CN102199210B

CN102199210B - Anti-interleukin-8 antibody

Info

Publication number: CN102199210B
Application number: CN 201110075269
Authority: CN
Inventors: 华绍炳; 余乃绚
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-03-28
Filing date: 2011-03-28
Publication date: 2013-04-17
Anticipated expiration: 2031-03-28
Also published as: CN102199210A

Abstract

The invention relates to anti-interleukin-8 antibody, comprising a heavy chain variable region and a light chain variable region. The amino acid sequences of the three hypervariable regions of CDRH1, CDRH2 and CDRH3 in the heavy chain variable region are respectively GGSFSGYYM, SIYYSGSTYYNPSLKS and HDYYDFWSGYYDAFDI; and the amino acid sequences of the three hypervariable regions of CDRL1, CDRL2 and CDRL3 in the light chain variable region are respectively TGSSSDVGDYYYVS, DVNYRPS and SSYRDTQILG. The anti-interleukin-8 antibody provided by the invention has the advantages of whole humanization, high specificity, relatively simple preparation process and low cost.

Description

Anti-interleukin-8 antibody

Technical field

The present invention relates to biological technical field, relate in particular to a kind of anti-interleukin-8 antibody.

Background technology

Antibody (antibody) is body under antigenic substance stimulates, that the plasmocyte that is become by the B cytodifferentiation produces, can with the immunoglobulin (Ig) of corresponding antigens generation specific binding reaction.

As shown in Figure 1, antibody structure can resolve into several forms according to function: whole antibody (IgG), Fab (antigen-binding fragment, Fab) and single-chain antibody (scFv), every kind of antibody includes heavy chain (H chain) and light chain (L chain).

On heavy chain and the light chain variable region is arranged, the variable region is comprised of 4 framework regions (FRs) that mutually replace and 3 hypervariable regions (CDRs).The aminoacid sequence of framework region is relatively conservative, and the amino acid of hypervariable region forms and easier the changing that put in order, and has determined the specificity of antibodies antigen.The upper in addition constant region of IgG and Fab, in given species, the constant region of different antibodies molecule all has identical or almost identical aminoacid sequence.

Antibody-antigen-reactive is a kind of protein-protein interaction of uniqueness.One of method that Study on Protein-protein interaction is the most frequently used is yeast-two hybrid technique (the Marks JD by Fields and colleague's invention thereof, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. (1991) J Mol Biol.222:581-597.) (Fields, S and Song, O. (1989) Nature 340:245-247.), this technology only needs dna sequence dna and nonprotein just can be determined part (the Bartel PL of combination, Fields S (ed.) (1997) " The Yeast Two-Hybrid System (Advances in Molecular Biology) " Oxford University Press.) (Zhu L and Hannon (ed.) (2000) " Yeast Hybrid Technologies " Eaton Publishing, Natick, MA.) (Fields S, Sternglanz R. (1994) Trends Genet.10:286-292.).The principle of yeast-two hybrid technique is to utilize interactional two protein components that the transcription factor that merges with this two component is combined also to activate transcribing of nuclear reporter gene, make this technology be widely used in Study on Protein-protein interaction (De Jaeger G. et al (1997) FEBS Lett.403:116-122.) (Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A, Galeffi P. (2000) Nature.366:469-472.), or research (the Visintin M of intracellular antibody (intrabodies), Tse E, Axelson H, Rabbitts TH, Cattaneo A. (1999) Proc Natl Acad Sci U S is A.96:11723-11728.) (De Jaeger G. Fiers E, Eeckhout D, Depicker A. (2000) FEBS Lett.467:316-320.) (Portner-Taliana A, Russell M, Froning KJ, Budworth PR, Comiskey JD, Hoeffler JP. (2000) J Immunol Methods.238:161-172.).

Interleukin 8 (Interleukin-8, or claim IL-8) be a member in Gro-beta-T (Chemokine) family.Known IL-8 is strong chemotactic and incitant (the Schroder JM of a kind of neutrophilic granulocyte (Neutrophil), Mrowietz U, Morita E, Christophers E. (1987) JImmunol.139:3474-3483.) (Peveri P, Walz A, Dewald B, Baggiolini M. (1988) J Exp Med.167:1547-1559.).Think that at present IL-8 can strongly compile and activate neutrophilic granulocyte, widely biological activity (Schall TJ, Bacon KB. (1994) Curr Opin Immunol.6:865-873.) is arranged in many physiology and pathologic process.Excessive IL-8 can cause the autoimmune diseases (Baggiolini M, Clark-Lewis I. (1992) FEBS Lett.307:97-101) such as inflammation.IL-8 can be produced by the emiocytosis of multiple participation inflammatory reaction, such as scavenger cell, neutrophilic granulocyte, lymphocyte, endotheliocyte, keratinocyte etc., then act on the relevant cells of inflammation such as endotheliocyte, neutrophilic granulocyte and T lymphocyte, thereby form inflammatory reaction.A lot of human diseasess are unusual or excessive, unbalance relevant with the interior IL-8 expression and secretion of body.

Chinese patent 200410089202.4 disclose a kind of have in and the variable region sequences of the bioactive monoclonal antibody of interleukin 8 (IL-8) and this monoclonal antibody.This patent is take IL-8 as immunogenic, take mouse as immune object, by the immunization mouse, get the immune mouse spleen and prepare suspension, and make it to carry out cytogamy with the myeloma cell and obtain to express the hybridoma cell strain of anti-IL-8 monoclonal antibody to prepare anti-IL-8 monoclonal antibody.

Chinese patent application 01813639.7 discloses a kind of method of producing antibody library and screening antibodies in yeast, in yeast cell, express the test protein library, this test fused protein is included in the first polypeptide subunit of sequence variations in the library, its sequence is independent of the second polypeptide subunit of the first polypeptide subunit variation in the library, and is connected a joint peptide that connects the first and second polypeptide subunits; Express one or more target fused proteins in the yeast cell of expressing test protein, every kind of target fused protein comprises a target peptide or protein; Those express the yeast cell of reporter genes with screening, and the expression of this report gene is tested fused protein and is attached on the target fused protein and activates.

Summary of the invention

The invention provides the high anti-interleukin-8 antibody of a species specificity.

A kind of anti-interleukin-8 antibody comprises variable region of heavy chain and variable region of light chain, and 3 hypervariable region CDRH1, CDRH2 in the variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively GGSFSGYYW, SIYYSGSTYYNPSLKS, HDYYDFWSGYYDAFDI; The aminoacid sequence of 3 hypervariable region CDRL1, CDRL2, CDRL3 is respectively TGSSSDVGDYYYVS, DVNYRPS, SSYRDTQILG in its variable region of light chain.

A kind of anti-interleukin-8 antibody, its variable region of heavy chain have the described aminoacid sequence of SEQ ID NO.1; Described variable region of light chain has the described aminoacid sequence of SEQ ID NO.2.

Above-mentioned antibody can be whole antibody, antibody fragment or single-chain antibody.

The present invention also provides a kind of gene of above-mentioned Ro 24-7472/000-8 antibody of encoding, and the dna sequence dna of encoding heavy chain variable region and variable region of light chain is respectively shown in SEQ ID NO.3 and SEQ ID NO.4.

Above-mentioned antibody can be applicable to the medicine for the preparation for the treatment of relevant disease unusual, excessive with Ro 24-7472/000-8 expression and secretion or out of control.

Anti-interleukin-8 antibody specificity of the present invention is high, and preparation process is relatively simple, and is with low cost.

Description of drawings

Fig. 1 is the structural representation of 3 types of antibody;

Fig. 2 is the structural representation of single-chain antibody of the present invention;

Fig. 3 is #IL-8-31 antibodies specific test antibody concentration of the present invention and OD ₄₅₀Graph of relation;

Fig. 4 is #IL-8-21 antibodies specific test antibody concentration of the present invention and OD ₄₅₀Graph of relation;

Fig. 5 is the graph of relation of #IL-8-31 antibody concentration of the present invention and IL8 trend inhibiting rate;

Fig. 6 is the graph of relation of #IL-8-21 antibody concentration of the present invention and IL8 trend inhibiting rate.

Embodiment

The present invention adopts patent application 01813639.7 disclosed method to make up people's single-chain antibody library, and screens IL-8 antibody in people's single-chain antibody library, and specific implementation process is as follows:

Embodiment 1 reverse transcription and amplification obtain human antibody heavy chain and light chain DNA

Take from the polyA+RNA (available from Clontech) of people's marrow, people's tire liver, people's spleen and human peripheral leucocytes as template, and use the reverse transcriptase test kit of buying from Clontech, take oligo (dT) and random primer (random primers) as primer, the Methods Instruction that provides according to the Clontech test kit is with the reverse cDNA that is transcribed into of polyA+RNA.Take above-mentioned cDNA as template, use the primer of a series of identification human antibody heavy chain variable regions (VH) and variable region of light chain (VL) gene, use heavy chains all in the PCR method amplification people antibody and the variable region of light chain.The primer sequence of a series of identification human antibody heavy chains and chain variable region gene is as follows:

5 ' of the 1st group of human antibody heavy chain variable region (VH) gene-end primer

VH1b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTG?CAG?CTGCAG?GAG?TC(C/G)G

VH2b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTA?CAG?CTGCAG?CAG?TCA

VH3b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTG?CAG?CTACAG?CAG?TGG?G

VH4b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?GAG?GTG?CAG?CTG(G/T)TG?GAG(A/T)C(C/T)

VH5b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTC?CAGCT(G/T)GT(A/G)CAG?TCT?GG

VH6b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?(A/G)TC?ACCTTG?AAG?GAG?TCT?G

VH7b：5’- CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTG?CAG?CTGGTG(C/G)A(A/G)TCT?GG

3 ' of the 2nd group of human antibody heavy chain variable region (VH) gene-end primer

VH1f：5’- GCC?GCC?TGA?TCC?ACC?ACC?GCC?TGA?GGA?GAC?(A/G)GT?GAC?CAG?GGT?G

VH2f：5’- GCC?GCC?TGA?TCC?ACC?ACC?GCC?TGA?GGA?GAC?GGTGAC?CAG?GGT?T

VH3f：5’- GCC?GCC?TGA?TCC?ACC?ACC?GCC?TGA?AGA?GAC?GGTGAC?CAT?TGT

VH4f：5’- GCC?GCC?TGA?TCC?ACC?ACC?GCC?TGA?GGA?GAC?GGTGAC?CGT?GGT?CC

VH5f：5’- GCC?GCC?TGA?TCC?ACC?ACC?GCC?GGT?TGG?GGC?GGATGC?ACT?CC

VH6f：5’- GCC?GCC?TGA?TCC?ACC?ACC?GCC?(C/G)GA?TGG?GCCCTT?GGT?GGA(A/G)GC

5 ' of the 3rd group of people's antibody λ-variable region of light chain (V λ) gene-end primer

VL1b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?TCT?GT(C/G)(C/G/T)TG?ACG?CAG?CCG?CC

VL2b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?TCC?TAT?G(A/T)GCTG?AC(A/T)CAG?CCA?C

VL3b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?TCC?TAT?GAG?CTGA(C/T)(A/G)CAG?C(C/T)A?CC

VL4b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?CCT?GTG?CTGACT?CA(A/G)(C/T)C

VL5b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?(A/G/T)CT?GTGGTG?AC(C/T)CAG?GAG?CC

VL6b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?CC(A/T)G(G/T)G?CTG?ACT?CAG?CC(A/C)CC

VL7b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?TCC?TCT?GAG?CTGA(C/G)T?CAG?GA(C/G)CC

VL8b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?TCT?G(C/T)(C/T)CTGA(C/T)T?CAG?CCT

VL9b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?AAT?TTT?ATG?CTGACT?CAG?CCC?C

3 ' of the 4th group of people's antibody λ-variable region of light chain (V λ) gene-end primer

VL1f：5’- GGG?GTT?TTT?CAG?TAT?CTA?CGA?TAG?GAC?GGT(C/G)A(C/G)CTT?GGT?CC

VL2f：5’- GGG?GTT?TTT?CAG?TAT?CTA?CGA?GAG?GAC?GGT?CAGCTG?GGT?GC

5 ' of the 5th group of people's antibody κ-variable region of light chain (V κ) gene-end primer

VK1b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?GAC?ATC?C(A/G)G(A/G/T)TG?ACC?CAG?TCT?CC

VK2b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?GAA?ATT?GT(A/G)(A/T)TG?AC(A/G)CAG?TCT?CC

VK3b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?GAT?ATT?GTG(A/C)TG?AC(C/G/T)CAG(A/T)CT?CC

VK4b：5’- GGC?AGC?GGT?GGT?GGA?GGC?AGT?GAAACG?ACA?CTCACG?CAG?TCT?C

3 ' of the 6th group of people's antibody κ-variable region of light chain (V κ) gene-end primer

VK1f：5’- GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?GAT?TTC?CACCTT?GGT?CC

VK2f：5’- GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?GAT?CTCCA(C/G)CTT?GGT?CC

VK3f：5’- GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?GAT?ATC?CACTTT?GGT?CC

VK4f：5’- GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?AAT?CTC?CAGTCG?TGT?TC

The combination of using above-mentioned the 1st group of primer and the 2nd group of primer during variable region of heavy chain (VH) in the PCR method amplification people antibody namely has 42 PCR to react; Similarly, the combination of using above-mentioned the 3rd group of primer and the 4th group of primer during lambda light chain variable district (V λ) in the amplification people antibody namely has 18 PCR to react; The combination of using above-mentioned the 5th group of primer and the 6th group of primer during kappa light chain variable district (V κ) in the amplification people antibody namely has 16 PCR to react.

Include yeast two-hybrid carrier pACT2 (Hua SB in the 1st group of primer, Luo Y, Qiu M, Chan E, Zhou H, Zhu L. (1998) Gene.215:143-152.) (Hua SB, Qiu M, ChanE, Zhu L, Luo Y. (1997) Plasmid.38:91-96.) homologous sequence (underscore part) of multiple clone site upstream; Include the homologous sequence (underscore part) with yeast two-hybrid carrier pACT2 multiple clone site downstream in the 4th group and the 6th group of primer; And include the sequence (underscore part) of connection peptides (linker) in the 2nd group, the 3rd group and the 5th group of primer.Connection peptides is used for connecting the heavy chain of antibody and the variable region of light chain.

The reaction conditions of all heavy chain and variable region of light chain is as follows in the PCR method amplification people antibody:

5.0 μ l PCR damping fluid (10x)

1.0 μ l 3 '-end primer (10 μ M)

1.0 μ l 5 '-end primer (10 μ M)

5.0 μ l cDNA substrate

1.0μl?dNTP(10mM)

36 μ l water

1.0 the Taq of unit polysaccharase (Clontech production)

Above-mentioned each component mixed to be placed in the PCR instrument (Applied BioSystems company) react.The parameter of PCR reaction is for unwinding 94 ℃, 50 ℃ of 1 minutes, annealing, 1 minute, extend 72 ℃, 2.5 minutes, circulate 30 times.

The homologous sequence of embodiment 2 connection carrier multiple clone site and connection peptides sequence

Take the human antibody heavy chain variable region DNA of PCR gained described in the embodiment 1 as template, be respectively upstream primer and downstream primer with primer 7 and primer 8, adopt the PCR method to increase, reaction conditions is the same.Primer 7 sequences are the upstream homologous sequence of carrier pACT2 multiple clone site, and primer 8 sequences are connection peptides anti-chain sequence.

Primer 7 (pACT2 multiple clone site upstream homologous sequence)

5’-ACC?CCA?CCA?AAC?CCA?AAA?AAA?GAG?ATC?TGT?ATG?GCT TAC?CCA?TAC?GAT?GTT?CCA?GAT?TAC

Primer 8 (connection peptides anti-chain sequence)

5’-ACT?GCC?TCC?ACC?ACC?GCT?GCC?ACC?TCC?GCC?AGA?TCCTCC? GCC?GCC?TGA?TCC?ACC?ACC?GCC

Take the human antibody light chain variable region DNA of PCR gained described in the embodiment 1 as template, be respectively downstream primer and upstream primer with primer 9 and primer 10, adopt the PCR method to increase, reaction conditions is the same.Primer 9 is the downstream homologous sequence of carrier pACT2 multiple clone site, and primer 10 is that connection peptides is along chain-ordering.

Primer 9 (pACT2 multiple clone site downstream homologous sequence)

5’-GAG?ATG?GTG?CAC?GAT?GCA?CAG?TTG?AAG?TGA?ACT?TGC GGG?GTT?TTT?CAG?TAT?CTA?CGA

Primer 10 (connection peptides is along chain-ordering)

5’-GGC?GGT?GGT?GGA?TCA?GGC?GGC?GGA?GGA?TCT?GGC?GGAGGT? GGC?AGC?GGT?GGT?GGA?GGC?AGT

Embodiment 3 connects into single-chain antibody with human antibody heavy chain and light chain DNA by the connection peptides sequence

The homologous sequence that contains carrier pACT2 multiple clone site that pcr amplification among the embodiment 2 is obtained and the human antibody heavy chain variable region DNA of connection peptides sequence and variable region of light chain DNA mix.Take this hybrid dna as template, be respectively upstream primer and downstream primer with primer 7 and primer 9, carry out pcr amplification, obtain comprising single-chain antibody (scFv) DNA of human antibody heavy chain's dna sequence dna, light chain dna sequence dna, carrier pACT2 multiple clone site homologous sequence and connection peptides dna sequence dna, reaction conditions is with embodiment 1

Embodiment 4 makes up people's single-chain antibody (scFv) gene library

Above-described embodiment 3 described single-chain antibody (scFv) DNA both sides are connected to the homologous sequence of yeast two-hybrid carrier pACT2 (production of Clontech company) multiple clone site both sides.Method (the Yeast Protocol Handbook that above-described embodiment 3 described single-chain antibody (scFv) DNA and yeast two-hybrid carrier pACT2 after restriction enzyme (Bam HI and Eco RI) is processed are provided according to Clontech company, PT3024-1) its common conversion is entered yeast strain Y187 (genotype MAT α ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112

Gal80, URA3::GAL1UAS-GAL1TATA-lacZ) in, in cell, after the homologous recombination single-chain antibody (scFv) DNA is incorporated on the pACT2 carrier, thereby obtains yeast two-hybrid single-chain antibody (scFv) library.Gal4 active region on single-chain antibody (scFv) dna fragmentation and the pACT2 carrier (Activation Domain, AD) merges.

For checking the quality in this people's single-chain antibody (scFv) library, 30 clones have been chosen at random.The Insert Fragment sequencing analysis is shown that all clones comprise single-chain antibody (scFv) dna fragmentation (data do not show) that merges with Gal4, and all single-chain antibodies (scFv) dna sequence dna all is unique.

Above-mentionedly obtain antibody dna copy number nearly 1 * 10 in yeast two-hybrid single-chain antibody (scFv) gene library through homologous recombination ⁸Individual, can be applicable to yeast-two hybrid technique and screen specific antibody.

Embodiment 5 screening antibodies

The dna clone of encoding human IL-8 is entered carrier pGBKT7 (Clontech company) construct pGBK-IL-8.PGBK-IL-8 coding Gal4DNA calmodulin binding domain CaM (Binding Domain, BD) and the N end that merges at people IL-8.

After the dna sequence dna of encoding human IL-8 is verified, the pGBK-IL-8 plasmid DNA transform into yeast strain AH109 (genotype MAT α, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,112,

Gal80, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ) (Clontech company).AH109 yeast with the pGBK-IL-8 plasmid can be grown at the synthetic medium that does not contain tryptophane (SD/-W).

When carrying out co-cultivation, this two types cell can the mating combination with the MAT α type yeast cell (AH109 bacterial strain) that contains pGBK-IL-8 of equivalent and the MAT α type yeast cell (Y187 bacterial strain) that comprises single-chain antibody (scFv) gene library.

The pACT2 carrier that is loaded with single-chain antibody (scFv) library contains the LEU2 gene, and pGBK-IL-8 comprises the TRP1 gene.The yeast cell that comprises above-mentioned two kinds of plasmids can be grown in the yeast synthetic medium (SD/-LW) that does not contain leucine and Serine.Exist the interactional cell of scFv/IL-8 can activate reporter gene ADE2 and the HIS3 that is incorporated in the strain gene group, thereby so that yeast cell can be grown in the shortage VITAMIN B4, Histidine, on the substratum of leucine and tryptophane (SD/-AHLW), and form bacterium colony at this plate culture medium.

On above-mentioned screening culture medium (SD/-AHLW), altogether pick out 20 bacterium colonies.Then these bacterium colonies are done further to analyze.

The method of at first, pressing Clontech company uses the beta galactosidase enzyme detection method to detect the lacZ expression.Another reporter gene lacZ that exists the interactional cell of scFv/IL-8 also can activate to be incorporated in the strain gene group, thus can record the beta galactosidase enzyme of expressing in the yeast cell.The method that detects the yeast cell beta galactosidase enzyme is as follows:

(1) above-mentioned yeast is in the dull and stereotyped growth of screening culture medium (SD/-AHLW).

(2) yeast colony of survival is transferred on No. five filter paper of Whatman.

(3) will immerse among the liquid nitrogen with the filter paper of yeast cell bacterium colony, make cell rupture.

(4) filter paper is taken out from liquid nitrogen, and place in 30 ℃ the baking oven.

(5) repeating step 3 and 4 secondaries.

(6) filter paper is laid in an amount of X-gal solution, and placed in 37 ℃ the incubator about 15 minutes.If show blueness with the bacterium colony place, be indicated as the beta galactosidase enzyme positive.

The X-gal solution formula is as follows:

Detected result: have 12 to be the beta galactosidase enzyme positive in above-mentioned 20 bacterium colonies picking out, show that another reporter gene lacZ also has been activated in the cell of these bacterium colonies.

Secondly, the scFv of the positive bacterium colony of above-mentioned 12 beta galactosidase enzymes is carried out specificity analyses, whether be combined with the IL-8 part specifically to determine scFv, rather than and other parts.The pACT2 plasmid DNA that will contain the scFv gene extracts from the yeast of above-mentioned 12 beta galactosidase enzyme positives.And respectively with the pGBKT empty carrier, or with the plasmid pGBKT-Lam (Clontech company) that contains coding and merge Gal4-DNA land (BD) and human nuclear fabric layer albumen C encoding sequence, jointly be transformed in the AH109 yeast cell.Yeast cell after the conversion is laid on first the upper growth of plate culture medium (SD/-LW), then transfers on the plate culture medium (SD/-AHLW), and further uses the beta galactosidase enzyme analysis.

After aforesaid method carries out specificity analyses to scFv, obtain 3 people IL-8 had specific single-chain antibody (scFv).

At last, to above-mentioned 3 people IL-8 is had specific single-chain antibody (scFv) and carry out sequencing analysis, use the ABI automatic sequencer to measure the dna sequence dna of scFv.

The dna sequence dna of analyzing these single-chain antibodies (scFv) clone finds to have the single-chain antibody (scFv) of two different anti-human interleukin 8s.Wherein identical with clone #IL-8-31 have 2, and another is clone #IL-8-21.

The aminoacid sequence of single-chain antibody heavy chain (VH) variable region of clone #IL-8-31 is shown in SEQ IDNO.1, and the aminoacid sequence of single-chain antibody heavy chain (VL) variable region is shown in SEQ ID NO.2; The dna sequence dna of single-chain antibody heavy chain (VH) variable region of coding clone #IL-8-31 is shown in SEQ IDNO.3, and the dna sequence dna of single-chain antibody light chain (VL) variable region of coding clone #IL-8-31 is shown in SEQ ID NO.4.

The aminoacid sequence (SEQ ID NO.1) of single-chain antibody heavy chain (VH) variable region of #IL-8-31

QVQLQESGPGLVKPSETLSLTCAVY GGSFSGYYWSWIRQPPGKGLEWIG SIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR HDYYDFWSGYYDAFDIWGQGTMVTVSS

Underscore partly is followed successively by three hypervariable region CDRH1, CDRH2 in the variable region of heavy chain and the aminoacid sequence of CDRH3.

The aminoacid sequence (SEQ ID NO.2) of single-chain antibody light chain (VL) variable region of #IL-8-31

QSALIQPASVSGPPGQSITISC TGSSSDVGDYYYVSWYQQHPGKAPKLLIY DVNYRPSGVSSRFSGSKSGNTASLTISGLQAEDEADYYC SSYRD TQILGVFGGGTKLTVL

Underscore partly is followed successively by three hypervariable region CDRL1, CDRL2 in the variable region of light chain and the aminoacid sequence of CDRL3.

The aminoacid sequence of single-chain antibody heavy chain (VH) variable region of clone #IL-8-21 is shown in SEQ IDNO.5, and the aminoacid sequence of single-chain antibody heavy chain (VL) variable region is shown in SEQ ID NO.6; The dna sequence dna of single-chain antibody heavy chain (VH) variable region of coding clone #IL-8-21 is shown in SEQ IDNO.7, and the dna sequence dna of coding single-chain antibody light chain (VL) variable region is shown in SEQ ID NO.8.

The aminoacid sequence (SEQ ID NO.5) of single-chain antibody heavy chain (VH) variable region of #IL-8-21

EVQLLETGGGLVQPGGSLRLSCSAS GFSFSSFEMNWVRQAPGKGLEWLS YISKSGFTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SSWAQDFDWLLPFDWWGQGTLVTVSS

The aminoacid sequence (SEQ ID NO.6) of single-chain antibody light chain (VL) variable region of #IL-8-21

ETTLTQSPSSVSAYVGDRVTITC RASQGIRNYLAWYQQKPGKVPNLLIY AASTLQSGVPSRFSGSGSETDFTLTISSLQPEDVATYYC QKYNSVPFTFGPGTKVDIK

Embodiment 6 antibody expressions and purifying

With single-chain antibody (scFv) dna clone of the anti-human IL-8 that obtains of screening among the embodiment 5 pET27b (+) (Novagen company) and obtain pET27b-aIL-8 in the protein expression carrier.PET27b-aIL-8 behind the clone is pelB sequence (the scFv protein secreting after expressing can being arrived in the pericentral siphon chamber (periplasmic space) of expressing bacterium E.coli) at the N of scFv end, and the C end contains the sequence of one section coding HSV marker and a 6x His (Histidine) marker (can be used for protein purification).PET27b-aIL-8 is transformed into protein expression bacterium E.coli bacterial strain BL21DE3 (Novagen company).And the method that provides according to the said firm is with IPTG (concentration 0.5mM) abduction delivering and the single-chain antibody scFv protein that separates anti-human IL-8.

Because the C of scFv protein end contains a 6x His marker, the method that we can use Qiagen company provides uses Ni-NTA post (Qiagen company) to come purifying scFv protein easily.Protein SDS-PAGE gel electrophoresis analysis before and after the purifying.

The scFv characteristic of the anti-IL-8 of embodiment 7 elisa immunoassays (ELISA) test

We have tested the characteristic of the anti-IL-8 single-chain antibody behind the purifying.First characteristic is to measure the immune affine of antibody and IL-8 with elisa immunoassay (ELISA).

The protein of recombinant human IL-8 is available from Pierce company.The 96-orifice plate (the MaxiSorp plate is available from Nunc company) that is used for elisa immunoassay (ELISA).Method is as follows:

(1) above-mentioned 96-orifice plate is at first coated with IL-8.

(2) the 96-orifice plate after coated uses SuperBlock (available from Pierce company) to make sealing treatment again.

(3) sublimed anti-IL-8 single-chain antibody is done to add behind the serial dilution in 0.02%BSA and has been coated with in the 96-hole of IL-8, is combined with IL-8.

(4) with after the cleaning of 96-orifice plate, the antibody (available from Novagen company) that adds again the mouse-anti HSV marker that has diluted 5000 times in the 96-hole is used for detecting the single-chain antibody (containing a HSV marker at the C of scFv end) that combines.

(5) the 96-orifice plate is cleaned after, in the 96-hole, add again the conjugate (available from Sigma company) that the goat dynamics that diluted 10000 times and coupling have HRP (horseradish peroxidase).

(6) with after the final cleaning of 96-orifice plate, use and make color development treatment available from the HRP substrate TMB reagent of Sigma company.

(7) last, reaction stops with the sulfuric acid of 0.5M, and detects the absorption spectrum of 450nm.

The result as shown in Figure 3 and Figure 4, presentation of results clone #IL-8-31 and two single-chain antibodies of clone #IL-8-21 can both be combined IL-8 effectively.

Embodiment 8 test I L-8 single-chain antibodies are to the chemotaxis of IL-8

Adopt anti-IL-8 single-chain antibody behind micropore cell (follow-on Boyden Chamber) the chemotactic experiment test purifying to the chemotactic restraining effect of IL-8, experimental procedure is as follows:

(1) the anti-IL-8 single-chain antibody behind the protein of recombinant human IL-8 (concentration is 10ng/ml) and the purifying or mouse-anti people IL-8 antibody (available from Pharmingen company) or be blended in (concentration of these three kinds of antibody is 1 ug/ml or 10 ug/ml) in the substratum with the incoherent anti-p-53 single-chain antibody of IL-8, and place the bottom compartment of follow-on Boyden Chamber (available from New Probe company).

(2) neutrophilic granulocyte (2 * 10 after the separation ⁵Cell) is added on the upper chamber of follow-on Boyden Chamber.There is the poly-carbon film of aperture 3.0 μ m to be separated by between the upper and lower chamber.

(3) add the Boyden Chamber that puts behind the cell at 37 ℃ CO ₂Insulation is one hour in the incubator.

(4) neutrophilic granulocyte that moves to lower chamber is observed at microscopically.

The chemotactic experimental results as shown in Figure 5 and Figure 6, two single-chain antibodies of presentation of results clone #IL-8-31 and clone #IL-8-21 can both suppress IL-8 effectively to the chemotaxis of cell.

Claims

1. anti-interleukin-8 antibody, comprise variable region of heavy chain and variable region of light chain, it is characterized in that 3 hypervariable region CDRH1, CDRH2 in the variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively GGSFSGYYW, SIYYSGSTYYNPSLKS, HDYYDFWSGYYDAFDI; The aminoacid sequence of 3 hypervariable region CDRL1, CDRL2, CDRL3 is respectively TGSSSDVGDYYYVS, DVNYRPS, SSYRDTQILG in its variable region of light chain.

2. anti-interleukin-8 antibody according to claim 1, it is characterized in that: described variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO.1; Described variable region of light chain has the aminoacid sequence shown in the SEQ ID NO.2.

3. anti-interleukin-8 antibody according to claim 1 and 2, it is characterized in that: described antibody is single-chain antibody.

4. gene of claim 1 or 2 described anti-interleukin-8 antibodies of encoding.