CN102250243A - Antibody to interleukins-15 - Google Patents
Antibody to interleukins-15 Download PDFInfo
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- CN102250243A CN102250243A CN2011101842802A CN201110184280A CN102250243A CN 102250243 A CN102250243 A CN 102250243A CN 2011101842802 A CN2011101842802 A CN 2011101842802A CN 201110184280 A CN201110184280 A CN 201110184280A CN 102250243 A CN102250243 A CN 102250243A
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Abstract
The invention discloses an antibody to interleukins-15. The antibody comprises heavy chain variable zones and light chain variable zones, wherein amino acid sequences of three hypervariable regions of the heavy chain variable zones, namely, CDRH1, CDRH2 and CDRH3, are respectively GFTFSSYAM, AISGSGGTTYYADSVKG and AWAPFVNSIVVVNDL, and amino acid sequences of three hypervariable regions of the light chain variable zones, namely, CDRL1, CDRL2 and CDRL3, are respectively RASQGISTWLA, AASSLQS and QQANSFPI. The antibody to interleukins-15 provided in the invention has the characteristics of high specificity, a simple preparation process and low cost.
Description
Technical field
The present invention relates to a kind of antibody, relate in particular to a kind of anti-IL-8-15 antibody.
Background technology
(rheumatoid arthritis is a kind of common disease RA) to rheumatoid arthritis, is distributed in all race and ethnic origin, and patient's extend over the entire globe sees that with temperate zone, subtropics and refrigerant latitudes the torrid areas is rare more.Change countries and regions such as big Northern Europe, the U.S., Britain, Sweden, Italy, France, Russia, Finland in temperature and humidity more.According to the investigation in U.S. health center, the whole America has 400-500 ten thousand people to suffer from rheumatoid arthritis approximately.The sickness rate of foreign statistic is 0.5%-3%.Existing China and alliance of international rheumatology association have cooperated to carry out the epidemiology survey of rheumatoid arthritis, ankylosing spondylitis, and PRELIMINARY RESULTS is that the morbidity of China's rheumatoid arthritis is about 0.8-1.0%; The morbidity of Han nationality is apparently higher than other nationalitys; The women is ill more than the male sex.By this rough calculation, China nearly 1000-1400 all creation rheumatic arthritis patient.China surpasses 9,000,000 person-times because of RA prescription on individual diagnosis number every year, and inpatient is above 250,000 times.The financial loss that DB caused that RA causes, and bring white elephant to family.
On pathology, rheumatoid arthritis is to be the systemic autoimmune disease of principal character with chronic villous poiyarthritis, bone and cartilage destruction, and its item key is a leukocyte infiltration in the synovial membrane.Show as symmetric multiarticulate swelling, pain, morning deadlock, lose weight and the getting involved of whole body multisystem.This inflammatory process can stimulate anti-apoptosis to become the propagation of fiber-like synovial cell (FLS).So directly cause forming pannus and joint injury and intraarticular and cause inflammatory cytokine, as the expression of TNF-α (tumour necrosis factor).The pathogeny of rheumatoid arthritis does not still have final conclusion.Research is at present thought, many cytokines are (as TNF-α, interleukin 15 (interleukin-15, IL-15), Thr6 PDGF BB (platelet-derived growth factor, PDGF) etc.) plays an important role in the pathogenic process of RA.
In China, the medicine of treatment RA mainly contains NSAID (non-steroidal anti-inflammatory drug) (NSAIDS), chronic antirheumatic (DMARDS) and Chinese medicine class.Few part patient can use expensive biotechnological formulation class pharmacological agent rheumatoid arthritis.
Methotrexate (MTX) can suppress Tetrahydrofolate dehydrogenase and transformylase activity as the classical medicine of treatment rheumatoid arthritis.Leflunomide is a kind of different azole immunosuppressor with anti-proliferative activity, and its mechanism of action mainly is the de novo synthesis that suppresses the activity blocking-up pyrimidine of dihydroorate dehydrogenase.In addition, Pa Fulin also has certain curative effect to rheumatoid arthritis.But the problem of this class medicine is the patient and can produces many untoward reactions to medicine that most patient all will experience a chronic course of disease repeatedly simultaneously, thereby the state of an illness how more effectively to control rheumatoid arthritis patients is still a big problem.And the validity of Chinese medicine (as trypterygine, Stem of Orientoine etc.) treatment rheumatoid arthritis thorny problem especially.
In American-European countries, rheumatoid arthritis patients then can obtain three kinds of antibody class medicine etanercept (trade(brand)name Enbrel), infliximab (trade(brand)name Remicade), effective treatment of adalimumab (trade(brand)name Humira).These three kinds of antibody class medicines all act on this target of TNF-α.Many patients there is very gratifying curative effect.Many patients through the treatment after, the symptom completely dissolve.Therefore, antibody class pharmacological agent rheumatoid arthritis has its distinctive feature.
But above-mentioned three kinds of medicines are still inoperative to about 50% patient, and the pathogeny reason that this part patient is described is not TNF-α.Therefore, the antibody class medicine of researching and developing different targets has very profound significance.Our independent intellectual property right is monopolized, obtains in the blockade on new techniques of breaking American-European countries on the one hand, we also can enter and carve up American-European market by this on the other hand, again our treatment rheumatoid arthritis total man source antibody drug that production and selling and China's patient's level of consumption adapt that can reduce cost on the one hand.
(interleukin-15 IL-15) is a kind of very important inflammatory cytokine that causes to Interleukin-15, is produced by scavenger cell, dendritic cell, keratinocyte and epithelial cell.It belongs to 4-alpha-helix bundle cell factor family.IL-15 can activate the panimmunity cell, stimulates their propagation and prolong its life-span: IL-15 can breed and production of cytokines by inducing T cell, stimulates the migration and the chemotactic of normal T cell, and protects their to exempt apoptosis; Can strengthen the cytotoxicity of NK cell and rely on antibody-mediated cytotoxic effect, prolong the life-span of NK cell and promote NK emiocytosis IFN-γ, GM-CSF and TNF-α; Can induce the propagation and the type of the same clan conversion (the effect B cell is to plasmacytic conversion) of B cell; IL-15 can also promote the generation of CTL (cytotoxic T lymphocyte) memory cell.A large amount of experiments show that the interaction between IL-15 and the IL-15R in the various kinds of cell (being the IL-15 acceptor) can be induced and be produced the cascade signal transduction.Activation comprising Janus kinases (Jak) and STA T approach transcription activator.
IL-15 has the important inflammatory reaction effect that causes in numerous disease reacts as allergy, graft-rejection, autoimmune disorder.Britain McInnes study group, Harada and colleague, Bykovskaia etc. and Gonzalez-Alvaro etc. report that all rheumatoid arthritis patients can significantly detect high-caliber IL-15 in synovial membrane liquid, synovial membrane and serum.It may be to work by TNF-α that IL-15 causes the mechanism part of RA, and report IL-15 such as Ziolkowska cause rheumatoid arthritis by regulating IL-17.
Hand-in-gloves such as the McInnes study group at Denmark Genmab biological medicine company and Britain Glasgow university sacroiliitis center, Denmark Rigs hospital, Denmark Frederiksberg hospital, London Guy hospital and Sweden Karolinska hospital, utilize the monoclonal antibody of total man source IL-15 to carry out the clinical trial of treatment rheumatoid arthritis, the result gets the effect of making us satisfied.
Summary of the invention
The invention provides a specific specificity high anti-IL-8-15 antibody.
A kind of anti-IL-8-15 antibody, comprise variable region of heavy chain and variable region of light chain, three hypervariable region CDRH1, CDRH2 of described variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively: GFTFSSYAM, AISGSGGTTYYADSVKG, AWAPFVNSIVVVTANDL, three hypervariable region CDRL1, CDRL2 of described variable region of light chain, the aminoacid sequence of CDRL3 are respectively: RASQGISTWLA, AASSLQS, QQANSFPI.
The aminoacid sequence of described variable region of heavy chain is shown in SEQ ID NO.1.
The aminoacid sequence of described variable region of light chain is shown in SEQ ID NO.2.
Described antibody can be whole antibody, antibody fragment or single-chain antibody.
The present invention also provides the gene of the above-mentioned antibody of encoding.
The present invention also provides the application of above-mentioned anti-IL-8-15 antibody in preparation inhibition IL-15 inductive T cell proliferation medicine
Anti-IL-8 of the present invention-15 antibodies specific height, preparation process is simple relatively, and is with low cost.
Description of drawings
Fig. 1 is the structural representation of anti-IL-8 of the present invention-15 single-chain antibody;
Fig. 2 is the gel electrophoresis figure before and after the embodiment 6 abduction delivering product purifications, before the 1st road is purifying, after the 2nd road is purifying;
Fig. 3 is embodiment 7 antibody concentration of the present invention and OD
450Graph of relation;
Fig. 4 is distributed number figure after T cell and each the antibody mixed culture among the embodiment 8, and reading is CPMx10
3#IL15-3 is an anti-IL-15 single-chain antibody of the present invention; Ab16 is non-specific single-chain antibody; MAb is that mouse-anti human IL-15 antibody is (available from R﹠amp; D Systems company).
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:
With the polyA+RNA (available from Clontech) from people's marrow, people's tire liver, people's spleen and human peripheral leucocytes is template, and use the reverse transcriptase test kit of buying from Clontech, with oligo (dT) and random primer (random primers) is primer, the Methods Instruction that is provided according to the Clontech test kit is with the reverse cDNA that is transcribed into of poly A+RNA.With above-mentioned cDNA is template, uses the primer of a series of identification human antibody heavy chain variable regions (VH) and variable region of light chain (VL) gene, uses the 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:
First group: 5 ' of human antibody heavy chain variable region (VH) gene-end primer
VH1b：5’
CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTG?CAG?CTG?CAG?GAG?TC(C/G)G
VH2b：5’-CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTA?CAG?CTG?CAG?CAG?TCA
VH3b：5’-CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTG?CAG?CTA?CAG?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?CAG?CT(G/T)GT(A/G)CAG?TCT?GG
VH6b：5’-CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG(A/G)TC?ACC?TTG?AAG?GAG?TCT?G
VH7b：5’-CCA?TAC?GAT?GTT?CCA?GAT?TAC?CAG?GTG?CAG?CTG?GTG(C/G)A(A/G)TCT?GG
Second group: 3 ' 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?GGT?GAC?CAG?GGT?T
VH3f：5’-GCC?GCC?TGA?TCC?ACC?ACC?GCC?TGA?AGA?GAC?GGT?GAC?CAT?TGT
VH4f：5’-GCC?GCC?TGA?TCC?ACC?ACC?GCC?TGA?GGA?GAC?GGT?GAC?CGT?GGT?CC
VH5f：5’-GCC?GCC?TGA?TCC?ACC?ACC?GCC?GGT?TGG?GGC?GGA?TGC?ACT?CC
VH6f：5’-GCC?GCC?TGA?TCC?ACC?ACC?GCC?(C/G)?GA?TGG?GCC?CTT?GGT?GGA?(A/G)?GC
The 3rd group: 5 ' 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)G?CTG?AC(A/T)?CAG?CCA?C
VL3b：5’-GGC?AGC?GGT?GGT?GGA?GGC?AGT?TCC?TAT?GAG?CTG?A(C/T)(A/G)?CAG?C(C/T)A?CC
VL4b：5’-GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?CCT?GTG?CTG?ACT?CA(A/G)(C/T)C
VL5b：5’-GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?(A/G/T)CT?GTG?GTG?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?CTG?A(C/G)T?CAG?GA(C/G)CC
VL8b：5’-GGC?AGC?GGT?GGT?GGA?GGC?AGT?CAG?TCT?G(C/T)(C/T)CTG?A(C/T)T?CAG?CCT
VL9b：5’-GGC?AGC?GGT?GGT?GGA?GGC?AGT?AAT?TTT?ATG?CTG?ACT?CAG?CCC?C
The 4th group: 3 ' 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?CAG?CTG?GGT?GC
The 5th group: 5 ' of people's antibody k-variable region of light chain (Vk) 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?GAA?ACG?ACA?CTC?ACG?CAG?TCT?C
The 6th group: 3 ' of people's antibody k-variable region of light chain (Vk) gene-end primer
VK1f：5’-
GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?GAT?TTC?CAC?CTT?GGT?CC
VK2f：5’-GGG?GTT?TTT?CAGTAT?CTA?CGA?TTT?GAT?CTC?CA(C/G)CTT?GGT?CC
VK3f：5’-GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?GAT?ATC?CAC?TTT?GGT?CC
VK4f：5’-GGG?GTT?TTT?CAG?TAT?CTA?CGA?TTT?AAT?CTC?CAG?TCG?TGT?CC
The combination of using above-mentioned first group of primer and second group of primer during variable region of heavy chain (VH) in the PCR method amplification people antibody promptly has 42 PCR to react.Similarly, the combination of using above-mentioned the 3rd group of primer and the 4th group of primer during λ-variable region of light chain (V λ) in the amplification people antibody promptly has 18 PCR to react.The combination of using above-mentioned the 5th group of primer and the 6th group of primer during k variable region of light chain (Vk) in the amplification people antibody promptly has 20 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, Chan E, Zhu L, Luo Y. (1997) Plasmid 38:91-96.) homologous sequence (underscore part) of multiple clone site upstream; Include homologous sequence (underscore part) in the 4th group and the 6th group of primer with yeast two-hybrid carrier pACT2 multiple clone site downstream; 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 to connect the variable region of the heavy chain and the light chain of antibody.
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
Human antibody heavy chain variable region DNA with PCR gained among the embodiment 1 is a template, is upstream primer and downstream primer with primer 7 and primer 8, adopts the PCR method to increase, and 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 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 sequence anti-chain)
5’-ACT?GCC?TCC?ACC?ACC?GCT?GCC?ACC?TCC?GCC?AGA?TCC?TCC?
GCC?GCC?TGA?TCC?ACC?ACC?GCC
Human antibody light chain variable region DNA with PCR gained among the embodiment 1 is a template, is respectively downstream primer and upstream primer with primer 9 and primer 10, adopts the PCR method to increase, and 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 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 (the connection peptides sequence is along chain)
5’-GGC?GGT?GGT?GGA?TCA?GGC?GGC?GGA?GGA?TCT?GGC?GGA?GGT?
GGC?AGC?GGT?GGT?GGA?GGC?AGT
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.With this hybrid dna is 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
The foregoing description 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 the foregoing description 3 described single-chain antibody (scFv) DNA and yeast two-hybrid carrier pACT2 after restriction enzyme (Bam HI and Eco RI) is handled 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, the gal4 Δ, the gal80 Δ, 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.(Activation Domain AD) merges Gal4 active region on single-chain antibody (scFv) dna fragmentation and the pACT2 carrier.
For checking the quality in this people's single-chain antibody (scFv) library, 21 clones have been chosen at random.All comprise single-chain antibody (scFv) dna fragmentation (data not shown) that merges with Gal4 to inserting all clones of sequencing fragment analysis revealed, 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 the specific antibody of yeast-two hybrid technique screening.
Embodiment 5 screening antibody
End user's Interleukin-15 (IL15) is screened yeast two-hybrid single-chain antibody scFv library as antigen.The dna clone of coding human IL-15 is entered carrier pGBKT7 (Clontech company) and constructs pGBK-IL15.PGBK-IL15 coding Gal4 DNA calmodulin binding domain CaM (Binding Domain, i.e. BD) also merges human IL-15 at its C end.
After the dna sequence dna of coding human IL-15 was verified, the pGBK-IL15 plasmid DNA transformed into yeast strain AH109 (genotype MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,112, gal4 Δ, gal80 Δ, LYS2::GAL1
UAS-GAL1
TATA-HIS3, GAL2
UAS-GAL2
TATA-ADE2, URA3::MEL1
UAS-MEL1
TATA-lacZ) (Clontech company).The AH109 yeast that has the pGBK-IL15 plasmid can be gone up growth 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 MATa type yeast cell (AH109 bacterial strain) that contains pGBK-IL15 of equivalent and the MAT α type yeast cell (Y187 bacterial strain) that comprises single-chain antibody scFv library.The pACT2 carrier that is loaded with the scFv library contains the LEU2 gene, and pGBK-IL15 comprises the TRP1 gene.The yeast cell that comprises 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-15 can activate reporter gene ADE2 and the HIS3 that is incorporated in the strain gene group, thereby make yeast cell can be grown in the shortage VITAMIN B4, Histidine, on the substratum of leucine and tryptophane (SD/-AHLW), and on this plate culture medium, form bacterium colony.
We pick out 23 bacterium colonies altogether on above-mentioned screening culture medium (SD/-AHLW).Then, these bacterium colonies are further analyzed.
At first, the method for 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-15 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 grown on screening culture medium (SD/-AHLW) flat board.
(2) yeast colony is transferred on No. five filter paper of Whatman.
(3) will have among the filter paper immersion liquid nitrogen of yeast cell bacterium colony, make cell rupture.
(4) filter paper is taken out from liquid nitrogen, and place in 30 ℃ the baking oven.Repeating step 3 and 4 secondaries.
(5) filter paper is laid in an amount of X-gal solution, and placed in 37 ℃ the incubator about 15 minutes.Show blueness if having the bacterium colony place, be indicated as the beta galactosidase enzyme positive.
The X-gal solution formula is as follows:
16.1g/L Na
2HPO
4·7H
2O
5.50g/L NaH
2PO
4·H
2O
0.75g/L KCl
0.246g/L?MgSO
4·7H
2O
35mg/L X-gal
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
The 5mM beta-mercaptoethanol
pH7.0
Detected result: have 11 to be the beta galactosidase enzyme male in above-mentioned 23 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 11 beta galactosidase enzymes is carried out specificity analyses, determining that single-chain antibody scFv combines with the IL-15 part specifically, rather than and other parts.The pACT2 plasmid DNA that will contain scFv extracts from above-mentioned 11 beta galactosidase enzyme male yeast.And respectively with pGBKT empty carrier DNA, or with contain the DNA that coding merges the plasmid pGBKT-Lam (Clontech company) of Gal4-DNA land (BD) and human nuclear fabric layer albumen C encoding sequence, be transformed into jointly in the AH109 yeast cell.Yeast cell after the conversion is laid on earlier on the SD/-LW plate culture medium grows, and transfers to then on the SD/-AHLW plate culture medium.And further use the beta galactosidase enzyme analysis.
After aforesaid method carried out specificity analyses to scFv, we obtained 5 human IL-15 are had specific single-chain antibody scFv.
Then, to above-mentioned 5 human IL-15 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 sequence of analyzing these single-chain antibodies scFv clone shows the single-chain antibody that two different anti-Ro 24-7472/000s-15 are arranged.Wherein identical with clone #IL15-3 has 2, and identical with clone #IL15-7 then has 3.
The aminoacid sequence of the VH of the single-chain antibody of clone #IL15-3 is shown in SEQ ID NO.1, and dna sequence dna is shown in SEQ ID NO.3; The aminoacid sequence of VL is shown in SEQ ID NO.2, and dna sequence dna is shown in SEQ ID NO.4.
Weight chain variable region amino acid sequence (SEQ ID NO.1)
EVQLLESGGGLVQPGGSLRLSCAAS
GFTFSSYAMSWVRQAPGKGL?EWVS
AISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNNLRVEDTAV?YYCAK
AWAPFVNSIVVVTANDLWGQGTLVTVSS
Underscore partly is followed successively by three hypervariable region CDRH1, CDRH2 in the variable region of heavy chain and the aminoacid sequence of CDRH3.
Light chain variable region amino acid sequence (SEQ ID NO.2)
DIQLTQSPS?SVSASVGDRVTITC
RASQGISTWLAWYQQKPGKAPKL?LIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFC
QQANSFPITF?GQGTRLEIK
Underscore partly is followed successively by three hypervariable region CDRL1, CDRL2 in the variable region of light chain and the aminoacid sequence of CDRL3.
Embodiment 6 antibody expressions and purifying
With the dna clone of the single-chain antibody scFv of anti-human IL-15 pET27b (+) (Novagen company) and obtain pET27b-IL15 in the protein expression carrier.PET27b-IL15 behind the clone, at the N of scFv end is pelB sequence (can with the scFv protein secreting after expressing in the pericentral siphon chamber periplasmic space that expresses bacterium E.coli), and the C end contains the sequence of one a section coding HSV marker and a 6x His marker (can be used for protein purification).PET27b-IL15 is transformed into protein expression bacterium E.coli bacterial strain BL21 DE3 (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-15.
Because the proteinic C end of scFv contains a 6x His marker, the method that can use Qiagen company is provided uses Ni-NTA post (Qiagen company) to come purifying scFv protein easily.The protein analysis of SDS-PAGE gel electrophoresis before and after the purifying, the result as shown in Figure 2.
The scFv characteristic of the anti-IL15 of embodiment 7 elisa immunoassays (ELISA) test
The protein of recombinant human IL-15 is available from U.S. R﹠amp; D Systems company.The 96-orifice plate (MaxiSorp plate) that is used for elisa immunoassay (ELISA) is available from Nunc company.Method is as follows:
(1) above-mentioned 96-orifice plate at first wraps quilt with IL-15.
(2) the 96-orifice plate behind the bag quilt uses SuperBlock (available from Pierce company) to make sealing treatment again.
(3) add after sublimed anti-IL-15 single-chain antibody is made serial dilution in 0.02% BSA and be coated with in the 96-hole of IL-15, combine with IL-15.
(4) with after the cleaning of 96-orifice plate, the antibody (available from Novagen company) that adds the mouse-anti HSV marker that has diluted 5000 times in the 96-hole again is used for detecting the single-chain antibody (annotating: contain a HSV marker at the C of scFv end) that combines.
(5) the 96-orifice plate is cleaned after, in the 96-hole, add the conjugate (available from Sigma company) that the mountain sheep anti-mouse igg antibody that diluted 10000 times and coupling have HRP (horseradish peroxidase) again.
(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, presentation of results clone #IL15-3 single-chain antibody can be effectively in conjunction with IL-15.
Embodiment 8 test scFv suppress IL-15 inductive T cell proliferation
The anti-IL-15 single-chain antibody of test behind the purifying IL-15 is induced CD8
+The restraining effect of T cell proliferation.Experimental procedure is as follows:
(1) every hole 2.5 * 10
4CD8 after the individual separation
+The T cell cultures is in 96 porocyte culture plates.Substratum is RPMI-1640+10% FCS (foetal calf serum).Anti-IL-15 single-chain antibody (1nM) behind the protein of recombinant human IL-15 (concentration is 10pM) and the above-mentioned purifying or mouse-anti human IL-15 antibody are (available from R﹠amp; D Systems company) (1nM) is blended in the substratum.
(2) at 37 ℃ CO
2Cultivated 72 hours in the incubator.
(3) add in every hole 0.5 μ Ci [
3H]-thymus pyrimidine.CO at 37 ℃
2Continue in the incubator to cultivate 6 hours.
(4) with the RPMI-1640 substratum give a baby a bath on the third day after its birth all over after, with cell harvesting, with liquid glimmer instrument counting, triplicate is averaged again.
The result as shown in Figure 4, the anti-IL-15 single-chain antibody #IL15-3 that is obtained can effectively suppress IL-15 and induce CD8
+The T cel l proliferation.
Claims (6)
1. anti-IL-8-15 antibody, comprise variable region of heavy chain and variable region of light chain, it is characterized in that, three hypervariable region CDRH1, CDRH2 of described variable region of heavy chain, the aminoacid sequence of CDRH3 are respectively: GFTFSSYAM, AISGSGGTTYYADSVKG, AWAPFVNSIVVVTANDL, three hypervariable region CDRL1, CDRL2 of described variable region of light chain, the aminoacid sequence of CDRL3 are respectively: RASQGISTWLA, AASSLQS, QQANSFPI.
2. anti-IL-8-15 antibody according to claim 1 is characterized in that the aminoacid sequence of described variable region of heavy chain is shown in SEQ ID NO.1.
3. anti-IL-8-15 antibody according to claim 1 is characterized in that the aminoacid sequence of described variable region of light chain is shown in SEQ ID NO.2.
4. anti-IL-8-15 antibody according to claim 1 is characterized in that described antibody is single-chain antibody.
5. the gene of coding claim 1～4 arbitrary described anti-IL-8-15 antibody.
6. the application of the arbitrary described anti-IL-8-15 of claim 1～4 antibody in preparation inhibition IL-15 inductive T cell proliferation medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106661106A (en) * | 2014-07-02 | 2017-05-10 | 克里普索生物科技公司 | Antibodies to IL-15 |
| CN106699887A (en) * | 2015-07-15 | 2017-05-24 | 杭州贝颐药业有限公司 | Anti-human PD-1 protein antibody as well as encoding gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1571836A (en) * | 2001-08-23 | 2005-01-26 | 根马布股份公司 | Human antibodies specific for interleukin 15 (IL-15) |
| CN1780856A (en) * | 2003-02-26 | 2006-05-31 | 根马布股份公司 | Human antibodies specific for interleukin 15 (IL-15) |
| CN102010471A (en) * | 2003-11-10 | 2011-04-13 | 先灵公司 | Interleukin-10 antibodies |
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2011
- 2011-07-01 CN CN 201110184280 patent/CN102250243B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1571836A (en) * | 2001-08-23 | 2005-01-26 | 根马布股份公司 | Human antibodies specific for interleukin 15 (IL-15) |
| CN1780856A (en) * | 2003-02-26 | 2006-05-31 | 根马布股份公司 | Human antibodies specific for interleukin 15 (IL-15) |
| CN102010471A (en) * | 2003-11-10 | 2011-04-13 | 先灵公司 | Interleukin-10 antibodies |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106661106A (en) * | 2014-07-02 | 2017-05-10 | 克里普索生物科技公司 | Antibodies to IL-15 |
| CN106661106B (en) * | 2014-07-02 | 2021-03-16 | 克里普索生物科技公司 | anti-IL-15 antibodies |
| CN106699887A (en) * | 2015-07-15 | 2017-05-24 | 杭州贝颐药业有限公司 | Anti-human PD-1 protein antibody as well as encoding gene and application thereof |
| CN106699887B (en) * | 2015-07-15 | 2021-02-12 | 杭州贝颐药业有限公司 | Anti-human PD-1 protein antibody and coding gene and application thereof |
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