CN106279414A - Humanized's anti-amylin antibody and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of humanized's anti-amylin antibody and preparation method thereof, belong to immunoglobulin domain.The heavy chain variable amino acid sequence of antibody of the present invention is as shown in SEQ ID NO:1, and chain variable region amino acid sequence is as shown in SEQ ID NO:2；Variable region of heavy chain DNA sequence is as shown in SEQ ID NO:3, and variable region of light chain DNA sequence is as shown in SEQ ID NO:4；Heavy chain gene is IgG1 subclass VH3 gene family, and light chain gene is Lambda gene family.The present invention has filtered out humanized's amylin antibody by the antibacterial storehouse of the phage setting up high power capacity, and this antibody has good antigenic specificity and antigen-binding activity, provide guarantee for further producing amylin detection kit, have broad application prospects.

Description

Humanized's anti-amylin antibody and preparation method thereof

Technical field

The invention belongs to immunoglobulin domain, specifically a kind of humanized's anti-amylin antibody and preparation method thereof.

Background technology

Amylin (Amylin) is the hormone of B cell secretion, is stored in the secretory granule of B cell altogether with insulin In, both are discharged into outside B cell together with certain proportion under glucose and non-saccharide hormonal stimulation.Internal metabolism process class Being similar to C-peptide, research finds, amylin is slow far beyond insulin at the accretion rate of liver, and amylin is mainly arranged through kidney Let out.Have now found that amylin is a pleiotropic hormone, take part in material and the energy metabolism of body with multiple player, but its Definite biological function is unclear.The main composition composition of islet amyloid (IA) is amylin, and postmortem finds 90% Above type Ⅱdiabetes mellitus person's islets of langerhans has the deposition of IA.Research shows, solubility, has Cytotoxic amylin aggregation and is Early stage intermediate product in islet amyloid modified fibre forming process, this aggregation is referred to as median size toxicity Amyloid granule, can destroy islet cells bilayer lipid membrane, causes the disorder of intracellular environment, the changing of ion concentration Become, cause response to oxidative stress, final islet cells generation apoptosis.

Amylin increasingly comes into one's own with the relation of type Ⅱdiabetes mellitus and progressively confirms, there is also many disputes simultaneously, And the definite biological function of amylin is not yet fully apparent from, preparing this hormone high-purity antibody is to study this hormone further The key of physiological and pathological effect.Little yet with the molecular weight of this hormone, in-vivo content is low, uses monoclonal antibody immunity method Detect its method complicated, antibody deficiency stability and specificity；The method preparing antibody by tradition, such as hybridoma technology and antigen It is few that the method for immunity prepares the antibody that antibody obtains, and intricate operation, and the cycle is longer, the antibody deficiency stability prepared and Specificity, brings resistance therefore to research this hormone physiological and pathological roles widely in vivo, therefore prepares this hormone High-purity antibody is the key studying this hormone further.

Antibody technique development experience three phases: first on behalf of antiserum polyclonal antibody (Polyclonal Antibody, PcAb), second utilizes B lymph on behalf of 70 mid-nineties 90 German scholar Kohler and British scholar milstein Cell hybridoma technique (technology of cell engineering generation antibody) is prepared for authentic monoclonal antibody (Monoclonal Antibody, McAb), one of this milestone being the discovery that biotech development, diagnosing, treat, the aspect such as prevention the most aobvious Show important function.But, easily cause due to M-band human anti-mouse antibody (Human anti-mouse antibody, HAMA) reaction, and people's hybridoma technology is difficult to break through, and therefore occurs in that third generation antibody, both genetic engineering antibody.Gene work Engineered antibody includes three kinds, i.e. chimeric antibody (chimeric antibody), reshaped antibody (reshaping antibody) and Antibody prepared by Antibody library.Wherein the above two belong to part-humanised antibody, and antibody prepared by Antibody library belongs to full people Source antibody.I.e. clone out by repertoire antibody (Repertoire) heavy chain and chain variable region gene with gene clone technology, Restructuring is to prokaryotic expression carrier, by directly expressing the Antibody molecule fragments of function escherichia coli, screen specific can Become gene.Engineered method is used to eliminate the screening process that hybridoma technology is numerous and diverse, it might even be possible to obtain without immunity Must significantly advance the progress of antibody engineering for the acquisition of the specific antibody of any antigen, particularly human antibody, and solve Animal derived antibody of having determined produces a difficult problem for allos reaction when human body therapy, has widened the range of application of antibody.

The preparation appearing as human matural antibodies of phage antibody library technique opens new approach, is used for for a large amount of preparations The human antibody of the high-efficiency high-quality of diagnosis and treatment provides feasibility, and it makes to screen potential people's antibody fragment and is possibly realized Property, preferably resolve problem prepared by antibody difficulty.This technology achieves breakthrough in immunotherapy of tumors.

Summary of the invention

The present invention is contemplated to solve existing amylin test kit costly, and humanized's pancreas prepared by traditional method Opsonin antibody easily causes allergic reaction, the problem such as Antibody stability and poor specificity, the anti-amylin of a kind of humanized proposed Antibody and preparation method thereof.

The present invention realizes according to techniques below scheme.

A kind of humanized's anti-amylin antibody, the heavy chain variable amino acid sequence such as SEQ ID NO:1 institute of described antibody Showing, chain variable region amino acid sequence is as shown in SEQ ID NO:2.

The variable region of heavy chain DNA sequence of above-mentioned antibody as shown in SEQ ID NO:3, variable region of light chain DNA sequence such as SEQ Shown in ID NO:4；Heavy chain gene is IgG1 subclass VH3 gene family, and light chain gene is Lambda gene family.

The preparation method of a kind of above-mentioned humanized anti-amylin antibody, comprises the following steps:

I. the PCR amplification of human IgG Fab fragment gene: separate the lymphocyte in human peripheral, extract total serum IgE, by extract RNA reverse transcription becomes cDNA, and employment specific IgG Kappa chain, Lambda chain and heavy chain Gamma strand primer are respectively to people's light chain PCR amplification is carried out with heavy chain gene；

II. the foundation of phage antibody library: the PCR product obtained in step I is mixed respectively by Fd, κ chain and λ chain；Will be double Light chain PCR mixture and phasmid after enzyme action are attached reaction, connect product electricity and are transformed into competent cell, and amplification is shaken Light chain gene storehouse is built after bacterium；The light chain storehouse phagemid dna built and heavy chain Fd section PCR mixture are carried out double digestion, even Connect rear Electroporation-competent cells, add culture medium culturing, add helper phage and continue to cultivate；Add after centrifugal collection supernatant Entering PEG precipitated liquid, ice bath, centrifugal supernatant the resuspended precipitation of abandoning, recentrifuge collection supernatant is phage antibody library；

III. the specific enrichment of phage antibody library and screening: be coated elisa plate, BSA-PBS solution with the amylin after dilution Add phage antibody library after closing, hatch, first wash with PBST solution, use Gly-HCl buffer solution elution then, and use Tris Buffer neutralizes, and infects and cultivates the escherichia coli to exponential phase, and after taking gradient dilution, liquid bed board measures titre, calculates every The output capacity of wheel screening；Remaining bacterium adds helper phage infection and carries out the enrichment isolation of next round, 3-8 time repeatedly；

IV. the Phage-ELISA detection of anti-amylin antibody: random picking multiple clone preparation from the bacterial clump after screening Monoclonal phage-antibodies, is coated elisa plate with the amylin after dilution, and BSA-PBS solution adds phage supernatant after closing, Wash with PBST solution after hatching；Add HA-Tag Mouse mAb, wash with PBST solution after hatching；Add HRP-goat Anti-mouse IgG, washs with PBST solution after hatching, and adds the colour developing of TMB solution, measures A450 value；

V. after variable region determined dna sequence: ELISA detection, screening positive clone carries out DNA sequencing.

Present invention obtains following beneficial effect.

The present invention effectively solves existing amylin test kit costly, and humanized's pancreas prepared by traditional method Opsonin antibody easily causes allergic reaction, the problem such as Antibody stability and poor specificity.The present invention establishes the phage of high power capacity Antibacterial storehouse, has filtered out humanized's amylin antibody, and this antibody has good antigenic specificity and antigen-binding activity, Provide guarantee for further producing amylin detection kit, have broad application prospects.

Accompanying drawing explanation

Fig. 1 is heavy chain Fd fragment gene PCR purified product electrophoretogram of the present invention；

Fig. 2 is light chain gene PCR purified product electrophoretogram of the present invention；

Fig. 3 is that light chain storehouse of the present invention is through Sac I and Xba I double digestion electrophoretogram；

Fig. 4 is that phage antibody library of the present invention is through Sac I and Spe I double digestion electrophoretogram；

Fig. 5 is that Phage-ELISA method of the present invention detects the phage antibody combination activity figure to amylin antigen.

Fig. 6 is the secondary structure figure of C1 heavy chain of the present invention；

Fig. 7 is the secondary structure figure of C1 light chain of the present invention.

Detailed description of the invention

One, experiment material

Phagemid vector pComb3XSS(4900bp) buy in U.S.'s Scripps institute；Escherichia coli XL1-Blue and auxiliary Helper phage VCSM13 is purchased from Stratagene company of the U.S.；TRNzol-A⁺ Total RNA Reagent、 Quantscript RT Kit, 2 × Taq PCR MasterMix, plain agar sugar gel recovery test kit, high-purity plasmid are little Carry middle amount test kit and be purchased from TIANGEN company；It is public that restricted enzyme Sac I, Xba I, Xho I, Spe I are purchased from TaKaRa Department；Amylin fragment (8-37) is purchased from Sigma company；HA-Tag Mouse mAb(26D11) purchased from Abmart company, HRP-goat anti-mouse IgG (H+L) is purchased from Beijing Ding Guo company；HRP labelling goat-anti human Fab's IgG antibody is purchased from Sigma company； Other reagent are domestic analytical pure.

Two, experimental technique

1. the PCR amplification of human IgG Fab fragment gene:

From the peripheral blood of normal healthy people, separate lymphocyte with lymphocyte separation medium, use TRNzol-A⁺Extraction cell is total RNA, with Oligo(dT)₁₅For primer, the RNA of extraction is become cDNA by Reverse Transcriptase kit reverse transcription, (reference literature Kang AS, Burton DR, Jones TM, et al. Combinatorial immunoglubulin libraries in phage λ. Methods: Comp Methods Enzymol,1991;2 (2): 111-118. and Marks JD, Hoogenboom HK, Bonnert TP, et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol, 1991;222 (3): 581-597.) divide Not with human specific IgG Kappa chain, Lambda chain and heavy chain Gamma chain 5 ' end and various combination (the primer sequence of 3 ' end primers Arrange as follows) people's light chain (VC+HC) and heavy chain (VH+CH1) gene are carried out PCR amplification, PCR condition is 94 DEG C of denaturations 3min, 94 DEG C of 1min, 54 DEG C ~ 58 DEG C (54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C) 50s, 72 DEG C of 1min, after 30 circulations, 72 DEG C Extend 10min.

(1) human immunoglobulin heavy chain's (Gamma chain) Fd section variable region 5 ' end primer:

VH1a:5 '-CAG GTG CAGCTC GAGCAG TCT GGG-3 ',

VH1f:5 '-CAG GTG CAG CTGCTC GAGTCT GGG-3 ',

VH2f:5 '-CAG GTG CAG CTACTC GAGTCG GG-3 ',

VH3a:5 '-CAG GTG CAGCTC GAG GAGTCT GGG-3 ',

VH3f:5 '-GAG GTG CAG CTGCTC GAGTCT GGG-3 ',

VH4f:5 '-CAG GTG CAG CTGCTC GAGTCG GG-3 ',

VH6a:5 '-CAG GTA CAGCTC GAGCAG TCA GG-3 ',

VH6f:5 '-CAG GTG CAG CTACTC GAG TGG GG-3’。

Human immunoglobulin heavy chain's (Gamma chain) CH1 district 3 ' end primer:

CG1z:5 '-GCA TGTACT AGTTTT GTC ACA AGA TTT GGG-3 ',

CG2a:5 '-CTC GACACT AGTTTT GCG CTC AAC TGT CTT-3 ',

CG3a:5 '-TGT GTGACT AGTGTC ACC AAG TGG GGT TTT-3 ',

CG4a:5 '-GCA TGAACT AGTTGG GGG ACC ATA TTT GGA-3 ',

(2) human normal immunoglobulin Kappa chain variable region 5 ' end primer:

V κ 1a:5 '-GAC ATCGAG CTCACC CAG TCT CCA-3 ',

V κ 1s:5 '-GAC ATCGAG CTCACC CAG TCT CC-3 ',

V κ 2a:5 '-GAT ATTGAG CTCACT CAG TCT CCA-3 ',

V κ 3a:5 '-GAA ATTGAG CTCACG CAG TCT CCA-3 ',

V κ 3b:5 '-GAA ATTGAG CTC ACG CAG TCT CCA-3’。

Human normal immunoglobulin's Kappa chain constant region 3 ' end primer:

C κ 1d:5 '-GCG CCGTCT AGA ATT AAC ACT CTC CCC TGT TGA AGC TCT TTG TGA CGG GCG AAC TCA G-3’。

(3) human normal immunoglobulin Lambda chain variable region 5 ' end primer:

VL1:5 '-AAT TTTGAG CTCACT CAG CCC CAC-3 ',

VL2:5 '-TCT GCCGAG CTCCAG CCT GCC TCC GTG-3 ',

VL3:5 '-TCT GTGGAG CTCCAG CCG CCC TCA GTG-3 ',

VL4:5 '-TCT GAAGAG CTCCAG GAC CCT GTT GTG TCT GTG-3 ',

VL5:5 '-CAG TCTGAG CTCACG CAG CCG CCC-3 ',

VL6:5 '-CAG ACTGAG CTCACT CAG GAG CCC-3 ',

VL7:5 '-CAG GTTGAG CTCACT CAA CCG CCC-3 ',

(4) human normal immunoglobulin Lambda chain constant region 3 ' end primer:

C λ 2:5 '-CGC CGTCTA GAA TTA TGA ACA TTC TGT AGG-3’

CTC GAGFor restriction endonuclease Xho I recognition site；ACT AGTFor restriction endonuclease Spe I recognition site；GAG CTCFor restriction endonuclease Sac I recognition site；TCT AGAFor restriction endonuclease Xba I recognition site.

2. the foundation of phage antibody library:

Above-mentioned PCR product after agarose gel purification reclaims (680bp), is mixed by Fd, κ chain and λ chain respectively respectively.Will be light Chain PCR mix products and phasmid pComb3XSS with Xba I and Sac I double digestion, reclaim through purified after electrophoresis respectively.Then The two is attached reaction with T4 ligase, connects product 10 μ l electricity and be transformed into 300 μ l competent cell XL1-Blue, electricity The condition of turning is: Bio-Red electroporation, 0.2cm electricity revolving cup, 2.5kV, and amplification builds light chain gene storehouse after shaking bacterium.

Light chain storehouse phagemid dna and the heavy chain Fd section PCR mix products Spe I and Xho I of extraction are carried out double digestion, even Electricity transformed competence colibacillus bacterium XL1-Blue after connecing.Electricity adds 3ml after turning and preheats SOC culture medium, adds after 37 DEG C of shaken cultivation 1h The SB-A of 10ml preheating⁺T⁺(Amp 50 μ g/ml, Tet 10 μ g/ml) culture medium, adds Amp to 100 after 37 DEG C of shaken cultivation 1h μ g/ml, 37 DEG C of shaken cultivation 1h, add helper phage VCSM13 about 10¹² It is incorporated to 100ml SB-A after pfu/ml mixing⁺T⁺ (Amp 50 μ g/ml, Tet 10 μ g/ml) culture medium, adds shaken cultivation after Kan to 70 μ g/ml after 37 DEG C of shaken cultivation 2h Overnight.Adding PEG precipitated liquid after centrifugal collection in secondary morning supernatant, be centrifuged and abandon supernatant after ice bath 30min, 2ml 1% BSA-PBS is resuspended After precipitation, recentrifuge is collected supernatant and is phage antibody library.

Wherein

SOC culture medium:

Tryptone 20g

Yeast extract 5 g

NaCl 0.5 g

KCl(1mol/L) 2.5ml

It is dissolved in 800ml distilled water, adjusts PH to 7.0 with 5mol/L NaOH, mend distilled water to 1000ml, autoclave sterilization After, 4 DEG C of preservations.Add degerming glucose before using to 20mmol/L and degerming MgCl₂To 10mmol/L.

SB culture medium:

Tryptone 30g

Yeast extract 20 g

MOPS 10 g

It is dissolved in 800ml distilled water, adjusts PH to 7.0 with 5mol/L NaOH, mend distilled water to 1000ml, autoclave sterilization After, 4 DEG C of preservations.

Experimental result: take the fresh peripheral blood of blood normal healthy people, separates peripheral blood lymphocyte, extracts cell total rna And reverse transcription synthesis cDNA, with 13, light chain primer and 21 pairs of heavy chain Fd section primers are carried out PCR amplification, occur at 680bp Specific band (see Fig. 1,2；Wherein, in Fig. 1,1-5 swimming lane is can by human immunoglobulin heavy chain's Gamma chain Fd section respectively Become district 5 ' end primer and CH1 district 3 ' end primer carries out the product after PCR as primer: VH1a+CG2a, VH1f+CG2a, VH2f+ CG2a、VH3a+CG2a、VH4f+CG2a；In Fig. 2,1-5 swimming lane is to hold with human normal immunoglobulin Lambda chain variable region 5 ' respectively Primer and human normal immunoglobulin's Lambda chain constant region 3 ' end primer carry out the product after PCR as primer: VL3+ C λ 2, VL4 + C λ 2, VL5+ C λ 2, VL6+ C λ 2, VL7+ C λ 2).XL1-Blue antibacterial is proceeded to through enzyme action electricity after the mixing of light chain PCR primer, Set up light chain gene storehouse (4380bp).With Sac I and Xba I double digestion, light chain (680bp) gene proceeded to is scaled off, electrophoresis (seeing Fig. 3, wherein 1-10 swimming lane is that light chain storehouse is through Sac I and Xba I to obtain two specific bands at 3700bp and 680bp Product after double digestion, M1 is Marker DL15000, be followed successively by from top to bottom 15000bp, 10000bp, 7500bp, 5000bp、3000bp、1500bp、1000bp、500bp；M2 is Marker DL2000, be followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp).By equal to light chain storehouse phagemid dna and the heavy chain Fd section PCR product of extraction With Xho I and Spe I double digestion, after connection, electricity transformed competence colibacillus bacterium XL1-Blue, establishes phage antibody gene bank (4760bp), storage capacity is 8.4 × 10⁷.With Sac I and Spe I double digestion by proceed to containing light chain (680bp) and heavy chain Fd (680bp) common fragment scales off, and electrophoresis obtains two specific bands at 3260bp and 1500bp and (sees Fig. 4, wherein 1-10 swimming lane is phage antibody library product after Sac I and Spe I double digestion；M1 is Marker DL15000, from top to bottom It is followed successively by 15000bp, 10000bp, 7500bp, 5000bp, 3000bp, 1500bp, 1000bp, 500bp；M2 is Marker DL2000, is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom；3-7,9,10 is positive gram Grand, discharge the genetic fragment of 1500bp, for Fab fragment).

3. the specific enrichment of phage antibody library and screening: (reference literature Griffiths AD, Duncan AR. Strategies for selection of antibodies by phage display [J] . Curr Opin Biotechnol, 1998;9 (1): 102-108, and Barbas C III, Kang AS, Lenner RA, et al. Assembly of combinatorial antibody libraries on phage surfaces : the gene Ⅲ site. Proc Natl Acad Sci USA, 1991;89:10164-10168, and Barbas C III, Lenner RA, Combinatorial immunoglobulin libraries on the surface of phage (phabs) : rapid selection of antigen specific Fabs. Methods: Comp Methods Enzymol, 1991;2 (2): 119-124.) diluting amylin to concentration with bicarbonate buffer (pH8.6) is 50 μ g/ml, every hole 50 μ l is coated elisa plate, and in wet box, 4 DEG C overnight；The phosphate buffer (PBS) of secondary daily 3% BSA-PBS(bovine serum albumin) Close 1h, add the phage antibody library (100 μ l/ hole) of fresh preparation, wet box hatches 2 hours for 37 DEG C；Abandon antibody library liquid, use PBS(PBST containing 0.05% Tween-20) (first round screens to wash away non-specific binding or the weak phage antibody of adhesion Time wash 1 time；Second takes turns and washes 5 times；Third round washes 10 times), every hole is with the 0.1mol/L Gly-HCl buffer of 50 μ l pH2.2 Phage antibody specific binding with amylin under eluting, and neutralize with the 2M Tris buffer of 8 μ l pH9.6, infect 2ml fresh cultured, to the E.colistrain XL1 blue of exponential phase (OD600 is 0.6 ~ 1.0), hatches 30 min for 37 DEG C.Take After 10 μ l gradient dilutions, bed board measures titre, calculates the output capacity often taking turns screening；Remaining bacterium carries out next after adding VCSM13 infection The enrichment isolation of wheel, such " absorption-eluting-amplification " 3-8 time repeatedly, preferably 5 times.

Wherein PBS composition and compound method:

800ml distilled water dissolves

NaCl 8g

KCl 0.2g

Na₂HPO₄ 1.44g

KH₂PO₄ 0.24g

Add water to 7.4 with the pH value of HCl regulation solution and be settled to 1L, steam sterilization 30min under high pressure.It is stored in room temperature.

Experimental result: as antigen, phage antibody library has been carried out 3 with immobilised people Amylin and taken turns screening, calculated every One takes turns productivity (yield%) is shown in Table 1, although washing intensity increases by wheel, but the specific bacteriophage antibody titer eluted Substantially increasing, productivity also increases, and specific bacteriophage antibody has obtained obvious enrichment.After 3 take turns screening the titre of enrichment be 4 × 10⁹。

*: productivity (%)=(the phagocytosis scale of construction of output)/(the phagocytosis scale of construction of input) × 100%

The Phage-ELISA detection of the most anti-amylin antibody:

From the bacterial clump after screening, random 28 clones of picking prepare monoclonal phage-antibodies, by Amylin antigen diluent It is coated elisa plate to 10 μ g/ml, sets empty carrier pComb3XSS phage supernatant, irrelevant antigen BSA as negative right simultaneously According to.3% BSA-PBS adds above-mentioned phage supernatant after closing, and hatches 2h for 37 DEG C.PBST washes 6 times, each 5min.Every hole adds 50 μ l HA-Tag Mouse mAb(26D11) (1:600), 37 DEG C hatch 1 h after PBST wash 6 times；Every hole adds 50 μ l HRP-goat anti-mouse IgG (H+L) (1:2000), hatches 1 h for 37 DEG C, and PBST adds TMB colour developing after washing plate, and microplate reader surveys A₄₅₀ Value (see Table 2).

C10: empty carrier phage supernatant negative control；C11: irrelevant antigen BSA comparison；C12: blank

Select the bacterial strain of positive colony to serve sea Invitrogen company after ELISA detection and complete DNA sequencing.

Experimental result: wherein have 4 clones (A12, B11, C1, C2) to be positive (with OD₄₅₀> negative control value 2.1 times sentences It is set to the positive), positive rate is 14.3%(table 2).One factor analysis of variance, positive colony group is carried out with SPSS13.0 statistical software Compare with empty vector control group, difference statistically significant (P< 0.01)；Positive colony group nothing to do with antigen BSA matched group ratio Relatively, difference statistically significant (P< 0.01).The above results shows that the antibody of positive colony has the special knot of amylin antigen Close activity and (see Fig. 5, wherein^bP < 0.01 vs empty vector control group；^dP < 0.01 vs BSA matched group).

After taking C1 positive colony amplification culture, extract phasmid DNA, it is carried out sequencing analysis.With in NCB1 NucleotidemBlast and Blast Analysis of Immunoglobulin Sequences analyzes software to its sequence It is analyzed finding: the variable region of light chain of C1 positive colony and human normal immunoglobulin's λ chain have the homology of 93%, weight chain variable District's gene and human normal immunoglobulin IgG heavy chain VH3 have the homology of 88%.Lambda light chain V gene belongs to V λ 2 subgroup, and J gene belongs to J λ3；Heavy chain V gene belongs to VH3-15 subgroup, and D gene is from D3-16, J gene from JH5.

5. amino acid sequence translation

Positive colony C1 light chain and heavy chain Fd section nucleotide sequence are translated into aminoacid sequence and to it by correct reading frame Determinant complementary region (CDR) is positioned.With analyzing software, the aminoacid sequence of positive colony light chain and heavy chain Fd section is entered Having gone Sketch of secondary structure, result display both meets antibody secondary structure configuration (Fig. 6,7).

SEQUENCE LISTING

<110>General Hospital of Tianjin Medical Univ.

<120>humanized's anti-amylin antibody and preparation method thereof

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Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

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Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

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ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag gacgaggctg 120

attattactg cagctcacat acaagcagca gaacttgggt gttcggcgga gggaccaagc 180

tgaccgtcct aggtcagccc aaggctgccc cctcggtcac tctgttcccg ccctcctctg 240

aggagcttca agccaacaag gccacactgg tgtgtctcat aagtgacttc tacccgggag 300

ccgtgacagt ggcctggaag gcagatagca gccccgtcaa ggcgggagtg gagaccacca 360

caccctccaa acaaagcaac aacaagtacg cggccagcag ctacctgagc ctgacgcctg 420

agcagtggaa gtcccacaaa agctacagct gccaggtcac gcatgaaggg agcaccgtgg 480

agaagacagt ggcccctaca gaatgttcat aattctagat aattaattag gaggaattta 540

aaatgaaata cctattgcct acggcagccg ctggattgtt attactcgct gcccaaccag 600

ccatggccga ggtgcagctg ctcgagtctg ggggaggctt ggtaaagcct ggggggtccc 660

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ctccaggctg aggacgaggc tgattattac tgcagctcac atacaagcag cagaacttgg 60

gtgttcggcg gagggaccaa gctgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 120

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 180

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 240

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 300

agctacctga gcctgacgcc tgagcagtgg aagtcccaca aaagctacag ctgccaggtc 360

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc ataattctag 420

ataattaatt aggaggaatt taaaatgaaa tacctattgc ctacggcagc cgctggattg 480

ttattactcg ctgcccaacc agccatggcc gaggtgcagc tgctcgagtc tgggggaggg 540

ttggtaaagc ctggggggtc ccttagactc tcctgtgcag cctctggatt cgctttcaga 600

gccgcctgga tgaactgggt ccgccaggct ccagggaagg gctggagtgg ttggccgcat 660

taacagacaa attatg 676

SEQUENCE LISTING

<110>General Hospital of Tianjin Medical Univ.

<120>humanized's anti-amylin antibody and preparation method thereof

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 60

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221>C1 heavy chain variable amino acid sequence

<222> (1)..(60)

<400> 1

Glu Glu Phe Lys Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu

1 5 10 15

Leu Leu Leu Ala Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu

20 25 30

Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys

35 40 45

Ala Ala Ser Gly Phe Thr Phe Ser Thr Ala Trp Met

50 55 60

<130> 2

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 104

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221>C1 chain variable region amino acid sequence

<222> (1)..(104)

<400> 1

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Ala

100

<130> 3

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 679

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221>C1 variable region of heavy chain DNA sequence

<222> (1)..(679)

<400> 1

aactcataat ttataatgtc agtcatcagc cctcaggggt ttctagtcgc ttctctggct 60

ccaagtctgg caacacggcc tccctgacca tctctgggct ccaggctgag gacgaggctg 120

attattactg cagctcacat acaagcagca gaacttgggt gttcggcgga gggaccaagc 180

tgaccgtcct aggtcagccc aaggctgccc cctcggtcac tctgttcccg ccctcctctg 240

aggagcttca agccaacaag gccacactgg tgtgtctcat aagtgacttc tacccgggag 300

ccgtgacagt ggcctggaag gcagatagca gccccgtcaa ggcgggagtg gagaccacca 360

caccctccaa acaaagcaac aacaagtacg cggccagcag ctacctgagc ctgacgcctg 420

agcagtggaa gtcccacaaa agctacagct gccaggtcac gcatgaaggg agcaccgtgg 480

agaagacagt ggcccctaca gaatgttcat aattctagat aattaattag gaggaattta 540

aaatgaaata cctattgcct acggcagccg ctggattgtt attactcgct gcccaaccag 600

ccatggccga ggtgcagctg ctcgagtctg ggggaggctt ggtaaagcct ggggggtccc 660

tcagactctc ctgtgcagc 679

<130> 4

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 676

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221>C1 variable region of light chain DNA sequence

<222> (1)..(676)

<400> 1

ctccaggctg aggacgaggc tgattattac tgcagctcac atacaagcag cagaacttgg 60

gtgttcggcg gagggaccaa gctgaccgtc ctaggtcagc ccaaggctgc cccctcggtc 120

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 180

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 240

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 300

agctacctga gcctgacgcc tgagcagtgg aagtcccaca aaagctacag ctgccaggtc 360

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc ataattctag 420

ataattaatt aggaggaatt taaaatgaaa tacctattgc ctacggcagc cgctggattg 480

ttattactcg ctgcccaacc agccatggcc gaggtgcagc tgctcgagtc tgggggaggg 540

ttggtaaagc ctggggggtc ccttagactc tcctgtgcag cctctggatt cgctttcaga 600

gccgcctgga tgaactgggt ccgccaggct ccagggaagg gctggagtgg ttggccgcat 660

taacagacaa attatg 676

Claims (3)

1. humanized's anti-amylin antibody, it is characterised in that: the heavy chain variable amino acid sequence such as SEQ of described antibody Shown in ID NO:1, chain variable region amino acid sequence is as shown in SEQ ID NO:2.

A kind of humanized's anti-amylin antibody the most according to claim 1, it is characterised in that: the weight chain variable of described antibody District's DNA sequence is as shown in SEQ ID NO:3, and variable region of light chain DNA sequence is as shown in SEQ ID NO:4；Heavy chain gene is IgG1 Subclass VH3 gene family, light chain gene is Lambda gene family.

3. the preparation method of humanized's anti-amylin antibody described in claim 1-2, it is characterised in that: include following step Rapid:

I. the PCR amplification of human IgG Fab fragment gene: separate the lymphocyte in human peripheral, extract total serum IgE, by extract RNA reverse transcription becomes cDNA, and employment specific IgG Kappa chain, Lambda chain and heavy chain Gamma strand primer are respectively to people's light chain PCR amplification is carried out with heavy chain gene；

II. the foundation of phage antibody library: the PCR product obtained in step I is mixed respectively by Fd, κ chain and λ chain；Will be double Light chain PCR mixture and phasmid after enzyme action are attached reaction, connect product electricity and are transformed into competent cell, and amplification is shaken Light chain gene storehouse is built after bacterium；The light chain storehouse phagemid dna built and heavy chain Fd section PCR mixture are carried out double digestion, even Connect rear Electroporation-competent cells, add culture medium culturing, add helper phage and continue to cultivate；Add after centrifugal collection supernatant Entering PEG precipitated liquid, ice bath, centrifugal supernatant the resuspended precipitation of abandoning, recentrifuge collection supernatant is phage antibody library；

III. the specific enrichment of phage antibody library and screening: be coated elisa plate, BSA-PBS solution with the amylin after dilution Add phage antibody library after closing, hatch, first wash with PBST solution, use Gly-HCl buffer solution elution then, and use Tris Buffer neutralizes, and infects and cultivates the escherichia coli to exponential phase, and after taking gradient dilution, liquid bed board measures titre, calculates every The output capacity of wheel screening；Remaining bacterium adds helper phage infection and carries out the enrichment isolation of next round, 3-8 time repeatedly；

IV. the Phage-ELISA detection of anti-amylin antibody: random picking multiple clone preparation from the bacterial clump after screening Monoclonal phage-antibodies, is coated elisa plate with the amylin after dilution, and BSA-PBS solution adds phage supernatant after closing, Wash with PBST solution after hatching；Add HA-Tag Mouse mAb, wash with PBST solution after hatching；Add HRP-goat Anti-mouse IgG, washs with PBST solution after hatching, and adds the colour developing of TMB solution, measures A450 value；

V. after variable region determined dna sequence: ELISA detection, screening positive clone carries out DNA sequencing.