CN101092457A - Human source Fab antibody for anti recombined alkalescent fibroblast growth factor and application - Google Patents

Abstract

This invention discloses anti-recombinant alkaline fibroblast growth factor human-derived Fab antibody and its application. The antibody segment genome comprises: heavy chain Fd chain and light chain kappa chain. This invention utilizes phage surface display technique to successfully construct kappa chain and Fd chain genes of human-derived Fab antibody that can neutralize recombinant human bFGF or FGF-2. Functional neutralizing antibody that can specifically bind bFGF or FGF-2 is obtained in prokaryotic cells, yeast cells, insect cells and eukaryotic cells, which can be used to prepare antibody drugs that can diagnose and treat tumors, and inhibit kidney, liver and lung fibrosis. The bFGF or FGF-2 human-derived Fab antibody has high affinity and specificity, and can be directly used to develop antibody drugs.

Description

Anti recombined alkalescent fibroblast growth factor human source Fab antibody and application thereof

Technical field

The invention belongs to biological technical field, particularly a kind of anti recombined alkalescent fibroblast growth factor (or is called fibroblast growth factor 2, FGF-2) human source Fab antibody and application thereof.

Background technology

Prostatropin (basic fibroblast growth factor, bFGF) have another name called fibroblast growth factor 2 (fibroblast growth factor 2, FGF-2) be fibroblast growth factor (fibroblast growth factor, the factor that FGF) family finds the earliest among known 23 members at present, research is maximum.BFGF has the various biological effect, comprises promotes growth, division, differentiation function; Inoblast had the short proliferation function of intensive.

BFGF is one of cytokine of the most important stimulation angiogenesis after VEGF.In close relations with the generation of tumour, development, bFGF participates in the growth of kinds of tumors (as neurospongioma, rhabdomyoma, leukemia, kidney, pneumonocyte knurl, melanoma, liver cancer), has the tumour of considerable part the high expression level of bFGF and bFGF acceptor to occur.Known its effect link mainly contains (1) and promotes tumor vascular new life, thereby increases the blood supply of tumour, provides oxygen and nutritive substance for tumor growth.(2) bFGF can promote the secretion of multiple somatomedin, as VEGF, and TGF, aFGF etc., these factors are regulated mutually, promote the generation of tumour jointly; (3) there is the high expression level of bFGF acceptor in tumour cell, usually higher more than 10 times than normal cell, bFGF can by autocrine and (or) paracrine action directly stimulates tumour cell, and the various proteolytic enzyme of tumor tissues and collagenic acid secretion are increased, thereby quicken invading profit and shifting of tumour cell; (4) bFGF and oncogene are in close relations, have found that some oncogenes (hst.int-2 hst/k-fgf) expressed protein and bFGF are 39%～50% with originality.And, the closely-related Ras of bFGF and cytodifferentiation, Akt, C-fos, c-myc etc. have close ties, and these genes all have the tumorigenic effect of promotion.(5) kinds of tumor cells self high expression level bFGF can detect the bFGF of pg/mL level in the tear of tumour patient, blood, urine.BFGF can also directly act on tumour cell, promotes the propagation of tumour cell and quickens the transfer of tumour cell.

In addition, bFGF also participates in Fibrotic formation such as liver, lung, kidney, and bFGF is also relevant with the resistance of tumour.The expression of bFGF obviously strengthens in these pathological tissues, and therefore one of approach of controlling and treating this class disease is exactly to utilize anti-bFGF neutralizing antibody to block its effect.

Matsuzaki etc. have begun the antitumor action research of bFGF mouse source monoclonal antibody as far back as 1989, two strain monoclonal antibodies of their development-plain binding growth factor of anti-beef liver and anti-bFGF monoclonal antibody, external, all show the strong inhibition ox venous endothelial cell propagation and the effect of solid tumor resisting.Aonuma etc. study the antitumor action of the two strain monoclonal antibodies (2G11 and 1E6) of the different epi-positions of bFGF respectively, find, have diverse biologic activity at the monoclonal antibody of the different epi-positions of bFGF.External to have a heparin-binding active but do not have anti-tumor activity in vivo at the monoclonal antibody (2G11) of bFGF heparin land.And do not show very strong antitumor action with the heparin competition anti-bFGF monoclonal antibody of bonded (1E6).Domestic, Lin Wei etc. (2005) utilize ovarian cancer nude mice subcutaneous transplantation knurl model, inquired into bFGF in the ovarian epithelium tumour expression intensity and the relation between the tumor-blood-vessel growth, bFGF mouse source monoclonal antibody is to the influence of human ovarian cancer transplanted tumor vasculogenesis and tumor growth.Xie Qingxiang etc. have studied bFGF and antibody thereof to nude mice subcutaneous transplantation bladder cancer growth effect.

The inhibition neonate tumour blood vessel of bFGF monoclonal antibody and antineoplastic action have obtained the confirmation of Chinese scholars and have affirmed, but up to the present, also do not see other document and patent reports of studying about anti-bFGF human antibody both at home and abroad.

Summary of the invention

In order to solve above-mentioned the deficiencies in the prior art part, primary and foremost purpose of the present invention is to provide a kind of anti recombined alkalescent fibroblast growth factor (bFGF) (or to be called fibroblast growth factor 2, FGF-2) human source Fab antibody is in this antibody capable specificity and people bFGF (or FGF-2).

Another object of the present invention is to provide the application of the human source Fab antibody of above-mentioned anti recombined alkalescent fibroblast growth factor (bFGF) or fibroblast growth factor 2 (FGF-2).

Purpose of the present invention realizes by following technical proposals: a kind of anti recombined alkalescent fibroblast growth factor (bFGF) (or is called fibroblast growth factor 2, FGF-2) human source Fab antibody, described antibody fragment gene comprise the Fd chain and the light chain κ chain of heavy chain; The gene order of described heavy chain Fd chain is as follows:

CAG TCT GGG GGA GGC TTG GTA CAG CCT GGC AGG TCC CTG AGA CTC ACC TGT

GCA GCC TCT GGA TTC ACC TTT GAA GAT TAT GGC ATG CAC TGG GTC CGG CAA

GCT CCA GGG AAG GGC CTG GAG TGG GTC TCA GGT ATT AGC TGG AAT AGT GGT

AGT ATA GGC TAT GCG GAC TCT GTG AAA GGC CGC TTC ACC ATC TCC AGA GAC

AAC GCC AAG AAT TCC CTG TAT TTG CAA ATG AAC AGT CTG AGA GCT GAG GAC

ACG GCC TTG TAT TAC TGT GCA AAA GAC AGG CGC TTC GGT GAC TAC GTC CAC

TCC TAC TAC GGT ATG GAC GTC TGG GGC CCA TCG GTC TTC CCC CTG GCA CCC

TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC AAG

GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC

AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC

TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG AGC ACC CAG

ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC

AAG AGA GTT GAG CCC AAA TCT TGT GAC AAA；

The gene order of described light chain κ chain is as follows:

GAT ATT GAG CTC ACT CAG TCT CCA TCC TCC CTG TCT GCA TCT ATA GGA GAC

ACA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAC ATC TAT GTA AAT

TGG TAT CAG CAC AAA GCA GGC AAA GTC CCT AAA CTC CTA ATG CAT ACT GCA

TCC AGT TTA GAA AGT GGG GTC CCA CCA AGG TTC AGT GGC AGT GGC TCT GGG

ACA GAT TTC ACT CTC ACT ATC AGC GGT CTG CAA CCT GAA GAT TTT GCA ACT

TAC TAC TGT CAA CAG AGT TAT CGT CTT GTC AGT TTC GGC CCT GGG ACC AAA

GTG GAT ATT AGA CGA ACT GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA

TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT

AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC

CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC

AGC ACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG

AAA CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGT TCG CCC

GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT。

The aminoacid sequence of described heavy chain Fd chain is as follows:

Q S G G G L V Q P G R S L R L T C

A A S G F T F E D Y G M H W V R Q

CDR1

A P G K G L E W V S G I S W N S G

CDR2

S I G Y A D S V K G R F T I S R D

N A K N S L Y L Q M N S L R A E D

T A L Y Y C A K D R R F G D Y V H

CDR3

S Y Y G M D V W

P K S C D K；

The aminoacid sequence of described light chain κ chain is as follows:

D I E L T Q S P S S L S A S I G D

T V T I T C R A S Q S I D I Y V N

CDR1

W Y Q H K A G K V P K L L M H T A

S S L E S G V P P R F S G S G S G

CDR2

T D F T L T I S G L Q P E D F A T

Y Y C Q Q S Y R L V S F G P G T K

CDR3

V D I R

Described heavy chain Fd chain is by 633 based compositions, 211 amino acid of encoding, and (promptly from GPSV to KRVE, 95 amino acid belong to constant 1 district to the 111st～205 amino acids by the variable region of heavy chain of antibody and constant 1 district (CH1); The 206th～211 amino acids belongs to the hinge region part) constitute, 3 CDR (complementary determinant) district is contained in the variable region.CDR1 5 amino acid of encoding, CDR2 7 amino acid of encoding, CDR3 17 amino acid of encoding.The homology of the framework region of variable region and other people endogenous antibody then is a specific sequence up to 97.6%, 3 CDR district.Have than big-difference with other people endogenous antibody Fd chain.

Described light chain κ chain is by 639 based compositions, and 213 amino acid of encoding are made of the variable region of heavy chain of antibody and constant region (CL) (the 107th～213 amino acids, promptly from RTVA to ending), and 3 CDR (complementary determinant) district is contained in the variable region.CDR1 11 amino acid of encoding, CDR2 17 amino acid of encoding, CDR3 18 amino acid of encoding.The homology of the framework region of variable region and other people endogenous antibody is that 96.7%, 3 CDR district then is a specific sequence.Have than big-difference with other people endogenous antibody κ chain.

Anti recombined alkalescent fibroblast growth factor (bFGF) or the proteic function of fibroblast growth factor 2 (FGF-2) human source Fab antibody are present in specificity nucleotide sequence decision among the complementary decision of antibody weight chain variable region antigen family (complementarity determing regions CDRs) CDR1, CDR2, the CDR3 (being functionally active of the present invention district), and its amino acid sequence corresponding has constituted antibodies specific bFGF or (FGF-2) antigen binding domain.

The purposes of above-mentioned anti recombined alkalescent fibroblast growth factor (bFGF) or fibroblast growth factor 2 (FGF-2) human source Fab antibody:

This specificity is in conjunction with bFGF or functional neutralizing antibody gene (FGF-2), this recombinant antibodies is to be determined by the specific gene order in hypervariable region (CDRs) that is present in light chain of antibody and the variable region of heavy chain, based on above-mentioned weight chain variable region gene, can adopt gene engineering method, the small molecules genetic engineering antibody that makes up and be expressed as various ways is (as Fab antibody, F (ab) ₂, single-chain antibody (scFv), nano antibody (Nanobody), antibody fusion protein) and IgG whole antibody.

Any other gene that contains this antibody gene variable region after prokaryotic cell prokaryocyte, yeast cell, insect cell and eukaryotic cell obtain that this antibody gene is expressed or serve as the basis reconstruction with this gene.During acquisition has and the antibody product of people bFGF, (1) is used for the diagnosis of tumour clinically, and (2) suppress neonate tumour blood vessel and suppress growth of tumor and transfer, and (3) suppress the process of liver, lung, renal fibrosis.

Above-mentioned anti recombined alkalescent fibroblast growth factor (bFGF) or fibroblast growth factor 2 (FGF-2) human source Fab antibody are used for diagnosing and treating the application of tumour and Fibrotic antibody drugs such as inhibition kidney, liver or lung in preparation.

The present invention compared with prior art, have following advantage and beneficial effect: anti-bFGF or FGF-2 human source Fab antibody that the present invention obtains, this antibody is the total man source, be from people's blood lymphocyte isolation of RNA, reverse transcription cDNA, people's phage antibody library of structure, and the antibody that therefrom screens, have high-affinity and specificity,, can directly be developed as the antibody drug of human because be full humanized's antibody.The advantage of the anti-bFGF antibody of exploitation humanized is mainly reflected in: (1) humanized's antibody bFGF antibody has solved mouse source monoclonal antibody can't avoid human antimouse antibody (HAMA) reaction, can directly develop antibody drug and be used for human body therapy.(2) bFGF is people's source protein, and itself can not cause people's immune response, and bFGF and tumour are in close relations, so can not directly develop vaccine with it; (3) directly the lymphocyte of personnel selection is the gene constructed antibody library of material clonal antibody, the anti-bFGF antibody of the humanized who screens has the characteristics of specificity in conjunction with bFGF, in can be effectively and the bFGF that transition is expressed in tumour and the fibrosis patient body, can effectively suppress growth of tumor and transfer, also can be used for suppressing Fibrotic formation such as liver, lung or kidney.

Description of drawings

Fig. 1 is the Fd chain gene and the aminoacid sequence of humanized's antibody (Fab) of bFGF or FGF-2.

Fig. 2 is the κ chain gene and the aminoacid sequence of humanized's antibody (Fab) of bFGF or FGF-2.

Fig. 3 is the ELISA figure as a result of different bacteriophages antibody cloning.

Wherein, 1～6 be respectively different phage antibody clone (Clone2, Clone6, Clone15, Clone16, Clone25, Clone26), 7 is blank, 8 negative contrasts.

Fig. 4 is the ELISA detected result figure of the expression product crude extract of different Fab antibody clonings.

Wherein, 1～3: the expression product crude extract that is respectively pComb3H-Fab-6, pComb3H-Fab-25, pComb3H-Fab-26; 4: empty carrier pComb3H expression product crude extract.

Fig. 5 is the SDS-PAGE detected result figure of Fab antibody cloning expression product.

Wherein, 1 negative contrast, 2～4 are respectively the SDS-PAGE result into pComb3H, pComb3H-Fab-6, pComb3H-Fab-25, pComb3H-Fab-26, and M is a molecular weight marker.

Fig. 6 is the Western blot detected result figure of Fab antibody cloning expression product.

Wherein, 1 is that 1pComb3H-Fab-6,2 is the Western blot result of pComb3H-Fab-26 for pComb3H-Fab-25,3,4 negative contrasts.

Embodiment

The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.

Embodiment 1

The construction process of anti recombined alkalescent fibroblast growth factor (bFGF) or fibroblast growth factor 2 (FGF-2) human source Fab antibody:

1, the structure in the clone of Fd chain, κ chain gene and κ chain library

From high titre bFGF or (FGF-2) autoantibody patient peripheral blood isolated lymphocytes.Extract cell total rna with Trizol (TAKARA company, RNA extracts reagent) reagent, adopt the Olig-dt primer, cDNA is synthesized in ReverTra Ace-a (seeing TOYOBO company test kit product description for details) test kit reverse transcription.(Fd-6 chain 3 ' primer is that VH2 and 5 ' primer are VH6 with the Fd of a group-specific IgG Fab and κ strand primer (seeing Table 1); κ-6 chain 3 ' primer is V κ 1, and κ-6 chain 5 ' primer is V κ 3) Fd and the κ chain of human source Fab carried out pcr amplification (reaction system sees Table 2), the PCR condition is: 94 ℃ of 5min, [94 ℃ of 45 Sec, 52 ℃ of 40 Sec, 72 ℃ of 50 Sec] * 29,72 ℃ of 10min.

The primer of table 1 pcr amplification people monoclonal antibody fragment gene

People's antibody Fd chain 3 ' primer
		VH1
5′-GCATGT ACTAGTTTTGTCACAAGATTTGGG-3′ VH2 5′-CTCGAC ACTAGTTTTGCGCTCAACTGTCTT-3′ VH3 5′-TGTGTG ACTAGTGTCACCAAGTGGGGTTTT-3′ VH4 5′-GCATGA ACTAGTThe AGGTGCAG of TGGGGGACCATATTTGGA-3 ' people antibody Fd chain 5 ' primer VH5 5 '-(C/G) CTCGAGTCTGGG-3′ VH6 5′-(C/G)AGGTGCAG CTCGAG(C/G) AGTCTGGG-3 ' people antibody κ chain 3 ' primer V κ 15 '-GCGCCG TCTAGAATTAACACTCTCCCCTGTTG AAGCTCTTTGTGACGGGCGAACTCAG-3 ' people antibody κ chain 5 ' primer V κ 25 '-GACATC GAGCTCACCCAGTCTCCA-3′	(Spe I) (Spe I) (Spe I) (Spe I) (Xho I) (Xho I) (Xba I) (Sac I)

Vκ3 5′-GATATT GAGCTCACTCAGTCTCCA-3′ Vκ4 5′-GAAATT GAGCTCACGCAGTCTCCA-3′	(Sac I) (Sac I)
		Annotate: underscore partly is a restriction enzyme site

The pcr amplification condition of table 2 Fd chain and κ chain

Reagent	The Fd chain	The κ chain	Positive control
				Tri-distilled water 10 times of PCR buffer solution 10 pmol/LdNTPs, 5 ' primers (20pmol/L) 3 ' primers (20pmol/L) cDNA Hot Start Taq (5U/ μ L)	33.75μL 5μL 4μL 1μL 1μL 5μL 0.25μL	33.75μL 5μL 4μL 2μL 2μL 3μL 0.25μL	38.25μL 5μL 4μL 1μL 1μL 0.5μL 0.25μL

Above-mentioned PCR product reclaims test kit (company) through sepharose and reclaims purifying, obtains Fd chain, the κ chain PCR product of the humanized's antibody about 650～700bp.

2, the structure of phage antibody library

Light chain κ chain PCR purified product is mixed, use Sac I and Xba I double digestion respectively, product reclaims with cleaning PCR test kit (Xgene company product), be inserted into pComb3 carrier (Babas with Sac I and Xba I double digestion, et al.Proc.Natl.Acas.Sci.USA.1991,89:10164-10168) in.Connector reclaims through ethanol sedimentation, electricity transformed competence colibacillus XL1-Blue bacterium (U.S. Stretigene company) (electric commentaries on classics condition: the BTX electroporation, 0.2cm electricity revolving cup, 2.0kv), electricity transforms the back rapid 1mL of adding SOC substratum, and (compound method is: the 20g Tryptones, the 5g yeast extract, 0.5g NaCl, 950mL deionized water, 20mL 0.25mol/LKCl, with 5mol/LNaOH the pH value is transferred to 7.0, adds deionized water and be settled to 1L, autoclaving deposit 4 ℃ standby.The 2mmol/L MgCl that adds the 5mL sterilization before using ₂1mol/L glucose solution with the 20mL degerming.), 37 ℃ of shaking culture 1h, get 100 μ L and behind 10 times of doubling dilutions, spread the dull and stereotyped storage capacity that detects of LB, the remaining access in the 50mL LB liquid nutrient medium (contains the 10mg/L tsiklomitsin, the 50mg/L penbritin), 37 ℃ of overnight incubation, next day is the upgrading grain from the bacterium of cultivating, and obtains light chain kappa gene storehouse pComb3-L.Fd chain PCR purified product and pComb3-L use Xho I and Spe I double digestion respectively, and the method same by light chain connects, and electricity transforms XL1-Blue.Electricity transforms the back and adds 1mL SOC substratum rapidly, 37 ℃ of shaking culture 1h, get 100 μ L and behind 10 times of doubling dilutions, spread the dull and stereotyped storage capacity that detects of LB, (containing the 10mg/L tsiklomitsin, the 50mg/L penbritin) 37 ℃ of overnight incubation in the remaining access 50mL LB liquid nutrient medium.Above-mentioned overnight culture is got 10mL divide 10 to guarantee and stay bacterial classification, get 80mL bacterium liquid alkaline lysis method of extracting recombinant plasmid dna, this plasmid is the band segmental pComb3 of Fab (being the Fab gene pool), called after pComb3-Fab.Be stored in-30 ℃.Incorporate above-mentioned residue bacterium liquid into 100mL LB (containing 50mg/L penbritin and 25mg/L tsiklomitsin), 37 ℃ of shaking culture are to OD ₆₀₀≈ 0.6.Add 400 μ L (about 10 ¹²Pfu) phage VCSM13,37 ℃ of shaking culture 2h.Add 70 μ g/ml kantlex then, 37 ℃ of shaking culture are spent the night.Inoculum is at 4 ℃, and the centrifugal 15min of 4000rpm puts supernatant in another aseptic triangular flask, adds 4%PEG ₈₀₀₀(polyoxyethylene glycol) and 3%NaCl, treat PEG and NaCl the dissolving after, put triangular flask in ice bath, the precipitation 30min.4 ℃ of centrifugal 30min of 8500rpm.Abandon supernatant, centrifuge tube is inverted in 10min on the aseptic thieving paper, remove PEG liquid.With the resuspended phage precipitation of 2mL PBS pH7.2, instantaneous centrifugal, promptly get the original storehouse of phage Fab antibody phage.Packing 0.4mL/ pipe is deposited in-20 ℃.Be used for therefrom screening the human source Fab antibody clone of high-affinity.

3, the screening of phage antibody library

Recombination human basic fibroblast growth factor (bFGF) or fibroblast growth factor 2 (FGF-2) (Bio-engineering Institute of Jinan University provides) are diluted to 1mg/L with 50mmol/L sodium bicarbonate (pH10.0), and bag is by elisa plate; 1%BSA-PBS sealing back adds the original storehouse of phage Fab antibody phage (about 10 of 100 μ L preparation ¹¹Pfu), hatch 2h, PBST (the PBS damping fluid adds 0.1% tween 20) liquid is washed 1 time, and (the 2nd takes turns and washes 5 times, the 3rd takes turns and washes 10 times, each 5min), reclaim phage with 200 μ L elutriants, the Tris damping fluid of pH8.0 is regulated pH to neutral, and the intestinal bacteria XL1-Blue bacterial strain that infects logarithmic phase carries out enrichment.Add 10mL LB substratum (containing 20mg/L penbritin and 10mg/L tsiklomitsin) after changing triangular flask over to, get 10 μ L respectively and 1 μ L is coated with LB (Amp ⁺) plate, the phage that titration elutes, remaining is in 37 ℃ of shaking culture 1h.Add the above-mentioned substratum of 100mL, 37 ℃ of shaking culture are to OD ₆₀₀=0.6, add VCSM13 (about 10 ¹²Pfu) carry out the phage antibody library activation, carry out the next round screening behind the mensuration CFU (plaque-forming unit).Undertaken 3 by same procedure and take turns elutriation.Obtain positive bacteriophage Fab antibody cloning.

4, the evaluation of phage antibody and sequential analysis

Picking 16 strains are inoculated in the LB nutrient solution that contains 50 μ g/mL penbritins through 3 positive colonies of taking turns the screening acquisition, and 37 ℃ are cultured to OD ₆₀₀=0.3～0.4, add helper phage (VCSM13) and activate the enrichment phage.Each phage clone of enrichment adopts the ELISA method to carry out specificity and identifies, every hole bag is added 70 μ L phage antibodies by people's recombinant bfgf 100ng, the mouse-anti M13 antibody colour developing of HRP mark.The results are shown in Figure 3.

Positive bacteriophage antibody plasmid with 3 strain specificity high-affinities is a template respectively, and pcr amplification goes out corresponding Fd chain and κ chain (called after Fd-6 chain, κ-6 chain successively; Fd-25 chain, κ-25 chain; Fd-26 chain, κ-26 chain), be connected respectively to difference construction recombination plasmid pMD18-T-Fd-6, pMD18-T-Fd-25, pMD18-T-Fd-26 on the T carrier; PMD18-T-κ-6 and pMD18-T-κ-25, pMD18-T-κ-26.Analyze through order-checking (serving the order-checking of marine life Engineering Co., Ltd).Measure sequence through its behaviour endogenous antibody gene of database (http://www.ncbi.nlm.nih.gov/igblast/) analysis revealed, and variable region sequences is a specific sequence.PMD18-T-Fd-6, pMD18-T-κ-6 are the recombinant plasmid of Fd-6 chain, κ-6 chain building, antibody promptly of the present invention, and Fd-6 chain, κ-6 chain-ordering sequencing result are as shown in Figure 1 and Figure 2.

The SDS-PAGE detected result of expression product: give birth to the molecular weight that worker's sequencing result is predicted expression product according to Shanghai, the molecular weight of albumen of Fd-6, Fd-25 and Fd-26 is about 25kD, and the molecular weight of albumen of κ-6, κ-25 and κ-26 is about 22 kD.Detect through SDS-PAGE and Western blot, contrast empty carrier pComb3H, the expression product crude extract band respectively occurs at 22kD and 25kD place, corresponding κ chain of difference and Fd chain, (seeing Fig. 5, Fig. 6) conforms to expected results.

5, solubility bFGF monoclonal antibody segment table reaches the structure of carrier

Plasmid pMD18-T-Fd-6, pMD18-T-Fd-25 and pMD18-T-Fd-26 use Xho I and Spe I double digestion respectively, after reclaiming the test kit specification sheets and reclaim Fd-6, Fd-25 and Fd-26 gene fragment respectively by dna gel, be connected to and pass through on the pComb3H carrier of same Xho I and Spe I double digestion, connect, transform XL1-Blue.Plasmid pMD18-T-κ-6, pMD18-T-κ-25 and pMD18-T-κ-26 use Sac I and Xba I double digestion respectively, after reclaiming the test kit specification sheets and reclaim κ-6, κ-25 and κ-26 gene fragment respectively by dna gel, respectively with corresponding same pComb3H-Fd-6, pComb3H-Fd-25 and pComb3H-Fd-26 carrier through Sac I and Xba I double digestion, connect, transform XL1-Blue.The plasmid that makes up is respectively through Nhe I and Spe I double digestion, removes the gene III sheet recirculation of having no progeny, and promptly is built into pComb3H-Fab-6, pComb3H-Fab-25 and pComb3H-Fab-26 solubility expression plasmid.

6, the expression of solubility bFGF monoclonal antibody section

With recombinant soluble expression plasmid pComb3H-Fab-6, pComb3H-Fab-25 and pComb3H-Fab-26 difference transformed into escherichia coli XL1-Blue, (this culture medium prescription is: 5g yeast water, 5g Tryptones, 2.5g NaCl at the 2YT substratum, 1000mL, pH7.0) 37 ℃ of overnight incubation in, be forwarded to 300mL 2YT (contain 20mg/L penbritin and 10mg/L tsiklomitsin) substratum with 1: 100 (volume ratio) next day, and 37 ℃ are cultured to OD ₆₀₀, add IPTG (final concentration is 1mmol/L), 30 ℃ of abduction deliverings 12～15 hours at=0.3～0.6 o'clock.4 ℃ of centrifugal thalline 15min of 4100rpm, with the resuspended thalline of 6mL0.02mol/L PBS (pH7.4), multigelation 3 times, ultrasonication 10min, the centrifugal 20min of 8000rpm, supernatant is the expression product crude extract.

7, the activity of expression product detects

Bag is by recombinant human bfgf 1 μ g/ hole, 4 ℃ are spent the night, 37 ℃ of sealings of 1%BSA 1h, PBS-T washes 4 times, sample well adds expression product crude extract 100 μ L/ holes, negative control hole adds the empty carrier pComb3H bacterium liquid ultrasonication supernatant 100 μ L/ holes behind the abduction delivering, hatch 1h for 37 ℃, PBS-T washes 5 times, adds through 1: 40, the special goat anti-human igg antibody of alkali phosphatase enzyme mark Fab of 000 dilution, 1h is hatched for 37 ℃ in 100 μ L/ holes, and PBS-T washes 5 times, adds the substrate O-Phenylene Diamine, hatch 30min, read A for 25 ℃ ₄₀₅Value.The results are shown in Figure 4.The expression product crude extract of 3 strain clones (pComb3H-Fab-6, pComb3H-Fab-25 and pComb3H-Fab-26) detects A through ELISA ₄₀₅All more than 2.5 of negative value times.Therefore, the expression product of three strain clones can both combine (as shown in Figure 4) with bFGF is specific.

The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

SEQUENCE LISTING

＜110〉Ji'nan University

＜120〉anti recombined alkalescent fibroblast growth factor human source Fab antibody and application thereof

<130>28

<160>4

<170>PatentIn version 3.2

<210>1

<211>633

<212>DNA

<213>Artificial sequence

<220>

＜223〉gene order of heavy chain Fd chain

<400>1

cagtctgggg gaggcttggt acagcctggc aggtccctga gactcacctg tgcagcctct 60

ggattcacct ttgaagatta tggcatgcac tgggtccggc aagctccagg gaagggcctg 120

gagtgggtct caggtattag ctggaatagt ggtagtatag gctatgcgga ctctgtgaaa 180

ggccgcttca ccatctccag agacaacgcc aagaattccc tgtatttgca aatgaacagt 240

ctgagagctg aggacacggc cttgtattac tgtgcaaaag acaggcgctt cggtgactac 300

gtccactcct actacggtat ggacgtctgg ggcccatcgg tcttccccct ggcaccctcc 360

tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 420

gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 480

gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 540

agcttgagca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 600

gacaagagag ttgagcccaa atcttgtgac aaa 633

<210>2

<211>639

<212>DNA

<213>Artificial sequence

<220>

＜223〉gene order of light chain κ chain

<400>2

gatattgagc tcactcagtc tccatcctcc ctgtctgcat ctataggaga cacagtcacc 60

atcacttgcc gggcaagtca gagcattgac atctatgtaa attggtatca gcacaaagca 120

ggcaaagtcc ctaaactcct aatgcatact gcatccagtt tagaaagtgg ggtcccacca 180

aggttcagtg gcagtggctc tgggacagat ttcactctca ctatcagcgg tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttatcgtc ttgtcagttt cggccctggg 300

accaaagtgg atattagacg aactgtggct gcaccatctg tcttcatctt cccgccatct 360

gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420

agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480

agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540

agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600

agttcgcccg tcacaaagag cttcaacagg ggagagtgt 639

<210>3

<211>211

<212>PRT

<213>Artificial sequence

<220>

＜223〉aminoacid sequence of heavy chain Fd chain

<400>3

Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Thr

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr Gly Met His Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Ser Trp

35 40 45

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

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Asp Arg Arg

85 90 95

Phe Gly Asp Tyr Val His Ser Tyr Tyr Gly Met Asp Val Trp Gly Pro

100 105 110

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

115 120 125

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

130 135 140

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

145 150 155 160

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

165 170 175

Val Pro Ser Ser Ser Leu Ser Thr Gln Thr Tyr Ile Cys Asn Val Asn

180 185 190

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

195 200 205

Cys Asp Lys

210

<210>4

<211>213

<212>PRT

<213>Artificial sequence

<220>

＜223〉aminoacid sequence of light chain κ chain

<400>4

Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly

1 5 10 15

Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ile Tyr

20 25 30

Val Asn Trp Tyr Gln His Lys Ala Gly Lys Val Pro Lys Leu Leu Met

35 40 45

His Thr Ala Ser Ser Leu Glu Ser Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Set Tyr Arg Leu Val Ser

85 90 95

Phe Gly Pro Gly Thr Lys Val Asp Ile Arg Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

Claims (3)

1, a kind of anti recombined alkalescent fibroblast growth factor human source Fab antibody, described antibody fragment gene comprise heavy chain Fd chain and light chain κ chain, it is characterized in that:

The gene order of described heavy chain Fd chain is as follows:

CAG TCT GGG GGA GGC TTG GTA CAG CCT GGC AGG TCC CTG AGA CTC ACC TGT

GCA GCC TCT GGA TTC ACC TTT GAA GAT TAT GGC ATG CAC TGG GTC CGG CAA

GCT CCA GGG AAG GGC CTG GAG TGG GTC TCA GGT ATT AGC TGG AAT AGT GGT

AGT AIA GGC TAT GCG GAC TCT GTG AAA GGC CGC TTC ACC ATC TCC AGA GAC

AAC GCC AAG AAT TCC CTG TAT TTG CAA ATG AAC AGT CTG AGA GCT GAG GAC

ACG GCC TTG TAT TAC TGT GCA AAA GAC AGG CGC TTC GGT GAC TAC GTC CAC

TCC TAC TAC GGT ATG GAC GTC TGG GGC CCA TCG GTC TTC CCC CTG GCA CCC

TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC AAG

GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC

AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC

TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG AGC ACC CAG

ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC

AAG AGA GTT GAG CCC AAA TCT TGT GAC AAA；

The gene order of described light chain κ chain is as follows:

GAT ATT GAG CTC ACT CAG TCT CCA TCC TCC CTG TCT GCA TCT AIA GGA GAC

ACA GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAC ATC TAT GTA AAT

TGG TAT CAG CAC AAA GCA GGC AAA GTC CCT AAA CTC CTA ATG CAT ACT GCA

TCC AGT TTA GAA AGT GGG GTC CCA CCA AGG TTC AGT GGC AGT GGC TCT GGG

ACA GAT TTC ACT CTC ACT ATC AGC GGT CTG CAA CCT GAA GAT TTT GCA ACT

TAC TAC TGT CAA CAG AGT TAT CGT CTT GTC AGT TTC GGC CCT GGG ACC AAA

GTG GAT ATT AGA CGA ACT GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA

TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT

AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC

CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC

AGC ACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG

AAA CAC AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGT TCG CCC

GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT。

2, according to the described a kind of anti recombined alkalescent fibroblast growth factor human source Fab antibody of claim 1, it is characterized in that: the aminoacid sequence of described heavy chain Fd chain is as follows:

Q S G G G L V Q P G R S L R L T C

A A S G F T F E D Y G M H W V R Q

CDR1

A P G K G L E W V S G I S W N S G

CDR2

S I G Y A D S V K G R F T I S R D

N A K N S L Y L Q M N S L R A E D

T A L Y Y C A K D R R F G D Y V H

CDR3

S Y Y G M D V W G P S V F P L A P

S S K S T S G G T A A L G C L V K

D Y F P E P V T V S W N S G A L T

S G V H T F P A V L Q S S G L Y

S L S S V V T V P S S S L S T Q

T Y I C N V N H K P S N T K V D

K R V E P K S C D K；

The aminoacid sequence of described light chain κ chain is as follows:

D I E L T Q S P S S L S A S I G D

T V T I T C R A S Q S I D I Y V N

CDR1

W Y Q H K A G K V P K L L M H T A

S S L E S G V P P R F S G S G S G

CDR2

T D F T L T I S G L Q P E D F A T

Y Y C Q Q S Y R L V S F G P G T K

CDR3

V D I R R T V A A P S V F I F P P

S D E Q L K S G T A S V V C L L N

N F Y P R E A K V Q W K V D N A L

Q S G N S Q E S V T E Q D S K D

S T Y S L S S T L T L S K A D Y E

K H K V Y A C E V T H Q G L S S P

V T K S F N R G E C。

3, the application that is used for diagnosing and treat tumour and suppresses the antibody drug of kidney, liver or pulmonary fibrosis in preparation according to the described a kind of anti recombined alkalescent fibroblast growth factor human source Fab antibody of claim 1.

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