interferon-' alpha ' homologue new and/or through improveing and this homologue of coding, for example produces new treatment or prevention characteristic etc.; Be used for polymerase chain reaction (PCR) or cloning process, for example comprise digestion or ligation, to identify new and/or through the natural or non-natural IFN-'alpha ' nucleic acids of improvement with by its encoded polypeptides.Optional polynucleotide, or the complement of these polynucleotide is applied to cell with code book invention Interferon, rabbit homologue, finishing the method for treatment or prevention usefulness, or in vivo, ex vivo or vivoexpression treatment product.Comprise in the body or the application of ex vivo, for example these application of gene therapy comprise multiple technologies, can change genetic expression in the cell by them.Described method for example comprises: import to express for example to treat or prevent and use polypeptide, as the gene of Interferon, rabbit homologue of the present invention.Described method for example comprises: infect with the retrovirus that contains polynucleotide of the present invention and/or polypeptide.Randomly, retrovirus also contains other external source, for example treatment or preventative gene construct sequence.On the one hand, such as hereinafter detailed description, the invention provides experimenter by treating to needs, comprise biology or Mammals, comprise for example people, primate, mouse, pig, milk cow, goat, rabbit, rat, cavy, hamster, horse, sheep; Or non-mammalian vertebrates, as birds (as chicken or duck) or fish, or in one or more cell paste of invertebrates, ex vivo or the external nucleic acid of using one or more inventions described herein, the gene therapy method of prevention or the described experimenter's of therapeutic treatment disease.

On the other hand, such as hereinafter detailed description, the invention provides in one or more cell paste of experimenter by treating (comprise defined herein those) to needs, ex vivo or the external polypeptide of using one or more inventions described herein, the method for prevention or the described experimenter's of therapeutic treatment disease. Expression of polypeptides

Use the well-known technology of those skilled in the art, the polynucleotide of the Interferon, rabbit homologue polypeptide of the present invention of encoding in the body or the treatment of ex vivo or prophylactic applications particularly useful.For example, (DNA or RNA) transforms the culturing cell of ex vivo with polynucleotide, then the cell through transforming is back in patient's body.Also can in the body or the cell of ex vivo transform, with respectively in vivo or ex vivo ground express polypeptide.

Known multiple virus vector is suitable for organism in vivo or ex vivo ground transduction and expressing.Described carrier comprises that retroviral vector is (referring to up-to-date microbiology of Miller (1992) and immunology proposition 158:1-24; Salmons and Gunzburg (1993) human gene therapy 4:129-141; Miller etc. (1994) Enzymology method 217:581-599) and gland-related viral vectors (referring to the up-to-date biotechnology viewpoint of Carter (1992) 3:533-539; Up-to-date microbiology of Muzcyzka (1992) and immunology proposition 158:97-129).Operable other virus vector comprises adenovirus carrier, herpesvirus vector and sindbis virus carrier, and general description is in for example JollY (1994) gene therapy for cancer 1:51-64; Latchman (1994) molecular biotechnology 2:179-195; With (1995) nucleic acids research 23:1495-1501 such as Johanning.

Gene therapy provides several different methods to be used to resist chronic infectious disease, and (for example HIV infects, viral hepatitis, hsv (HSV), hepatitis B virus (HepB), dengue virus etc.), and non--communicable disease, comprise the birth defects of cancer and allergic disease and some forms, as enzyme defect.Used several method that nucleic acid is imported in the body, ex vivo and external cell.These methods comprise: based on gene transmission (Debs and Zhu (1993) WO93/24640 and the United States Patent (USP) 5,641,662 of liposome; Mannino and Gould-Fogerite (1988) biotechnology 6 (7): 682-691; Rose, United States Patent (USP) 5,279,833; Brigham (1991) WO91/06309; With (1987) Proc.NatlAcad.Sci.USA84:7413-7414 such as Felgner); Brigham etc. (1989) United States Medicine science magazine 298:278-281; Nabel etc. (1990) science 249:1285-1288; Hazinski etc. (1991) Am.J.Resp.Cell Molec.Biol 4:206-209; And Wang and Huang (1987) Proc.NatlAcad.Sci. (USA) 84:7851-7855); Adenovirus vector-mediated gene transmission for example is used for the treatment of cancer (referring to (1994) Proc.Natl Acad.Sci.USA 91:3054-3057 such as for example Chen; Tong etc. (1996) Gynecol.Oncol.61:175-179; Clayman etc. (1995) cancer research 5:1-6; O ' Malley etc. (1995) cancer research 55:1080-1085; Hwang etc. (1995) Am.J.Respir.Cell Mol.Biol.13:7-16; Up-to-date microbiology of Haddada etc. (1995) and immunology proposition 199 (Pt.3): 297-306; Addison etc. (1995) Proc.Natl Acad.Sci.USA 92:8522-8526; Colak etc. (1995) brain research 691:76-82; Crystal (1995) science 270:404-410; Elshami etc. (1996) human gene therapy 7:141-148; Vincent etc. (1996) Neurological Surgery magazine 85:648-654) and much other disease.In addition, used to duplicate-damaged retroviral vector, it carries the part of curative polynucleotide sequence as the reverse transcription virus gene group, wherein should mention simple MuLV carrier especially, referring to (1990) molecular cytobiology 10:4239 (1990) such as for example Miller; (1991) human gene therapy 2:215 such as Kolberg (1992) NIH research magazine 4:43 and Cornetta).In addition, also used and part-specific the nucleic acid delivery (Wu and Wu (1988) journal of biological chemistry 263:14621-14624) that combines based on cationic movement system.Also described exposed DNA expression vector (Nabel etc. (1990), document is the same; Wolff etc. (1990) science 247:1465-1468).Usually, the nucleic acid by the Interferon, rabbit homologue described herein of will encoding mixes suitable carrier, can make these methods be adapted to the present invention.

Describe gene therapy method, comprise by nucleic acid importing patient of the present invention is adapted to common textbook of the present invention: Robbins (1996) gene therapy method, Humana press, NJ, and Joyner (1993) gene target: working method, IRL press, the Oxford, Britain. Antisense technology

Except expressing nucleic acid of the present invention, in case when no longer needing express nucleic acid in the cell, described nucleic acid also can be used for having justice and the inhibition expression of antisense ground, for example reduces expression of nucleic acids of the present invention as the gene substitution nucleic acid.Similarly, also can use nucleic acid of the present invention, or its subsequence or antisense sequences are blocked natural homologue expression of nucleic acids.Multiple justice and antisense technology are arranged is known in the art, for example can be referring to Nellen (1997) antisense technology: working method, IRL press, the Oxford University, the Oxford, Britain and Agrawal (1996) antisense therapy agent Humana press, NJ and the reference of wherein mentioning. Pharmaceutical composition

Polynucleotide of the present invention and polypeptide (comprising the carrier that for example contains polynucleotide of the present invention or polypeptide, cell, antibody etc.) can make up to be used for the treatment of and preventive use with suitable pharmaceutical carrier.Described composition contains polynucleotide of the present invention or polypeptide and the medicine acceptable carrier or the vehicle of treatment or prevention significant quantity.The medicine acceptable carrier comprises the pharmaceutical carrier of any standard, damping fluid and vehicle.Described carrier or vehicle include but not limited to: salt solution, buffer saline (as phosphoric acid buffers saline solution), glucose, water, glycerine, ethanol, emulsion (as oil/water or water/oil-emulsion), polytype wetting agent and/or adjuvant, and combination.Suitable pharmaceutical carrier and reagent are described in REMINGTON ' S pharmaceutical science (Mack publishing company, Easton, the 19th edition, 1995).Manner of formulation should adapt with the mode of administration of promoting agent (as Nucleotide, polypeptide, carrier, cell etc.).Administration of nucleic acid, polypeptide, carrier, cell, antibody and method of protein are well-known in the art, and further discuss hereinafter. As probe

This paper also wishes to comprise generally have at least 12 bases, preferred at least 15 bases, more preferably at least 20, the purposes of the polynucleotide of 30 or 50 bases (this paper also is referred to as oligonucleotide), described polynucleotide can be hybridized with the polynucleotide sequence of above-mentioned Interferon, rabbit homologue under the condition of at least highly tight (or super-highly tight or super-super-highly tight condition).According to the method described in the above-mentioned document, polynucleotide can be used as probe, and primer has justice and antisense reagent etc. Sequence variations Silent variant

Those skilled in the art should understand: owing to the degeneracy of genetic code, can produce the nucleotide sequence of a variety of codings Interferon, rabbit homologue of the present invention polypeptide, wherein some and this paper are clear, and disclosed nucleotide sequence has very low sequence homology.

Table 1The password sublist

Amino acid code

L-Ala Ala A GCA GCC GCG GCU

Halfcystine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

L-glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine Ile I AUA AUC AUU

Methionin Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine(Met) Met M AUG

L-asparagine Asn N AAC AAU

Proline(Pro) Pro P CCA CCC CCG CCU

Glutamine Gln Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Xie Ansuan Val V GUA GUC GUG GUU

Tryptophane Trp W UGG

Tyrosine Tyr Y UAC UAU

For example, by password sublist (table 1) as can be seen: codon AGA, AGG, CGA, CGC, CGG and CGU be the coded amino acid arginine.Therefore, each is refered in particular to by a codon and is arginic position in nucleic acid of the present invention, codon can be changed in the above-mentioned corresponding codon any, and can not change encoded polypeptides.Should understand U in the RNA sequence and be equivalent to T in the dna sequence dna.

For example, use is equivalent to the nucleotide sequence TGT GATCTG CCT CAG of the Nucleotide 1-15 among the SEQ ID NO:1, the silent variant of this sequence comprises TGC GAC TTA CCA CAA, and two sequences of this of encoding amino acid sequence CDLPQ are equivalent to the amino acid/11-5 among the SEQ ID NO:36.

This " silent variant " is a kind of " through the conservative variation of modifying " that will discuss hereinafter.One skilled in the art will recognize that: can be by each codon (except AUG, it is the unique codon of methionine(Met) normally) in the standard technique modification of nucleic acids with the identical polypeptide of encoding function.Therefore, each silent variant that in any described sequence, implies nucleic acid encoding.Each and each that the invention provides the nucleotide sequence of code book invention polypeptide may make a variation, and they are by selecting to make up according to possible codon.The used standard triplet genetic code (as shown in table 1) of nucleotide sequence according to coding Interferon, rabbit homologue polypeptide of the present invention prepares these combinations.By considering sequence and genetic code, specifically provide and described above-mentioned all varients of each nucleic acid of this paper. Conservative variations

" through the conservative variation of modifying " of specific nucleic acid sequence or abbreviate " conservative variations " as and refer to: those nucleic acid of the identical or identical in fact aminoacid sequence of encoding, or, refer to substantially the same sequence when nucleic acid not during encoding amino acid sequence.Those skilled in the art think: change, the single amino acids in interpolation or the disappearance encoding sequence or the amino acid of little per-cent (generally are lower than 5%, more preferably less than 4%, 3%, 2% or 1%) each replacement, disappearance or to add be " through conservative variation of modifying " wherein changes to cause aminoacid deletion, aminoacid addition or with aminoacid replacement amino acid like the chemofacies.

It is well-known in the art that intimate amino acid whose conservative replacement table is provided.Table 2 has been listed 6 groups, and it contains can " the conservative replacement " another amino acid whose amino acid.

Table 2The conservative group that replaces

????1	L-Ala (A) Serine (S) Threonine (T)
????1	L-Ala (A) Serine (S) Threonine (T)	????2	Aspartic acid (D) L-glutamic acid (E)
????3	L-asparagine (N) glutamine (Q)	????2	Aspartic acid (D) L-glutamic acid (E)
????3	L-asparagine (N) glutamine (Q)	????4	Arginine (R) Methionin (K)
????5	Isoleucine (I) leucine (L) methionine(Met) (M) Xie Ansuan (V)	????4	Arginine (R) Methionin (K)
????5		????6	Phenylalanine (F) tyrosine (Y) tryptophane (W)

Therefore, " through the conservative variation that replaces " of the listed peptide sequence of the present invention or abbreviate " the conservative replacement " as and comprise with the medium and small per-cent of aminoacid replacement peptide sequence of the conservative selection of warp of identical conservative replacement group generally is lower than 5%, more preferably less than 4%, 3%, 2% or 1% amino acid.

For example, the conservative replacement variation meeting that is accredited as the polypeptide of SEQ ID NO:36 in this article contains with good grounds above defined 6 groups " conservative replacements " in about 8 or 9 residues (promptly about 5% amino acid) of the polypeptide of 166 amino acid longs.

In other example, if 4 conservative zones that are positioned at the amino-acid residue 141-166 that is equivalent to SEQ ID NO:36 that replace, according to the conservative replacement that table 2 is listed, the example of the conservative replacement variation of this zone WEVVR AEIMRSFSFS TNLQK RLRRKE comprises: WEVVR SEIMRSFS YS TNLQ RRLRRK DAnd WE LVR AEI VR SFSFS TNL NK RLR KKE etc. (in above-mentioned example, underscore replace) for conservative.The protein sequence that this paper lists provides all through the conservative proteinic tabulation that replaces in conjunction with above-mentioned replacement table.

At last, add the active sequence of coding that can not change nucleic acid molecule, for example adding non--functional sequence also is the conservative variations of basic nucleic acid.

Those skilled in the art should understand: a variety of conservative variations of disclosed nucleic acid construct have produced construct identical on the function.For example, as discussed above, because the degeneracy of genetic code, " the reticent replacement " (being the replacement that can not change coded polypeptide in the nucleotide sequence) is the feature that the nucleotide sequence of each coded amino acid implies.Similarly, also can easily be accredited as similar with one or several amino acid whose " conserved amino acid replacement " in the different aminoacids substituted-amino acid sequence with height similar characteristics to disclosed construct height.This conservative variations of each open sequence also is a feature of the present invention. Nucleic acid hybridization

When mutually combining (generally carrying out) in solution, nucleic acid is " hybridization ".Nucleic acid hybridization is that as hydrogen bond, solvent repels, base stacking etc. owing to there is the multiple physical-chemical power of having been identified in detail.The detailed guidance of related nucleic acid hybridization can referring to Tijssen (1993) biological chemistry and Molecular Biology Lab's technology-with nucleic acid probe hybridization, part i, the 2nd chapter, " hybridization principle and nucleic acid probe test strategy summary ", and Ausubel, document is the same.Hames and Higgins (1995) gene probe 1, the IRL press of Oxford University Press, the Oxford, Britain (Hames and Higgins 1) and Hames and Higgins (1995) gene probe 2, the IRL press of Oxford University Press, Oxford, it is relevant synthetic that Britain (Hames and Higgins 2) provides, mark detects and quantitative DNA and RNA, comprises the details of oligonucleotide.

The nucleic acid hybridization experiment is depended on sequence as " tight hybridization wash conditions " in Southern and the Northern hybridization context, also is different under different environmental parameters.The detailed guidance of related nucleic acid hybridization can be referring to Tijssen (1993), document the same and Hames and Higgins 1 and Hames and Higgins2, and document is the same.

For the purposes of the present invention, usually will " highly tight " hybridize and wash conditions is chosen to be the bit sequencing and is listed in definite ionic strength and the pyrolysis chain temperature (T under the pH _m) low about 5 ℃ or lower (as mentioned below, also can with the tight condition of suitable term description height).T _mTemperature (under ionic strength of determining and pH) when being 50% cycle tests with the probe hybridization of accurate coupling.Very tight condition is chosen to be the T with particular probe _mEquate.

T _mBe the temperature of nucleic acid duplex, under this temperature, duplex is 50% sex change under specified criteria, and this temperature can directly be measured the stability of nucleic acid heterozygote.Therefore, T _mBe equivalent to from being threaded to the curling at random pairing temperature of transformation mid point; For one section long nucleotide sequence, it depends on length, and Nucleotide is formed and ionic strength.

After the hybridization, can remove the not nucleic acid substances of hybridization, can adjust the tight degree of washing according to required result by a series of washing.The wash conditions of low stringency (for example using higher salt concn and lower temperature) increases susceptibility, but can produce non-specific hybridization signal and strong background signal.The condition of higher tight degree (for example use lower salt concn and near the comparatively high temps of hybridization temperature) can reduce background signal, generally only stays specific signals.Referring to Rapley, R and Walker, J.M compiles, molecular biology method handbook (Humana publishing company 1998) (hereinafter being referred to as " Rapley and Walker "), it lists this paper in as a reference in full.

Use following equation can estimate the T of DNA-DNA duplex _m:

T _m(℃)=81.5 ℃+16.6 (log ₁₀M)+0.41 (%G+C)-0.72 (%f)-500/n, wherein M is the weight-molality of univalent cation (normally Na+), (%G+C) be the per-cent of guanosine (G) and cytidine (C) Nucleotide, %f is the per-cent of methane amide, n is the nucleotide base number (being length) of heterozygote, referring to Rapley and Walker, document is the same.

Press following formula and estimate the T of RNA-DNA duplex _m:

T _m(℃)=79.8 ℃+18.5 (log ₁₀M)+0.58 (%G+C)-11.8 (%G+C) ²-0.56 (%f)-820/n, wherein M is the weight-molality of univalent cation (normally Na+), (%G+C) is the per-cent of guanosine (G) and cytidine (C) Nucleotide, and %f is the per-cent of methane amide, n is the nucleotide base number (being length) of heterozygote, and document is the same.

Equation

1 and 2 general only accurate to the heterozygosis duplex of being longer than 100-200 Nucleotide, document is the same.

Can be shorter than the T of the nucleotide sequence of 50 Nucleotide by the following formula estimation _m:

T _m(℃)＝4(G+C)+2(A+T)，

A (VITAMIN B4) wherein, C, T (thymus pyrimidine) and G are the numbers of corresponding nucleotide.

The tight hybridization conditions example that has the complementary nucleic acid of complementary residue more than 100 in hybridization on Southern or the Northern blotting membrane is: containing in 50% formalin of 1mg heparin, 42 ℃ of hybridization are spent the night.The example of tight wash conditions is: 0.2 * SSC, 65 ℃ of washings 15 minutes (description of relevant SSC damping fluid can be referring to Sambrook, and document is the same).Washing through using low stringency always substitutes the washing of high tight degree, to remove the background probe signals.The example of low stringency washing is 2 * SSC, and 40 ℃ were washed 15 minutes.

Usually, in specific cross experiment, if 2.5 times-5 times of signal/noise ratios to (or being higher than) with the observed respective value of uncorrelated probe, then show to detect specific hybrid.In the context of the present invention, detect that tight at least hybridization then shows between two sequences: have relative higher structural similarity or homology with the nucleic acid of the present invention that provides in this paper sequence table for example.

As mentioned above, will " highly tight " condition be chosen to be the bit sequencing and be listed in definite ionic strength and the pyrolysis chain temperature (T under the pH _m) low about 5 ℃ or lower.Can under highly tight condition, identify and the closely related or identical target sequence of interested nucleotide sequence (for example " probe ").Condition than low stringency is applicable to complementary relatively poor sequence, and referring to for example Raoley and Walker, document is the same.

Can use relatively hybridization to identify nucleic acid of the present invention, this relatively hybrid method is a preferred method of distinguishing nucleic acid of the present invention.In the context of the present invention, detect that highly tight hybridization shows between two sequences: have relative higher structural similarity/homology with the nucleic acid that provides in this paper sequence table for example.Highly tight hybridization has been illustrated ratio by the detected high structure of tight hybridization conditions between two nucleotide sequences, and nucleotide base is formed, and arranges or similarity or homology level in proper order.Particularly, in the context of the present invention, detect highly tight hybridization and show: have higher structural similarity or structural homology (for example nucleotide structure, based composition is arranged or order) with the nucleic acid that provides in this paper sequence table for example.For example, need to identify the nucleic acid that tried of the nucleic acid hybridization that can under stringent condition, be exemplified with this paper.

Therefore, a criterion to stringent condition is: can be in highly tight condition (or very tight condition, or super-highly tight hybridization conditions, or super-super-highly tight hybridization conditions) under, hybridizes with one of listed nucleic acid (for example nucleic acid sequence SEQ ID NO:1 to SEQ ID NO:35 and SEQ ID NO:72 to SEQID NO:78 and complementary polynucleotide sequence thereof).For any nucleic acid that tried, all can easily determine stringent condition (comprising for example highly tight, super-highly tight or super-super-highly tight hybridization conditions) and wash conditions by rule of thumb.

For example, when determining highly tight hybridization and wash conditions, can improve hybridization and wash conditions gradually (for example by elevated temperature in hybridization or washing process, reduce salt concn, improve detergent concentration and/or improve organic solvent, concentration as formalin), until reaching the selected standard of a cover.For example, improve hybridization and wash conditions gradually, until probe can (this target nucleic acid contains one or more and is selected from SEQ ID NO:1 to SEQ ID NO:35 in conjunction with the complementary target of accurately mating, the nucleotide sequence of SEQ ID NO:72 to SEQ IDNO:78 and complementary polynucleotide sequence thereof), described probe also contains one or more and is selected from SEQ ID NO:1 to SEQ ID NO:35, the nucleotide sequence of SEQ ID NO:72 to SEQ ID NO:78 and complementary polynucleotide sequence thereof, at least 2.5 times of the signal/noise ratios of this moment, optional 5 times to or the respective value of high power when viewed probe is hybridized with unmatched target more.At this moment, unmatched target is the nucleic acid corresponding to known interferon-alpha, when for example submitting the application, as GenBank ^TMPublic database in the interferon-alpha nucleic acid that exists.The example of this unmatched target nucleic acid comprises the nucleic acid that for example has following GenBank registration number: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, and V00549 (α-2a) and I08313 (α-Con1).Those skilled in the art can identify other this type of sequence in GenBank.Human interferon gene and proteinic name are discussed at (1996) Interferon, rabbit and cytokine research magazines such as Diaz respectively, (1996) Interferon, rabbit such as 16:179-180 and Allen and cytokine research magazine, 16:181-184 lists this paper in as a reference all in full.

Be at least when the hybridization degree of being tried nucleic acid and probe nucleic acid probe and the accurate complementary target of coupling the hybridization degree 1/2, it is 1/2 o'clock that signal/noise ratio is at least probe and target hybridization, we can say and tried nucleic acid and probe nucleic acid specific hybrid, wherein under the condition of described hybridization, accurately the probe of coupling combines with the complementary target of accurately mating, its signal/noise ratio is at least about 2.5 times-10 times, general 5 times-10 times respective value when viewed probe and the hybridization of any unmatched target nucleic acid, described unmatched target nucleic acid is the nucleic acid with following GenBank registration number: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), and X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, and M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other the similar interferon-' alpha ' sequence that provides among the GenBank.

Super-highly tight hybridization and wash conditions is condition as described below, wherein improve the tight degree of hybridization and wash conditions, until probe and accurate at least 10 times of respective value when viewed probe is hybridized with any unmatched target nucleic acid of complementary target nucleic acid bonded signal/noise ratio of coupling, described unmatched target nucleic acid is the nucleic acid with following GenBank registration number: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), and X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, and M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other the similar IFN-α sequence that provides among the GenBank.If target nucleic acid can be under this condition and probe hybridization, and signal/noise ratio be at least accurate coupling complementary target nucleic acid 1/2, we can say that promptly this target nucleic acid and probe can combine under super-highly tight condition.

Similarly, by improving the hybridization and/or the wash conditions of relevant cross experiment gradually, can determine even higher tight level.For example, can identify following condition, wherein improve the tight degree of hybridization and wash conditions, until probe and at least 10 times of the complementary target nucleic acid bonded signal/noise ratios of accurately mating, 20 times, 50 times, 100 times or 500 times or the respective value of high power when viewed probe and any unmatched target nucleic acid hybridization more, described unmatched target nucleic acid is the nucleic acid with following GenBank registration number: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), and X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, and M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other the similar interferon-' alpha ' sequence that provides among the GenBank.If target nucleic acid can be under this condition and probe hybridization, and signal/noise ratio be at least accurate coupling complementary target nucleic acid 1/2, we can say that promptly this target nucleic acid and probe can combine under super-super-highly tight condition.

Can be at height, super-height and super-super-height stringent condition are features of the present invention with the target nucleic acid of nucleic acid hybridization shown in SEQ ID NO:1 to SEQ IDNO:35 and SEQ ID NO:72 to the SEQ ID NO:78 down.The example of described nucleic acid comprises with given nucleotide sequence to be compared, and has the nucleic acid that one or several reticent or conservative nucleic acid replaces.

If nucleic acid encoded polypeptide that can not the phase mutual cross under stringent condition is substantially the same, can think that described nucleic acid is also substantially the same.For example, when the use maximum codon degeneracy generation nucleic acid that genetic code allowed copies, perhaps when among SEQ ID NO:36 to SEQ ID NO:70 and SEQID NO:79 to the SEQ ID NO:85 one or more and this situation can appear when producing antiserum(antisera), wherein reduce described antiserum(antisera) with known interferon-' alpha ' sequence encoded polypeptide, described polypeptide comprises for example by the following interferon-' alpha ' nucleic acids sequence among the GenBank, be that the GenBank registration number is: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (other similar interferon-' alpha ' sequence encoded polypeptide that provides among the nucleotide sequence of α-Con1) or the GenBank.Can find the details of the Immunological Identification of relevant polypeptide of the present invention hereinafter.In addition,, can use TMAC1 hybrid method well known by persons skilled in the art for the duplex of distinguishing sequence less than about 100 Nucleotide, referring to for example Sorg, U etc., nucleic acids research (1991,9,11) 19 (17), it lists this paper in as a reference in full.

On the one hand, the invention provides a kind of nucleic acid, it contains unique subsequence in the nucleic acid that is selected from SEQ ID NO:1 to SEQ IDNO:35 or SEQ ID NO:72 to SEQ ID NO:78.With compare corresponding to the nucleic acid of any known disturbances element-'alpha ' nucleic acids sequence, the subsequence of this uniqueness is unique, and described nucleotide sequence comprises the known array that for example has following GenBank registration number: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), and V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other the similar interferon-' alpha ' sequence that provides among the GenBank.By any and a whole set of nucleic acid among SEQ ID NO:1 to SEQ ID NO:35 or SEQ ID NO:72 to the SEQ ID NO:78 are carried out sequence alignment, can measure the subsequence of this uniqueness, described a whole set of nucleic acid is corresponding to (the α-D) of known disturbances element-'alpha ' nucleic acids sequence: the J00210 with for example following GenBank registration number, J00207 (α-a), X02958 (α-6), X02956 (α-5), and V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other the similar interferon-' alpha ' sequence that provides among the GenBank.The BLAST algorithm that use is set to default parameter can carry out sequence alignment.The subsequence of any uniqueness all can be used as probe to identify nucleic acid of the present invention.

Similarly, the present invention includes a peptide species, it contains unique amino acid subsequence in the polypeptide that is selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85.Compare with the amino acid subsequence of known disturbances element-α peptide sequence, the subsequence of this uniqueness is unique, (the α-D) by the amino acid subsequence corresponding to the coded polypeptide of other similar interferon-' alpha ' nucleic acids that provides among the known disturbances element-'alpha ' nucleic acids of following arbitrary GenBank registration number or the GenBank: J00210 for example is provided described amino acid subsequence, J00207 (α-A), X02958 (α-6), X02956 (α-5), and V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or the amino acid subsequence of other similar polypeptide sequence that provides among the GenBank.Polypeptide and a whole set of known interferon-alpha polypeptides sequence are carried out sequence alignment, described polypeptide is for example by corresponding to other similar interferon-' alpha ' nucleic acids encoded polypeptide: the J00210 that provides among the nucleic acid of following GenBank registration number or the GenBank (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), (being called " contrast polypeptide ") (notes working as sequence corresponding to non--translation sequences, during as pseudogene, only need can produce corresponding polypeptide, wherein read frame and be chosen to be reading frame corresponding to homology interferon-alpha nucleic acid by in computer, nucleotide sequence being translated into aminoacid sequence) or GenBank in other similar polypeptide sequence of providing.

In addition, the invention provides target nucleic acid, it is hybridized with unique oligonucleotides coding down in tight at least or highly tight condition (or condition of higher tight degree), described oligonucleotide coding is selected from the unique subsequence in the polypeptide of SEQ IDNO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85, wherein with GenBank in known disturbances element-α peptide sequence of showing the amino acid subsequence or compare corresponding to the polypeptide of any contrast polypeptide, the subsequence of this uniqueness is unique.By the sequence of measuring uniqueness mentioned above.

In one embodiment, select stringent condition so that can hybridize with oligonucleotides coding with the accurate complementary oligonucleotide of oligonucleotides coding, and its signal/noise ratio than accurate complementary oligonucleotide with corresponding to the signal/noise ratio of the contrast nucleic acid hybridization of any contrast polypeptide at least about high 5 to 10 times.But selection condition to be observing the higher signal noise ratio in employed special test, for example about 15 times, and 20 times, 30 times, 50 times or high power more.In this embodiment, target nucleic acid and unique oligonucleotides coding hybridization, and its signal/noise ratio will be compared the signal/noise ratio of hybridizing according to nucleic acid and oligonucleotides coding at least about high 2 times.In addition, can select the higher signal noise ratio, for example about 2.5 times, about 5 times, about 10 times, about 20 times, about 30 times, about 50 times or high power more.Specific signal will depend on the mark that correlation test is used, fluorescent mark for example, colorimetric mark, radio-labeling etc.

On the other hand, the invention provides a peptide species, this polypeptide contains unique subsequence in the polypeptide that is selected from SEQ ID NO:36 to SEQID NO:70 or SEQ ID NO:79 to SEQ ID NO:85, wherein with corresponding to known disturbances element-α polypeptide, compare as the interferon-alpha polypeptides polypeptide of sequence sequence that provides among the GenBank, the subsequence of this uniqueness is unique. The substrate and the form of sequence reorganization

Polynucleotide of the present invention can be used as multiple reorganization and return reorganization (for example DNA reorganization) reaction, and other diversity generation technique, comprise for example Ausubel, Berger and Sambrook, substrate in induced-mutation technique that document is same as above and the standard cloning process promptly is used to produce other interferon-' alpha ' homologue with desired characteristic.According to used screening or system of selection, can produce and isolate reorganization, for example through reorganization interferon-' alpha ' homologue polypeptide, described polypeptide is endowed multiple desired characteristic, enhanced antiviral activity for example, the enhanced antiproliferative activity is at the enhanced growth-inhibiting of particular target cell, static and/or the cytotoxic activity of cell, the immunogenicity of reduction etc.

Can use varied property generation method, comprise nucleic acid reorganization method, these methods are described in detail in the art.Can separately and/or unite these methods of using, producing one or more variants of a kind of nucleic acid or a cover nucleic acid, and coded proteinic variant.Can provide strong with these methods of uniting use separately, widely used approach produces diversified nucleic acid and nucleic acid makes up (set) (comprising for example nucleic acid library), be used for for example transforming or develop out fast and have nucleic acid new and/or improved characteristics, protein, approach, cell and/or organism.

Although in the process of the property the illustrated discussion that will carry out method is distinguished below and classified, should understand technology often is to contain mutually.In fact, can be separately or associating, parallel or use several different methods successively, to produce various sequence variants.

The result of any diversity generation method as herein described produces one or more nucleic acid, can select or filter out the proteinic nucleic acid that coding has or give desired characteristic therein.By one or more methods as herein described, or operable other method of those skilled in the art generates after the diversity, can in any nucleic acid that produces, select required activity or characteristic, enhanced antiviral activity for example, the enhanced antiproliferative activity, enhanced resists-angiogenic activity, at the enhanced growth-inhibiting of particular target cell, static and/or the cytotoxic activity of cell, the immunogenicity of reduction etc.It is antiviral that mensuration has enhanced, antiproliferative, and growth-inhibiting, the method for the immunogenic nucleic acid of cell static and/or cytotoxic activity or reduction comprises those methods as herein described.Described method can comprise: by any test of this area, identify any activity that can detect with for example automatization or automatable mode.Can estimate multiple relevant (or even incoherent) characteristic successively or abreast according to operator's suggestion.

Following publication has been described varied property generation method, comprise the recurrence recombination method, and/or produce the method for modified nucleotide sequence, they can be used for method of the present invention, described publication comprises following publication and the reference of wherein mentioning: Soong, N.W etc. (2000) " the molecule breeding of virus ", Nature Genetics25:436-439; Stemmer, W etc. (1999) " are used for the viruses molecule breeding of target and other clinical characteristics ", cancer target 4:1-4; Ness etc. (1999) " the DNA reorganization of subtilisin subgene group sequence ", Nature Biotechnology17:893-896; Chang etc. (1999) " use the reorganization of DNA family to develop cytokine ", Nature Biotechnology17:793-797; Minshull and Stemmer (1999) " develop by molecule breeding carrying out protein ", Current Opinionin Chemical Biology3:284-290; Christians etc. (1999) " use the directed used thymidine kinase of AZT phosphorylation that develops of DNA family reorganization ", Nature Biotechnology 17:259-264; Crameri etc. (1998) " carry out DNA reorganization to the gene family from a plurality of species and can promote directed the differentiation ", natural 391:288-291; Crameri etc. (1997) " by DNA reorganization the arsenate detoxification pathways being carried out molecule develops ", Nature Biotechnology15:436-438; Zhang etc. (1997) " develop into effective fucosidase by DNA reorganization and screening with the tilactase orientation ", Proc.Natl Acad Sci.USA 94:4504-4509; Patten etc. (1997) " application of DNA reorganization in medicine and vaccine ", Current Opinion in Biotechnology8:724-733; Crameri etc. (1996) " make up and develop antibody-phage library by DNA reorganization ", natural drug 2:100-103; Crameri etc. (1996) " develop the improvement green fluorescent protein by the molecule that uses DNA reorganization ", Nature Biotechnology14:315-319; Gates etc. (1996) " by being showed in lac repressor ' cap dimer ' " with affine optionally isolating ligands from peptide library, molecular biology magazine 255:373-386; Stemmer (1996) " sexual PCR and assembling PCR ", molecular biology encyclopedia, VCH Publishers, New York, p447-457; Crameri and Stemmer (1995) " make up the conversion that multiple cassette mutagenesis produces all sudden changes and wild flask ", biotechnology 18:194-195; Stemmer etc. (1995) " single stage assembled base because of and whole plasmid form a large amount of oligodeoxyribonucleotides ", gene 164:49-53; Stemmer (1995) " differentiation that divides subalgorithm ", science 270:1510; Stemmer (1995) " search sequence is at interval ", biotechnology 13:549-553; Stemmer (1994) " reorganizes at external quick differentiation protein by DNA ", natural 370:389-391; And Stemmer (1994) " carry out DNA reorganization by random fragmentation with ressembling: vitro recombination develops to carry out molecule ", Proc.Natl Acad.Sci.USA91:10747-10751.

Other details of relative dna reorganization and other diversity generation method is found in following United States Patent (USP), PCT application and EP application: the USPN5 of Stemmer, 605,793 (on February 25th, 1997), " vitro recombination method "; The USPN5 of Stemer etc., 811,238 (on September 22nd, 1998), " producing the method for polynucleotide " with required feature by repeating to select and recombinating; The USPN5 of Stemmer etc., 830,721 (on November 3rd, 1998), " by random fragmentation and ressemble carry out DNA mutagenesis "; The USPN5 of Stemmer, 834,252 (on November 10th, 1998), " terminal complementary polymeric enzyme reaction "; The USPN5 of Minshull, 837,458 (on November 17th, 1998), " method and composition of cell and metabolic engineering "; WO95/22625, Stemmer and Crameri, " by random fragmentation and ressemble carry out mutagenesis "; WO96/33207, Stemmer and Lipschutz, " terminal complementary polymerase chain reaction "; WO97/20078, Stemmer and Crameri, " producing the method for polynucleotide " with required feature by repeating to select and recombinating; WO97/35966, Minshull and Stemmer, " method and composition of cell and metabolic engineering "; WO99/41402, Punnonen etc., " target of genetic vaccine vector "; WO99/41383, Punnonen etc., " antigen library immunity "; WO99/41369, Punnonen etc., " genetic vaccine vector engineering "; WO99/41368, Punnonen etc., " optimization of dna gene vaccine modulation characteristic "; EP752008, Stemmer and Craneri, " by random fragmentation and ressemble carry out DNA mutagenesis "; EP0932670, Stemmer, " changing the cell DNA absorption " by returning the sequence reorganization; WO99/23107, Stemmer etc., " by viral genome reorganization modification virus preferendum and host range "; WO99/21979, Apt etc., " human papilloma virus poisonous carrier "; WO98/31837, Del Cardayre etc., " changing whole cell and organism " by returning the sequence reorganization; WO98/27230, Patten and Stemmer, " method and composition of polypeptide engineering "; EP0946755, Patten and Stemmer, " method and composition of polypeptide engineering "; And WO98/13487, Stemmer etc., " by returning sequence reorganization and selecting the optimization gene therapy methods "; WO00/00632, " producing the method for height diverse libraries "; WO00/09679, " obtaining method and institute's calling sequence " through the polynucleotide sequence storehouse of vitro recombination; WO98/42832, Arnold etc., " the primer recombination of polynucleotide sequence of using at random or determining "; WO99/29902, Arnold etc., " producing the method for polynucleotide and peptide sequence "; WO98/41653, Vind, " in vitro method in constructed dna library "; WO98/41622, Borchert etc., " using DNA reorganization to make up the method in library "; And WO98/42727, Pati and Zarling, " using homologous recombination to change sequence ".

Some U. S. application provides other details of relative dna reorganization and correlation technique and other diversity generation method, these applications comprise: Patten equals (USSN60/102 on September 29th, 1998,362), " gene that the reorganization codon changes to some extent " submitted on January 29th, 1999 (USSN60/117,729) and on September 28th, 1999; Del Cardayre equals on July 15th, 1998 (USSN09/166,188) and submission on July 15th, 1999 (USSN 09/354,922) and " changes whole cell and organism by returning the sequence reorganization "; Crameri equals on February 5th, 1999 (USSN60/118,813), " the oligonucleotide mediated nucleic acid reorganization " that on June 24th, 1999 (USSN60/141,049) and on September 28th, 1999 (USSN09/408,392) submit to; " using oligonucleotide to synthesize reorganization " that Welch equals to submit on September 28th, 1999 (USSN 09/408,393) based on codon; Selifonov and Stemmer are in " preparation has the character chain of desired characteristic, the method for polynucleotide and polypeptide " of on February 5th, 1999 (USSN 60/118854) and submission on October 12nd, 1999 (USSN 09/416,375); Patten equals on March 5th, 1999 (USSN 60/122,943), and on July 2nd, 1999, (USSN 60/142,299), " the modified nucleic acid that reorganization is inserted " submitted on November 10th, 1999 (USSN60/164,618) and on November 10th, 1999 (USSN 60/164,617); With the USSN60/186 that Affholter submitted on March 2nd, 2000,482 " reorganization of single-chain nucleic acid template mediation separates with nucleic acid fragment ".

As above-mentioned publication, patent, disclosed foreign application and U.S. Patent application comment disclosed, can implement the diversity method of generationing by the multiple method of having set up, for example reorganization (or " returning (recursive) recombinates ") nucleic acid is providing the new nucleic acid with desired characteristic.In these methods any all is applicable to the present invention, can be used for forming interferon-alpha discussed in this article, produces the new interferon-alpha homologue with new features or improved characteristics.Two kinds of methods that prepare the above-mentioned Interferon, rabbit (as the IFN homologue) that produces by these methods also are features of the present invention.Briefly, the sequence modification method of the big classification of several differences can be used for the present invention as reorganization, and these methods are described in for example above-mentioned reference.At first, can be at any recombinant nucleic acid in the external multiple technologies of discussing in by above-mentioned reference, for example comprise with dnase digestion nucleic acid to be recombinated, connect then and/or ressemble nucleic acid by PCR.The second, by recombinating between the nucleic acid that for example makes the cell Shen, in vivo or ex vivo ground return recombinant nucleic acid.The 3rd, can use full genome recombination method, wherein the complete genome group of cell or other organism is recombinated, and the optional admixture that comprises has the genome reorganization mixture of the required library composition gene of approach of the present invention (for example corresponding to).The 4th, can use synthetic recombination method, wherein synthetic and reassembly in PCR or ligation corresponding to the oligonucleotide of required target, thus producing new recombinant nucleic acid, described oligonucleotide comprises the oligonucleotide corresponding to an above parental generation nucleic acid.Can pass through the Nucleotide additive process of standard, or prepare oligonucleotide by for example three-Nucleotide synthesis method.The 5th, can implement in silico recombination method, wherein in computer, use genetic algorithm, with the sequence chain of reorganization corresponding to homology (or even non--homology) nucleic acid.Optional by synthetic nucleic acid corresponding to recombination sequence, for example unite use oligonucleotide synthetic/the gene technology of reassemblying, and change gained recombination sequence chain into nucleic acid.Can repeat in the above-mentioned general recombination form any, have more multifarious recombinant nucleic acid to produce a cover.The 6th, can use increases natural multifarious method, and for example by making the hybridization of various nucleic acid or nucleic acid fragment and single-stranded template, then polymerization and/or connection are to regenerate full length sequence, randomly, degrade again template and reclaim the modified nucleic acid of gained.Above-mentioned reference provides these and other basic recombinant forms and multiple through the change method based on these forms.Regardless of used recombinant forms, nucleic acid of the present invention can (mutually, or with the nucleic acid of relevant (or even not relevant)) reorganization, overlaps and possesses multifarious recombinant nucleic acid to produce one, comprises for example homologous nucleic acid.In general, sequence recombinant technology as herein described provides special benefit, because they make SEQ ID NO:1 to SEQ ID NO:35 and SEQ IDNO:72 to SEQ ID NO:78, or can recombinate in any operable mode between its fragment or the variant, thereby a kind of method very fast is provided, has influenced required result's mode in order to study different combined sequence.

After the reorganization, can in any nucleic acid that is produced, screen or select required activity.In the context of the present invention, comprise, for example detect and identify any activity that to measure in automatable mode by any test known in the art.In addition, also can select following useful characteristic, as reduced immunogenicity, the half life of increase, the solvability of improvement, characteristic that can be oral etc.Can use any available to test to detect the characteristic of multiple alpha-interferon relevant (or even not relevant).

DNA mutagenesis and reorganization provide strong, can widely used method in order to produce diversity, described diversity can be used for transforming out the protein with improved characteristics, approach, cell and biology.Except above-mentioned basic skills, also need sometimes to unite and use reorganization methodology and other to produce multifarious technology.Varied property generation method can be united use (or separating use with it) with the reorganization method, and can be in system of the present invention The selection result (being various nucleic acid populations).By can cause each Nucleotide in abutting connection with or the method that changes of the Nucleotide group of adjacency not, i.e. mutafacient system can import other diversity.Multiple mutafacient system is described in the above-mentioned reference; Other details of relevant mutafacient system can be referring to following reference.

Producing multifarious mutafacient system for example comprises: reorganization (PCT/US98/05223; Publication number WO98/42727); Site-directed mutagenesis (Ling etc. (1997) " Approaches to DNA mutagenesis:anoverview, " Anal.Biochem.254 (2): 157-178; Dale etc. (1996) " Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, " Methods Mol.Biol.57:369-374; Smith (1985) " In vitro mutagenesis, " Ann.Rev.Genet.19:423-462; Botstein ﹠amp; Shortle (1985) " Strategies andapplications of in vitro mutagenesis, " Science 229:1193-1201; Carter (1986) " Site directed mutagenesis, " Biochem.J.237:1-7; And Kunkel (1987) " Theefficiency of oligonucleotide directed mutagenesis, " Nucleic Acids ﹠amp; MolecularBiology (D.M.J compiles for Eckstein, F and Lilley, Springer Verlag, Berlin)); Use contains mutagenesis (Kunkel (1985) " Rapid and efficient site-specific mutagenesiswithout phenotypic the selection, " Proc.Nat ' l Acad.Sci.USA 82:488-492 of the template of uridylic; Kunkel etc. (1987) " Rapid and efficient site-specific mutagenesis without phenotypicselection, " Results Probl.Cell Differ.154,367-382; With (1988) " Mutant Trprepressors with new DNA-binding specificities, " Science 242:240-245 such as Bass); Oligonucleotide mediated mutagenesis (Results Probl.Cell Differ.100:468-500 (1983); Results Probl.Cell Differ.154:329-350 (1987); Zoller ﹠amp; Smith (1982) " Oligonucleotide-directed mutagenesis using M13-derived vectors:an efficient and generalprocedure for the production of point mutations in any DNA fragment, " NucleicAcids Res.10:6487-6500; Zoller ﹠amp; Smith (1983) " Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, " Results Probl.CellDiffer.100:468-500; With Zoller ﹠amp; Smith (1987) " Oligonucleotidedirectedmutagenesis:a simple method using two oligonucleotide primers and a singlestranded DNA template, " Results Probl.Cell Differ.154:329-350); DNA mutagenesis (Taylor etc. (1985) " The use of phosphorothioate-modified DNAin restriction enzyme reactions to prepare nicked DNA, " Nucl.Acids Res.13:8749-8764 that thiophosphatephosphorothioate is modified; Taylor etc. (1985) " The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, " Nucl.AcidsRes.13:8765-8787 (1985); Nakamaye ﹠amp; Eckstein (1986) " Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis, " Nucl Acids Res.14:9679-9698; Sayers etc. (1988) " Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, " Nucl.Acids Res.16:791802; With (1988) " Strand specific cleavage of phosphorothioate-containing DNA byreaction with restriction endonucleases in the presence of ethidium bromide, " NuclAcids Res.16:803-814 such as Sayers); Use mutagenesis (Kramer etc. (1984) " Thegapped duplex DNA approach to oligonucleotide-directed mutation construction, " the Nucl.Acids Res.12:9441-9456 of gapped duplex DNA; Kramer ﹠amp; Fritz (1987) " Oligonucleotidedirected construction of mutations via gapped duplex DNA, " Results Probl.CellDiffer.154:350-367; Kramer etc. (1988) " Improved enzymatic in vitroreactions in the gapped duplex DNA approach to oligonucleotide-directedconstruction of mutations, " Nucl.Acids Res.16:7207; With (1988) " Oligonucleotide-directed construction of mutations:a gapped duplex DNAprocedure without enzymatic reactions in vitro, " Nucl.Acids Res.16:6987-6999 such as Fritz).

Other appropriate means comprise a mispairing reparation (Kramer etc. (1984) " Point MismatchRepair; " Cell 38:879-887), use mutagenesis (Carter etc. (1985) " Improved oligonucleotide site-directed mutagenesis using M13 vectors, " the Nucl.Acids Res.13:4431-4443 of reparation-damaged host's strain; And Carter (1987) " Improved oligonucleotide-directedmutagenesis using M13 vectors, " Results Probl.Cell Differ.154:382-403), deletion mutagenesis (Eghtedarzadeh ﹠amp; Henikoff (1986) " Use of oligonucleotides to generate largedeletions; " Nucl.Acids Res.14:5115), restricted-as to select and restricted-select and restricted-purifying (Wells etc. (1986) " Importance of hydrogen-bond formation in stabilizing thetransition state of subtilisin; " Phil.Trans.R.Soc.Lond.A317:415-423), by synthetic mutagenesis (Nambiar etc. (1984) " Total synthesis and cloning of a genecoding for the ribonuclease S the protein, " Science223:1299-1301 of carrying out of total gene; Sakamar and Khorana (1988) " Total synthesis and expression of a gene for the a-subunit ofbovine rod outer segment guanine nucleotide-binding protein (transducing), " Nucl.Acids Res.14:6361-6372; Wells etc. (1985) " Cassette mutagenesis:an efficientmethod for generation of multiple mutations at defined sites, " Gene34:315323; With (1985) " Oligonucleotide-directed mutagenesis by microscale ' shot-gun ' gene synthesis; " such as Grundstrom Nucl.Acids Res.13:3305-3316), double-strand break repair (Mandecki (1986) " Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli:a method for site-specific mutagenesis; " Proc.Nat ' lAcad.Sci.USA, 83:7177-7181).Other details of relevant multiple aforesaid method can referring to: Methods in Enzymology, Vol.154 has wherein also described the useful control device to the problem that makes one nervous in the multiple mutafacient system.

Also can utilize use to add or degenerate oligonucleotide at random or half-random mutagenesis (Arkin and Youvan (1992) " Optimizing nucleotide mixtures to encode specific subsets ofamino acids for semi-random mutagenesis, " Biotechnology 10:297300; Reidhaar-Olson etc. (1991) " Random mutagenesis of protein sequences usingoligonucleotide cassettes, " Methods Enzvmol.208:564-86; Lim and Sauer (1991) " The role of internal packing interactions in determining the structure and stabilityof a protein, " J.Mol.Biol.219:359-76; Breyer and Sauer (1989) " Mutationalanalysis of the fine specificity of binding of monoclonal antibody 51F to lambdarepressor, " J.Biol.Chem.264:13355-60); " step is moved mutagenesis " (Crea, R; United States Patent (USP) 5,830,650 and 5,798,208 and EP patent 0527809B1) produce diversity.

In one aspect of the invention, can use fallibility PCR to produce the nucleic acid variant.Use this technology, under the low condition of the copy fidelity of archaeal dna polymerase, carry out PCR, make to obtain high-frequency point mutation along total length PCR product.The example of this technology can be referring to above-mentioned reference, and example is seen (1992) PCR Methods Applic.2:28-33 such as (1989) Technique1:11-15 such as Leung and Caldwell.Similarly, assemble in the method for PCR product, can use assembling PCR at the mixture that relates to by little dna fragmentation.Can be in identical tubule parallelly carry out a plurality of different PCR reactions, cause the product of another reaction with the product of a reaction.Can use sexual (Sexual) PCR mutagenesis, wherein by make the dna molecular random fragmentation based on sequence homology, then in the PCR reaction, carry out crossover fixation by primer extension, and external different, but between the relevant dna molecular of dna sequence dna homologous recombination takes place.This method is described in above-mentioned reference, and example is seen Stemmer (1994) Proc.Nat ' lAcad.Sci.USA 91:10747-10751.Can use to return group (ensemble) mutagenesis, wherein use protein mutagenesis algorithm to produce the diversity colony of phenotypic correlation mutant, the aminoacid sequence of described group member is different.This method is used the many continuously wheel combinations of Feedback mechanism control cassette mutagenesis.The example of this method sees Arkin ﹠amp; Youvan (1992) Proc.Nat ' l Acad.Sci.USA89:7811-7815.

As mentioned above, in allowing in any interested nucleotide sequence, to produce the method for site-specific mutant, can use oligonucleotide mediated mutagenesis.The example of this technology can be referring to above-mentioned reference, (1988) Science such as Reidhaar-Olson for example, 241:53-57.Similarly, can use cassette mutagenesis in the method for the zonule that substitutes double chain DNA molecule with synthetic oligonucleotide box, described oligonucleotide box is different from native sequences.Oligonucleotide can contain native sequences for example complete and/or incomplete randomization.

(or ex vivo) mutagenesis is used in the method that produces random mutation among any clone's the required DNA in the body, and described method for example is included in breeds DNA in the coli strain, and one or more DNA reparation approach of described bacterial strain carry sudden change.For the wild-type parental generation, these " mutators " have higher random mutation rate.Propagation DNA produces random mutation the most at last in bacterial strain in DNA therein.

Can use the mutagenesis of index group to have the combinatorial library of peculiar, the functional mutants of high per-cent with generation, wherein group's residue is by randomization abreast, causes producing the amino acid of functional protein to identify each position that changes to some extent.The example of this method can be referring to Delegrave ﹠amp; Youvan (1993) Biotechnology Research 11:1548-1552.Similarly, can use at random and site-directed mutagenesis.The example of this method can be referring to Arnold (1993) Current Opinion in Biotechnology4:450-455.

Be used for mutagenesis, the test kit of library construction and other diversity generation method also can be purchased.For example, test kit can be available from Stratagene for example (as QuickChange ^TMThe site-directed mutagenesis test kit; And Chameleon ^TMDouble-stranded, the site-directed mutagenesis test kit), Bio/Can Scientific, Bio-Rad (for example using above-mentioned Kunkel method), Boehringer Mannheim Corp., Clonetech Laboratories, DNATechnologies, Epicentre Technologies (as 5prime 3prime test kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs, PharmaciaBiotech, Promega Corp., Quantum Biotechnologies, Amersham Intemational plc (for example using above-mentioned Eckstein method) and Anglian Biotechnology Ltd (for example using above-mentioned Carter/Winter method).

Any described reorganization or induced-mutation technique can be united use, described genome such as bacterium, fungi, animal or plant genome with other multifarious method of importing in genome.For example, except aforesaid method, also suggestion is used and can be produced the polymeric technology of chimeric nucleic acid, and described polymer is suitable for being converted into a plurality of species (referring to for example Schellenberger United States Patent (USP) 5,756,316 and above-mentioned reference).When this chimeric polymer by the genomic constitution of divergent (for example by natural diversity or use site-directed mutagenesis, fallibility PCR, generations such as mutagenesis bacterial isolates approach) each other, and when being converted into suitable host, can provide nucleic acid diversity source so that the DNA variation.

The chimeric polymer that is converted into host species is suitable for use as in the body substrate of (or ex vivo) reorganization method.Perhaps, the multiple polynucleotide in shared portion sequence similarity or homology zone can be converted into host species, and by host cell in vivo (or ex vivo ground) recombinate.Can use ensuing many wheel cells divisions to produce the library, the member in described library is contained single, uniformly monomer or concentrated nucleic acid population.Perhaps, can reclaim monomer nucleic acid, and return reorganization with any described reorganization method by standard technique.

Also suggestion is used and can be generated multifarious chain termination method (referring to for example United States Patent (USP) 5965408 and above-mentioned reference).In this method, when existing or lack gene-specific primer, the double-stranded DNA corresponding to one or more genes is mixed and sex change, described gene is shared sequence similarity or homology zone.The strand polynucleotide of annealing then, and polysaccharase and chain termination reagent (as ultraviolet ray, γ or x-ray bombardment; Ethidium bromide or other intercalator; DNA is conjugated protein, as single strand binding protein, and activating transcription factor or histone; Polycyclic aromatic hydrocarbons; Trivalent chromium or chromic salt; Or by the cripetura polymerization of rapid thermal cycles mediation etc.) existence insulation down, cause producing part duplex molecule.Then ensuing many wheels duplicate or part duplicate in sex change and annealing portion duplex molecule again, the described molecule that for example contains the part extended chain, cause producing polynucleotide, described polynucleotide are shared sequence similarity or homology in various degree, and are chimeric polynucleotide with regard to initial dna molecular colony.Randomly, can be at the one or more stage amplified productions of this method or the part gleanings of product.The polynucleotide that produce by for example above-mentioned chain termination method are suitable substrates of the diversity method of generationing (for example RSR, DNA reorganizes) according to any described form.

Can further not increase diversity by use based on the method and the DNA reorganization (as described in above-mentioned publication and application, it can be based on homology or not based on homology, and this depends on definite form) of homology.For example, the increment brachymemma (ITCHY) that can use being used to of describing among (1999) " A combinatorial approach to hybridenzymes independent of DNA homology " Nature Biotech.17:1205 such as Ostermeier to produce heterozyme produces initial reorganization library, this library can be used as take turns or take turns more external, the substrate of diversity generation method (for example RSR or reorganization method) in ex vivo or the body.

Described the method that produces many species expression library (for example United States Patent (USP) 5,783,431; 5,824,485 and above-mentioned reference), the someone proposes these methods and can be used for identifying required protein active (United States Patent (USP) 5,958,672 and above-mentioned reference).In general, many species expression library contains from the cDNA of a plurality of species or bacterial strain or genome sequence, and described sequence can be operated with suitable adjusting sequence in expression cassette and be linked to each other.Choose wantonly cDNA and/or genome sequence are connected at random with further increase diversity.Carrier can be to be suitable for transforming more than a kind of host living beings, as bacterial species, and eukaryotic cell and the shuttle vectors of expressing therein.In some cases, by selecting the coding desired protein in advance, or make the library have bias with the sequence of required nucleic acid hybridization.Any this type of library all can become the substrate of any method described herein.

In some applications, before reorganization, need to select in advance or screen (Kuo Zeng library for example, library in advance, genomic library, the cDNA library, normalization method library etc.) or other substrate nucleic acid, perhaps make substrate be partial to the nucleic acid of encoding function product (itself also has these effects the reorganization method).For example, when antagonist is transformed, can generate diversity (for example DNA reorganization) by any described method before, utilize interior (or ex vivo or the external) recombination event of body to make the reorganization method be partial to have the antibody of functional antigen binding site.For example, generating diversity (for example DNA reorganization) according to any method described herein before, can increase and derive from the recombinant C DR of B cell cdna library, and it is assembled to frame area (for example (1998) " Exploiting sequence space:shuffling in vivo formedcomplementarity determining regions into a master framework, " Gene 215:471 such as Jirholt).

The proteinic nucleic acid that coding has required activity (for example binding affinity, enzymic activity, antiviral activity, the ability of induce immune response, antiproliferative activity, adjuvant characteristic etc.) can be partial in the library.For example, from the library, identify after the clone with given activity, the method mutagenesis clone that can use any known importing DNA to change, described method includes but not limited to the reorganization of recurrence sequence or the diversity method of formation of DNA reorganization or other form.Containing the required activity of screening in the library of the homologue of mutagenesis then, described activity can be identical or different with initial given activity.An example of this method can be referring to United States Patent (USP) 5,939,250.Can identify required activity by any method known in the art.For example, WO99/10539 mentions: by extract that mixes gene library and the component that derives from the vigorous cell of metabolism, and identify and show required active combination, and can the screening-gene library.Also the someone proposes (for example WO98/58085): insert the library sample by the substrate with biologically active, and use fluorescence analyser, flow cytometer for example, CCD, photofluorometer or spectrophotometer detect the biological activity fluorescence corresponding to the product with required activity, can identify to have required active clone.

The nucleic acid with special characteristic also can be partial in the library, for example with the nucleic acid of selected nucleic acid probe hybridization.For example, application WO99/10539 proposes: (enzymic activity for example is as lipase can to identify the required activity of coding as follows from genomic dna sequence; esterase, proteolytic enzyme, Glycosylase; glycosyltransferase, Phosphoric acid esterase, kinases; oxygenase, peroxidase, lytic enzyme; hydratase; nitrilase, transaminase, Ntn hydrolase or acyltransferase) polynucleotide.Make in the genomic dna colony single strand dna with the probe hybridization of part-put together.Genomic dna can be hung oneself and be cultivated or uncultivated microorganism, or derives from environmental sample.Perhaps, genomic dna can derive from multicellular organism or its tissue.

Can directly carry out the synthetic of second chain by hybridization probe used in catching, described probe can discharge from catching medium in advance, also can not discharge, perhaps also can be by synthetic second chain of multiple other strategy known in the art.Perhaps, can make isolating strand genomic dna colony segmentization, and further not clone, and they are directly used in the gene based on-reorganization method of reassemblying.In a kind of these class methods, make from the segment colony of genomic library and part, or often be that the ssDNA or the RNA of about total length anneals corresponding to opposite strand.By this colony complicated mosaic gene of assembling is by removing the not segment end of hybridization based on ribozyme, polymerization to be to mend the breach between flat these segments, carries out strand subsequently and connects and mediate.By digestion (if RNA or contain uridylic), under the sex change condition, carry out magnetic and separate (if to help this isolating mode mark) or other operable separation/purification method, can remove the parental generation chain.Perhaps, optional with the parental generation chain with chimeric chain purifying, in subsequently screening and procedure of processing, remove the parental generation chain again.As Affholter (USSN60/186,482, submission on March 2nd, 2000) " Single-stranded nucleic acid template-mediated recombination andnucleic acid fragment isolation ", and the WO98/27230 of Patten and Stemmer, " Methods and Compositions for Polypeptide Engineering " is described, also can use the reorganization of single-stranded template and required nucleic acid, wherein said nucleic acid combines with the part of template.

In one approach, single chain molecule is changed into double-stranded DNA (dsDNA), and the dsDNA molecule combines with solid support by the combination of part-mediation.Isolate after the unconjugated DNA, discharge selected dna molecular from upholder, and with its import suitable host cell with produce can with the library enrichment sequence of probe hybridization.The library of Chan Shenging provides desirable substrate for any reorganization reaction as herein described in this way.

" non--at random " method that produces nucleic acid and polypeptide is described in Short, J. " Non-StochasticGeneration of Genetic Vaccines and Enzymes " WO00/46344.Comprise proposition non--polynucleotide reassembly and these class methods of gene locus saturation mutagenesis at random, and the synthetic connection polynucleotide method of reassemblying as herein described also can be used for the present invention.

Understandablely be: be applicable to that above-mentioned any technology of enriched library also can be used for screening product or the product library that produces by the diversity method of formation before variation.

The recombinant nucleic acid that produces by recurrence reorganization one or more polynucleotide of the present invention and one or more other nucleic acid also constitutes a part of the present invention.One or more other nucleic acid can comprise another kind of polynucleotide of the present invention; Randomly, perhaps, or in addition, one or more other nucleic acid can comprise for example encode natural interferon-α or its subsequence, or any homologous interferon-' alpha ' sequence or its subsequence, or the nucleic acid of interferon-beta sequence or its subsequence (for example interferon-' alpha ' or the interferon-beta sequence of describing in GenBank or other the obtainable document), perhaps for example any other homology or nonhomologous nucleic acid (above-mentioned some recombinant forms, the particularly reorganization of carrying out with synthetic or in silico method do not need homology).

As describing in detail in the above-mentioned reference, reconstitution steps can be in vivo, carries out in the ex vivo, external or in silico.The present invention also comprises the cell that contains any gained recombinant nucleic acid, generate by diversity, reorganization or return reorganization nucleic acid as herein described and the nucleic acid library that produces, contain described library or contain the cell colony of any recombinant nucleic acid, carrier, virus, plasmid etc., wherein said recombinant nucleic acid generate by diversity or reorganization (or returning reorganization) nucleic acid as herein described produces with another kind of this type of nucleic acid or other nucleic acid.Corresponding sequence chain in the database that exists in computer system or the computer-readable medium also is a feature of the present invention. Other polynucleotide compositions

The present invention also comprises the composition (for example as the reorganization substrate) that contains two or more polynucleotide of the present invention.Composition can contain the recombinant nucleic acid library, and its Chinese library contains at least 2,3, and 5,10,20 or 50 or more kinds of nucleic acid.Optional with nucleic acid clone to expression vector, expression library is provided.

The present invention also comprises by using restriction endonuclease, one or more polynucleotide of the present invention of RNA enzyme or dnase digestion (carrying out in above-mentioned some recombinant forms) and the composition that produces; With by with mechanical system (for example supersound process, vortex etc.) segmentization or shear one or more polynucleotide of the present invention and the composition that produces, said composition also can be used to provide the reorganization substrate in aforesaid method.Similarly, cover also can be used as the reorganization substrate corresponding to the composition of the oligonucleotide of more than one nucleic acid of the present invention more than containing, and also is feature of the present invention.For convenience's sake, through segmentization, shearing or oligonucleotide synthetic mixture are called the nucleic acid cover of segmentization with these.

The present invention also comprise by ribonucleotide-or deoxyribonucleotide triphosphoric acid and nucleic acid polymerase in the presence of the composition that produces of the nucleic acid cover of one or more segmentizations of insulation.Resulting composition forms the reorganization mixture, can be used for above-mentioned multiple recombinant forms.Nucleic acid polymerase can be a RNA polymerase, the archaeal dna polymerase (for example " reversed transcriptive enzyme ") of archaeal dna polymerase or RNA-mediation; Polysaccharase can be a thermostable DNA polymerases (for example VENT, TAQ etc.) for example. Interferon, rabbit homologue polypeptide

The invention provides the interferon-' alpha ' homologue polypeptide of separation or reorganization, this paper also is referred to as " interferon-' alpha ' homologue " or " Interferon, rabbit homologue " or " IFN-alpha homologues " or " IFN homologue ".The Interferon, rabbit homologue polypeptide of separation or reorganization of the present invention comprises: contain the polypeptide of sequence that is selected from SEQ ID NO:36 to SEQ ID NO:70 and SEQ ID NO:79 to SEQ ID NO:85, and through the conservative variant of modifying, and segment, described segment for example has antiproliferative activity in the test (or other similar test) based on people Daudi clone, and/or for example has antiviral activity in the test (or other similar test) based on mouse cell line/EMCV.Fig. 1 provides the sequence alignment of the Interferon, rabbit homologue peptide sequence of the present invention that exemplifies.Those skilled in the art use public database and sequence alignment program easily peptide sequence of the present invention to be compared mutually, or compare with known natural interferon-α sequence.

The present invention also provides a peptide species, its contain among SEQ ID NO:36-70 or the SEQ ID NO:71 any at least about 100,120,130,140,150,155,160,163,165 or 166 successive amino acid.On the one hand, described aminoacid sequence contains amino acid Lys160 and Glu166, wherein corresponding one by one among the amino acid in sequence numbering and the SEQ ID NO:36.

The sequence of the present invention (Fig. 1) that exemplifies is compared with being derived from people and inhuman known natural interferon-α and other I type Interferon, rabbit (comprising β, δ, ω, τ-Interferon, rabbit) sequence, can draw several conclusions.These sequences can easily derive from a plurality of sources, are Pfam (protein families) database of http://www.sanger.ac.uk/Software/Pfam/index.Shtml as GenBank and network address.

What need special instruction is: have following amino-acid residue (being expressed as " I group " residue) in Interferon, rabbit homologue peptide sequences more of the present invention, these residues can not appear on the equivalent site of known natural human or inhuman I type Interferon, rabbit sequence.

I group: Asp11; Pro14; Arg50; Phe55; Asp75; Asn80; Pro111; Leu124; Glu134; Ser140 and Ala143; The residue numbering is corresponding to the ripe Interferon, rabbit homologue sequence of identifying among the SEQ ID NO:36.

What need special instruction is: have following amino-acid residue (being expressed as " II group " residue) in Interferon, rabbit homologue peptide sequences more of the present invention, these residues can not appear on the equivalent site of known natural human interferon-alpha hypotype sequence.

II group: Pro9; (Lys, Ser) 12; (Thr, Val) 24; Gln34; Arg40; Ser45; Arg47; Leu56; Ile60; Phe67; Ala79, Gly88; His90; Arg91; Glu95; Val101; (Gly, Ala) 104; Val112; Gly114; Pro116; Lys133 and His136.

In other embodiments, Interferon, rabbit homologue polypeptide contains among the SEQ ID NO:36-70 any at least 20,50,100,150,155 or 160 or more a plurality of successive amino acid, and/or following one or more amino acid Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein amino acid whose numbering is corresponding to the numbering among the SEQ IDNO:36, or following one or more amino acid Pro9, (Lys or Ser) 12, (Thr or Val) 24, Gln34, Arg40, Ser45, Arg47, Leu56, Ile60, Phe67, Ala79, Gly88, His90, Arg91, Glu95, Val101, (Gly, Ala) 104, Val112, Gly114, Pro116, Lys133 and His136, the amino acid numbering in the wherein said peptide sequence is corresponding to each amino acid whose numbering in the aminoacid sequence of SEQ ID NO:36.Therefore, for example, in this embodiment, interferon polypeptides contains aminoacid sequence, this sequence contains proline residue at the 9th amino acids place of sequence, contains Methionin or serine residue at the 12nd, contains Threonine or Xie Ansuan residue at the 24th, contain glutamine residue at the 34th, contain arginine residues etc. at the 40th.This peptide species shows antiproliferative activity and (for example is at least about 8.3 * 10 in the proliferation test based on people Daudi clone ⁶Individual unit/mg), and/or in the test based on people WISH cell/EMCV, show antiviral activity and (be at least about 2.1 * 10 ⁷Individual unit/mg).Some these peptide species are in conjunction with the human alpha interferon acceptor.The length of some these peptide species is 166 amino acid.On the other hand, this peptide species can contain the sequence that is selected among SEQ ID NO:36 to the SEQ ID NO:54 any.

The antiproliferative activity of any polypeptide of the present invention is general and polypeptide causes cell or the growth of its part or apace, the ability that often repeatedly produces new cell growth is relevant.

The present invention also comprises polypeptide (for example any among SEQ ID NO:36-71 or the SEQ ID NO:79-85) or nucleic acid encoding (for example any among SEQ ID NO:1-35 or the SEQ ID NO:72-78), wherein resist-the vasculogenesis test determination by those skilled in the art are well-known, described polypeptide has anti--angiogenic activity.

The present invention also comprises:

(a) contain any interferon-alpha polypeptides that above-mentioned one or more I organizes amino-acid residues.

(b) proper manners Interferon, rabbit sequence (promptly demonstrating the sequence of high-caliber similarity or homology) with human interferon, or to appended sequence table in any sequence of listing or its segment height is similar or homology (promptly has and is at least about 80%, 90%, 95%, 96%, 97%, 98% or higher sequence homology or sequence identity per-cent) the context of sequence in contain any interferon-alpha polypeptides of above-mentioned one or more II group amino-acid residues.

(c) contain any interferon-alpha polypeptides that following residue makes up, described residue is positioned at or near known or be considered to and the interactional interferon-' alpha ' molecular domains of I type Interferon Receptors, wherein said combined sequence (primitive) can not appear on the equivalent site of any known natural human or inhuman I type Interferon, rabbit:

(i) (Tyr or Gln) 34; Add one or more among Ile132 or the Arg134; Or

(ii) Asp78, Glu79 or (Asp or Thr) 80; Add one or more among Ile132 or the Arg134.

In another embodiment, the invention provides and contain sequence: CDLPQTHSLG-X shown in the SEQ ID NO:71 ₁₁-X ₁₂-RA-X ₁₅-X ₁₆-LL-X ₁₉-QM-X ₂₂-R-X ₂₄-S-X ₂₆-FSCLKDR-X ₃₄-DFG-X ₃₈-P-X ₄₀-EEFD-X ₄₅-X ₄₆-X ₄₇-FQ-X ₅₀-X ₅₁-QAI-X ₅₅-X ₅₆-X ₅₇-HE-X ₆₀-X ₆₁-QTFN-X ₆₇-FSTK-X ₇₂-SS-X ₇₅-X ₇₆-W-X ₇₈-X ₇₉-X ₈₀-LL-X ₈₃-K-X ₈₅-X ₈₆-T-X ₈₈-L-X ₉₀-QQLN-X ₉₅-LEACV-X ₁₀₁-Q-X ₁₀₃-V-X ₁₀₅-X ₁₀₆-X ₁₀₇-X ₁₀₈-TPLMN-X ₁₁₄-D-X ₁₁₆-ILAV-X ₁₂₁-KY-X ₁₂₄-QRITLYL-X ₁₃₂-E-X ₁₃₄-KYSPC-X ₁₄₀-WEVVRAEIMRSFSFSTNLQKRLRRKE, or its conservative variant that replaces, wherein X ₁₁Be N or D; X ₁₂Be R, S or K; X ₁₅Be L or M; X ₁₆Be I, M or V; X ₁₉Be A or G; X ₂₂Be G or R; X ₂₄Be I or T; X ₂₆Be P or H; X ₃₄Be H, Y or Q; X ₃₈Be F or L; X ₄₀Be Q or R; X ₄₅Be G or S; X ₄₆Be N or H; X ₄₇Be Q or R; X ₅₀Be K or R; X ₅₁Be A or T; X ₅₅Be S or F; X ₅₆Be V or A; X ₅₇Be L or F; X ₆₀Be M or I; X ₆₁Be I or M; X ₆₇Be L or F; X ₇₂Be D or N; X ₇₅Be A or V; X ₇₆Be A or T; X ₇₈Be E or D; X ₇₉Be Q or E; X ₈₀Be S, R, T or N; X ₈₃Be E or D; X ₈₅Be F or L; X ₈₆Be S or Y; X ₈₈Be E or G; X ₉₀Be Y, H, N; X ₉₅Be D, E or N; X ₁₀₁Be I, M or V; X ₁₀₃Be E or G; X ₁₀₅Be G or W; X ₁₀₆Be V or M; X ₁₀₇Be E, G or K; X ₁₀₈Be E or G; X ₁₁₄Be V, E or G; X ₁₁₆Be S or P; X ₁₂₁Be K or R; X ₁₂₄Be F or L; X ₁₃₂Be T, I or M; X ₁₃₄Be K or R; And X ₁₄₀Be A or S; Or the pulsating interferon alpha homologue of described SEQ ID NO:71.On the other hand, the Interferon, rabbit homologue polypeptide of SEQ ID NO:71 or its segment show antiproliferative activity and (are at least about 8.3 * 10 in the proliferation test based on people Daudi clone ⁶Individual unit/mg), and/or in the test based on people WISH cell/EMCV, show antiviral activity and (be at least about 2.1 * 10 ⁷Individual unit/mg).Hereinafter will go through this two kinds of tests.This peptide species can contain the aminoacid sequence that is selected from SEQ ID NO:36 to SEQ ID NO:54, perhaps can be by being selected from the nucleotide sequence coded of SEQ IDNO:1 to SEQ ID NO:19.

The segment of Interferon, rabbit homologue polypeptide as herein described also is a feature of the present invention.Interferon alpha homologue segment of the present invention generally contains Interferon, rabbit homologue polypeptide, its contain among SEQ ID NO:36-71 or the SEQID NO:79-85 any at least about 20,25 or 30, generally at least about 40,50,60,70,80,90 or 100 successive amino acid.In other embodiments, segment contain usually among SEQID NO:36-71 or the SEQ ID NO:79-85 any at least about 100,110,120,125,130,140,150,155,158,160,162,163,164 or 165 successive amino acid.This polypeptide fragments has antiproliferative activity in the test based on people Daudi clone, and/or has antiviral activity in the test based on people or mouse cell line/EMCV.

In other embodiments, the invention provides length is 166 amino acid whose polypeptide, in some these type of embodiments, described polypeptide has antiproliferative activity in the test (or other similar test) based on people Daudi clone, comprise for example being at least about 8.3 * 10 ⁶Individual unit/mg, and/or in test (or other similar test), have antiviral activity based on people WISH cell/EMCV, comprise for example being at least about 2.1 * 10 ⁷Individual unit/mg.

In other embodiments, the invention provides polypeptide, it contains by proteinic 100 of coded polynucleotide sequence encoding at least, 150,155 or 160 successive amino acid, described coded polynucleotide sequence contain any in following: (a) SEQ ID NO:1 to SEQ ID NO:35 or SEQ IDNO:72 to SEQ ID NO:78; (b) coding is selected from the coded polynucleotide sequence of first polypeptide of any among SEQ ID NO:36 to SEQ ID NO:70 or SEQID NO:79 to the SEQ ID NO:85; (c) under the hybridization conditions of at least highly tight (or super-highly tight or super-super-height stringent condition), with (a) or the complementary polynucleotide sequence of the hybridization of polynucleotide sequence (b) of total length basically.This peptide species has antiproliferative activity in the test (or other similar test) based on people Daudi clone, and/or has antiviral activity in the test (or other similar test) based on people WISH cell/EMCV.This polypeptid specificity ground more of the present invention are in conjunction with the human alpha interferon acceptor.Polypeptide of the present invention and nucleic acid needn't with target molecule or associated molecule, the polypeptide or its segment (being included in those that have antiviral or antiproliferative activity in the test as herein described) that comprise among the SEQ ID NO:36-71 any, or the nucleic acid or the corresponding sequence of its segment (being included in those that have antiviral or antiproliferative activity in the test as herein described) of any is identical among the SEQ ID NO:1-35, but can be substantially the same with it.Can carry out multiple change to polypeptide, insert as conservative or non-conservation ground, disappearance and replacement, wherein this change can be its use benefit is provided.Can modify polypeptide of the present invention with several different methods, as long as that they contain is substantially the same with sequence natural or the plain peptide molecule of known disturbances (such as hereinafter definition), or get final product with sequence that this sequence has the identity of certain percentage.

Comparison generally is easy with more relative short aminoacid sequence (being less than about 30 residues).Relatively long sequence needs more complicated method to obtain the best comparison of two kinds of sequences.Local homology's algorithm by Smith and Waterman (1981) Adv.Appl.Math.2:482, homology alignment algorithm by Needleman and Wunsch (1970) J.Mol.Biol48:443, similarity retrieval method by Pearson and Lipman (1988) Proc.Nat ' l Acad.Sci. (USA) 85:2444, by using these algorithms of computer run (GAP, BESTFIT, FASTA and TFASTA, Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI), or by checking the optimal sequence comparison of the comparison window of can comparing, the best comparison of selecting to produce (promptly cause the sequence similarity per-cent in the comparison window the highest) by several different methods.

Term sequence identity refers to: two polynucleotide sequences are identical (promptly being right after on the basis of Nucleotide at Nucleotide) in comparison window.Term " sequence identity per-cent " calculates by the following method, the sequences that promptly in comparison window, compare two best comparisons, measure the positional number that occurs identical residue in two sequences to produce the matched position number, with the matched position number divided by the total number of positions in the comparison window (being window size), the result be multiply by 100, promptly draw sequence identity per-cent.On the one hand, the invention provides Interferon, rabbit homologue nucleic acid, the nucleic acid of any or its segment have and are at least about 80%, 85%, 90% among it and SEQ ID NO:1 to SEQ ID NO:35 or SEQ IDNO:72 to the SEQ ID NO:78,95%, 96%, 97%, 98%, 99%, 99.5% or the sequence identity of higher per-cent.

When being used for polypeptide, term is substantially the same to refer to: when the program GAP by for example using default breach weighted value (hereinafter will describe in detail) or BESTFIT carry out the best comparison, two peptide sequences are enjoyed the sequence identity at least about 80%, preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity or higher (sequence identity) as 97,98 or 99%.Preferably, make different residue positions different by conservative aminoacid replacement.Conservative aminoacid replacement refers to the interchangeableness of the residue with similar side chain.For example, one group of amino acid with aliphatic lateral chain is glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; One group of amino acid with aliphatics-hydroxyl side chain is Serine and Threonine; One group of amino acid with amide containing side chain is l-asparagine and glutamine; One group of amino acid with aromatic series side chain is phenylalanine, tyrosine and tryptophane; One group of amino acid with basic side chain is Methionin, arginine and Histidine; One group of amino acid with sulfur-containing side chain is halfcystine and methionine(Met).Preferred conserved amino acid replacement group is: Val-Leu-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan and l-asparagine-glutamine.On the one hand, the invention provides Interferon, rabbit homologue polypeptide, the polypeptide of any or its fragment have and are at least about 80%, 85% among it and SEQ ID NO:36-71 or the SEQID NO:79-85,90%, 95%, 96%, 97%, 98%, 99%, 99.5% or the sequence identity of higher per-cent.

The preferred example that is suitable for measuring the algorithm of sequence identity per-cent and sequence similarity is a fasta algorithm, and it is described in Pearson, W.R.﹠amp; Lipman, D.J., 1988, Proc.Nat ' l Acad.Sci.USA85:2444.Also can be referring to W.R.Pearson, 1996, Methods Enzymol.266:227-258.The preferred parameter that uses in the dna sequence dna FASTA comparison of calculating identity per-cent is optimised BL50 matrix 15:-5, k-tuple=2; Connect point penalty=40, optimization=28; Breach point penalty-12, notch length point penalty=-2; And width=16.

The preferred example that another kind is suitable for measuring the algorithm of sequence identity per-cent and sequence similarity is BLAST and BLAST2.0 algorithm, it is described in Altschul etc. respectively, and 1977, Nuc.Acids Res.25:3389-3402 and Altschul etc., 1990, J.Mol.Biol.215:403-410.Use has the BLAST of parameter described herein and BLAST2.0 to measure nucleic acid of the present invention and proteinic sequence identity per-cent.The public can obtain to carry out the software that BLAST analyzes from National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).This algorithm comprises: the short word joint (word) that at first is tested and appraised length W in the inquiry sequence identifies that the sequence of high scoring is to (HSP), when with database sequence in the byte of equal length when comparing, described HSP can mate or be fit to part positive number threshold value T.T is called as adjacent byte scoring threshold value (Altschul etc., document is the same).These original adjacent byte focuses (hit) can start retrieval as clue, to find to contain their longer HSP.The focus byte is extended on both direction along each sequence, up to increasing cumulative comparison scoring.For nucleotide sequence, but operation parameter M (the prize branch of a pair of coupling residue; Always＞0) and the N (point penalty of mispairing residue; Always＜0) calculate the accumulation scoring.For aminoacid sequence, use rating matrix to calculate the accumulation scoring.When following situation occurring, stop at and extend the focus byte on each direction: compare with its accessible maximum value, accumulation comparison scoring slippage is X; Because one or more scorings are the accumulation of the residue comparison of negative value, the accumulation scoring is zero or is lower than zero; Perhaps arrived the end of sequence.BLAST algorithm parameter W, T and X can measure the susceptibility and the speed of sequence alignment.BLASTN program (at nucleotide sequence) is used following default value: word length (W) is 11, and expected value (E) is 10, M=5, and N=-4, and two chains are all compared.For aminoacid sequence, the BLASTP program is used following default value: word length is 3, and expected value (E) is 10, and the BLOSUM62 rating matrix is (referring to Henikoff ﹠amp; Henikoff (1989) Proc.Nat ' l Acad.Sci.U.S.A.89:10915) sequence alignment (B) is 50, and expected value (E) is 10, M=5, and N=-4, and two chains are all compared.

The BLAST algorithm also carries out statistical analysis (referring to for example Karlin ﹠amp to the similarity between two sequences; Altschul (1993) Proc.Nat ' l Acad.Sci.U.S.A.90:5873-5787).The similarity measurement value that the BLAST algorithm provides is minimum summation possibility (P (N)), and it provides the accidental indication that the possibility of coupling takes place between two Nucleotide or the aminoacid sequence.For example,,, most preferably be lower than approximately 0.001, can think that then nucleic acid is similar to canonical sequence more preferably less than about 0.01 if to find that minimum summation possibility is lower than with reference to nucleic acid the time about 0.2 relatively being tried nucleic acid.

Another example of useful algorithm is PILEUP.PILEUP uses progressive paired sequence alignment to produce a plurality of sequence alignments by one group of correlated series, to show relation and sequence identity per-cent.This algorithm also can be drawn tree derivation, shows the aggregative relationship that is used to produce sequence alignment.PILEUP uses Feng﹠amp; The simplified method of the described progressive sequences comparison of Doolittle (1987) J.Mol.Evol.35:351-360 method.Method therefor is similar to Higgins ﹠amp; The described method of Sharp (1989) CABIOS5:151-153.This program can be compared nearly 300 sequences, and the maximum length of each sequence is 5000 Nucleotide or amino acid.Multiple comparison method starts from the paired comparison of two similar sequences, and it produces two troop (cluster) of aligned sequences.Then this is trooped with next maximally related sequence or trooping of aligned sequences carry out sequence alignment.By the paired sequence alignment of two independent sequences of simple extension, two sequences are trooped carry out sequence alignment.Obtain final sequence alignment by a series of progressive paired sequence alignments.By particular sequence and amino acid or the nucleotide coordinate that indicates the sequence comparison domain, and, get final product working procedure by indicating program parameter.Use PILEUP, utilize following parameters comparison canonical sequence and other to be tried sequence and concern: default breach weighted value (3.00), the terminal breach of default notch length weighted value (0.10) and weighting to measure sequence identity per-cent.PILEUP can derive from GCG sequence analysis software bag, for example version 7.0 (Devereaux etc. (1984) Nuc.Acids Res.12:387-395)).

The preferred example that another kind is suitable for the algorithm of a plurality of DNA and aminoacid sequence comparison is CLUSTALW program (Thompson, J.D. etc. (1994) Nucl.Acids.Res.22:4673-4680).ClustalW carries out multiple paired comparison between many group sequences, and according to homology they is assembled into a plurality of sequence alignments.Breach is opened and breach extension point penalty is respectively 10 and 0.05.For the aminoacid sequence comparison, the BLOSUM algorithm can be used as protein weighting matrix (Henikoff and Henikoff (1992) Proc.Nat ' l Acad.Sci.U.S.A.89:1091510919). Prepare polypeptide of the present invention

Above described and produced and the recombination method that separates Interferon, rabbit homologue polypeptide of the present invention.Except recombinant production, can by use solid phase technique direct peptide synthesis production of polypeptides in next life (referring to (1969) Solid-Phase Peptide Synthesis such as Stewart, W.H.Freeman Co, San Francisco; Merrifield, J. (1963) J.Am.Chem.Soc.85:2149-2154).Can use the artificial technology or carry out peptide synthetic by automatization.For example, (Perkin Elmer, Foster City Calif.) can carry out synthesizing automatically the explanation use Applied Biosystems431A Peptide Synthesizer that provides according to manufacturer.For example, can distinguish the chemosynthesis subsequence, and use chemical process that they are combined so that total length Interferon, rabbit homologue to be provided.The fragment of the Interferon, rabbit homologue polypeptide of the present invention that above goes through also is a feature of the present invention, can synthesize these fragments by using method mentioned above.

By in cell colony, importing nucleic acid of the present invention (this nucleic acid can be operated with the adjusting sequence that can effectively produce coded polypeptide and link to each other), culturing cell is to produce polypeptide in substratum, and choose isolated polypeptide from cell or substratum wantonly, can produce polypeptide of the present invention.

On the other hand, by in cell colony, importing the recombinant expression vector (wherein at least a nucleic acid can be operated with the adjusting sequence that can effectively produce coded polypeptide and link to each other) that contains at least a nucleic acid of the present invention, under proper condition in substratum culturing cell to produce polypeptide by expression vector codes, and choose isolated polypeptide from cell or substratum wantonly, can produce polypeptide of the present invention. Use polypeptide Antibody

In another aspect of this invention, use Interferon, rabbit homologue polypeptide production antibody of the present invention, described antibody has for example diagnosis, prevention and therepic use, and described purposes and for example activity of Interferon, rabbit homologue, it is relevant with expression to distribute.

Can produce antibody by method well-known in the art at Interferon, rabbit homologue of the present invention.Described antibody includes but not limited to: polyclone, and mono-clonal, chimeric, humanization, single-chain antibody, Fab fragment and the fragment that produces by the Fab expression library.For treatment or preventive use, the antibody of blocking-up receptors bind is particularly preferred.

Be used to induce the Interferon, rabbit homologue polypeptide of antibody to need not biologically active; Yet polypeptide or oligopeptides must possess antigenicity.Be used to induce the peptide of specific antibodies can have, the aminoacid sequence that preferred at least 15 or 20 amino acid are formed by at least 10 amino acid.One section short Interferon, rabbit homologue polypeptide can with another kind of protein, merge as keyhole chirp hemocyanin (keyhole limpet hemocyanin), and produce antibody at chimeric molecule.

It is well known by persons skilled in the art producing polyclone and monoclonal antibody method, and a lot of antibody are available.Referring to for example Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; And Harlow and Lane (1989) Antibodies:A LaboratoryManual, Cold Spring Harbor Press, NY; Stites etc. (volume) Basic and ClinicalImmunology (4th ed.) Lange Medical Publications, Los Altos, CA and the reference of wherein mentioning; Goding (1986) Monoclonal Antibodies:Principles and Practice (2ded.) Academic Press, New York, NY; And Kohler and Milstein (1975) Nature256:495-497.Other suitable antibody production techniques is included in selects the recombinant antibodies library in phage or the similar substrates, referring to (1989) Science 246:1275-1281 such as Huse; With (1989) Nature341:544-546 such as Ward.Specific mono-clonal and polyclonal antibody and antiserum(antisera) be usually to be at least about 0.1 μ M, preferably is at least about 0.01 μ M or better, and be the most common and be preferably 0.001 μ M or better K _DIn conjunction with.

The detailed method for preparing chimeric (humanization) antibody is found in United States Patent (USP) 5,482,856.About other details of humanization and other antibody producing and renovation technique can be referring to Borrebaeck (volume) (1995) Antibody Engineering, 2 ^NdEdition, Freeman and Company, NY (Borrebaeck); McCafferty etc. (1996) Antibody Engineering, A Practical Approach, IRL at OxfordPress, Oxford, England (McCafferty), and Paul (1995) Antibody EngineeringProtocols, Humana Press, Towata, NJ (Paul).

In a useful embodiment, the invention provides the antibody at Interferon, rabbit homologue of the present invention of full-length human.In antibody is used as people patient's body and in the application of the prevention of ex vivo and therapeutical agent, humanized antibody caters to the need especially.People's antibody is made up of distinctive human normal immunoglobulin sequence.Can use a variety of methods to produce people's antibody of the present invention (referring to for example Larrick etc., United States Patent (USP) 5,001,065 and Borrebaeck McCafferty and Paul, document is the same, relevant comment).In one embodiment, originally in the trioma cell, produce people's antibody of the present invention.Then with the gene clone of encoding antibody to other cell, as the non-human mammal cell, and express therein.The general method of producing people's antibody by the trioma technology is described in (1983) such as Ostberg, Hybridoma2:361367, Ostberg, United States Patent (USP) 4,634,664 and Engelman etc., United States Patent (USP) 4,634,666.The antibody producing cells system that obtains by this method is called as trioma, because they are three cells; The i.e. offspring of two human-like cells and a mouse cell.Found to compare with the common hybridoma for preparing from people's cell, trioma can more stably produce antibody. Adjuvant

On the one hand, Interferon, rabbit homologue polypeptide of the present invention or its fragment when with antigen or after transmitting antigen or before when using, can be used as adjuvant to stimulate enhancing, the immunne response that reinforcement or expansion are relevant with antigen.On the other hand, the invention provides the method for using peptide more than one or more inventions described herein to the experimenter. Therapeutical agent and preventive

As hereinafter described in detail, Interferon, rabbit homologue polypeptide of the present invention or its fragment can be used for the multiple disease of preventing and/or treating property treatment.

For example, the invention provides interferon-' alpha ' homologue polypeptide (with the interferon-' alpha ' homologue nucleic acid of this peptide species of coding), described polypeptide has antiviral and antiproliferative activity in test as herein described.On the one hand, the invention provides interferon-' alpha ' homologue polypeptide (with the interferon-' alpha ' homologue nucleic acid of this peptide species of coding), wherein the ratio of antiviral activity and antiproliferative activity is greater than mentioned other known disturbances element-α of this paper, as the corresponding ratio of listed those among the GenBank.Described polypeptide (with their nucleic acid of coding) can be used for the multiple disease for the treatment of and/or preventing property treatment, for example is used for hepatitis B, and third liver is in the treatment plan of HIV and HSV.In this treatment plan, some these type of polypeptide (with their nucleic acid of coding), for known disturbances element-α compound, has significant advantage as interferon-' alpha ' homologue 2BA8, because with respect to known interferon-' alpha ' compound, as interferon-' alpha ' 2a, they may show lower side effect after using, higher effectiveness, therefore only need lower dosage, and can cause less immunogenicity consequence. Sequence variations Through conservative variation of modifying

Interferon, rabbit homologue polypeptide of the present invention comprises one or more variations through conservative modification (or " conservative variations " or " the conservative replacement ") to SEQ ID NO:36 to SEQ ID NO:70 or the disclosed peptide sequence of SEQ ID NO:79 to SEQ ID NO:85.The conservative variation of modifying of this warp comprises replacement, add or disappearance, change in described variation any in SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQID NO:85, the amino acid that adds or lacked single amino acids or little per-cent (generally is lower than about 5%, more preferably less than about 4%, 2% or 1%).

For example, the length of conservative modification variation (for example disappearance) of polypeptide that is accredited as 166 amino acid longs of SEQ ID NO:36 in this article is at least about 157 or 158 amino acid, preferably be at least about 159 or 160 amino acid, more preferably be at least about 162 or 163 amino acid, still more preferably be at least about 164 or 165 amino acid, they are equivalent to respectively lack and are lower than about 5%, 4%, 2% or 1% peptide sequence.

Another conservative example about 8 residues (promptly being lower than about 5%) in the polypeptide of 166 amino acid longs of modifying variation (for example " through the conservative variation that replaces ") that are accredited as SEQ ID NO:36 in this article locate to contain " the conservative replacement " of 6 replacement groups shown in the with good grounds table 2 (seeing above).

Interferon, rabbit homologue peptide sequence of the present invention, comprise the part existence that can be used as big peptide sequence through the conservative sequence that replaces, this situation for example occurs in, (the poly Histidine section for example that adds one or more structural domains so that protein purification, FLAG epi-position section etc.), for example proteinic interferon-' alpha ' activity is partly had slight influence or do not have influence when other functional structure territory, perhaps by the procedure of processing after synthetic, for example by can remove other structural domain the time with protease treatment.

In another embodiment, Interferon, rabbit homologue polypeptide of the present invention contains the following sequence that is accredited as SEQ ID NO:71 in this article: CDLPQTHSLG-X ₁₁-X ₁₂-RA-X ₁₅-X ₁₆-LL-X ₁₉-QM-X ₂₂-R-X ₂₄-S-X ₂₆-FSCLKDR-X ₃₄-DFG-X ₃₈-P-X ₄₀-EEFD-X ₄₅-X ₄₆-X ₄₇-FQ-X ₅₀-X ₅₁-QAI-X ₅₅-X ₅₆-X ₅₇-HE-X ₆₀-X ₆₁-QTFN-X ₆₇-FSTK-X ₇₂-SS-X ₇₅-X ₇₆-W-X ₇₈-X ₇₉-X ₈₀-LL-X ₈₃-K-X ₈₅-X ₈₆-T-X ₈₈-L-X ₉₀-QQLN-X ₉₅-LEACV-X ₁₀₁-Q-X ₁₀₃-V-X ₁₀₅-X ₁₀₆-X ₁₀₇-X ₁₀₈-TPLMN-X ₁₁₄-D-X ₁₁₆-ILAV-X ₁₂₁-KY-X ₁₂₄-QRITLYL-X ₁₃₂-E-X ₁₃₄-KYSPC-X ₁₄₀-WEVVRAEIMRSFS FSTNLQKRLRRKE, or its conservative variant that replaces, wherein X ₁₁Be N or D; X ₁₂Be R, S or K; X ₁₅Be L or M; X ₁₆Be I, M or V; X ₁₉Be A or G; X ₂₂Be G or R; X ₂₄Be I or T; X ₂₆Be P or H; X ₃₄Be H, Y or Q; X ₃₈Be F or L; X ₄₀Be Q or R; X ₄₅Be G or S; X ₄₆Be N or H; X ₄₇Be Q or R; X ₅₀Be K or R; X ₅₁Be A or T; X ₅₅Be S or F; X ₅₆Be V or A; X ₅₇Be L or F; X ₆₀Be M or I; X ₆₁Be I or M; X ₆₇Be L or F; X ₇₂Be D or N; X ₇₅Be A or V; X ₇₆Be A or T; X ₇₈Be E or D; X ₇₉Be Q or E; X ₈₀Be S, R, T or N; X ₈₃Be E or D; X ₈₅Be F or L; X ₈₆Be S or Y; X ₈₈Be E or G; X ₉₀Be Y, H, N; X ₉₅Be D, E or N; X ₁₀₁Be I, M or V; X ₁₀₃Be E or G; X ₁₀₅Be G or W; X ₁₀₆Be V or M; X ₁₀₇Be E, G or K; X ₁₀₈Be E or G; X ₁₁₄Be V, E or G; X ₁₁₆Be S or P; X ₁₂₁Be K or R; X ₁₂₄Be F or L; X ₁₃₂Be T, I or M; X ₁₃₄Be K or R; And X ₁₄₀Be A or S; Or the fragment of described SEQ ID NO:71.As hereinbefore defined, the conservative modification variation of SEQ ID NO:71 sequence can comprise about altogether 8 amino acid whose disappearances in 166 amino acid whose polypeptide, insert or conservative the replacement, wherein do not morph in the position that is called as X in SEQ ID NO:71, their correspondences be the amino acid of explication.

For example, if 4 conservative subsequences that are arranged in corresponding to the amino acid/11 41-166 of SEQ ID NO:71 that replace, the example of the conservative replacement variation of this subsequence WEVVR AEIMR SFSFS TNLQK RLRRKE comprises: WEVVR SEIMR SFS YS TNLQ RRLRRK DAnd WE LVRAEI VR SFSFS TNL NK RLR KKE etc., wherein underscore replaces for conservative.

Feature of the present invention is an Interferon, rabbit homologue polypeptide, its contain among SEQ ID NO:36-71 or the SEQ IDNO:79-85 any at least about 20, be at least about 25 usually, be at least about 30,40,50,60,70,80,90 or 100 successive amino acid usually.In other embodiments, polypeptide generally contain among SEQ ID NO:36-70 or the SEQ ID NO:79-85 any at least about 100,110,120,125,130,140,150,155,158,160,163,164 or 165 successive amino acid.

In other embodiments, Interferon, rabbit homologue polypeptide of the present invention contains aminoacid sequence, and described sequence contains following one or more amino-acid residue (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein amino acid whose numbering is numbered corresponding to the amino acid among the aminoacid sequence SEQ ID NO:36.In preferred embodiments, Interferon, rabbit homologue polypeptide contains aminoacid sequence, described sequence contains among the SEQ ID NO:36-70 any at least 150,155 or 166 successive amino-acid residues, and further containing Lys160 and Glu166, wherein amino acid whose numbering is numbered corresponding to the amino acid among the aminoacid sequence SEQ ID NO:36.Some these peptide species show in the test based on people Daudi clone and are at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/mg, or in the test based on people WISH cell/EMCV, show and be at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg. By immunoreactivity definition polypeptide

Owing to compare with other interferon-alpha homologue, polypeptide of the present invention provides multiple new peptide sequence, and this polypeptide also provides new constitutional features, and described feature for example can be identified in the immunological testing.Specificity is in conjunction with the sero-fast generation of polypeptide of the present invention, and also is feature of the present invention by this antiserum(antisera) bonded polypeptide.

The present invention includes interferon-' alpha ' homologue polypeptide, described polypeptide can combine or take place with it specific immune response with antibody that produces at immunogen or antiserum(antisera) specificity, described immunogen contains and is selected from SEQ ID NO:36 to SEQ ID NO:70, the one or more aminoacid sequence among SEQ ID NO:71 and SEQ ID NO:79 to the SEQ IDNO:85.In order to eliminate and other interferon-alpha polypeptides, cross reactivity as known disturbances element-α polypeptide, with obtainable known interferon-alpha subduction antibody or antiserum(antisera), described known interferon-alpha for example comprises the polypeptide by the nucleic acid encoding with following GenBank registration number: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), and X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, and M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), perhaps any other known interferon-alpha polypeptides (being commonly referred to as " contrast interferon-alpha polypeptide ").When registration number during, produce polypeptide, and use it for subduction antibody/antiserum(antisera) by nucleic acid encoding corresponding to nucleic acid.When nucleic acid corresponding to non--encoding sequence, for example during pseudogene, produce (for example synthetic the generation) and read the amino acid of frame corresponding to nucleic acid, perhaps its modification of carrying out minimum degree is used for recombinant production to comprise initiator codon.

In a typical method, immunoassay is used the polyclonal antiserum that produces at one or more polypeptide, described polypeptide contains one or more aminoacid sequences, described aminoacid sequence is corresponding to SEQID NO:36 to SEQ ID NO:70, one or more among SEQ ID NO:71 and SEQ ID NO:79 to the SEQ IDNO:85, or its basically subsequence (be the full length sequence that provided at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% or longer).Derive from SEQ IDNO:36 to SEQ ID NO:70, the one or more a complete set of potential polypeptide immunogen among SEQ ID NO:71 and SEQ ID NO:79 to the SEQ ID NO:85 is collectively referred to as " immunogenic polypeptide " hereinafter.Choose wantonly and in the gained antiserum(antisera), select contrasting the antiserum(antisera) that the plain polypeptide of interferon-alpha polypeptide and/or other known disturbances has low cross reactivity, remove any cross reactivity by carrying out immunosorption with one or more contrast interferon-alpha polypeptide, then, in immunoassay, use polyclonal antiserum.

In order to produce antiserum(antisera) to be used for immunoassay, by production described herein and one or more immunogenic polypeptide of purifying.For example, can in mammal cell line, produce recombinant protein.Use is mixed with standard adjuvant, as the immunogenic polypeptide of formula adjuvant not, with the mouse immune method of standard (referring to Harlow and Lane (1998) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, wherein standard has been described the generation of antibody, the form of immunoassay and the condition that is used to measure specific immune response) immune mouse inbred strain (why using inbred strain in this test is because the substantial gene identity of mouse makes test-results have more reproducibility).Perhaps, put together, again as immunogen deriving from one or more synthetic of sequence described herein or the polypeptide and the carrier protein of reorganization.

Collect polyclonal serum, and, for example one or more immunogenic polypeptide are fixed in the solid-phase immunoassay on the solid support immunogenic polypeptide is carried out titration in immunoassay.Select, collecting titre is 10 ⁶Or higher polyclonal antiserum, and with contrast interferon-alpha polypeptide it is reduced to produce through subduction, collect and titrating polyclonal antiserum.

Detection is through subduction, and collection and titrating polyclonal antiserum are to the cross reactivity of contrast interferon-alpha polypeptide.Preferably in this measures, use at least two kinds of immunogenic interferon-alpha polypeptide, preferably unite use, to identify the antibody of specificity binding immunoassay originality polypeptide with at least two kinds of contrast interferon-alpha polypeptide.

In this comparison test, mensuration is through subduction, the distinctiveness of titrating polyclonal antiserum is in conjunction with condition, and described condition causes through titrating polyclonal antiserum and immunogenicity interferon-alpha bonded signal/noise ratio with respect to Yan Yaogao is at least about 5-10 doubly with contrast interferon-alpha bonded signal/noise ratio.That is, by adding nonspecific competitor, as albumin and skim-milk, perhaps by regulating the salt condition, temperature waits the tight degree of regulating association reaction.Whether use these to be tried polypeptide in conjunction with condition with mensuration in test subsequently combines with the polyclonal antiserum specificity of collecting through subduction.Particularly, shine 2-5 times at least of polypeptide height distinctive in conjunction with signal/noise ratio comparison under the condition, compare with immunogenic polypeptide, signal/noise ratio is at least about the former 1/2 polypeptide that tried and compares with known interferon-alpha, sharing higher structural similarity or homology with immunogenic polypeptide, is polypeptide of the present invention therefore.

In another example, use the immunoassay of competing combining form to detect and tried polypeptide.For example, as mentioned above,, from the anti-serum mixture of collecting, remove the antibody of cross reaction by carrying out immunosorption with contrast interferon-alpha polypeptide.Then immunogenic polypeptide is fixed in and is exposed to through subduction, on the solid support in the antiserum(antisera) of collection.Add in test tried protein with competition and collection through reducing sero-fast the combination.To compare with immobilized protein, be tried protein competition in conjunction with the immunogenic polypeptide competition bonded ability that in reducing sero-fast ability and test, adds of collecting compare (immunogenic polypeptide can effectively with sero-fast combination of immobilization immunogenic polypeptide competition) with collection.Use canonical algorithm to calculate the per-cent that is tried the protein cross reactivity.

In parallel test, compare in conjunction with sero-fast ability with immunogenic polypeptide competition, measure the control protein competition in conjunction with collect through reducing sero-fast ability.Use canonical algorithm to calculate the per-cent of contrast polypeptide cross reactivity.When high at least 5-10 times of the cross reactivity per-cent that is tried polypeptide, we can say and tried combining of polypeptide and collection through subduction antiserum(antisera) specificity.

In general, can in competition binding immunoassay assay method as herein described, use antiserum(antisera) through immunosorption and collection with more any polypeptide and immunogenic polypeptide of being tried.In order to carry out this comparison, can detect each in two peptide species of wide range of concentrations, and use the measured by standard techniques will be through the amount that combines the required every peptide species of inhibition 50% of subduction antiserum(antisera) and immobilized protein.If the required amount of being tried polypeptide is hanged down twice than the amount of required immunogenic polypeptide, just we can say and tried polypeptide energy specificity that condition is that described amount is compared high approximately 5-10 times according to polypeptide at least in conjunction with the antibody that produces at immunogenic polypeptide.

What measure at last is specificity, optional with immunogenic polypeptide (rather than contrast polypeptide) the collected antiserum(antisera) of immunosorption fully, combine or detect until the faint of used immunogenic polypeptide in the antiserum(antisera) set of immunogenic polypeptide subduction and immunosorption that only can detect gained less than combination.Detect antiserum(antisera) and the reactivity of being tried polypeptide then through complete immunosorption.If only observing faint reactivity or anergy (promptly compares with the combination of immunogenic polypeptide with the antiserum(antisera) of the complete immunosorption of viewed warp, signal/noise ratio is no more than 2 times), so, tried the polypeptide antiserum(antisera) that combination is produced by immunogenic protein with regard to the energy specificity. The antiproliferative properties of Interferon, rabbit homologue

It is described to press embodiment 1, checks the effect of Interferon, rabbit homologue cell growth in based on the test of people Daudi clone.Fig. 2 has shown and the contrast Interferon, rabbit that humanIFN-2a compares with total humanIFN-(Con1), and the antiproliferative activity of the Interferon, rabbit homologue that the present invention exemplifies, described homologue contain the aminoacid sequence of SEQ ID NO:36 to SEQ ID NO:54.Shown among the figure with humanIFN-2a and compared with the humanIFN-who has, activity unit's number (Y-axis) of every milligram of (mg) interferon receptor 2 test agent of the interferon alpha homologue that one cover exemplifies, wherein each homologue sample all has a title (clone's title) on X-axis.These results show that the composition that contains interferon-' alpha ' homologue of the present invention can be used for suppressing or reduces in the method for tumor cell proliferation, described tumour cell includes but not limited to: human cancer cell, the hematopoiesis cancer cells, human leukemia cell, human lymphoma cell and human melanoma cell.Inhibition can be in external (being used for for example multiple proliferation test), and (as for example treatment or preventive) carries out in ex vivo or the body.

Interferon-' alpha ' homologue of the present invention demonstrates the various binding modes (referring to for example embodiment 2) at multiple cancerous cell line.Use body outer cell line screening (being described in for example Monks, A. etc. (1991) J.Nat ' l Cancer Inst.83:757-766) to detect the selective growth restraining effect of interferon-' alpha ' homologue of the present invention and/or to the cell killing effect of particular cancer clone.Screened human carcinoma cell line (referring to for example embodiment 2, table 3) comprises leukemia, melanoma and lung cancer, colorectal carcinoma, the cancer of the brain, central nervous system cancer, ovarian cancer, mammary cancer, prostate cancer and kidney.

In the cancerous cell line screening, measure 3 reactivity parameters: 1) GI50 (" 50% growth-inhibiting "), be the active observed value of growth-inhibiting, GI50 be cell growth be suppressed 50% o'clock the interferon receptor 2 test agent (IFN alpha homologues or contrast IFN α) concentration, described inhibition is by when holding stage finishes, with observed comparing in control cells (no given the test agent), the increase of clean protein/polypeptide reduction by 50% is measured in the interferon receptor 2 test agent; 2) TGI (" total growth-inhibiting "), be the static active observed value of cell, TGI is the concentration of the cell growth of specific cells system interferon receptor 2 test agent when being suppressed fully, and wherein the amount of cell protein equals the amount of cell protein when soak begins when soak finishes; With 3) LC50, observed value for cytotoxic activity, LC50 is that viewed cell protein quality is compared when beginning with soak, when insulation finishes, the amount of the cell protein of measuring reduces the concentration of 50% o'clock interferon receptor 2 test agent, and it has shown the net loss of cell after adding the interferon receptor 2 test agent.Other details of this test and data analysing method is provided among the embodiment 2.

Compare with interferon-' alpha ' Con1 and human interferon-alpha 2a contrast, the interferon-' alpha ' homologue 3DA11 that exemplifies (SEQ ID NO:40) is shown in Fig. 3 A, 3B and 3C at the reactivity parameter of multiple cancerous cell line.

About growth inhibitory activity, homologue 3DA11 and contrast interferon-' alpha ' Con1 demonstrate the activity that is at most of subject cells especially, interferon-' alpha ' Con1 generally shows higher activity, interferon-' alpha ' 2a generally shows lower overall activity, and only goes out activity (Fig. 3 A) at the inferior concentrated expression of clone.

What form contrast especially with it is, with Interferon, rabbit-Con1 with human interferon-alpha 2a contrast compare, in the cell toxicant of homologue 3DA11 and the static activity of cell, observe significant difference.In the concentration range of being tried, homologue 3DA11 demonstrates the static activity of remarkable cell at the cell colony that contains 11 clones, and Interferon, rabbit-Con1 only demonstrates the activity at the cell colony that contains 1 clone, and homologue 3DA11 also has at this group cell activity (Fig. 3 B).On the other hand, IFN-α 2a is not have activity to any subject cell in this test.Therefore, homologue 3DA11 has the static profile of activity of wideer cell than total human interferon-alpha (Con1) and human interferon-alpha 2a.

Compare with Interferon, rabbit-Con1 and human interferon-alpha 2a contrast, homologue 3DA11 also demonstrates significant cytotoxic activity (Fig. 3 C).Surprisingly, homologue 3DA11 demonstrates the cytotoxic activity at the cell colony that contains 8 clones, and Interferon, rabbit-Con1 and interferon-' alpha ' 2a do not show the activity that can survey to impinging upon in the used concentration range of test to the cell colony that contains any clone.Therefore, compare with human interferon-alpha 2a with Interferon, rabbit-Con1, homologue 3DA11 also has the cytotoxic activity distribution plan of broad.

Fig. 4 A-4D has illustrated the cell static activity (by the reflection of TGI value) of the interferon-' alpha ' homologue that the present invention exemplifies.In each figure, draw multiple interferon-' alpha ' homologue and mutually pair cell static activity (be expressed as-log TGI) figure of two kinds of contrast Interferon, rabbit (Interferon, rabbit-Con and human interferon-alpha 2a) at particular cancer cell line cell colony.

Exemplifying in the homologue of being tried, homologue 1D3 (SEQ ID NO:54) and 3DA11 (SEQ IDNO:40), rather than contrast any in Interferon, rabbit to show in the experimental concentration scope at the leukemia cell be the static activity of remarkable cell (Fig. 4 A) of RPMI-8226 cell colony.In this embodiment, for the static activity of cell that any contrast (Interferon, rabbit-Con1 or interferon-' alpha ' 2a) at the leukemia cell is cell colony, 1D3 and 3DA11 homologue demonstrate at cell colony at least about high 25 times corresponding activity (corresponding) with the TGI difference that is at least about 1.4 log units.

Homologue 1D3,2G5 (SEQ ID NO:45), 6CG3 (SEQ ID NO:52) and 3DA11, rather than contrast any in Interferon, rabbit demonstrates the static activity of remarkable cell (Fig. 4 B) at lung cancer cell line NCI-H23.In this embodiment, with respect to Interferon, rabbit-Con1 or interferon-' alpha ' 2a at for the static activity of cell of lung cancer cell line cell colony, 1D3,2G5,6CG3 and 3DA11 homologue demonstrate at the lung cancer cell line cell colony at least about high 12 times corresponding activity (corresponding) with the TGI difference that is at least about 1.1 log units.

Homologue 1D3,2G5 and 3DA11, rather than contrast any in Interferon, rabbit demonstrates the static activity of remarkable cell (Fig. 4 C) at renal carcinoma cell line ACHN cell colony.In this embodiment, with respect to Interferon, rabbit-Con1 or interferon-' alpha ' 2a at for the static activity of cell of renal carcinoma cell line cell colony, 1D3,2G5 and 3DA11 homologue demonstrate at described renal carcinoma cell line cell colony at least about high 35 times corresponding activity (corresponding) with the TGI difference that is at least about 1.55 log units.

Homologue 1D3,2G5,3DA11,2CA5 (SEQ ID NO:42) and 2DB11 (SEQ IDNO:41), and Interferon, rabbit-Con1 contrast, rather than interferon-' alpha ' 2a contrast demonstrates the static activity of remarkable cell (Fig. 4 D) at ovarian cancer cell line OVCAR-3 cell colony.In this embodiment, with regard to regard to the static activity of cell of each group ovarian cancer cell line cell, homologue 1D3 demonstrates than Interferon, rabbit-Con1 at least about high 2 times corresponding activity (corresponding with the TGI difference that is at least about 0.3 log unit), and 1D3,2G5,3DA11,2CA5 demonstrates than interferon-' alpha ' 2a at least about high 40 times corresponding activity (corresponding with the TGI difference that is at least about 1.6 log units) with the 2DB11 homologue.

Can find out significantly from the data that exemplify provided herein: interferon-' alpha ' homologue of the present invention demonstrates the static profile of activity of various kinds of cell, and described distribution plan is significantly different with interferon-' alpha ' Con1 and Intederon Alpha-2a.

The present invention includes the interferon-' alpha ' homologue, for human interferon-alpha 2a or total human interferon-alpha Con1, it has the static activity of cell of increase.In a plurality of embodiments, the interferon-' alpha ' homologue to the corresponding activity of the static specific activity human interferon-alpha of the cell 2a of cancerous cell line cell colony at least about high 2 times (being that the TGI value is at least about low 2 times), perhaps the interferon-' alpha ' homologue to the corresponding activity of the static specific activity Interferon, rabbit-Con1 of cell of the cell colony that is selected from following one or more cancerous cell lines at least about high 2 times: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; Central nervous system (CNS) cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.

In other embodiments, the interferon-' alpha ' homologue to the corresponding activity of the static specific activity human interferon-alpha of the cell 2a of cancerous cell line cell colony to high approximately 5 times (being that TGI value is at least about hanging down 5 times), perhaps the interferon-' alpha ' homologue to the corresponding activity of the static specific activity Interferon, rabbit-Con1 of cell of the cell colony that is selected from following one or more cancerous cell lines at least about high 5 times: the leukemia cell is; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; Central nervous system (CNS) cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.In other embodiments, the corresponding activity of the static specific activity human interferon-alpha of the cell of cancerous cell line cell colony 2a is at least about high 10 times (TGI value at least about low 10 times) during the interferon-' alpha ' homologue, perhaps the interferon-' alpha ' homologue to the corresponding activity of the static specific activity Interferon, rabbit-Con1 of cell of the cell colony that is selected from following one or more cancerous cell lines at least about high 10 times: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; Central nervous system (CNS) cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.

The present invention includes the interferon-' alpha ' homologue, for human interferon-alpha 2a or Interferon, rabbit-Con1, it has the cytotoxic activity of increase.In a plurality of embodiments, the interferon-' alpha ' homologue to the cytotoxic activity of the cell colony that is selected from following one or more cancerous cell lines than the corresponding activity of human interferon-alpha 2a at least about high 2 times (being that the LC50 value is at least about low 2 times), at least high 5 times, or high at least 10 times: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; The CNS cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.In other embodiments, the interferon-' alpha ' homologue to the cytotoxic activity of the cell colony that is selected from following one or more cancerous cell lines than the corresponding activity of Interferon, rabbit-Con1 at least about high 2 times (being that the LC50 value is at least about low 2 times), at least about high 5 times, or at least about high 10 times: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; The CNS cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.

The present invention includes the interferon-' alpha ' homologue, for human interferon-alpha 2a or Interferon, rabbit-Con1, it has the growth inhibitory activity of increase.In a plurality of embodiments, the interferon-' alpha ' homologue to the growth inhibitory activity of the cell colony that is selected from following one or more cancerous cell lines than the corresponding activity of human interferon-alpha 2a at least about high 2 times (being that the GI50 value is at least about low 2 times), at least about high 5 times, or at least about high 10 times: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; The CNS cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.In other embodiments, the interferon-' alpha ' homologue to the growth inhibitory activity of the cell colony that is selected from following one or more cancerous cell lines than the corresponding activity of Interferon, rabbit-Con1 at least about high 2 times (being that the GI50 value is at least about low 2 times), at least about high 5 times, or at least about high 10 times: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; The CNS cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line.

Discovery as herein described is to develop Interferon, rabbit (interferon-' alpha ' homologue as described herein), modification or reorganization are to demonstrate the various active distribution plan, this is found to be differentiation and generation customization, specific Interferon, rabbit homologue provides chance, described homologue can be used for treating multiple specific disease, comprises for example multiple cancer or relative disease.For example, through optimization so that also can be optimised at the effectiveness enhanced of particular target cancer cells type Interferon, rabbit homologue of the present invention, so that its (advantageously) has the toxicity at non-target cell of reduction, therefore, the experimenter (for example patient) who uses homologue is produced lower side effect.

The present invention further provides at taking from experimenter's subgroup (as Mammals or people patient), or even take from the chance of each experimenter's (as Mammals or people patient) tumour cell optimization Interferon, rabbit homologue, so that treatment or the prophylactic treatment scheme that aims at each experimenter's customization to be provided.Optimized Interferon, rabbit homologue of the present invention can provide at cancer or relative disease, or the disease of other available interference extract for treating, or to Interferon, rabbit or the unresponsive treatment of diseases of other treatment plan or the preventive effect of present use. The ntiviral characteristic of Interferon, rabbit homologue

It is described to press embodiment 1, estimates the antiviral activity of Interferon, rabbit homologue of the present invention in people's WISH cell/EMCV test.Fig. 2 has shown the antiviral activity of the Interferon, rabbit homologue that the present invention of containing aminoacid sequence SEQ ID NO:36 to SEQ IDNO:54 exemplifies.

The extracorporeal antivirus effect activity through improvement that has confirmed the IFN-alpha homologues that the present invention exemplifies can be kept in mouse model system body.The someone confirmed in the past: two kinds of IFN-alpha homologues CH2.2 of the present invention and CH2.3 (being respectively SEQ ID NO:84 and 85) are in the test based on the mouse cell, for humanIFN-2a, have about 206 respectively, 000-is doubly with 138,000-is the antiviral activity through improveing doubly, and in identical test, compare with natural mouse Interferon, rabbit, have significantly higher activity (Chang etc. (1999) Nature Biotechnol.17:793-797).As described in embodiment 3 hereinafter, give the IFN-alpha homologues CH2.2 and the CH2.3 that use various dose with the Balb/c mouse of vesicular stomatitis virus (VSV) attack of lethal dose, natural mouse Interferon, rabbit Mu-IFN α 4 and humanIFN-2a.High external activity and observed activity in vivo interrelated preferably (Fig. 5).CH2.2 and CH2.3 homologue can be protected mouse completely effectively, make it avoid the virus attack that causes death, and the natural mouse Interferon, rabbit part of same dose effectively, and humanIFN-2a is invalid fully.These results show: the composition that contains Interferon, rabbit homologue of the present invention can be used for suppressing by in the method for the intravital virus replication of experimenter of virus infection, described virus includes but not limited to: human immunodeficiency virus (HIV), hepatitis C virus (HCV), hsv (HSV) and hepatitis B virus (HBV).Can be in external (being used for for example multiple antivirus test), (in the body of this paper discussion, be used as in the method and for example treat or preventive) in ex vivo (in the method for the ex vivo of this paper discussion, being used as for example treatment or preventive) or the body and suppress. The Interferon, rabbit homologue is used for the treatment of autoimmunization and other immunity-relative disease

Composition of the present invention can be used for treatment or prophylactically treatment, thereby alleviates panimmunity system-relative disease, and described disease is characterised in that too high or too low active function of immune system or further feature.Described disease comprises the too high and autoimmune disease of allergenicity, as multiple sclerosis, and I type (insulin-dependent) diabetes, lupus erythematosus, the sclerosis of amyotrophic lateral sclerosis side, CrohnShi disease, rheumatoid arthritis, stomatitis, asthma, transformation reactions, psoriasis etc. Treatment and prevention composition

According to method well-known in the art, suitable external, detect treatment or the prophylactic compositions that contains one or more Interferon, rabbit homologue polypeptide of the present invention or nucleic acid in ex vivo and the body in the disease animal model, to confirm effectiveness, tissue metabolism also estimates dosage.Especially, by in correlation test, comparing the activity of interferon-alpha homologue and existing interferon-alpha therapeutical agent or preventive, can measure dosage.On the one hand, the invention provides method, it comprises as hereinafter described in detail, give Mammals or non--lactation vertebrates, use one or more Interferon, rabbit homologue Nucleotide of the present invention mentioned above or polypeptide (or its fragment) as bird (as chicken or duck), described Mammals comprises for example people, primate, mouse, pig, milk cow, goat, rabbit, rat, cavy, hamster, horse, sheep.Described composition generally contains one or more Interferon, rabbit homologue Nucleotide of the present invention or polypeptide (or its fragment) and vehicle, for example pharmaceutically-acceptable excipients.

On the one hand, by using restriction endonuclease, one or more nucleic acid of the present invention of RNA enzyme or dnase digestion (or its fragment) produce composition of the present invention.

In another aspect of this invention, provide by at deoxyribonucleotide triphosphoric acid and nucleic acid polymerase, under the existence as the polysaccharase of thermostability, be incubated one or more nucleic acid mentioned above and the composition that produces.

The present invention also comprises the composition that contains two or more nucleic acid mentioned above.Described composition can contain nucleic acid library, and its Chinese library contains at least about 5,10, and 20,50,100,150 or 200 or more kinds of nucleic acid.

Molecule is imported by being usually used in, finally any approach that contacts with blood or histocyte carries out administration.Can preferably use interferon-' alpha ' homologue of the present invention in any suitable manner with the medicine acceptable carrier.The proper method of using the Interferon, rabbit homologue in the context of the invention to the patient is operable, although can use more than a kind of approach to use specific composition, specific approach often can provide than the more direct and more effective reaction of another kind of approach.

Medicine acceptable carrier part is by the particular composition of being used, and uses the used ad hoc approach of composition and decide.The prescription that therefore, a variety of suitable pharmaceutical compositions of the present invention are arranged.

Can use peptide composition by number of ways, to be used for prevention as herein described, in any in treatment and the diagnostic method, described approach includes but not limited to: oral, and intravenously, intraperitoneal, intramuscular, through skin, subcutaneous, the part, the hypogloeeis, vagina or rectum approach, or by sucking.Also can use Interferon, rabbit homologue peptide composition via liposome.Described route of administration and appropriate formulations generally are known in the art.

Interferon, rabbit homologue polypeptide or nucleic acid also can be made into aerosol (be them can by " spraying ") via inhalation separately or with other suitable component associating.Aerosol can be placed in the propelling agent accepted of pressurization, and as Refrigerant 12, propane is in the nitrogen etc.

Be suitable for the preparation of parenterai administration, for example by intraarticular, intravenously, intramuscular, through skin, the preparation of intraperitoneal and subcutaneous route administration comprises moisture and water-free isotonic sterile injection solution, it contains antioxidant, damping fluid, fungistat and the isoosmotic solute of blood and the moisture and water-free sterile suspensions that make preparation and administration acceptor, it can comprise suspension agent, solubilizing agent, thickening material, stablizer and sanitas.Nucleic acid preparation through packing may reside in the sealed vessel of unitary dose or multiple doses, in peace berkelium and tubule.

Parenterai administration and intravenous administration are preferred medications.Particularly, existing interferon-alpha therapeutical agent or the already used route of administration of preventive, and the preparation that uses at present is preferred route of administration, and the preferred formulation of interferon-alpha homologue polypeptide of the present invention and nucleic acid.

In ex vivo or body in the context of therapy, also can be by the cell of Interferon, rabbit homologue transduction mentioned above by intravenously mentioned above or parenterai administration.Should understand that it is routine techniques that cell is passed to experimenter (for example people patient), for example is passed to blood via intravenously or intraperitoneal administration with cell.

In the context of the present invention, being administered to experimenter's (for example patient) the Interferon, rabbit homologue polypeptide of the present invention or the dosage of nucleic acid is enough within a certain period of time, in experimenter (for example patient), produce favourable treatment or prophylactic response, perhaps be enough to suppress the infection of pathogenic agent, this depends on concrete application.By effectiveness and the activity of used Interferon, rabbit homologue and patient's the situation of specific support or preparation, and patient's to be treated body weight or surface-area are determined dosage.By in particular patient, using specific support, preparation, the existence of any adverse side effect that transducer cell type etc. accompanies, characteristic and degree are determined the size of dosage.

In the treatment and prophylactic treatment method of invention described herein, the general range of the significant quantity (for example nucleic acid dosage) of interferon-' alpha ' nucleic acids of the present invention (for example DNA or mRNA) for example is: about 0.05 microgram/kilogram (kg) is about 0.005-5mg/kg usually to about 50mg/kg.Yet, should understand can be according to mode well known to those skilled in the art, change the significant quantity of nucleic acid (for example nucleic acid dosage) and/or polypeptide (for example polypeptide dosage) according to a plurality of factors, described factor comprises: the active or effectiveness of polypeptide, any nucleic acid construct of being used (carrier for example, promotor, expression system) active or render a service disease to be treated (for example specific cancer) and the experimenter who is transmitted nucleic acid.

In order to transmit some polypeptide by for example transmitting nucleic acid encoding, use-case is as being about 0.005mg/kg can reach enough levels to the nucleic acid dosage of about 5mg/kg translation and/or expression.Those skilled in the art can determine easily that according to factor mentioned above other has the active polypeptide of the known organism dosage of (with their nucleic acid of coding).Other known disturbances element-α provides guide at the used dosage of specified disease for dosage and the treatment plan of determining nucleic acid of the present invention or polypeptide.The significant quantity scope of interferon-' alpha ' homologue polypeptide is extremely about 1 milligram of about 1 microgram, and more preferably about 1 microgram is to about 100 micrograms.

The composition that is used for the treatment of invention described herein and method of prophylactic treatment for example can contain, and concentration is of the present invention interferon-' alpha ' homologue nucleic acid (for example DNA or mRNA) and the medicine acceptable carrier (for example aqueous carrier) of about 0.1 mcg/ml (ml) to about 20mg/ml.

The composition that is used for the treatment of invention described herein and method of prophylactic treatment for example can contain concentration as mentioned with interferon-' alpha ' homologue polypeptide of the present invention as herein described and medicine acceptable carrier (for example aqueous carrier).

When treatment or preventing cancer or virus disease, determine the significant quantity of carrier, in the process of cell type or the preparation used, the doctor needs the evaluation cycle blood plasma level, the toxicity of carrier/cell/preparation/Interferon, rabbit homologue, the process of disease and anti--carrier/Interferon, rabbit homologue production of antibodies.

The interferon-' alpha ' that the dosage range of using to for example 70 kilograms patient equals to use is at present treated or preventative proteinic dosage, calculate the carrier of generation Interferon, rabbit homologue sequence or the dosage of cell, with Interferon, rabbit homologue nucleic acid that produces same amount or the protein that gives expression to.By any known routine treatment, comprise cytotoxic agent, nucleotide analog (for example when being used for the treatment of the HIV infection), biological response modifier etc., but the disease of carrier supplement therapy cancer of the present invention and virus-mediation.

When being applied to patient's healthy state total and integral body, in order to carry out administration, should use Interferon, rabbit homologue of the present invention and transducer cell with certain ratio, described ratio is the LD50 by Interferon, rabbit homologue polypeptide or nucleic acid, carrier, or the cell type of transduction and the Interferon, rabbit homologue polypeptide or the nucleic acid of various concentration, the side effect of carrier or cell type is measured.Can carry out administration via single dose or the dosage that separates.

For the cell that will have been transduceed by recombinant nucleic acid imports experimenter (for example patient), before perfusion, obtain blood sample, and preserve blood sample to analyze.Poured into 1 * 10 at 60-200 minute in the internal jugular vein ⁶To 1 * 10 ¹²Individual cell through transduction.Monitor vital sign closely and monitor oxygen saturation by pulse oximeter.Obtained blood sample in back 5 minutes and 1 hour in perfusion, preserve this blood sample to analyze subsequently.Choose wantonly in 4 to 6 treatments altogether in 1 year, repeated leukopheresis every 2 to 3 months, transduction and perfusion again.After the treatment,, mainly the outpatient is poured into for the first time according to clinician's judgement.If the outpatient is poured into again,, monitor the participant after preferred 8 hours treatment at least 4 hours.Preparation is poured into used through transducer cell again according to the method set up.Referring to (1991) J.Clin.Apheresis 6:48-53 such as Abrahamsen; Carter etc. (1988) J.Clin.Arpheresis 4:113-117; Aebersold etc. (1988), J.Immunol.Methods 112:1-7; (1987) Transfusion27:362-365 such as (1987) J.Immunol.Methods101:171181 such as Muul and Carter.After cultivating about 2 to 4 weeks, cell number should reach 1 * 10 ⁶To 1 * 10 ¹²Individual.In this regard, the growth characteristics of cell different and different with patient's difference and cell type.Before pouring into transducer cell more about 72 hours, take out the per-cent of aliquots containig with the cell of analyzing phenotype and expression treatment or preventive.

Fever occurs if stand the dabbling experimenter of carrier or transducer cell or protein formulation (for example patient), be afraid of cold or myalgia, can take the Asprin of suitable dosage, Ibuprofen BP/EP, acetaminophen or other ntipyretic analgesic medicine.Stand perfusion reaction, as fever, myalgia and the experimenter (for example patient) who is afraid of cold can take Asprin in preceding 30 minutes in advance in perfusion, acetaminophen, or diphenhydramine for example.Dolantin can be used for can not more serious being afraid of cold and myalgia of quick response to febrifuge and antihistaminic agent.According to the severity of reaction, can slow down or cancel the cell perfusion. Treatment and preventative-therapeutic method

The present invention also comprises the treatment or the method for prophylactic treatment disease, and described method comprises in the subject or ex vivo ground uses the nucleic acid of one or more inventions mentioned above or the polypeptide composition of pharmaceutically-acceptable excipients and one or more described nucleic acid or polypeptide (or contain), and described experimenter comprises for example Mammals, comprise for example people, primate, mouse, pig, milk cow, goat, rabbit, rat, cavy, hamster, horse, sheep; Or non--mammalian vertebrates, as birds (as chicken or duck) or fish or invertebrates.

In one aspect of the invention, in the method for ex vivo, obtain or take out one or more interesting cells or cell colony (tumour cell for example, neoplasmic tissue sample in the subject, the organ cell, blood cell, skin, lung, heart, muscle, brain, mucous membrane, liver, intestines, spleen, stomach, lymphsystem, uterine neck, vagina, prostate gland, oral cavity, the cell of tongue etc.), and with a certain amount of polypeptide of the present invention that can effectively prevent or treat disease contact.To return or be passed to the site that obtains cell in the subject, another interesting site (for example comprising above defined those sites) in the subject perhaps to be treated through the cell of contact then.In case of necessity, can use standard and well-known implantation technique, to be transplanted to tissue interesting in the subject through the cell of contact, organ or system site (comprising all sites mentioned above) perhaps for example use the transmission of standard or implantttion technique that it is passed to blood or lymphsystem.

The present invention also provides method in the body, and wherein one or more interesting cells or the cell colony with the experimenter contacts with a certain amount of polypeptide of the present invention that can effectively prevent or treat disease directly or indirectly.In direct contact/form of medication, generally, comprise topical by in the several different methods any, injection (for example by using syringe needle or syringe), or vaccine or particle gun transmission push tissue, organ or skin site directly are applied to polypeptide or are transferred to cell to be treated or interested tissue site (tumour cell for example, neoplasmic tissue sample, the organ cell, blood cell, skin, lung, heart, muscle, brain, mucous membrane, liver, intestines, spleen, stomach, lymphsystem, uterine neck, vagina, prostate gland, the oral cavity, the cell of tongue etc.).Can be by intramuscular for example, intracutaneous, subcutaneous, oral, intraperitoneal, in the sheath, intravenously transmits polypeptide, or polypeptide placed in the body cavity (comprises for example at surgical procedures), or by suction or vagina or rectal administration transmission polypeptide.

In vivo in indirect contact/form of medication, generally can promote one or more cells or the cell colony for the treatment of by polypeptide of the present invention directly being contacted or being applied to, and polypeptide is applied to indirectly or is transferred to cell to be treated or interested tissue site, comprise those sites mentioned above (skin cells for example, tract, lymphsystem or blood cell system etc.).For example, by with blood or lymphsystem, the cell of skin or organ contacts with the polypeptide of q.s, so that polypeptide is passed to interested site (for example intravital tissue, organ or interested cell or blood or lymphsystem), and cause effectively prevention or therapeutic action, can treat the intravital tumour cell of experimenter.By using one or more route of administration or pattern mentioned above, generally can carry out this contact, administration or transfer.

On the other hand, the invention provides the method for ex vivo, wherein obtain or take out one or more interesting cells or cell colony (tumour cell for example in the subject, neoplasmic tissue sample, the organ cell, blood cell, skin, lung, heart, muscle, brain, mucous membrane, liver, intestines, spleen, stomach, lymphsystem, uterine neck, vagina, prostate gland, oral cavity, the cell of tongue etc.), and by described one or more cells or cell colony are contacted with polynucleotide constructs transform these cells, described construct contains the target nucleic acid sequence of the present invention of the required biologically active polypeptides of coding (polypeptide for example of the present invention), and described polypeptide can effectively prevent or treat disease.One or more cells or cell colony are contacted with the described nucleotide sequence expression promoter of control with the polynucleotide constructs of q.s, so that cell is taken in polynucleotide constructs (and promotor), give full expression to target nucleic acid sequence of the present invention, produce a certain amount of biologically active polypeptides that can effectively prevent or treat disease.Polynucleotide constructs can comprise control nucleotide sequence expression promoter sequence of the present invention (for example CMV promoter sequence) and/or, in case of necessity, also comprise one or more other nucleotide sequence, at least a or multiple another kind of polypeptide of the present invention of described sequence encoding, cytokine, adjuvant or common-stimulation molecule, or other required polypeptide.

After the transfection, will return, transmit or be transferred to the tissue site or the system that obtain cell in the subject, intravital another site of experimenter perhaps to be treated (tumour cell for example through cell transformed, neoplasmic tissue sample, organ cell, blood cell, skin, lung, heart, muscle, brain, mucous membrane, liver, intestines, spleen, stomach, lymphsystem, uterine neck, vagina, prostate gland, oral cavity, the cell of tongue etc.).In case of necessity, can use standard and well-known implantation technique, cell is transplanted to tissue interesting in the subject, skin, organ or body system perhaps for example use the transmission of standard or implantttion technique that it is passed to blood or lymphsystem.By using one or more route of administration or pattern mentioned above, generally can transmit, use or shift through cell transformed.The expression of target nucleic acid can spontaneous generation, perhaps also can be induced (hereinafter will describe in detail), and the amount of expressed coded polypeptide should be enough to and treat effectively the disease at site or tissue system place.

On the other hand, the invention provides method in the body, wherein one or more interesting cells or cell colony (for example comprising those cells mentioned above and cell system and experimenter) are transformed in the subject, conversion is to realize by making cell or cell colony contact (or use one or more route of administration mentioned above or pattern to be applied to or be transferred to cell or cell colony) with polynucleotide constructs, described construct contains the nucleotide sequence of the present invention of the required biologically active polypeptides of coding (polypeptide for example of the present invention), and described polypeptide can effectively prevent or treat disease.

Polynucleotide constructs directly can be applied to or be transferred to diseased cells (for example by using one or more route of administration or pattern mentioned above directly to contact).Perhaps, at first by using one or more route of administration mentioned above or pattern to make not ill cell or other diseased cells directly contact (nucleotide sequence and control nucleotide sequence expression promoter that described construct contains the encoding human active polypeptide) with the polynucleotide constructs of q.s, make cell take in polynucleotide constructs (and promotor), give full expression to target nucleic acid sequence of the present invention, produce a certain amount of biologically active polypeptides that can effectively prevent or treat disease, thereby the express polypeptide that makes polynucleotide constructs or gained can be naturally or initial transmission site in the subject automatically, system, tissue or organ metastasis ill site to the subject, tissue, organ or system (for example via blood or lymphsystem), and polynucleotide constructs is applied to indirectly or is transferred to diseased cells.The expression of target nucleic acid can spontaneous generation, perhaps also can be induced (hereinafter will describe in detail), so that the amount of expressed coded polypeptide is enough to and can treats effectively the disease at site or tissue system place.Polynucleotide constructs can comprise control nucleotide sequence expression promoter sequence (for example CMV promoter sequence) and/or, in case of necessity, also comprise one or more other nucleotide sequence, at least a or multiple another kind of polypeptide of the present invention of described sequence encoding, cytokine, adjuvant or common-stimulation molecule, or other required polypeptide.

In body mentioned above and in each of the methods of treatment of ex vivo, can use or transmit the composition that contains vehicle and polypeptide of the present invention or nucleic acid.On the one hand, by mentioned above, the composition that will contain pharmaceutically-acceptable excipients and polypeptide of the present invention or nucleic acid is applied to or is passed to the experimenter, and its consumption can effectively be treated disease.

On the other hand, in body mentioned above and in each of the methods of treatment of ex vivo, the amount that is administered to cell or experimenter's polynucleotide can be to be enough to make one or more cells of experimenter to take in described polynucleotide, and give full expression to described nucleotide sequence, produce and a certain amount ofly can effectively strengthen experimenter's immunne response, comprise amount by the biologically active polypeptides of immunogen (as antigen) inductive immunne response.On the other hand, for every kind of method, the polypeptide amount that is applied to cell or experimenter can be the immunne response that is enough to strengthen the experimenter, comprises the amount by immunogen (as antigen) inductive immunne response.

On the other hand, using the polynucleotide constructs composition of polynucleotide constructs (or contain) to transmit to the experimenter in the body of physiological active polypeptide or in the interior therapeutic method, can induce the expression of polynucleotide constructs by using derivable unlatching-and close-gene expression system.The example of this unlatching-and close-gene expression system comprises Tet-OnTM Gene Expression System and Tet-Off respectively ^TM(detailed description of relevant each system can be referring to for example ClontechCatalog2000, pg.110-111) for Gene Expression System.Other controlled or derivable unlatching-and close-gene expression system is well known by persons skilled in the art.Use this system, can be accurately, reversibly and quantitatively regulate the polynucleotide constructs expression of nucleic acids that hits.For example, can after organ or the system, induce the genetic expression of target nucleic acid again making through the cell transmission of stable transfection or being transferred to or contacting interested tissue site, wherein said cell contains polynucleotide constructs, and described construct contains target nucleic acid.This system is to methods of treatment and form particularly advantageous, and wherein it can advantageously postpone or accurately control the expression of target nucleic acid (for example to finishing surgical operation and/or postoperative rehabilitation with opportunity; Make free site to be treated, cell, system or the tissue of arriving of the polynucleotide constructs that contains target nucleic acid; Make the graft that contains the cell that is fabricated the body conversion mix the tissue of its institute's grafting or attaching or organ etc. if having time). Integrated system

The invention provides computer, computer-readable medium and integrated system contain in them corresponding to polypeptide described herein and nucleic acid, comprise for example listed those sequences and the multiple reticent character chain that replaces and guard the sequence information that replaces thereof of this paper.

Several different methods known in the art and genetic algorithm (GO) can be used for detecting homology or the similarity between the different characteristics chain, perhaps are used to exercise other required function, for example control output file, provide a description the basis of the information that comprises sequence etc.BLAST for example discussed above.

Therefore, can be in the integrated system of this paper the dissimilar homologys and the similarity of the multiple tight degree of detection and Identification and length.For example, design a variety of homology measuring methods, be used for the sequence of comparative analysis biological polymer, when word processing, checked spelling data searching from the several data storehouse.4 main interactions of examining two-paired complement of spiral in the bases of natural polynucleotide have been understood, also can be with the basis of the complementary homology polynucleotide chain annealed model of simulation as sequence alignment or other operation, described operation is generally carried out (for example word-processing operation on corresponding to the character chain of this paper sequence, structure contains the figure of sequence or subsequence character chain, output form etc.).Be used for sequence of calculation similarity or homology, and the example that contains the software package of GO is BLAST, by the character chain of input corresponding to this paper sequence, BLAST is applicable to the present invention.

Similarly, by the character chain of input corresponding to interferon alpha homologue of the present invention (nucleic acid or protein, or both), the desktop computer application software of standard, as Word (as Microsoft Word ^TMOr Corel WordPerfect ^TM) and database software (for example spreadsheet such as Microsoft Excel ^TM, Corel Quattro Pro ^TMOr database program, as Microsoft Access ^TMOr Parados ^TM) applicable to the present invention.For example, integrated system can comprise the above-mentioned software with suitable character chain information, for example is used for being connected with the operating characteristics chain with user interface (for example standard operation system such as Windows, the graphic user interface in Macintosh or the LINUX system).As mentioned above, also program such as the BLAST that is specifically designed to sequence alignment can be mixed system of the present invention with comparison nucleic acid or protein (or corresponding character chain).

Analyze used integrated system among the present invention and generally comprise the digital machine that the used GO software of aligned sequences is housed, and the data setting that can enter the software system that contain any sequence described herein.Computer can be PC (Intel x86 or Pentium chip compatible type DOS for example ^TM, OS2 ^TMWINDOWS ^TMWINDOWS NT ^TM, WINDOWS95 ^TM, WINDOWS98 ^TM, based on the machine of LINUX, MACINTOSH ^TM, Power PC, or based on UNIX (SUN for example ^TMMachine) or other commercially available common computer well known by persons skilled in the art workstation).The software that is used for sequence alignment or operating sequence can obtain, and perhaps those skilled in the art use the standard programming language, as Visualbasic, and Fortran, Basic, Java etc. can easily create these software.

Any controller or computer are optional to comprise watch-dog, and it often is cathode-ray electrode tube (" CRT ") indicating meter, flat-panel screens (for example active matrix formula liquid-crystal display, liquid-crystal display) or other indicating meter.Computer circuits often are placed in the box, comprise a plurality of integrated circuit (IC) chip in the box, as microprocessor, and storer, interface circuit etc.Also can choose wantonly in the box and comprise hard disk drive, floppy disk, the packaged type driving mechanism of heavy body is as CD-ROM and other the common peripheral equipment that can write.The optional sequence that the to be compared or operation for user's input and in being chosen in relevant computer system of input unit such as keyboard or mouse is provided.

Computer generally comprises the instruction that appropriate software is used to accept the user, and described instruction can be the form that the user imports the set of parameter field, for example in GUI, or the instruction type of programming in advance, for example be that multiple different specific operation is programmed in advance.Then, software changes these instructions into suitable language, and indicating not, the operation and the transfer control of fixed-direction carry out necessary operations.

Software also can comprise the comparison of sequence or this paper sequence (for example according to) control nucleic acid synthetic output element, or other occurs in the operation in sequence alignment downstream, or other operation of using the character chain corresponding to this paper sequence to carry out.

In one embodiment, the invention provides integrated system, it contains the computer or the computer-readable medium of database, has one or more sequential recordings in the described database.Each sequential recording contains one or more corresponding to the nucleic acid that is selected from SEQ ID NO:1 to SEQ ID NO:85 or the character chain of polypeptide or protein sequence.Integrated system also contains user's inputting interface, and it can make the user optionally check one or more sequential recordings.In a this integrated system, computer or computer-readable medium contain the comparison instruction set, can compare character chain and one or more other, corresponding to the character chain of nucleic acid or polypeptide or protein sequence.

A this integrated system comprises instruction set, and it contains in following at least one: local homology's comparative measurement, and the homology sequence comparison is measured, and similarity retrieval is measured and BLAST mensuration.In some embodiments, system also contains readable output element, and it can show the comparison result that produces by the sequence alignment instruction set.In another embodiment, computer or computer-readable medium also contain instruction set, it can translate into aminoacid sequence with at least a nucleotide sequence, and described nucleotide sequence contains the sequence that is selected from SEQ ID NO:1 to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78.This instruction set can be applied to be tried nucleotide sequence by the instruction set of codon being used instruction set or mensuration sequence identity and select nucleic acid.

The method of at least a institute inherent information of a plurality of sequential recordings that the present invention also provides the system of using a computer to present to store in the database.Each sequential recording contains at least a character chain corresponding to SEQ IDNO:1 to SEQ ID NO:85.This method comprises measures at least a character chain corresponding to one or more or its subsequence among SEQ IDNO:1 to the SEQ ID NO:85; It is any to determine that the user selects at least a character chain of listing; With every kind of selected character chain of demonstration, or compare every kind of selected character chain and further feature chain.This method also comprises sequence alignment and/or the display list that shows every kind of selected character chain and further feature chain. Test kit

In others, the invention provides the method as herein described that makes, composition, the test kit that system and instrument are specialized.Optional one or more the following components that comprises of test kit of the present invention: (1) instrument as herein described, system, system component or Instrument assembly; (2) implement methods described herein, and/or operate instrument described herein or Instrument assembly, and/or use the specification sheets of composition described herein; (3) one or more interferon-alpha homologue compositions (for example contain at least a interferon alpha homologue nucleic acid of the present invention or polypeptide or its segment, cell, the composition of carrier etc.) or component (interferon alpha homologue nucleic acid of the present invention or polypeptide or its segment, cell, carrier etc.); (4) place one or more article of the present invention, comprise the container of described component or composition; (5) wrapping material.

On the other hand, the invention provides any instrument as herein described, Instrument assembly, composition or test kit are used to implement the purposes of any method as herein described or test, and/or any instrument or test kit are used to implement the purposes of any test as herein described or method.

Embodiment Embodiment 1: preparation and screening are through the interferon-' alpha ' library of reorganization

By the segment (length is 25-60 base pair (bp)) of pcr amplification and about 20 the human interferon-alpha subclass genes of DNA enzyme Processing of Preparation, press Crameri A etc. (1998 basically; Nature15:288-291) the described reorganization is to produce through the ripe encoding sequence of the interferon-' alpha ' of reorganization.By preparing expression library through the ripe encoding sequence subclone of the interferon-' alpha ' of reorganization to intestinal bacteria secretion vector.Interferon polypeptides through reorganization is expressed as the mature protein that merges at C-terminal and E mark (Amersham-Pharmacia) so that quantitative and from periplasmic space purifying.Use the machine bacterium colony to select instrument (Q-Bot, Genetix Pharmaceuticals) the intestinal bacteria transformant is selected to the droplet plate, and preparation pericentral siphon extract.

Press Scarozza, A.M. etc. (1992) J.Interferon Res.12:35-42 is described, detects the antiproliferative activity of pericentral siphon extract to people Daudi clone.

The clone who shows antiproliferative activity in the Daudi test is screened again, and by using anti--E traget antibody (Amersham-Pharmacia) to measure expression level through the Western trace.Select to check order at expression level the most highly active standardized clone, and, this clone is taken turns the substrate of reorganization and screening as other by mentioned above.

To derive from first and second and take turns reorganization, and the clone's subclone that has higher relatively antiproliferative activity in Daudi test is to CHO expression vector (pDEI-1011), and wherein E-mark/6-His mark (Amersham-Pharmacia) merges with C-terminal through the Interferon, rabbit of reorganization.To clone transfection to Chinese hamster ovary celI, select stable clone with 1mg/ml G418.Go up the ripe Interferon, rabbit that purifying CHO-expresses at anti--E mark Sepharose post (Amersham-Pharmacia), and undertaken quantitatively by Bradford test (Biorad).By the antiproliferative activity through the reorganization Interferon, rabbit of Daudi analysis of experiments CHO-purifying, by hereinafter described, end user's WISH cell/EMCV analysis of experiments antiviral activity. People's WISH cell/EMCV antivirus test

100 μ l in 96 well culture plates are added with 10% foetal calf serum, in the RPMI substratum (Gibco-BRL) of penicillin (100 μ g/ml) and Streptomycin sulphate (100 μ g/ml), with 6 * 10 ⁴The density inoculation WISH cell of individual cells/well was in 37 ℃ of insulations 24 hours.In the hole, add the substratum (cumulative volume is 100 μ l) contain the interferon-alpha polypeptides sample, in 37 ℃ at 5%CO ₂Atmosphere in the insulation 3 hours.Adding volume in the hole was EMCV (encephalomyocarditis virus) diluent of 50 μ l, by above-mentioned insulation 24 hours.Carefully remove substratum, the hole is washed 2 times with warm phosphate-buffered saline (PBS).In the hole, add toluylene red (diluting 100 μ l/ holes by 1: 50), by above-mentioned insulation 2 hours with substratum.Add glutaraldehyde (0.5% PBS solution, 50 μ l/ holes), by above-mentioned insulation 30 minutes.With hole flushing 2 times, add 100 μ l/ holes, 50% methyl alcohol, the solution of 1% acetate with PBS.Use the droplet plate reader to measure the absorbance value that 540 nanometers (nm) are located.

Fig. 2 has shown with interferon-' alpha ' 2a and interferon-' alpha ' Con1 and has compared the antiproliferative activity and the antiviral activity of the Interferon, rabbit homologue that the present invention exemplifies.Shown activity unit's number (Y-axis) of every milligram of homologue of the interferon alpha homologue that a cover exemplifies among the figure, wherein each homologue sample all has a title on X-axis. Embodiment 2: the screening of cancer cell in vitro system

Use the screening of cancer cell in vitro system (to be described in for example Monks, (1991) J.Nat ' l CancerInst.83:757-766 (hereinafter being referred to as " Monks ") and http://dtp.nci.gov./branches/btb/ivclsp.html such as A. list this paper in as a reference all in full) analyze selective growth inhibition and/or the cell killing effect of interferon-' alpha ' homologue of the present invention to particular cancer clone.Used 60 kinds of cancerous cell lines (table 3) comprise leukemia, melanoma, and lung cancer, colorectal carcinoma, the cancer of the brain, ovarian cancer, mammary cancer, prostate cancer, central nervous system cancer, kidney system cancer and kidney.According to the method cultivator tumor cell line described in " Monks " and the http://dtp.nci.gov./branches/btb/ivclsp.html.

Screened human carcinoma cell line

Cancer types	Clone
Cancer types	Clone	Leukemia	CCRF-CEM，HL-60(TB)，K-562，MOLT-4，PRMI-8226，SR
Colorectal carcinoma	COLO205，HCC-2998，HCT-15，HCT-116，HT29，KM12，SW-620	Leukemia	CCRF-CEM，HL-60(TB)，K-562，MOLT-4，PRMI-8226，SR
Colorectal carcinoma	COLO205，HCC-2998，HCT-15，HCT-116，HT29，KM12，SW-620	The CNS cancer	SF-268，SF-295，SF-539，SNB-19，SNB-75，U251
Lung cancer	A549/ATCC，EKVX，HOP-62，HOP-92，NCI-H23，NCI-H226， NCI-H322M，NCI-H460，NCI-H522	The CNS cancer	SF-268，SF-295，SF-539，SNB-19，SNB-75，U251
Lung cancer		Mammary cancer	MCF-7，NCI/ADR?HS578T，MDA-MB-231/ATCC，MDA-MB-435， MDA-N，BT-549，T-47D
Melanoma	LOX?IMVI，M14，MALME-3M，SK-MEL-2，SK-MEL-5，SK- MEL28，UACC-62，UACC-257	Mammary cancer
Melanoma		Ovarian cancer	IGROV1，OVCAR-3，OVCAR-4，OVCAR-5，OVCAR-8，SK-OV- 3
Prostate cancer	DU-145，PC-3	Ovarian cancer	IGROV1，OVCAR-3，OVCAR-4，OVCAR-5，OVCAR-8，SK-OV- 3
Prostate cancer	DU-145，PC-3	Kidney	786-0，A498，ACHN，CAKI-1，RXF393，SN12C，TK-10，UO-31

Briefly, the growth characteristics according to specific cells system are inoculated in 96 hole droplet plates with cell with about 5000 density to about 40000 cells/well.After the inoculation, the droplet plate is incubated 24 hours, adds given the test agent (Interferon, rabbit homologue for example of the present invention or contrast Interferon, rabbit) then in 37 ℃.After 24 hours, with two flat boards of every kind of clone of Tricholroacetic Acid (TCA) anchored in place, (T when adding given the test agent to be determined at ₀) cell colony of every kind of clone.In remaining culture plate, add through 5 10 times of serial dilutions, concentration is 10 ^-0.8To 10 ^-4.8The Interferon, rabbit sample of μ g/ml (by Chinese hamster ovary celI supernatant liquor affinity purification).

Add after the sample, culture plate is incubated 6 days again.By adding the TCA termination test.

By measuring cell colony in conjunction with measuring cell protein in testing at the quantitative protein dyestuff.In each hole, add 1% acetic acid solution (100 μ l) that contains 0.4% (w/v) sulfo-rhodamine (sulforhodamine) B, then at room temperature be incubated 10 minutes.By washing 5 times with 1% acetate removing unconjugated dyestuff, and make culture plate air-dry.With the dyestuff of 10 mmoles (mM) Tris dissolving, on the culture plate reader of automatization, read the absorbance value that 515 nanometers (nm) are located with protein bound.

7 parts of absorbance value measurement results are got in the test of each dose-response, they corresponding to: (time is 0 before adding sample; T ₀) the cell protein quality, the amount of cell protein (contrast growth when soak finishes under the condition that lacks given the test agent, C), with 5 parts corresponding to when having the interferon receptor 2 test agent of 5 kinds of concentration, the measurement result of cell protein quality when soak finishes (growth when detecting the interferon receptor 2 test agent that has 5 concentration levels, T _i).Use these measurement results to calculate following 3 parameters of every kind of given the test agent:

GI50, or " 50% growth-inhibiting " is to make cell growth 50% be suppressed the concentration of required interferon receptor 2 test agent, this value is by when soak finishes, and the increase of clean protein/polypeptide reduces 50% and measures in the interferon receptor 2 test agent in the control cells (not containing given the test agent).GI50 is calculated as the concentration of given the test agent, wherein [(T _i-T _o)/(C-T _o)] x100=50.See Fig. 3 A.

TGI, or " total growth-inhibiting " make the cell growth be suppressed the concentration of required interferon receptor 2 test agent comprehensively, the amount of the cell protein the when amount of the cell protein when wherein soak finishes equals soak and begins.The concentration that produces the interferon receptor 2 test agent of total growth-inhibiting (TGI) is calculated as the concentration of given the test agent, wherein T _i=T _o

LC50 is that viewed cell protein quality is compared when beginning with soak, and when insulation finished, the amount of the cell protein of mensuration reduced the concentration of 50% o'clock interferon receptor 2 test agent, and it has shown the net loss of cell after adding the interferon receptor 2 test agent.LC50 is calculated as the concentration of given the test agent, wherein [(T _i-T _o)/T _o] * 100=-50.

If for specific given the test agent, in the concentration range of being tried, be not effective or effect very good, the value of described parameter is represented as and is greater than or less than maximum or the Cmin that is tried. The external activity of embodiment 3:IFN-alpha homologues is associated with effectiveness in the body

Reorganization human interferon-alpha gene segment, and it is described press (1999) Nature Biotechnol.17:793-797 such as Chang, screening activity in based on the antivirus test of mouse cell.Isolate higher by 10 at the antiviral activity of mouse cell than human interferon-alpha 2a ⁵Interferon-' alpha ' homologue doubly.The antiviral activity of multiple interferon-' alpha ' homologue even significantly surpass natural mouse interferon comprises the antiviral activity of Mu-IFN-α 4 (Chang etc., document is the same).The human interferon-alpha gene segment is returned sequence reorganization (for example DNA reorganization) to produce new interferon alpha homologue, screen this homologue at the mouse Interferon Receptors subsequently, the result can identify and isolate the interferon-' alpha ' homologue that activity is better than muroid closely-related with it.

In mouse, carry out dose-response research be determined at observation in vitro to high antiviral active whether also can keep in vivo.In this research, two kinds of mouse-optimization interferon-' alpha ' homologues that are called as CH2.2 and CH2.3 (being respectively SEQ ID NO:84 and 85) have in this article been used.CH2.2 and CH2.3 are in external mouse cell antivirus test, for human interferon-alpha 2a, have high approximately 138 respectively, 000-is doubly with 206,000-activity doubly, for natural mouse interferon-α 4, has the high 1.6 times activity (Chang etc., document is the same) of high approximately 2.5 times of peace treaties.

Continuous 4 days of several groups of Balb/c mouse are being every day that the subcutaneous dosage of 2,10 or 50 μ g (cumulative volume is 50 μ l) is accepted phosphate-buffered saline (PBS), interferon-' alpha ' homologue CH2.2, interferon-' alpha ' homologue CH2.3, mouse IFN-α 4 or human interferon-alpha 2a.At the 2nd day, mouse is exposed in the vesicular stomatitis virus (VSV) of deadly intranasal dose (10 times to LC50).Data are represented as the mouse number that can live the 21st day.

Fig. 5 shown the protection mouse and avoided comparing with natural mouse interferon Mu-IFN-α 4 aspect the VSV infection, and mouse-optimization interferon-' alpha ' homologue CH2.2 and CH2.3 are equally effectively or more effective.Avoid aspect the virus infection the protection mouse, the humanIFN-2a that is tried concentration is almost completely invalid.Therefore, effectiveness is more obvious with the dependency of observed antiviral activity in vitro tests in the body of interferon-' alpha ' homologue of the present invention.

Although for the purpose of illustrating and understanding, described foregoing invention in greater detail, those skilled in the art can know clearly by reading this content: the multiple change to form and details does not deviate from the real scope of the present invention.For example, can use all technology mentioned above, method, composition, instrument and system with multiple array mode.For all purposes, all publications of mentioning among the application, patent, patent application or other file are all listed this paper in as a reference in full, resemble each independent publication, patent, patent application or other file are mentioned individually for all purposes need list this paper in as a reference equally.

Sequence

Sequence numbering	Clone's numbering	Sequence
Sequence numbering	Clone's numbering	Sequence	SEQ?ID?NO：1	?2DH12	?TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATG ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGACC ?CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTAGGGGTGAAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：2	?2CA3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTGACAGGAGGGCCATGATA ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGAACTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGGAGAGACTCCCCTGATG ?AATGGGGACTCCATCCTGGCTGTGAAGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	SEQ?ID?NO：1	?2DH12
SEQ?ID?NO：2	?2CA3		SEQ?ID?NO：3	?4AB9	?TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCGGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：4	?2DA4	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATAGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGC?AAAA ?AGATTAAGGAGGAAGGAA	SEQ?ID?NO：3	?4AB9
SEQ?ID?NO：4	?2DA4		SEQ?ID?NO：5	?3DA11	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGGTA

		?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
			SEQ?ID?NO：6	?2DB11	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACTTG ?GAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：7	?2CA5	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCGGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	SEQ?ID?NO：6	?2DB11
SEQ?ID?NO：7	?2CA5		SEQ?ID?NO：8	?2G6	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACCCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：9	?3AH7	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGCGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATAGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTCACCAGCAACTGAATGAACTG	SEQ?ID?NO：8	?2G6

		?GAAGCATGTGTAGTACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACCTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
			SEQ?ID?NO：10	?2G5	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：11	?2BA8	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCCTGATA ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTAATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	SEQ?ID?NO：10	?2G5
SEQ?ID?NO：11	?2BA8		SEQ?ID?NO：12	?1F3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGGACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAACCTCTTCAGCACAAAGGACTCATCTGTTGCTTGGGATGAGAGG ?CTTCTAGACAAACTCTATACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGTGTGATGCAGGAGGTGTGGGTGGGAGGGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAGAAAATACTTCCAAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：13	?4BE10	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAGATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATAATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTG ?GAAGCATGTGTGATACAGGGGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCTTGGCTGTGAGGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	SEQ?ID?NO：12	?1F3

SEQ?ID?NO：14	?2DD9	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATG ?CTCCTGGCACAAATGGGAAGAATCTCCCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGGACTCTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：14	?2DD9		SEQ?ID?NO：15	?3CA1	?TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTACCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG ?GAAGCATGTGTGATACAGGAGGTTGGGATGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：16	?2F8	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	SEQ?ID?NO：15	?3CA1
SEQ?ID?NO：16	?2F8		SEQ?ID?NO：17	?6CG3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAAGAGGGCCATGATG ?CTCCTGGCACAAATGGGAAGAACCTCTCCTTTCTCCTGTCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAGGGCTCAAGCCATCTTTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATTTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAAGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
SEQ?ID?NO：18	?3CG7	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGTAGGGCCTTGATG ?CTCCTGGCACAAATGGGAAGAATCTCCCCTTTCTCCTGCCTGAAGGAC ?AGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAAC	SEQ?ID?NO：17	?6CG3

		?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG ?GAAGCATGTGTGATACAGGAGGTTGGGATGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
			?SEQ?ID?NO：19	?1D3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC ?AGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCCACCAGTTC ?CAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：20	?2G4	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCATGATG ?CTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCCTGTCTGATGGAC ?AGACATGACTTTGAATTTCCCCAGGAGGAATTTGATGATAAACAGTTC ?CAGAAGGCTCCAGCCATCTCTGTCCTCCATGAGGTGATTCAGCAGACC ?TTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGACC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTG ?GAAGCATGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATG ?AATGCGGACTCCATCTTGGCTGTGAGGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAAAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：19	?1D3
?SEQ?ID?NO：20	?2G4		?SEQ?ID?NO：21	?1A1	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTC ?CAGAAGGCCCAAGCCATCTCTGCCTTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTCACCAGCAACTGAATGACCTG ?GAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAGGAAATACTTTCAAAGAATCACT ?CTTTATCTAATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：22	?1D10	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC ?AGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCCACCAGTTC ?CAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA	?SEQ?ID?NO：21	?1A1

		?AGATTAAGGAGGAAGGAA
		?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：23	?1F6	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG ?ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGCTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTAACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：24	?2A10	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAGGAAATACTTTCAAAGAATCACT ?CTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：23	?1F6
?SEQ?ID?NO：24	?2A10		?SEQ?ID?NO：25	?2C3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTCC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGATACTTGGGATGCGACC ?CTTTTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：26	?2D1	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCGGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：25	?2C3
?SEQ?ID?NO：26	?2D1		?SEQ?ID?NO：27	?2D10	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAGTCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC

		?CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCGAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
			?SEQ?ID?NO：28	?2D7	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGCGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGAAGGAC ?AGACATGACTTCAGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTG ?GAAGCTTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCTATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：29	?2D9	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACT ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGGTG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：28	?2D7
?SEQ?ID?NO：29	?2D9		?SEQ?ID?NO：30	?2DA2	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGCCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACAGGACTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTCCACCAGCAACTGAATGAACTG ?GAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：31	?2DH9	?TGTGATCTGCCTCAGACCCACAGCCCTGGTAACAGGAGGGCCTTGATG ?CTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGGGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTCTACCGGCAGCTGAATGACCTG ?GAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT	?SEQ?ID?NO：30	?2DA2

		?CTTTATCTGACAGAGAAGAAGCATAGCCCTTGTTCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
			?SEQ?ID?NO：32	?2G11	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGACTTCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGATACTTGGGAACAGAGC ?CTCCTAGAAAAATTCTACATTGAACTTTTCCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAGAAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGGAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：33	?2G12	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG ?CTCATGGCACAAATGAGGAGAATCTCTCCTTTCCCCCGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCTATCTTCCTTTTCCATGAGATGATGCAGCAGACC ?TTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACTTG ?GAAGCCTGTGTGATGCAGGAGGGGAGGGTGGGAGAAACTCCCCTGATG ?AATGCGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGCTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：32	?2G11
?SEQ?ID?NO：33	?2G12		?SEQ?ID?NO：34	?2H9	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTA ?GAAGCCTGTGTGACACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCTATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：35	?6BC11	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGCTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGAGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：34	?2H9

?SEQ?ID?NO：36	?2DH12	?CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQTLLEKFSTELYQQLNDL ?EACVIQEVGVKETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：36	?2DH12		?SEQ?ID?NO：37	?2CA3	?CDLPQTHSLGDRRAMILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNEL ?EACVIQEVGVGETPLMNGDSILAVKKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：38	?4AB9	?CDLPQTHSLGNRRALILIAQMGRISPFSCLKDRHDFGFPREEFDGNQF ?QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNEL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：37	?2CA3
?SEQ?ID?NO：38	?4AB9		?SEQ?ID?NO：39	?2DA4	?CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDSNQF ?QKAQAISVLHEMMQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：40	?3DA11	?CDLPQTHSLGNRRALVLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：39	?2DA4
?SEQ?ID?NO：40	?3DA11		?SEQ?ID?NO：41	?2DB11	?CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：42	?2CA5	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRQDFGFPQEEFDGNRF ?QKAQAISVLHEMIQQTFNLFSTKNSSAAWEQSLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：41	?2DB11
?SEQ?ID?NO：42	?2CA5		?SEQ?ID?NO：43	?2G6	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL ?EACVIQEVGVEETPLMNVDPILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：44	?3AH7	?CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDSNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELHQQLNEL ?EACVVQEVGVEETPLMNEDSILAVKKYLQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：43	?2G6
?SEQ?ID?NO：44	?3AH7		?SEQ?ID?NO：45	?2G5	?CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：46	?2BA8	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRRE	?SEQ?ID?NO：45	?2G5
?SEQ?ID?NO：46	?2BA8		?SEQ?ID?NO：47	?1F3	?CDLPQTHSLGNRRALILLGQMGRISHFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSVAWDERLLDKLYTELYQQLNDL ?EACVMQEVWVGGTPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE

?SEQ?ID?NO：48	?4BE10	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEIMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNEL ?EACVIQGVGVEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCSWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：48	?4BE10		?SEQ?ID?NO：49	?2DD9	?CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTGLYQQLNDL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：50	?3CA1	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNNL ?EACVIQEVGMEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：49	?2DD9
?SEQ?ID?NO：50	?3CA1		?SEQ?ID?NO：51	?2F8	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQF ?QKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNEL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：52	?6CG3	?CDLPQTHSLGNKRAMMLLAQMGRTSPFSCLKDRHDFGFPQEEFDGNQF ?QRAQAIFVLHEMIQQTFNFFSTKDSSAAWEQSLLEKFSTELNQQLNDL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：51	?2F8
?SEQ?ID?NO：52	?6CG3		?SEQ?ID?NO：53	?3CG7	?CDLPQTHSLGNSRALMLLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISAFHEMIQQTFNLFSTKDSSAAWEQNLLEKFSTELYQQLNNL ?EACVIQEVGMEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：54	?1D3	?CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPQEEFDGHQF ?QKTQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDL ?EACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLMEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：53	?3CG7
?SEQ?ID?NO：54	?1D3		?SEQ?ID?NO：55	?2G4	?CDLPQTHSLGNRRAMMLLAQMSRISPSSCLMDRHDFEFPQEEFDDKQF ?QKAPAISVLHEVIQQTFNLFSTEDSSAAWEQTLLEKFSTELYQQLNDL ?EACVMQEERVGETPLMNADSILAVRKYFQRITLYLTKKKYSPCSWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：56	?1A1	?CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQF ?QKAQAISAFHEMMQQTFNLFSTEDSSAAWEQSLLEKFSTELHQQLNDL ?EACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLMEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：55	?2G4
?SEQ?ID?NO：56	?1A1		?SEQ?ID?NO：57	?1D10	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFRFPQEEFDGNQL ?QKTQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL ?EACVIQGVGVEETPPMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：58	?1F6	?CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL ?EACVIQEAGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：57	?1D10
?SEQ?ID?NO：58	?1F6		?SEQ?ID?NO：59	?2A10	?CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQF ?QKAQAISAFHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNL ?EACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLMEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE

?SEQ?ID?NO：60	?2C3	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQS ?QKAQAISVLHEMIQQTFNLFSTKDSSDTWDATLLEKFSTELNQQLNDL ?EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：60	?2C3		?SEQ?ID?NO：61	?2D1	?CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISAFHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNNL ?EACVIQEVGMEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：62	?2D10	?CDLPQTHSLGNRRALILLAQMGRVSPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISAFHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNL ?EACVIQEVGVEETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：61	?2D1
?SEQ?ID?NO：62	?2D10		?SEQ?ID?NO：63	?2D7	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFRFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNL ?EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：64	?2D9	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL ?EACVIQEVGVEETPLVNVDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：63	?2D7
?SEQ?ID?NO：64	?2D9		?SEQ?ID?NO：65	?2DA2	?CDLPQTHSLGNRRPLILLAQMGRISPFSCLKDRQDFGFPQEEFDGNQF ?QKAQAISVLHEMMQQTFNLFSTKNSSAAWEQSLLEKFSTELHQQLNEL ?EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLIERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：66	?2DH9	?CDLPQTHSPGNRRALMLLAQMGRISPFSCLKDRYDFGFPQGEFDGNQF ?QKAQAISVLHEMMQQTFNLFSTKDSSAAWEQSLLEKFSTELYRQLNDL ?EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLTEKKHSPCSWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：65	?2DA2
?SEQ?ID?NO：66	?2DH9		?SEQ?ID?NO：67	?2G11	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQF ?QKTQAISVLHEMIQQTFNLFSTKDSSDTWEQSLLEKFYIELFQQLNDL ?EACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLTEEKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：68	?2G12	?CDLPQTHSLGNRRTLMLMAQMRRISPFPRLKDRYDFGFPQEVFDGNQF ?QKAQAIFLFHEMMQQTFNLFSTKNSSAAWDETLLDKFYTELYQQLNDL ?EACVMQEGRVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEAV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：67	?2G11
?SEQ?ID?NO：68	?2G12		?SEQ?ID?NO：69	?2H9	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDL ?EACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：70	?6BC11	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQL ?QKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELNQQLNDL ?EACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTERKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：69	?2H9
?SEQ?ID?NO：70	?6BC11		?SEQ?ID?NO：71	?t19bb	?CDLPQTHSLGXXRAXXLLXQMXRXSXFSCLKDRXDFGXPXEEFDXXXF ?QXXQAIXXXHEXXQQTFNXFSTKXSSXXWXXXLLXKXXTXLXQQLNXL ?EACVXQXVXXXXTPLMNXDXILAVXKYXQRITLYLXEXKYSPCXWEVV ?RAEIMRSFSFSTNLQKRLRRKE

?SEQ?ID?NO：72	?CH1.1	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGATGGAC ?AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGACAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAACAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA ?CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：72	?CH1.1		?SEQ?ID?NO：73	?CH1.2	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGGCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCCATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGC ?CTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTG ?GAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCGAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：74	?CH1.3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG ?ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA ?CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATG ?AATGTGGACTCTATCCTGACTGTGAGAAAATACTTTCGAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：73	?CH1.2
?SEQ?ID?NO：74	?CH1.3		?SEQ?ID?NO：75	?CH1.4	?TGTGATCTGCCTCAGACCCACAGCCTGGGTAATAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTCCCCCAGGAGGAGTTTGGTGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGATGAGACC ?CTCCTAGACAAATTCTACATTGAACTTTTCCAGCAACTGAATGACCTG ?GAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATG ?AATGCGGACTCCATCTTGGCTGTGAAGAAATACTTCCAAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：76	?CH2.1	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG ?ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC	?SEQ?ID?NO：75	?CH1.4

		?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA ?CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGTATGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATG ?AATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
			?SEQ?ID?NO：77	?CH2.2	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATA ?CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGATGGAC ?AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGACAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAACAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA ?CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATG ?AATGTGGACTCTATCCTGACTGTGAAGAAATACTTCCGAAGAATCACT ?CTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA
?SEQ?ID?NO：78	?CH2.3	?TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATG ?ATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGAC ?AGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC ?CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACC ?TTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACA ?CTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTG ?GAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATG ?AATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACT ?CTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTC ?AGAGCAGAAATCATGAGATCTTTCTCTTTCTCAACAAACTTGCAAAAA ?AGATTAAGGAGGAAGGAA	?SEQ?ID?NO：77	?CH2.2
?SEQ?ID?NO：78	?CH2.3		?SEQ?ID?NO：79	?CH1.1	?CDLPQTHSLGNRRALILLAQMGRISPFSCLMDRHDFGFPQEEFDDNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL ?EACVIQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：80	?CH1.2	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQGISVLHEMIQQTFHLFSTKDSSATWEQSLLEKFSTELNQQLNDL ?EACVIQEVGVEETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：79	?CH1.1
?SEQ?ID?NO：80	?CH1.2		?SEQ?ID?NO：81	?CH1.3	?CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL ?EACMMQEVGVEDTPLMNVDSILTVRKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：82	?CH1.4	?CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFGGNQF ?QKAQAISVLHEMIQQTFNLFSTEDSSAAWDETLLDKFYIELFQQLNDL ?EACVMQEERVGETPLMNADSILAVKKYFQRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：81	?CH1.3


			?SEQ?ID?NO：83	?CH2.1	?CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL ?EACMIQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE
?SEQ?ID?NO：84	?CH2.2	?CDLPQTHSLGNRRALILLAQMGRISPFSCLMDRHDFGFPQEEFDDNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL ?EACMMQEVGVEETPLMNVDSILTVKKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE	?SEQ?ID?NO：83	?CH2.1
?SEQ?ID?NO：84	?CH2.2		?SEQ?ID?NO：85	?CH2.3	?CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQF ?QKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDL ?EACMMQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEVV ?RAEIMRSFSFSTNLQKRLRRKE

9 A compound according to claim 1 or a pharmaceutically acceptable salt or N- Oxide form thereof, wherein in the formula (I) compound is selected from: 7 - chloro-5 - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (benzyloxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (cyclobutyl methoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (ethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (hydroxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (n-propoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (isopropoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (butyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (methoxymethyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro -5 (S) - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro -5 (R) - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (2 - cyclopropyl-ethyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (2,2,2 - trifluoroethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (propargyloxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (ethyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (cyclopropylmethoxy) -2 - methyl-5, 10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] Two nitrogen naphthalene, 7 - chloro-5 - (n-butyl) -2 - methyl-5, 10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (2 - cyclopropyl-ethyl) -2 - methyl-5, 10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] Two nitrogen naphthalene, 7 - chloro-5 - (cyclopropylmethoxy) -5,10 - dihydro-2 - (methylthio) -5 - (trifluoromethyl) pyrimidine and [4,5-b] quinoline, 7 - chloro-5 - (isobutoxy) -5,10 - dihydro-2 - (methylthio) -5 - (trifluoromethyl) pyrimido [4,5 - b] quinoline, 7 - chloro-5 - (benzyloxy) -5,10 - dihydro-2 - (methylthio) -5 - (trifluoromethyl) pyrimido [4,5-b] Quinoline, 7 - chloro-5 - (2 - pyridyl-ylmethoxy) -5,10 - dihydro-2 - (methylthio) -5 - (trifluoromethyl) pyrimidine And [4,5-b] quinoline, 7 - chloro-5 - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline Morpholine, 7 - chloro-5 - (cyclopropylamino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (isopropylamino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro -5 - (N, N-dimethylamino ethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [B] [1,8] dinitrogen naphthalene, 7 - chloro -5 - (N-morpholino-ethylamino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] two Nitrogen naphthalene, 7 - chloro-5 - ((1 - methyl-cyclopropyl) methoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [B] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (3,3,3 - trifluoro-propan-1 - oxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] Two nitrogen naphthalene, 7 - chloro-5 - (cyclopropylmethyl-amino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (methylamino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (ethylamino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, (S) -7 - chloro-5 - (cyclopropyl-ethyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, (R) -7 - chloro-5 - (cyclopropyl-ethyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - fluoro-5 - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - fluoro-5 - (cyclopropyl-ethoxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - fluoro-5 - (allyloxy) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (phenylamino) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (cyclopropylmethoxy) -2 - methyl-5, 10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] Two nitrogen naphthalene, 7 - chloro-5 - (n-butyl) -2 - methyl-5, 10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5 - (cyclopropyl-ethyl) -2 - methyl-5, 10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] two Nitrogen naphthalene, 7 - chloro-5 - (cyclobutyl methoxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline Morpholine, 7 - chloro-5 - (methoxymethyl) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline, (S) -7 - chloro-5 - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline Morpholine, (R) -7 - chloro-5 - (cyclopropylmethoxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline Morpholine, 7 - chloro -5 - (N-piperidinyl-ethoxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline Morpholine, 7 - chloro -5 - (N-pyrrolidinyl-ethoxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] Quinoline, 7 - chloro-5 - ((4 - methyl-piperazin-1 - yl) propan-1 - oxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimidine And [4,5-b] quinoline, 7 - chloro-5 - (prop-1 - yloxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline, 7 - chloro -5 - (N, N-dimethylamino-prop-1 - yl) -5,10 - dihydro-5 - (trifluoromethyl) pyrimidine and [4,5-b] quinoline, 7 - chloro-5 - (benzyloxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline, 7 - chloro-5 - (3 - pyridylmethyl) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline Morpholine, 7 - chloro-5 - (allyloxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline, 7 - chloro-5 - (propargyloxy) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5-b] quinoline, 7 - chloro -5 - (N, N-dimethylaminoethyl) -5,10 - dihydro-5 - (trifluoromethyl) pyrimido [4,5 - b] quinoline, 7 - chloro-5 - cyclopropyl-methoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 5 - allyl-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen naphthalene, 7 - Fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous -5 - carbonitrile, 7 - Fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous -5 - ol, 5 - cyclopropyl-methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 7 - chloro-5 - prop-2 - ynyl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 7 - chloro-5 - (1 - methyl - cyclopropylmethoxy) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] Nitrous Naphthalene 1 - oxides, 7 - chloro-5 - (2 - cyclopropyl - ethoxy) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene-1 - oxide, (7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - isopropyl - amine, (7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - cyclobutylmethyl - Amines, 7 - chloro-5 - (2 - cyclopropyl - ethyl) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene dinitrogen 1 - oxide, 5 - cyclobutyl methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, (7 - fluoro-1 --5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - iso Base - amine, 5 - cyclobutyl methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthyridin-2 nitrous - Alcohol, 7 - chloro-5 - (pyridin-2 - ylmethoxy)-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene, 5 - butyl-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-1 --5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous -5 - ol, 7 - chloro-5 - cyclopropyl-methoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 7 - chloro-5 - pyridin-2 - yl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 7 - Fluoro-5 - pyridin-2 - yl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen naphthalene, 5 - cyclopropyl-methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 7 - chloro-5 - pyridin-2 - yl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen naphthalene, 3,7 - dichloro-5 - cyclopropyl-methoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene, 3,7 - dichloro-5 - cyclopropyl-methoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene-1 - oxide, 3,7 - dichloro-5 - pentyl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, 5 - (2 - cyclopropyl - ethyl) -7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene, 5 - (2 - cyclopropyl - ethyl) -7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene dinitrogen 1 - oxide, 3,7 - dichloro-5 - cyclopropyl-methoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene-1 - oxide, 5 - (2 - cyclopropyl - ethyl) -7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene dinitrogen 1 - oxide, 3 - chloro-5 - cyclopropyl-methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene, 3 - chloro-5 - cyclopropyl-methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene-1 - oxide, 7 - chloro-5 - isobutoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, 5 - butyl-7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide, (S) 3 - chloro-5 - cyclopropyl-methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] Nitrous Naphthalene 1 - oxides, (7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - methanol, 7 - fluoro-5 - isobutoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, 7 - fluoro-5 - isopropoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, Methanesulfonic acid 7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous -5 - diyl ester, 7 - chloro-5 - isopropoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, (7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - acetonitrile, 7 - Fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous -5 - formaldehyde, 3 - bromo -5 - cyclopropyl-methoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene-1 - oxide, 5 - butyl-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide, 5 - diisopropoxy-7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene, 7 - fluoro-5 - isopropoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, 7 - chloro-5 - isobutyl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, 7 - chloro-5 - propyl-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - oxide Material, (S) 7 - fluoro-5 - isobutoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, (R) 7 - fluoro-5 - isobutoxy-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] naphthalene nitrous 1 - Oxides, (7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - acetaldehyde, 7 - chloro-5 - (2,2 - diisopropoxy - ethyl) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] Two nitrogen naphthalene, 7 - chloro-5 - (2 - isopropoxy - ethyl) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene, 2 - (7 - chloro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - ethanol, 7 - chloro-5 - (2 - isopropoxy - ethyl) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen Naphthalene-1 - oxide, (R) 7 - fluoro-5 - (2 - isopropoxy - ethyl) -5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] Nitrous Naphthalene 1 - oxides, (7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - acetic acid tert-butyl Esters, (7 - fluoro-1 --5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - acetic acid T-butyl (7 - fluoro-5 - trifluoromethyl-5 ,10 - dihydro - benzo [b] [1,8] nitrous naphthyl -5 - yl) - acetic acid, 7 - chloro-5 - cyclopropyl-methoxy-2 - (methylthio) (sulfanyl) -5 - trifluoromethyl-5 ,10 - dihydro - ethyl Pyrido [4,5-b] quinoline, 7 - chloro-5 - isobutoxy-2 - methylthio-5 - trifluoromethyl-5 ,10 - dihydro - pyrimido [4,5-b] quinoline Morpholine, 5 - benzyloxy-7 - chloro-2 - methylthio-5 - trifluoromethyl-5 ,10 - dihydro - pyrimido [4,5-b] quinoline Morpholine, 7 - chloro -2 - (methylthio) -5 - (pyridin-2 - ylmethoxy)-5 - trifluoromethyl-5 ,10 - dihydro - pyrimido [4,5-b] quinoline, 7 - chloro-5 - cyclopropyl-methoxy-5 - trifluoromethyl-5 ,10 - dihydro - pyrimido [4,5-b] quinoline-1 - Oxides, 7 - chloro-5 - cyclopropyl-methoxy-5 - (1,1 - difluoro - ethyl) -5,10 - dihydro - benzo [b] [1,8] two Nitrogen Naphthalene 1 - oxides, 5 - cyclopropyl-methoxy-5 - (1,1 - difluoro - ethyl) -7 - fluoro-5 ,10 - dihydro - benzo [b] [1,8] two Nitrogen naphthalene, 5 - cyclopropyl-methoxy-5 - (1,1 - difluoro - ethyl) -7 - fluoro-5 ,10 - dihydro - benzo [b] [1,8] two Nitrogen Naphthalene 1 - oxides, 7 - chloro-5 - (1,1 - difluoro - ethyl) -5 - isobutoxy-5 ,10 - dihydro - benzo [b] [1,8]] dinitrogen Naphthalene, 7 - chloro-5 - (1,1 - difluoro - ethyl) -5 - isobutoxy-5 ,10 - dihydro - benzo [b] [1,8] naphthalene dinitrogen 1 - oxide, (R) 7 - chloro-5 - cyclopropyl-methoxy-5 - (1,1 - difluoro - ethyl) -5,10 - dihydro - benzo [b] [1,8] Nitrous Naphthalene 1 - oxides, (S) 7 - chloro-5 - cyclopropyl-methoxy-5 - (1,1 - difluoro - ethyl) -5,10 - dihydro - benzo [b] [1,8] Nitrous Naphthalene 1 - oxides, 3 - chloro-10 - methoxy-10 is not propyl - (trifluoromethyl) -9,10 - dihydro -1,8,9 - triazine - anthracene, 3 - chloro-10 - cyclopropyl-methoxy-10 - (trifluoromethyl) -9,10 - dihydro -1,8,9 - triazine - anthracene 8 - Oxides, 3,6 - dichloro -10 - cyclopropyl-methoxy-10 - (trifluoromethyl) -9,10 - dihydro -1,8,9 - triazine - Anthracene, 3 - chloro-10 - isobutoxy -10 - (trifluoromethyl) -9,10 - dihydro -1,8,9 - triazine - anthracene, 3 - chloro-10 - isobutoxyphenyl -10 - (trifluoromethyl) -9,10 - dihydro -1,8,9 - triazine - anthracene 8 - Oxidation Material, 7 - chloro-5 - (difluoromethyl) -5 - isopropoxy-methyl-5 ,10 - dihydro - benzo [b] [1,8] dinitrogen naphthalene, 7 - chloro-5 - (difluoromethyl) -5 - isopropoxy-methyl-5 ,10 - dihydro - benzo [b] [1,8] quinoxaline 1 - Oxides, 7 - chloro -1,5 - dihydro -5 - (N-ethyl-aminomethyl) -5 - (trifluoromethyl) benzo [b] [1,8] dinitrogen Naphthalene, 7 - chloro-5, 10 - dihydro -5 - (N-isopropyl-aminomethyl) -5 - (trifluoromethyl) benzo [b] [1,8] two Nitrogen naphthalene, 7 - chloro-5, 10 - dihydro -5 - (N-isopropyl-N-ethyl aminomethyl) -5 - (trifluoromethyl) benzo [B] [1,8] dinitrogen naphthalene, 7 - chloro -5 - (N, N-diethyl-aminomethyl) -5,10 - dihydro-5 - (trifluoromethyl) benzo [b] [1,8] Two nitrogen naphthalene, 5 - (acetylamino)-7 - chloro-5, 10 - dihydro-5 - (trifluoromethyl) [b] [1,8] dinitrogen naphthalene, 5,10 - dihydro-7 - fluoro -5 - (N-methyl-methylsulfonyl) -5 - (trifluoromethyl) [b] [1,8] dinitrogen naphthalene, 5,10 - dihydro-7 - fluoro-5 - (isopropyl-methyl-amido) -5 - (trifluoromethyl) [b] [1,8] dinitrogen Naphthalene, 5,10 - dihydro-7 - fluoro-5 - (isopropyl guanadino methyl) -5 - (trifluoromethyl) [b] [1,8] two Nitrogen naphthalene, 1,5 - dihydro-7 - fluoro -5 - (N-isopropyl-methyl) -5 - (trifluoromethyl) [b] [1,8] naphthalene nitrous -1 - (N- Oxide), 5 - (N, N-diethyl-aminomethyl) -5,10 - dihydro-7 - fluoro-5 - (trifluoromethyl) [b] [1,8] dinitrogen Naphthalene -1 - (N-oxide), 5,10 - dihydro -5 - (N, N-dimethylaminomethyl)-7 - fluoro-5 - (trifluoromethyl) [b] [1,8] dinitrogen Naphthalene -1 - (N-oxide), 7 - chloro-5, 10 - dihydro -5 - (N-isopropyl-aminomethyl) -5 - (trifluoromethyl) [b] [1,8] naphthalene dinitrogen -1 - (N-oxide), 7 - chloro -5 - (N, N-diethylamino methyl) -5,10 - dihydro-5 - (trifluoromethyl) [b] [1,8] dinitrogen Naphthalene -1 - (N-oxide), and 7 - chloro-5, 10 - dihydro -5 - (N, N-dimethylaminomethyl) -5 - (trifluoromethyl) [b] [1,8] dinitrogen Naphthalene -1 - (N-oxide). ...

20??????????????????25??????????????????30Arg?Gln?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly?Val?Lys?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Val?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Ile?Glu?Arg?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val

130?????????????????135?????????????????140Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Phe?Ser?Thr?Asn?Leu?Gln?Lys145?????????????????150?????????????????155?????????????????160Arg?Leu?Arg?Arg?Lys?Glu

The description of 165＜210〉37＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2CA3＜400〉37Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asp Arg Arg Ala Met Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?Tyr?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Glu?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly?Val?Gly?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Gly?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

The description of 165＜210〉38＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 4AB9＜400〉38Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Arg?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu?His?Glu?Met?Met?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asn?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Glu?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Ash?Glu?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ser?Trp?Glu?Val?Val

The description of 165＜210〉39＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2DA4＜400〉39Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?Gln?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Ser?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉40＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 3DA11＜400〉40Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Val 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

100?????????????????105?????????????????110Asn?Glu?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

The description of 165＜210〉41＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2DB11＜400〉41Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

The description of 165＜210〉42＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2CA5＜400〉42Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?Gln?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Arg?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asn?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉43＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2G6＜400〉43Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Thr?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Asn?Gln?Gln?Leu?Asn?Asp?Leu

100?????????????????105?????????????????110Asn?Val?Asp?Pro?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val

The description of 165＜210〉44＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 3AH7＜400〉44Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Ser?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?His?Gln?Gln?Leu?Asn?Glu?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Val?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Glu?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Leu?Gln?Arg?Ile?Thr

The description of 165＜210〉45＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2G5＜400〉45Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

l30?????????????????135?????????????????140Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Phe?Ser?Thr?Asn?Leu?Gln?Lys145?????????????????150?????????????????155?????????????????160Arg?Leu?Arg?Arg?Lys?Glu

The description of 165＜210〉46＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2BA8＜400〉46Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

The description of 165＜210〉47＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 1F3＜400〉47Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Gly Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Val?Ala?Trp?Asp?Glu?Arg?65??????????????????70??????????????????75??????????????????80Leu?Leu?Asp?Lys?Leu?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Met?Gln?Glu?Val?Trp?Val?Gly?Gly?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

The description of 165＜210〉48＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 4BE10＜400〉48Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu?His?Glu?Ile?Met?Gln?Gln?Thr

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

The description of 165＜210〉49＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2DD9＜400〉49Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Gly?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉50＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 3CA1＜400〉50Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Leu?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asn?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asn?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly?Met?Glu?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Val?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

The description of 165＜210〉51＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2F8＜400〉51Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

The description of 165＜210〉52＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 6CG3＜400〉52Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Lys Arg Ala Met Met 15 10 15Leu Leu Ala Gln Met Gly Arg Thr Ser Pro Phe Ser Cys Leu Lys Asp

35??????????????????40??????????????????45Gln?Arg?Ala?Gln?Ala?Ile?Phe?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Phe?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Asn?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉53＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 3CG7＜400〉53Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Ser Arg Ala Leu Met 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Ala?Ile?Ser?Ala?Phe?His?Glu?Met?Ile?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Asn?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asn?Leu

The description of 165＜210〉54＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 1D3＜400〉54Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?His?Gln?Phe

35??????????????????40??????????????????45Gln?Lys?Thr?Gln?Ala?Ile?Ser?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Met?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val

130?????????????????????135?????????????????140Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Phe?Ser?Thr?Asn?Leu?Gln?Lys145?????????????????150?????????????????155?????????????????160Arg?Leu?Arg?Arg?Lys?Glu

The description of 165＜210〉55＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2G4＜400〉55Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Met Met 15 10 15Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Glu?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Asp?Lys?Gln?Phe

35??????????????????40??????????????????45Gln?Lys?Ala?Pro?Ala?Ile?Ser?Val?Leu?His?Glu?Val?Ile?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Glu?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Met?Gln?Glu?Glu?Arg?Val?Gly?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Ala?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Lys?Lys?Lys?Tyr?Ser?Pro?Cys?Ser?Trp?Glu?Val?Val

The description of 165＜210〉56＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 1A1＜400〉56Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?Tyr?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Val?Phe?Asp?Gly?Asn?Gln?Phe

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Ala?Ile?Ser?Ala?Phe?His?Glu?Met?Met?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Glu?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?His?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉57＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 1D10＜400〉57Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Arg?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Glu?Glu?Thr?Pro?Pro?Met

The description of 165＜210〉58＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 1F6＜400〉58Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met 15 10 15Ile Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Glu?Ala?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

The description of 165＜210〉59＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2Al0＜400〉59Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser His Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Thr?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asn?Leu

The description of 165＜210〉60＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2C3＜400〉60Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Ser

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Asp?Thr?Trp?Asp?Ala?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Asn?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉61＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2D1＜400〉61Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asn?Leu

The description of 165＜210〉62＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2D10＜400〉62Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile l 5 10 15Leu Leu Ala Gln Met Gly Arg Val Ser Pro Phe Ser Cys Leu Lys Asp

100?????????????????105?????????????????110Asn?Val?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr

The description of 165＜210〉63＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2D7＜400〉63Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Arg?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Glu?Arg?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val

The description of 165＜210〉64＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2D9＜400〉64Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Ile?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Val

The description of 165＜210〉65＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2DA2＜400〉65Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Pro Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asn?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?His?Gln?Gln?Leu?Asn?Glu?Leu

The description of 165＜210〉66＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2DH9＜400〉66Cys Asp Leu Pro Gln Thr His Ser Pro Gly Asn Arg Arg Ala Leu Met 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?Tyr?Asp?Phe?Gly?Phe?Pro?Gln?Gly?Glu?Phe?Asp?Gly?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Tyr?Arg?Gln?Leu?Asn?Asp?Leu

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Glu?Lys?Lys?His?Ser?Pro?Cys?Ser?Trp?Glu?Val?Val

The description of 165＜210〉67＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2G11＜400〉67Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Asp?Thr?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Tyr?Ile?Glu?Leu?Phe?Gln?Gln?Leu?Asn?Asp?Leu

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Glu?Glu?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val

The description of 165＜210〉68＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2G12＜400〉68Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met 15 10 15Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe Pro Arg Leu Lys Asp

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Ala?Ile?Phe?Leu?Phe?His?Glu?Met?Met?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asn?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Met?Gln?Glu?Gly?Arg?Val?Gly?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Ala?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Ala?Val

The description of 165＜210〉69＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 2H9＜400〉69Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Thr?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

The description of 165＜210〉70＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering 6BC11＜400〉70Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?Tyr?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Leu

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Asn?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165 <210> 71 <211> 166 <212> PRT <213> artificial sequence <220> <223> artificial sequence: synthetic amino acid <220> <223> clone numbering t19bb <220 <221> MOD_RES <222 > (11) <223> N or D <220 <221> MOD_RES <222> (12) <223> R; S; Or K <220> <221> MOD_RES <222> (15) <223> L or M <220 <221> MOD_RES <222> (16) <223> I; M or V <220> <221> MOD_RES <222> (19) <223> A or G <220 <221> MOD_RES <222> (22 ) <223> G or R <220 <221> MOD_RES <222> (24) <223> I or T <220 <221> MOD_RES <222> (26) <223> P or H <220 <221> MOD_RES < 222> (34) <223> H; Y or Q <220> <221> MOD_RES <222> (38) <223> F or L <220> <221> MOD_RES <222> (40) <223> Q, or R <220> <221> MOD_RES <222> (45) <223> G or S <220> <221> MOD_RES <222> (46) <223> N, or H <220> <221> MOD_RES <222> ( 47) <223> Q, or R <220> <221> MOD_RES <222> (50) <223> K or R <220> <221> MOD_RES <222> (51) <223> A or T <220> < 221> MOD_RES <222> (55) <223> S or F <220> <221> MOD_RES <222> (56) <223> V or A <220> <221> MOD_RES <222> (57) <223> L or F <220> <221> MOD_RES <222> (60) <223> M or I <220> <221> MOD_RES <222> (61) <223> I or M <220> <221> MOD_RES <222 > (67) <223> L or F <220> <221> MOD_RES <222> (72) <223> D or N <220> <221> MOD_RES <222> (75) <223> A or V <220 > <221> MOD_RES <222> (76) <223> A or T <220> <221> MOD_RES <222> (78) <223> E or D <220> <221> MOD_RES <222> (79) < 223> Q or E <220> <221> MOD_RES <222> (80) <223> S; R; T; Or N <220> <221> MOD_RES <222> (83) <223> E or D <220 <221> MOD_RES <222> (85) <223> F or L <220 <221> MOD_RES <222> (86) <223> S or Y <220 <221> MOD_RES <222> (88) <223> E or G <220 <221> MOD_RES <222> (90) <223> Y; H; N <220> <221> MOD_RES <222> (95) <223> D; E; Or N <220> <221> MOD_RES <222> (101) <223> I; M; Or V <220> <221> MOD_RES <222> (103) <223> E or G <220 <220 <221> MOD_RES <222> (105) < 223> G or W <220 <221> MOD_RES <222> (106) <223> V or M <220 <221> MOD_RES <222> (107) <223> E; G; Or K <220> <221> MOD_RES <222> (108) <223> E or G <220 <221> MOD_RES <222> (114) <223> V; E; Or G <220> <221> MOD_RES <222> (116) <223> S or P <220 <221> MOD_RES <222> (121) <223> K or R <220 <221> MOD_RES <222> (124) <223> F or L <220 <221> MOD_RES <222> (132 ) <223> T; I, or M <220> <221> MOD_RES <222> (134) <223> K or R <220 <221> MOD_RES <222> (140) <223> A or S <400 71Cys Asp Leu Pro Gln Thr His Ser Leu Gly Xaa Xaa Arg Ala Xaa Xaa 15 10 15Leu Leu Xaa Gln Met Xaa Arg Xaa Ser Xaa Phe Ser Cys Leu Lys Asp....

20??????????????????25??????????????????30Arg?Xaa?Asp?Phe?Gly?Xaa?Pro?Xaa?Glu?Glu?Phe?Asp?Xaa?Xaa?Xaa?Phe

35??????????????????40??????????????????45Gln?Xaa?Xaa?Gln?Ala?Ile?Xaa?Xaa?Xaa?His?Glu?Xaa?Xaa?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?Asn?Xaa?Phe?Ser?Thr?Lys?Xaa?Ser?Ser?Xaa?Xaa?Trp?Xaa?Xaa?Xaa?65??????????????????70??????????????????75??????????????????80Leu?Leu?Xaa?Lys?Xaa?Xaa?Thr?Xaa?Leu?Xaa?Gln?Gln?Leu?Asn?Xaa?Leu

85??????????????????90??????????????????95Glu?Ala?Cys?Val?Xaa?Gln?Xaa?Val?Xaa?Xaa?Xaa?Xaa?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Xaa?Asp?Xaa?Ile?Leu?Ala?Val?Xaa?Lys?Tyr?Xaa?Gln?Arg?Ile?Thr

115?????????????????120?????????????????125Leu?Tyr?Leu?Xaa?Glu?Xaa?Lys?Tyr?Ser?Pro?Cys?Xaa?Trp?Glu?Val?Val

165 <210> 72 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <223> Clone No. CH1.1 <400> 72tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60atgggaagaa tctctccttt ctcctgtctg atggacagac atgactttgg atttccccag 120gaggagtttg atgacaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180atccaacaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgcgtg 300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg 360aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 73 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <223> Clone No. CH1.2 <400> 73tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120gaggagtttg atggcaacca gttccagaag gctcaaggca tctctgtcct ccatgagatg 180atccagcaga ccttccatct cttcagcaca aaggactcat ctgctacttg ggaacagagc 240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg 300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg 360aagaaatact tccgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 74 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <223> Clone No. CH1.3 <400> 74tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ceatgagatg 180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300atgcaggagg ttggagtgga agacactcct ctgatgaatg tggactctat cctgactgtg 360agaaaatact ttcgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 75 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <223> Clone No. CH1.4 <400> 75tgtgatctgc ctcagaccca cagcctgggt aataggaggg ccttgatact cctggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag 120gaggagtttg gtggcaacca gttccagaag gctcaagcca tctctgtcct ceatgagatg 180atccagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggatgagacc 240ctcctagaca aattctacat tgaacttttc cagcaactga atgacctgga agcctgtgtg 300atgcaggagg agagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg 360aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 76 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <223> Clone No. CH2.1tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180atccagcaga ccttcaatct cttcagcaca aaggacteat ctgctacttg ggatgagaca 240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg 360aagaaatact tccgaagaat GaGtctctat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 77 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <220> <223> Clone No. CH2.2 <400> 77tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60atgggaagaa tctctccttt ctcctgtctg atggacagac atgactttgg atttccccag 120gaggagtttg atgacaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg 180atccaacaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300atgcaggagg ttggagtgga agacactcct ctgatgaatg tggactctat cctgactgtg 360aagaaatact tccgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 78 <211> 498 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : synthetic DNA <220> <223> Clone No. CH2.3 <400> 78tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ceatgagatg 180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca 240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg 300atgcaggagg ttggagtgga agacactcct ctgatgaatg aggactceat cttggctgtg 360aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc 420tgggaggttg tcagagcaga aatcatgaga tctttctctt tctcaacaaa cttgcaaaaa 480agattaagga ggaaggaa 498 <210> 79 <211> 166 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic amino acids <220> <223> Clone No. CH1.1 <400> 79Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Met Asp...

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Asp?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Thr?Trp?Asp?Glu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu

100?????????????????105?????????????????110Asn?Glu?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr

The description of 165＜210〉80＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering CH1.2＜400〉80Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

35??????????????????40??????????????????45Gln?Lys?Ala?Gln?Gly?Ile?Ser?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Thr

50??????????????????55??????????????????60Phe?His?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Thr?Trp?Glu?Gln?Ser?65??????????????????70??????????????????75??????????????????80Leu?Leu?Glu?Lys?Phe?Ser?Thr?Glu?Leu?Asn?Gln?Gln?Leu?Asn?Asp?Leu

The description of 165＜210〉81＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering CH1.3＜400〉81Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met 15 10 15Ile Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

85??????????????????90??????????????????95Glu?Ala?Cys?Met?Met?Gln?Glu?Val?Gly?Val?Glu?Asp?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Val?Asp?Ser?Ile?Leu?Thr?Val?Arg?Lys?Tyr?Phe?Arg?Arg?Ile?Thr

The description of 165＜210〉82＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering CH1.4＜400〉82Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

20??????????????????25??????????????????30Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Gly?Asn?Gln?Phe

50??????????????????55??????????????????60Phe?Asn?Leu?Phe?Ser?Thr?Glu?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?65??????????????????70??????????????????75??????????????????80Leu?Leu?Asp?Lys?Phe?Tyr?Ile?Glu?Leu?Phe?Gln?Gln?Leu?Asn?Asp?Leu

100?????????????????105?????????????????110Asn?Ala?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr

The description of 165＜210〉83＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering CH2.1＜400〉83Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met 15 10 15Ile Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

85??????????????????90??????????????????95Glu?Ala?Cys?Met?Ile?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

The description of 165＜210〉84＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering CH2.2＜400〉84Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 15 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Met Asp

85??????????????????90??????????????????95Glu?Ala?Cys?Met?Met?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met

100?????????????????105?????????????????110Asn?Val?Asp?Ser?Ile?Leu?Thr?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr

The description of 165＜210〉85＜211〉166＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜220〉＜223〉clone numbering CH2.3＜400〉85Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met 15 10 15Ile Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp

The description of 165＜210〉86＜211〉15＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic DNA＜400〉86tgcgacttac cacaa 15＜210〉87＜211〉26＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence description: synthetic amino acid＜400〉87Trp Glu Val Val Arg Ser Glu Ile Met Arg Ser Phe Ser Tyr Ser Thr 15 10 15Asn Leu Gln Arg Arg Leu Arg Arg Lys Asp

The description of 20 25＜210〉88＜211〉26＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence: synthetic amino acid＜223〉description of artificial sequence: synthetic amino acid＜400〉88Trp Glu Leu Val Arg Ala Glu Ile Val Arg Ser Phe Ser Phe Ser Thr 15 10 15Asn Leu Asn Lys Arg Leu Arg Lys Lys Glu

20??????????????????25

Claims

1. the nucleic acid of separation or reorganization, it contains and is selected from following polynucleotide sequence:

(a) SEQ ID NO:1 to SEQ ID NO:35, or its complementary polynucleotide sequence;

(b) coding is selected from the polynucleotide sequence of the polypeptide of SEQ ID NO:36 to SEQ ID NO:70, or its complementary polynucleotide sequence;

(c) under highly tight hybridization conditions, with the polynucleotide sequence of total length (a) or (b) polynucleotide sequence of hybridization basically; With

(d) contain (a), (b) or segmental polynucleotide sequence (c), described fragment coding has the polypeptide of antiproliferative activity in the test based on people Daudi clone.

2. the nucleic acid of separation or reorganization, it contains and is selected from following polynucleotide sequence:

(a) SEQ ID NO:72 to SEQ ID NO:78, or its complementary polynucleotide sequence;

(b) coding is selected from the polynucleotide sequence of the polypeptide of SEQ ID NO:79 to SEQ ID NO:85, or its complementary polynucleotide sequence;

(d) contain (a), (b) or segmental polynucleotide sequence (c), described fragment coding has the polypeptide of antiviral activity in the test based on mouse cell line/EMCV.

3. the nucleic acid of separation or reorganization, it contains the polynucleotide sequence of coded polypeptide, and described polypeptide contains following aminoacid sequence: CDLPQTHSLG-X ₁₁-X ₁₂-RA-X ₁₅-X ₁₆-LL-X ₁₉-QM-X ₂₂-R-X ₂₄-S-X ₂₆-FSCLKDR-X ₃₄-DFG-X ₃₈-P-X ₄₀-EEFD-X ₄₅-X ₄₆-X ₄₇-FQ-X ₅₀-X ₅₁-QAI-X ₅₅-X ₅₆-X ₅₇-HE-X ₆₀-X ₆₁-QTFN-X ₆₇-FSTK-X ₇₂-SS-X ₇₅-X ₇₆-W-X ₇₈-X ₇₉-X ₈₀-LL-X ₈₃-K-X ₈₅-X ₈₆-T-X ₈₈-L-X ₉₀-QQLN-X ₉₅-LEACV-X ₁₀₁-Q-X ₁₀₃-V-X ₁₀₅-X ₁₀₆-X ₁₀₇-X ₁₀₈-TPLMN-X ₁₁₄-D-X ₁₁₆-ILAV-X ₁₂₁-KY-X ₁₂₄-QRITLYL-X ₁₃₂-E-X ₁₃₄-KYSPC-X ₁₄₀-WEVVRAEIMRSFSFSTNLQKRLRRKE, or its conservative variant that replaces, wherein X ₁₁Be N or D; X ₁₂Be R, S or K; X ₁₅Be L or M; X ₁₆Be I, M or V; X ₁₉Be A or G; X ₂₂Be G or R; X ₂₄Be I or T; X ₂₆Be P or H; X ₃₄Be H, Y or Q; X ₃₈Be F or L; X ₄₀Be Q or R; X ₄₅Be G or S; X ₄₆Be N or H; X ₄₇Be Q or R; X ₅₀Be K or R; X ₅₁Be A or T; X ₅₅Be S or F; X ₅₆Be V or A; X ₅₇Be L or F; X ₆₀Be M or I; X ₆₁Be I or M; X ₆₇Be L or F; X ₇₂Be D or N; X ₇₅Be A or V; X ₇₆Be A or T; X ₇₈Be E or D; X ₇₉Be Q or E; X ₈₀Be S, R, T or N; X ₈₃Be E or D; X ₈₅Be F or L; X ₈₆Be S or Y; X ₈₈Be E or G; X ₉₀Be Y, H, N; X ₉₅Be D, E or N; X ₁₀₁Be I, M or V; X ₁₀₃Be E or G; X ₁₀₅Be G or W; X ₁₀₆Be V or M; X ₁₀₇Be E, G or K; X ₁₀₈Be E or G; X ₁₁₄Be V, E or G; X ₁₁₆Be S or P; X ₁₂₁Be K or R; X ₁₂₄Be F or L; X ₁₃₂Be T, I or M; X ₁₃₄Be K or R; And X ₁₄₀Be A or S.

4. the nucleic acid of claim 3, described polypeptide has antiproliferative activity in the cell proliferation test based on people Daudi clone, or has antiviral activity in the test based on people WISH cell/EMCV.

5. the nucleic acid of claim 3, wherein encoded polypeptide has in the test based on people Daudi clone and is at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/mg, or in the test based on people WISH cell/EMCV, have and be at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg.

6. the nucleic acid of claim 3, wherein encoded polypeptide contains the aminoacid sequence that is selected from SEQ ID NO:36 to SEQ ID NO:54.

7. the nucleic acid of claim 3, described nucleic acid contains the polynucleotide sequence that is selected from SEQ ID NO:1 to SEQ ID NO:19.

8. the nucleic acid of separation or reorganization, it contains the polynucleotide sequence of coded polypeptide, contains in the amino acid that described polypeptide contains: at least 20 the successive amino acid of any among the SEQ ID NO:36-70, and Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, one or more amino acid among Lys160 and the Glu166, wherein amino acid whose numbering is corresponding to the numbering among the SEQ ID NO:36.

9. the nucleic acid of claim 8, wherein the length of coded polypeptide is 166 amino acid.

10. the nucleic acid of claim 8, wherein encoded polypeptide has antiproliferative activity in the test based on people Daudi clone.

11. the nucleic acid of claim 8, wherein encoded polypeptide has antiviral activity in the test based on people WISH cell/EMCV.

12. the nucleic acid of claim 8, wherein encoded polypeptide contains amino acid Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166.

13. the nucleic acid of claim 8, wherein encoded polypeptide contains among the SEQ ID NO:36-70 at least 50 the successive amino-acid residues of any.

14. the nucleic acid of claim 8, wherein encoded polypeptide contains among the SEQ ID NO:36-70 at least 100 the successive amino-acid residues of any.

15. the nucleic acid of claim 8, wherein encoded polypeptide contains among the SEQ ID NO:36-70 at least 150 the successive amino-acid residues of any.

16. the nucleic acid of claim 8, wherein encoded polypeptide contains and is selected from following aminoacid sequence: SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:45 and SEQ ID NO:46.

17. the nucleic acid of claim 8, it contains and is selected from following polynucleotide sequence: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

18. the nucleic acid of separation or reorganization, it contains the polynucleotide sequence of coded polypeptide, described polypeptide contains: at least 155 the successive amino acid of any among the SEQ ID NO:36-70, described aminoacid sequence contains amino acid Lys160 and Glu166, and wherein amino acid whose numbering is corresponding to the numbering among the SEQ ID NO:36.

19. the nucleic acid of claim 18, wherein encoded polypeptide contains and is selected from following aminoacid sequence: SEQ ID NO:36, SEQ ID N0:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:45 and SEQ ID NO:46.

20. contain claim 1, the cell of 2,8 or 18 nucleic acid.

21. the cell of claim 20, wherein said cell expressing is by the polypeptide of described nucleic acid encoding.

22. contain claim 1, the carrier of 2,8 or 18 nucleic acid.

23. the carrier of claim 20, wherein said carrier comprises plasmid, clay, phage or virus.

24. the carrier of claim 22, wherein said carrier is an expression vector.

25. by the cell of the carrier transduction of claim 22.

26. contain claim 1,2,8 or 18 the nucleic acid and the composition of vehicle.

27. the composition of claim 26, wherein vehicle is a pharmaceutically-acceptable excipients.

28. by using restriction endonuclease, RNA enzyme or dnase digestion claim 1, one or more nucleic acid of 2,3,8 or 18 and the composition that produces.

29. the existence that the composition of Chan Shenging by the following method, described method are included in deoxyribonucleotide triphosphoric acid and nucleic acid polymerase is insulation claim 1 down, one or more nucleic acid of 2,3,8 or 18.

30. the composition of claim 29, wherein nucleic acid polymerase is the polysaccharase of thermostability.

31. the polypeptide of separation or reorganization, described polypeptide be by claim 1,2,3,8 or 18 nucleic acid encoding.

32. the polypeptide of the separation or reorganization of claim 31, it contains the sequence that is selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85.

33. the polypeptide of claim 31, it has in the test based on people Daudi clone and is at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/mg, or in the test based on people WISH cell/EMCV, have and be at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg.

34. the polypeptide of separation or reorganization, it contains following aminoacid sequence: CDLPQTHSLG-X11-X12-RA-X15-X ₁₆-LL-X ₁₉-QM-X ₂₂-R-X ₂₄-S-X ₂₆-FSCLKDR-X ₃₄-DFG-X ₃₈-P-X ₄₀-EEFD-X ₄₅-X ₄₆-X ₄₇-FQ-X ₅₀-X ₅₁-QAI-X ₅₅-X ₅₆-X ₅₇-HE-X ₆₀-X ₆₁-QTFN-X ₆₇-FSTK-X ₇₂-SS-X ₇₅-X ₇₆-W-X ₇₈-X ₇₉-X ₈₀-LL-X ₈₃-K-X ₈₅-X ₈₆-T-X ₈₈-L-X ₉₀-QQLN-X ₉₅-LEACV-X ₁₀₁-Q-X ₁₀₃-V-X ₁₀₅-X ₁₀₆-X ₁₀₇-X ₁₀₈-TPLMN-X ₁₁₄-D-X ₁₁₆-ILAV-X ₁₂₁-KY-X ₁₂₄-QRITLYL-X ₁₃₂-E-X ₁₃₄-KYSPC-X ₁₄₀-WEVVRAEIMRSFSFSTNLQKRLRRKE, or its conservative variant that replaces, wherein X ₁₁Be N or D; X ₁₂Be R, S or K; X ₁₅Be L or M; X ₁₆Be I, M or V; X ₁₉Be A or G; X ₂₂Be G or R; X ₂₄Be I or T; X ₂₆Be P or H; X ₃₄Be H, Y or Q; X ₃₈Be F or L; X ₄₀Be Q or R; X ₄₅Be G or S; X ₄₆Be N or H; X ₄₇Be Q or R; X ₅₀Be K or R; X ₅₁Be A or T; X ₅₅Be S or F; X ₅₆Be V or A; X ₅₇Be L or F; X ₆₀Be M or I; X ₆₁Be I or M; X ₆₇Be L or F; X ₇₂Be D or N; X ₇₅Be A or V; X ₇₆Be A or T; X ₇₈Be E or D; X ₇₉Be Q or E; X ₈₀Be S, R, T or N; X ₈₃Be E or D; X ₈₅Be F or L; X ₈₆Be S or Y; X ₈₈Be E or G; X ₉₀Be Y, H, N; X ₉₅Be D, E or N; X ₁₀₁Be I, M or V; X ₁₀₃Be E or G; X ₁₀₅Be G or W; X ₁₀₆Be V or M; X ₁₀₇Be E, G or K; X ₁₀₈Be E or G; X ₁₁₄Be V, E or G; X ₁₁₆Be S or P; X ₁₂₁Be K or R; X ₁₂₄Be F or L; X ₁₃₂Be T, I or M; X ₁₃₄Be K or R; And X ₁₄₀Be A or S.

35. the polypeptide of claim 34, it has in the test based on people Daudi clone and is at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/mg, or in the test based on people WISH cell/EMCV, have and be at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg.

36. the polypeptide of claim 34, it contains the sequence that is selected from SEQ ID NO:36 to SEQ ID NO:54.

37. a peptide species, it contains proteinic at least 100 the successive amino acid by a kind of coded polynucleotide sequence encoding, and wherein said polynucleotide sequence is selected from:

(a) SEQ ID NO:1 to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78;

(b) coding is selected from the coded polynucleotide sequence of first peptide species of SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQID NO:85; With

(c) under the height stringent condition, with the polynucleotide sequence of total length (a) or (b) the complementary polynucleotide sequence of hybridization basically.

38. the polypeptide of claim 37, described polypeptide has antiproliferative activity in the cell proliferation test based on people Daudi clone, or has antiviral activity in the test based on people WISH cell/EMCV.

39. the polypeptide of claim 37, wherein said polypeptid specificity is in conjunction with human's acceptor.

40. the polypeptide of claim 37, it contains coded proteinic at least 150 successive amino acid.

41. the polypeptide of separation or reorganization, it comprises the aminoacid sequence that contains at least 50 continuous amino acids of any among the SEQ ID NO:36-70, and comprises and contain following one or more amino acid: Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, the aminoacid sequence of Lys160 and Glu166, wherein amino acid whose numbering is corresponding to the numbering among the SEQ IDNO:36.

42. the polypeptide of claim 41, wherein said polypeptide is in conjunction with human's acceptor.

43. the polypeptide of claim 41, described polypeptide has antiproliferative activity in the cell proliferation test based on people Daudi clone, or has antiviral activity in the test based on people WISH cell/EMCV.

44. the polypeptide of claim 41, it has in the test based on people Daudi clone and is at least about 8.3 * 10 ⁶The antiproliferative activity of individual unit/mg, or in the test based on people WISH cell/EMCV, have and be at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg.

45. the polypeptide of claim 41, the length of wherein said polypeptide are 166 amino acid.

46. the polypeptide of claim 41, described polypeptide contains amino acid: Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, the amino acid numbering of wherein said polypeptide is numbered corresponding to the amino acid among the SEQ ID NO:36.

47. the polypeptide of claim 41, it contains among the SEQ ID NO:36-70 at least 100 the successive amino-acid residues of any.

48. the polypeptide of claim 41, it contains among the SEQ ID NO:36-70 at least 150 the successive amino-acid residues of any.

49. the polypeptide of claim 41, it contains among the SEQ ID NO:36-70 at least 155 the successive amino-acid residues of any.

50. the polypeptide of claim 41, it contains and is selected from following aminoacid sequence: SEQ IDNO:36, SEQ ID N0:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:45 and SEQ ID NO:46.

51. the polypeptide of separation or reorganization, contain in its aminoacid sequence that contains: at least 155 the successive amino acid of any among the SEQ ID NO:36-70, the polypeptide of this separation or reorganization contains amino acid Lys160 and Glu166, and wherein amino acid whose numbering is corresponding to the numbering among the SEQ ID NO:36.

52. the polypeptide of claim 51, it contains and is selected from following aminoacid sequence: SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:45 and SEQ ID NO:46.

53. having, the polypeptide of claim 51, described polypeptide be at least about 8.3 * 10 in the test based on people Daudi clone ⁶The antiproliferative activity of individual unit/mg, or in the test based on people WISH cell/EMCV, have and be at least about 2.1 * 10 ⁷The antiviral activity of individual unit/mg.

54. claim 31,34,37,41 or 51 polypeptide further contains secretion/positioning sequence.

55. claim 31,34,37,41 or 51 polypeptide further contains the peptide purification subsequence.

56. the polypeptide of claim 55, the sequence of wherein being convenient to purifying is selected from: epi-position mark, FLAG mark, poly histidine mark and GST fusions.

57. claim 31,34,37,41 or 51 polypeptide further contains methionine(Met) at N-terminal.

58. claim 31,34,37,41 or 51 polypeptide, it contains modified amino acid.

59. the polypeptide of claim 58, wherein modified amino acid is selected from: glycosylation amino acid, PEGization amino acid, farnesylation amino acid, ethanoyl amino acid and biotinylation amino acid.

60. contain claim 31,34,37,41 or 51 the polypeptide and the composition of vehicle.

61. the composition of claim 60, wherein vehicle is a pharmaceutically-acceptable excipients.

62. contain the polypeptide of claim 58 and the composition of pharmaceutically-acceptable excipients.

63. with anti-at least a antigenic polyclonal antiserum specificity bonded polypeptide, described at least a antigen contains at least a aminoacid sequence among SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to the SEQ IDNO:85, or its segment, wherein reduce described antiserum(antisera) with IFN-α polypeptide, described IFN-α polypeptide is by the nucleic acid encoding corresponding to following one or more GenBank registration numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, and V00549 (α-2a) and I08313 (α-Con1).

64. pass through to administration claim 31,34,37,41 or 51 polypeptide and the antibody or the antiserum(antisera) that produce, described antibody or antiserum(antisera) are specifically in conjunction with at least a antigen, contain at least a aminoacid sequence among SEQ ID NO:36 to SEQ ID NO:70 and SEQ IDNO:79 to the SEQ ID NO:85 in the polypeptide that described at least a antigen contains, or its segment, wherein antibody or antiserum(antisera) can not be specifically in conjunction with by the IFN-α polypeptide corresponding to the nucleic acid encoding of following one or more GenBank registration numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1).

65. the antibody of an energy specific binding polypeptide or antiserum(antisera), described polypeptide contains the sequence that is selected from SEQ IDNO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85, wherein said antibody or antiserum(antisera) can not be specifically in conjunction with by the IFN-α polypeptide corresponding to the nucleic acid encoding of following one or more GenBank registration numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, and V00549 (α-2a) and I08313 (α-Con1).

66. produce the method for polypeptide, described method comprises:

Import claim 1 in cell colony, 2,3,8 or 18 nucleic acid, described nucleic acid can be operated with the adjusting sequence that can effectively produce coded polypeptide and link to each other; With

In substratum, cultivate described cell to produce described polypeptide.

67. produce the method for polypeptide, described method comprises:

In cell colony, import and contain claim 1, the recombinant expression vector of 2,3,8 or 18 nucleic acid; With

Under the condition that is suitable for producing by the described polypeptide of described expression vector codes, in substratum, cultivate described cell.

68. suppress the method for tumour cell population growth, described method comprises:

With the claim 31 of tumour cell colony and significant quantity, the contact of 34,37,41 or 51 polypeptide, thus suppressing the growth of tumour cell in the described cell colony, wherein said significant quantity is enough to suppress the growth of tumour cell in the described tumour cell colony.

69. the method for claim 68, wherein said tumour cell is selected from: human cancer cell, human leukemia cell, people T-lymphoma cell and human melanoma cell.

70. the method for claim 68, wherein said tumour cell is in cultivation.

71. suppress by the method for at least one intracellular virus replication of virus infection, described method comprises:

Claim 31 with described at least one infected cells and significant quantity, 34,37,41 or 51 polypeptide contact, thereby suppress the virus replication in described at least one cells infected, wherein said significant quantity is enough to suppress the interior virus replication of described at least one cells infected.

72. the method for claim 71, wherein said virus is RNA viruses.

73. the method for claim 72, wherein said virus are human immunodeficiency virus or hepatitis C virus.

74. the method for claim 71, wherein said virus is dna virus.

75. the method for claim 74, wherein said virus is hepatitis B virus.

76. the method for claim 71, wherein said cell is through cultivating.

77. treatment patient's the method for autoimmune disease, described method comprises: use the claim 31 of significant quantity, 34,37,41 or 51 polypeptide to the patient.

78. the method for claim 77, wherein autoimmune disease is selected from multiple sclerosis, rheumatoid arthritis, lupus erythematosus and type i diabetes.

79. treatment is by using the method for the disease that interferon-' alpha ' can treat to the experimenter, described modification method comprises: use the claim 31 of significant quantity, 34,37,41 or 51 polypeptide to the experimenter.

80. the method for claim 79 wherein is selected from sclerosis, rheumatoid arthritis, lupus erythematosus and type i diabetes by using the disease that interferon-' alpha ' can treat.

81. prepare the method for nucleic acid modified or reorganization, described method comprises:

Make claim 1, the sequence of one or more nucleic acid of 2,3,8 or 18 and the reorganization of the serial regression of one or more other nucleic acid, every kind of sequence encoding interferon-' alpha ' or its amino acid subsequence of described one or more other nucleic acid.

82. the method for claim 81, at least a library that wherein said recurrence reorganization has produced recombinant interferon-alpha homologues nucleic acid.

83. the nucleic acid library that produces by the method for claim 82.

84. contain the cell colony in the library of claim 83.

85. the recombinant interferon-alpha homologues nucleic acid that produces by the method for claim 82.

86. contain the cell of the nucleic acid of claim 85.

87. the method for claim 81 wherein returns and is binned in external carrying out.

88. the method for claim 81, wherein return reorganization in vivo or ex vivo carry out.

89. contain claim 1, the composition of two or more nucleic acid in 2,3,8 or 18.

90. the composition of claim 89, wherein said composition contains the library that comprises at least 10 kinds of nucleic acid.

91. produce the method for the interferon-' alpha ' homologue nucleic acid of modified or reorganization, described method comprises makes claim 1,2,3,8 or 18 nucleic acid mutation.

92. the interferon-' alpha ' homologue nucleic acid of the modified or reorganization of producing by the method for claim 91.

93. contain the computer or the computer-readable medium of database, described database contains sequential recording, described sequential recording contains one or more corresponding to the nucleic acid that is selected from SEQ ID NO:1 to SEQ ID NO:85 or the character chain of protein sequence.

94. integrated system, it contains the computer or the computer-readable medium of database, contain one or more sequential recordings in the described database, each described sequential recording contains one or more corresponding to the nucleic acid that is selected from SEQ ID NO:1 to SEQ ID NO:85 or the character chain of protein sequence, this integrated system also contains user's inputting interface, and it can make the user optionally check described one or more sequential recording.

95. the integrated system of claim 94, wherein computer or computer-readable medium contain the comparison instruction set, can compare character chain and one or more other, corresponding to the character chain of nucleic acid or protein sequence.

96. the integrated system of claim 95, wherein instruction set contains one or more in following: local homology's comparative measurement, and the homology sequence comparison is measured, and similarity retrieval is measured and BLAST mensuration.

97. the integrated system of claim 95, it further contains the readable output element of user, can show the comparison result that produces by the sequence alignment instruction set.

98. the integrated system of claim 94, wherein computer or computer-readable medium also contain a kind of instruction set, the near few a kind of nucleotide sequence of this instruction energy collecting is translated into aminoacid sequence, and described nucleotide sequence contains the sequence that is selected from SEQ ID NO:1 to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78.

99. the integrated system of claim 94, wherein computer or computer-readable medium also contain a kind of instruction set, the near few a kind of aminoacid sequence reverse translation of this instruction energy collecting becomes nucleotide sequence, and described aminoacid sequence contains the sequence that is selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ IDNO:85.

100. the integrated system of claim 99, wherein this instruction set can be applied to be tried nucleotide sequence by the instruction set of codon being used instruction set or mensuration sequence identity and select nucleotide sequence.

101. the system of using a computer presents the method for at least a institute inherent information of a plurality of sequential recordings that store in the database, each described sequential recording contains at least a character chain corresponding to SEQ ID NO:1 to SEQ ID NO:85, and this method comprises:

Determine tabulation corresponding at least a character chain of one or more or its subsequence among SEQ ID NO:1 to the SEQ ID NO:85;

It is any to determine that the user selects at least a character chain of described tabulation; With

Show every kind of selected character chain, or every kind of selected character chain and a kind of additional features chain are compared.

102. the method for claim 101, it further comprises the sequence alignment that shows every kind of selected character chain and additional features chain.

103. the method for claim 101, it further comprises display list.

104. nucleic acid, it contains unique subsequence in the nucleic acid that is selected from SEQ ID NO:1 to SEQ ID NO:35 or SEQ IDNO:72 to SEQ ID NO:78, wherein compare with the nucleotide sequence of known disturbances element-'alpha ' nucleic acids sequence or corresponding to the nucleic acid of following any GenBank registration number, the subsequence of this uniqueness is unique: and J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1).

A 105. peptide species, it contains unique subsequence in the polypeptide that is selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ IDNO:79 to SEQ ID NO:85, with known disturbances element-α peptide sequence, or compare by sequence corresponding to the coded polypeptide of nucleic acid of following any GenBank registration number, the subsequence of this uniqueness is unique: and J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, and V00549 (α-2a) and I08313 (α-Con1).

106. target nucleic acid, it is hybridized with the oligonucleotides coding of uniqueness under stringent condition, described oligonucleotide coding is selected from the unique subsequence in the polypeptide of SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ IDNO:85, wherein with known disturbances element-α peptide sequence, or compared by the sequence corresponding to the coded polypeptide of nucleic acid of following any GenBank registration number, the subsequence of this uniqueness is unique: and J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1).

107. the nucleic acid of claim 106, wherein select stringent condition so that can hybridize with the oligonucleotides coding of uniqueness with the accurate complementary oligonucleotide of the oligonucleotides coding of uniqueness, and its signal/noise ratio than accurate complementary oligonucleotide with corresponding to the signal/noise ratio of following any GenBank number contrast nucleic acid hybridization at least about high 5 times: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14), V00545 (IFN-1B), X03125 (α-8), X02957 (α-16), V00540 (α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), the oligonucleotides coding hybridization of wherein said target nucleic acid and described uniqueness, and its signal/noise ratio will be compared the signal/noise ratio of photograph nucleic acid and the hybridization of this oligonucleotides coding at least about high 2 times.

108. claim 1,2,3, each nucleic acid in 8 or 18, nucleic acid encoding interferon-' alpha ' homologue wherein, with respect to the growth inhibitory activity of human interferon-alpha 2a for cancer cell population, described homologue strengthens to some extent to the growth inhibitory activity of described cancer cell population.

109. containing, the nucleic acid of claim 108, the cancer cells of wherein said cancer cell population is selected from following cancerous cell line: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; Central nervous system (CNS) cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line, growth inhibitory activity is measured as the concentration (GI50 value) that the growth 50% that makes cancerous cell line is suppressed required interferon-' alpha ' homologue, and wherein the GI50 value of interferon-' alpha ' homologue is than low at least 2 times of the GI50 value of human interferon-alpha 2a.

110. the nucleic acid of claim 109, the GI50 value of wherein coded interferon-' alpha ' homologue is hanged down at least 5 times than the GI50 value of human interferon-alpha 2a.

111. the nucleic acid of claim 107, the GI50 value of wherein coded interferon-' alpha ' homologue is hanged down at least 10 times than the GI50 value of human interferon-alpha 2a.

112. claim 1,2,3, each nucleic acid in 8 or 18, wherein said nucleic acid encoding interferon-' alpha ' homologue, with respect to the cell static activity of human interferon-alpha 2a for cancer cell population, described homologue strengthens to some extent to the static activity of cell of described cancer cell population.

113. the nucleic acid of claim 112, wherein cancer cells contains and is selected from following cancerous cell line: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; The CNS cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line, the static activity of cell is measured as the concentration (TGI value) of the interferon-' alpha ' that the growth that causes clone is subjected to suppressing required fully, and wherein the TGI value of interferon-' alpha ' homologue is than low at least 2 times of the TGI value of human interferon-alpha 2a.

114. the nucleic acid of claim 112, the TGI value of wherein coded interferon-' alpha ' homologue is hanged down at least 5 times than the TGI value of human interferon-alpha 2a.

115. the nucleic acid of claim 112, the TGI value of wherein coded interferon-' alpha ' homologue is hanged down at least 10 times than the TGI value of human interferon-alpha 2a.

116. claim 1,2,3, each nucleic acid in 8 or 18, wherein said nucleic acid encoding interferon-' alpha ' homologue, with respect to the cytotoxic activity of human interferon-alpha 2a for cancer cell population, described homologue strengthens to some extent to the cytotoxic activity of described cancer cell population.

117. the nucleic acid of claim 116, wherein cancer cells contains and is selected from following cancerous cell line: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; Central nervous system (CNS) cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line, cytotoxic activity is measured as: make the amount of cell protein in the clone of measuring after the soak reduce the concentration (LC50 value) of 50% required interferon-' alpha ', wherein the LC50 value of interferon-' alpha ' homologue is hanged down at least 2 times than the LC50 value of human interferon-alpha 2a.

118. the nucleic acid of claim 116, the LC50 value of wherein coded interferon-' alpha ' homologue is hanged down at least 5 times than the LC50 value of human interferon-alpha 2a.

119. the nucleic acid of claim 116, the LC50 value of wherein coded interferon-' alpha ' homologue is hanged down at least 10 times than the LC50 value of human interferon-alpha 2a.

120. claim 31, each polypeptide in 34,37,41 or 51, active for the inhibition of cancer cell population with respect to human interferon-alpha 2a, described polypeptide strengthens to some extent to the growth inhibitory activity of described cancer cell population.

121. the polypeptide of claim 120, wherein cancer cell population contains and is selected from following cancerous cell line: leukemia cell system; Melanoma cell series; Lung cancer cell line; Colon carcinoma cell line; The CNS cancerous cell line; Ovarian cancer cell line; Breast cancer cell line; Prostate cancer cell line and renal carcinoma cell line, growth inhibitory activity is measured as the concentration (GI50 value) that the growth 50% that causes clone is suppressed required polypeptide or human interferon-alpha 2a, and wherein the GI50 value of polypeptide is than low at least 2 times of the GI50 value of human interferon-alpha 2a.

122. the nucleic acid that produces by the method for claim 81.

123. the interferon-alpha polypeptides or its amino acid subsequence that produce by the method for claim 81.