CN101531718B

CN101531718B - Anti-VEGFR-2 antibody chimeric Fab antibody fragment, preparation method and application thereof

Info

Publication number: CN101531718B
Application number: CN2009100263970A
Authority: CN
Inventors: 朱进; 冯振卿; 黄剑飞; 唐奇; 唐小军; 管晓虹
Original assignee: Inst Of Military Medicine Nanjing Military Area Pla; Nanjing Medical University
Current assignee: Inst Of Military Medicine Nanjing Military Area Pla; Nanjing University; Nanjing Medical University
Priority date: 2009-04-22
Filing date: 2009-04-22
Publication date: 2011-11-02
Anticipated expiration: 2029-04-22
Also published as: CN101531718A

Abstract

The invention relates to an anti-VEGFR-2 antibody chimeric Fab antibody fragment, a preparation method and application thereof. Anti-VEGFR-2 hybridoma cell strain 8H1 is reformed by genetic recombinant technology, and a variable region of a murine antibody is recombined with CH1 and Ck of human IgG1 and is connected with a prokaryotic expression vector in the form of VH-linker-VL so as to prepare the anti-VEGFR-2 antibody chimeric Fab antibody fragment. The anti-VEGFR-2 antibody hybridoma cell strain 8H1 was preserved in China General Microbiology Culture Collection Center on April 9, 2009, and the preservation number is CGMCC No. 3013. The anti-VEGFR-2 antibody chimeric Fab antibody fragment is used for preparing anti-VEGFR-2 diagnostic reagents and therapeutic medicaments.

Description

Anti-VEGFR-2 antibody chimeric Fab antibody fragment and preparation method thereof, application

Technical field:

What the present invention relates to is anti-VEGFR-2 antibody chimeric Fab antibody fragment and preparation method thereof, use, utilize gene recombination technology, 8H1 transforms to the anti-VEGFR-2 hybridoma cell strain, with the variable region of mouse endogenous antibody and human IgG1's CH1, C κ reorganization, with the VH-linker-VL form it is connected with prokaryotic expression carrier, the preparation anti-VEGFR-2 antibody chimeric Fab antibody fragment, to reduce the mouse derived component of antibody, potential diagnosis and the treatment that is used for relevant vascular diseases (tumour that comprises some high expression level VEGFR-2) the present invention relates to genetic engineering technique and immune target diagnosis and treatment field.

Background technology:

The effect of immune in recent years targeted therapies in clinic diagnosis had than much progress, vasculogenesis links to each other closely with relevant vascular diseases (comprising tumour), and the generation of blood vessel mainly relies on the regulation and control of angiogenesis factor (VEGF) and angiostatin, wherein the closest is vascular endothelial growth factor and acceptor (VEGFR), particularly VEGFR-2.If ideal targeted drug identification VEGR/VEGFR passage is arranged, then can play the effect of diagnosis and treatment disease.

Because the high degree of specificity of antibody, utilizing antibody is a kind of ideal approach with the drug targeting target cell, and antibody targeted pharmacological agent demonstrates stronger diagnosis and treatment effect in animal model and clinical trial.Current monoclonal antibody drug or antibody coupling medicine have become and have increased the fastest, one of the maximum market of making a profit in the pharmaceutical industry.Along with clinical treatment goes on the market in a large number with monoclonal antibody, the demand that following clinical treatment is made with monoclonal antibody is also considerable, and the case of antibody clinical treatment success is also of common occurrence.

Fast development along with modern biotechnology, modern biochemistry and Protocols in Molecular Biologies such as language function genomics, proteomics, information biology, in conjunction with technology such as genetically engineered, protein engineering, cell engineerings, climaxes appear repeatedly to make biotech drug research and development.It is that it has great potential and application prospect in infection, cardiovascular disorder, autoimmune disorder, oncotherapy based on the medicine of the antibody engineering technology preparation of cell engineering and genetic engineering technique that therapeutic antibodies becomes the focus antibody drug.Current, the therapeutic antibodies medicament research and development has become the focus in biotech drug field, and the humanization of the selectivity of antibody drug action target spot, antibody drug, miniaturization and high efficiency also are research emphasis from now on.The monoclonal antibody fragment is made up of heavy chain Fd section and complete light chain, is 1/3rd of complete antibody molecule, belongs to small molecular antibody, and the tool penetration power is strong, and the advantage that the transformation period is short is particularly useful for the target diagnosis and treatment of human body diseases.

Because the reaction of the generation human antimouse antibody of the clinical application of mouse resource monoclonal antibody (HAMA) and anaphylaxis, lowered the curative effect of medicine, thereby use genetic engineering technique that mouse source antibody is transformed, and reduce the HAMA reaction and improve result of treatment simultaneously, become the research focus in this field.

Summary of the invention

The present invention seeks at above-mentioned background, a kind of anti-VEGFR-2 antibody chimeric Fab antibody fragment and preparation method thereof is provided, uses, be to utilize gene recombination technology that the anti-VEGFR-2 antibody hybridoma cell strain 8H1 of mouse source property is transformed, respectively and human IgG with the variable region of heavy chain of anti-VEGFR-2 antibody, variable region of light chain ₁The CH of antibody ₁With C κ fragment reorganization, be the nucleotide fragments of the anti-VEGFR-2 chimeric Fab antibody fragment about 1.5kb by the overlapping extension PCR total length that increases again.

This anti-VEGFR-2 antibody hybridoma cell strain 8H1 is preserved in Chinese common micro-organisms culture presevation administrative center on April 9th, 2009, and deposit number is CGMCC No.3013.

The nucleotide fragments of chimeric Fab antibody fragment and prokaryotic expression plasmid pComb3XSS are respectively with being connected behind the SfiI digestion with restriction enzyme, make up the segmental prokaryotic expression carrier of chimeric Fab, change the chimeric Fab antibody fragment of intestinal bacteria TOP10F ' back abduction delivering over to, wherein chimeric Fd segment molecule amount is about 29kDa, chimeric L chain molecular weight is about 24kDa, both connect to form anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment by interchain disulfide bond, and made up the prokaryotic expression plasmid of chimeric Fab antibody fragment and carried out solubility expression, set up the purification process of expressing protein, obtained highly purified soluble proteins, antibody character and specificity that this albumen has kept mouse source property anti-VEGFR-2 antibody to possess have the potential application prospect at the target diagnosis and treatment medicine that is used for relevant vascular diseases (tumour that comprises some high expression level VEGFR-2).

Anti-VEGFR-2 antibody chimeric Fab antibody fragment and preparation method thereof, application take following technical scheme to realize:

Anti-VEGFR-2 antibody chimeric Fab antibody fragment, the i.e. heavy chain of mouse source property anti-VEGFR-2 antibody and variable region of light chain and human IgG ₁Heavy chain part constant region (CH ₁) and constant region of light chain (C κ) fusion rotein of recombinating and forming; The variable region of light chain and the human IgG of mouse source property anti-VEGFR-2 antibody ₁The chimeric L chain of constant region of light chain C κ reorganization and the variable region of heavy chain VH and the human IgG of mouse source property anti-VEGFR-2 antibody ₁CH ₁The chimeric Fd section of reorganization connects by leader sequence pelB in the middle of both, and leader sequence pelB was sheared when chimeric Fd section, L chain were secreted into bacterium pericentral siphon chamber, and both connect and compose chimeric Fab antibody fragment by interchain disulfide bond;

The aminoacid sequence of chimeric L chain is:

Glu Leu Asp Ile Val Met Thr Gln Ser Ala Phe Ser Asn Pro Val Thr Leu Gly Thr Ser Ala

Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp

Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser

Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg

Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe

Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

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

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

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

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Leu

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys ter Phe ter

The aminoacid sequence of chimeric Fd section is:

Val Lys Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

Thr Val Ser Gly Phe Ser Leu Thr Ser His Gly Val His Trp Val Arg Gln Ser Pro Gly Lys

Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu

Gln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn Arg Arg Gly Tyr Tyr Ala Met Asp

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

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

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

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

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr Ser Gly Gln Ala Gly Gln His His

His His His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser ter

The preparation method of anti-VEGFR-2 antibody chimeric Fab antibody fragment, this antibody fragment is to utilize the genetic engineering technique preparation, concrete grammar is as follows:

1) segmental amplification of antibody variable gene and checking:

Mouse source property anti-VEGFR-2 antibody hybridoma 8H1 is cultured to logarithmic phase, Trizol-chloroform-isopropanol method extracting cell total rna; With the mRNA among total RNA is template, with oligodT ₁₅Be primer, post transcription cloning obtains strand cDNA.

The variable region of heavy chain (VH) of design mouse source property anti-VEGFR-2 antibody, the amplimer of variable region of light chain (VL), the VH that increases respectively, VL fragment, the upstream primer of variable region of light chain (VL) is VLF:5 '-G GGCCCAGGCGGCCGAGCTCGACATTGTGATGA CACAGTC-3 ', downstream primer be VLR:5 '-

TTTCAGCTCCAGCTTGGTCCC-3 ', holding the enzyme of having introduced SfiI to cut recognition site at 5 of VLF ' is the underscore partial sequence, 5 of VLR ' end has been introduced and human IgG ₁C κ upstream primer 5 ' 21 bases of end complementary be the italicized item sequence, be beneficial to the VL and the human IgG of mouse source property anti-VEGFR-2 antibody ₁The segmental overlapping pcr amplification of C κ; The upstream primer of variable region of heavy chain (VH) be VHF:5 '- CTCGAGGTGAAGCTGGTGGAGTC-3 ', downstream primer be VHR:5 '-

TGAGGAGACGGTGACCGTGGT-3 ' has introduced and human IgG at 5 of VHF ' end ₁21 base sequences of C κ downstream primer complementary be the italicized item sequence, 5 of VHR ' end has been introduced and human IgG ₁CH ₁21 base sequences of upstream primer complementary, i.e. italicized item is beneficial to the VH and the human IgG of anti-VEGFR-2 antibody ₁CH ₁Segmental overlapping pcr amplification.

CDNA with post transcription cloning is a template respectively, with the VH gene fragment of primer VHF, VHR amplification mouse source property anti-VEGFR-2 antibody, with the VL gene fragment of primer VLF, VLR amplification mouse source property anti-VEGFR-2 antibody.Amplification condition be 95 ℃ 4 minutes; 95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes.Agarose gel electrophoresis, glue reclaim purifying amplification gene fragment, and VH, VL PCR glue recovery purified product carry out TA with the pMD-18T carrier respectively and be connected.Connect product transformed into escherichia coli XL1-Blue, coating contains the LB flat board of concentration 100 μ g/ml penbritins, 30 μ g/ml tsiklomitsins, puts 37 ℃ of 12-16h.Next day is picking transformed bacteria and empty plasmid conversion contrast bacterium at random, 37 ℃ are shaken bacterium and with the upstream and downstream special primer of VH, VL gene fragment bacterium liquid are carried out PCR respectively after 5 hours and identify, contain the bacterial strain that inserts VH gene fragment plasmid and amplify a band about 351bp, contain the bacterial strain that inserts VL gene fragment plasmid and amplify a band about 336bp.The bacterium liquid that bacterium liquid PCR checking amplifies the correct band of size send biotech firm to check order.

Wherein the nucleotides sequence of VH is classified as:

GTG AAG CTG GTG GAG TCA GGA CCT GGC CTA GTG CAG CCC TCA CAG AGC CTG TCC ATC ACC TGC

ACA GTC TCT GGT TTC TCA TTA ACT AGC CAT GGT GTA CAC TGG GTT CGC CAG TCT CCA GGA AAG

GGT CTG GAG TGG CTG GGA GTG ATA TGG AGT GGT GGA AGC ACA GAC TAT AAT GCA GCT TTC ATA

TCC AGA CTG AGC ATC AGC AAG GAC AAT TCC AAG AGC CAA GTT TTC TTT AAA ATG AAC AGT CTG

CAA GCT AAT GAC ACA GCC ATA TAT TAC TGT GCC AGA AAT AGG AGG GGG TAC TAT GCT ATG GAC

TAC TGG GGT CAA GGA ACC ACG GTC ACC GTC TCC TCA

The nucleotides sequence of VL is classified as:

GAC ATT GTG ATG ACA CAG TCT GCA TTC TCC AAT CCA GTC ACT CTT GGA ACA TCA GCT TCC ATC

TCC TGC AGG TCT AGT AAG AGT CTC CTA CAT AGT AAT GGC ATC ACT TAT TTG TAT TGG TAT CTG

CAG AAG CCA GGC CAG TCT CCT CAG CTC CTG ATT TAT CAG ATG TCC AAC CTT GCC TCA GGA GTC

CCA GAC AGG TTC AGT AGC AGT GGG TCA GGA ACT GAT TTC ACA CTG AGA ATC AGC AGA GTG GAG

GCT GAG GAT GTG GGT GTT TAT TAC TGT GCT CAA AAT CTA GAA CTT CCT CTC ACG TTC GGT GCT

GGG ACC AAG CTG GAG CTG AAA

2) the segmental amplification of chimeric Fab:

(1) human IgG ₁The CH of antibody ₁Amplification with C κ section:

With plasmid pComb3XTT is template, and human IgG increases respectively ₁The C κ and the CH of antibody ₁Nucleotide fragments; The upstream primer of C κ section is C κ F:5 '-CGAACTGTGGCTGCACCATCTGTC-3 ', and downstream primer is C κ R:5 '-GGCCATGGCTGGTTGGGCAGC-3 '; CH ₁Upstream primer be CH ₁F:5 '-GCCTCCACCAAGGGCCCATCGGTC-3 ', downstream primer are CH ₁R:AGAAGCGTAGTCCGGAACGTC-3 '.Amplification condition be 95 ℃ 4 minutes; 95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes, and agarose gel electrophoresis amplifies the band of about 393bp, 321bp respectively, and glue recovery purifying amplified band is dissolved in the deionized water, and-20 ℃ frozen standby;

(2) chimeric L chain, the segmental amplification of Fd fragment gene:

VL and human IgG with mouse source property anti-VEGFR-2 antibody ₁The pcr amplification product of the C κ of antibody is a template, carry out the overlapping extension PCR chimeric L chain that increases with upstream primer LF:5 '-GGGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ' and downstream primer LR:5 '-GGCCATGGCTGGTTGGGCAGC-3 ', amplification condition is 95 ℃, 4 minutes; 95 ℃, 30 seconds, 56 ℃, 30 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes.Agarose gel electrophoresis amplifies the band of about 657bp, and glue reclaims amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby.

VH and human IgG with mouse source property anti-VEGFR-2 antibody ₁The CH of antibody ₁Pcr amplification product be template, carry out the overlapping extension PCR chimeric Fd fragment that increases with upstream primers F dF:5 '-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGGTGGAGTC-3 ' and downstream primer FdR:5 '-AGAAGCGTAGTCCGGAACGTC-3 ', amplification condition is 95 ℃, 4 minutes; 95 ℃, 30 seconds, 56 ℃, 30 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes.Agarose gel electrophoresis amplifies the band of about 774bp, and glue reclaims amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby.

(3) amplification of chimeric Fab gene fragment:

With the chimeric Fd section of amplification acquisition and the glue recovery purified product of L chain nucleotide sequence is template, carry out overlapping extension PCR amplification with upstream primers F ab F:5 ' GGGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ' and downstream primer FabR:5 '-AGAAGCGTAGTCCGGAACGTC-3 ', during amplification, do not add earlier primer, 95 ℃ 4 minutes; 94 ℃ 30 seconds, 45 ℃ 30 seconds, 72 ℃ 90 seconds, 8 circulations; Then add behind FabF, the FabR primer 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 90 seconds, 22 circulations; 72 ℃ were extended 10 minutes, and agarose gel electrophoresis amplifies the band of about 1.5kb, and glue reclaims the purifying amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby.

3) chimeric Fab construction of prokaryotic expression vector and evaluation:

Extract plasmid pComb3XSS, the segmental PCR glue of plasmid and Fab reclaims product and cuts 12-16h with the SfiI restriction enzyme at 50 ℃ of enzymes, and glue reclaims the big fragment of plasmid that enzyme is cut behind the electrophoresis, is dissolved in the deionized water.Get the enzyme of pComb3XSS, Fab amplified production and cut product, in same centrifuge tube, connect 12-16h for 16 ℃ with the T4 ligase enzyme by 1: 4 mol ratio mixing.

To connect product transformed competence colibacillus intestinal bacteria TOP10F ', coating contains the LB flat board of 100 μ g/ml penbritins, puts 37 ℃ of 12-16h; Next day, picking transformed bacteria and empty plasmid transformed the contrast bacterium at random, and 37 ℃ were shaken bacterium after 5 hours, identified bacterium liquid being carried out pcr amplification with the special primer of Fab respectively, and the bacterial strain that contains insertion Fab fragment plasmid amplifies the band about a treaty 1.5kb; The bacterium liquid that bacterium liquid PCR checking amplifies the correct band of size send biotech firm to check order, dna sequence analysis confirms to contain the chimeric Fab fragment of structure in recombinant plasmid, sequence is correct, wherein horizontal line part A TG AAA TAC CTA TTG CCTACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCC ATG GCC is leader sequence pelB, TAG is chimeric Fd chain, L chain translation stop codon, GCC AGA GCG GCC, GGC CAG GCC GGC is for connecting the SfiI restriction enzyme site of chimeric Fab antibody fragment gene and carrier pComb3XSS, GAG CTC is the SacI restriction enzyme site of carrier pComb3XSS, CAG CAC CAT CAC CAT CAC CAT GGC GCA TAC CCG TAC GAC GTT CCG GAC TAC GCT TCT contains the His label for carrier pComb3XSS goes up sequence, ACT AGT is the SpeI restriction enzyme site, C TAG A is the XbaI enzyme cutting site, and CTC GAG is the XhoI restriction enzyme site;

GCC AGA GCG GCC GAG CTC GAC ATT GTG ATG ACA CAG TCT GCA TTC TCC AAT CCA GTC ACTCTT GGA ACA TCA GCT TCC ATC TCC TGC AGG TCT AGT AAG AGT CTC CTA CAT AGT AAT GGC ATCACT TAT TTG TAT TGG TAT CTG CAG AAG CCA GGC CAG TCT CCT CAG CTC CTG ATT TAT CAG ATGTCC AAC CTT GCC TCA GGA GTC CCA GAC AGG TTC AGT AGC AGT GGG TCA GGA ACT GAT TTC ACACTG AGA ATC AGC AGA GTG GAG GCT GAG GAT GTG GGT GTT TAT TAC TGT GCT CAA AAT CTA GAACTT CCT CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGA ACT GTG GCT GCA CCA TCTGTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CTGCTG AAT AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC CAA TCGGGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC AGC CTC AGC AGCACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC GCC TGC GAA GTC ACC CATCAG GGC CTG AGC TTG CCC GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT TAG TT C TAG ATA ATTAAT TAG GAG GAA TTT AAA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG GTG GAG TCA GGA CCT GGC CTA GTG CAGCCC TCA CAG AGC CTG TCC ATC ACC TGC ACA GTC TCT GGT TTC TCA TTA ACT AGC CAT GGT GTACAC TGG GTT CGC CAG TCT CCA GGA AAG GGT CTG GAG TGG CTG GGA GTG ATA TGG AGT GGT GGAAGC ACA GAC TAT AAT GCA GCT TTC ATA TCC AGA CTG AGC ATC AGC AAG GAC AAT TCC AAG AGCCAA GTT TTC TTT AAA ATG AAC AGT CTG CAA GCT AAT GAC ACA GCC ATA TAT TAC TGT GCC AGAAAT AGG AGG GGG TAC TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC ACG GTC ACC GTC TCC TCAGCC TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC TCC TCC AAG AGC ACC TCT GGG GGCACA GCG GCC CTG GGC TGC CTG GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AACTCA GGC GCC CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TACTCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATC TGC AACGTG AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCC AAA TCT TGT GAC AAA ACT AGT GGC CAG GCC GGC CAG CAC CAT CAC CAT CAC CAT GGC GCA TAC CCG TAC GAC GTT CCG GAC TAC GCT TCT TAG

The chimeric Fab fragment of L chain that the expression of recombinant plasmid that makes up is chimeric and the reorganization of Fd section, 22 amino acid of leader peptide sequences pelB translation between the two connect, leading peptide pelB was sheared when the protein fragments of translation was secreted to bacterium pericentral siphon chamber, and chimeric L chain is connected by interchain disulfide bond with the Fd section.

4) express the Screening and Identification of chimeric Fab bacterium and the purifying of expressing protein

The bacterium liquid that will contain correct recombinant plasmid changed in the 2ml SB solution by 1: 100, and the penbritin working concentration is 100 μ g/ml, and the glucose final concentration is 4%, 37 ℃ and shakes bacterium and spend the night; Centrifugal 15 minutes of the bacterium 10000g that spends the night, abandoning supernatant, then use 2ml SB resuspended, changing the bacterium liquid after resuspended over to 2ml by 1: 100 contains in the SB substratum that the penbritin final concentration is 100 μ g/ml, cultivate about OD600=1.0 for 37 ℃, add IPTG to final concentration be 1mmol/L, sucrose final concentration to 4%, 23 ℃ of shaking culture 20h, centrifugal collection thalline, handle culture supernatant respectively, ultrasonic supernatant of thalline and ultrasound precipitation carry out SDS-PAGE and western-blot and detect, and the chimeric Fab fragment is in culture supernatant, in ultrasonic supernatant of thalline and the ultrasound precipitation expression is arranged all, wherein soluble proteins mainly is in the ultrasonic supernatant of thalline, wherein chimeric Fd segment molecule amount is about 29kDa, and chimeric L chain molecular weight is about 24kDa, and the middle no target protein band of contrast bacterium TOP10F ';

Bacterium liquid 10000g is centrifugal 15 minutes behind a large amount of abduction deliverings of bacterium, abandons culture supernatant, and the 20mM phosphate balance damping fluid that adds original bacteria liquid 1/10 volume in the precipitation is resuspended with bacterium, contains 0.5M/L NaCl in this damping fluid, the 10mM imidazoles; Then bacterium liquid is carried out ultrasonicly, surpass 10 seconds, stopped 10 seconds, surpass altogether 90 times, then 4 ℃ of 12000rpm centrifugal 30 minutes, abandon precipitation, and ultrasonic supernatant carries out purifying with 5ml Histrap HP pillar then with 0.22 μ m membrane filtration.Earlier wash pillar with the speed of 2.5ml/min, then use the level pad balance pillar of at least 10 times of column volumes, then with sample on the speed of 2ml/min with water with 5 times of column volumes; With level pad balance nickel post to baseline, wash pillar with the damping fluid that contains 50mM, 100mM, 200mM, 300mM, 400mM, 500mM imidazoles of 5 times of column volumes respectively, and the elutriant of collection respective concentration imidazoles, carry out the 12%SDS-PAGE electrophoresis, observe the protein purification situation.The result contains the Fab albumen of seeing purifying in the elutriant of 400mM imidazoles, carries out desalination with the millpore ultrafiltration pillar of 10KD and concentrates, and PBS washes 3 times, promptly obtains the chimeric Fab antibody fragment of purifying.

5) activity identification of chimeric Fab antibody fragment:

The I euzymelinked immunosorbent assay (ELISA): with containing the 0.1M carbonate buffer solution, the coating buffer of pH9.6 dilutes recombinant human VEGF R-2 albumen, and this albumen is available from U.S. R﹠amp; D company, production code member 357-KD-050.By 96 hole elisa plates, every hole adds 100 μ l to recombinant human VEGF R-2 albumen with the concentration bag of 2 μ g/ml, and 4 ℃ are spent the night; After the PBST washing,, hatch 2h for 37 ℃ with 5% skimmed milk-lavation buffer solution sealing; After the PBST washing 5 times, add 100 μ l anti-VEGFR-2 antibody chimeric Fab antibody fragments in each hole, with 200 μ g/ml initial concentrations, 11 concentration gradient dilutions, 4 ℃ are spent the night; Goat-anti human Fab second antibody 100 μ l/ holes with dilution in 1: 5000 join in the hole, hatch 1h for 37 ℃; Peroxidase substrate colour developing liquid 100 μ l/ holes, with 2M sulfuric acid stopped reaction, last machine testing colorimetric adopts dual wavelength 450nm/630nm to room temperature after following 15 minutes.Chimeric Fab antibody fragment can play antigen-antibody ELISA reaction with recombinant human VEGF R-2 albumen as a result.

The II immunoblotting: it is natural antigen that people's navel endothelial venule cell (HUVEC) of high expression level VEGFR-2, mouse low or that do not have a VEGF expression R-2 become fiber (NIH3T3) total protein of cell.Cell is cultured to 1 * 10 respectively ⁷, remove substratum under 4 ℃ of conditions and clean with PBS, with the abundant lysing cell of 500 μ l RIPA lysates, 10000g got supernatant and total protein of cell in centrifugal 5 minutes, measured the concentration of each total protein of cell.With each total protein of cell concentration make after the 1mg/ml equal concentrations packing frozen-20 ℃ standby.

HUVEC, NIH3T3 total protein of cell are carried out the 10%SDS-PAGE electrophoresis and electricity forwards on the nitrocellulose membrane, with confidential reference items antibody β-actin of the anti-VEGFR-2 antibody chimeric Fab antibody fragment of this film and 50 μ g/ml and dilution in 1: 2000 simultaneously 37 ℃ hatch 2h, the PBST washing room is every 5 minutes 4 times, goat-anti human Fab two resists 37 ℃ and hatches 1h, the DAB result that develops the color, one thick bright wisp band, three filaments of sun bands appear in HUVEC total protein swimming lane from the top down, respectively successively corresponding to VEGFR-2 albumen three band 230kDa, 200kDa, 150kDa and confidential reference items β-actin band; Confidential reference items β-actin band of 43kDa then only appears in NIH3T3 total protein of cell swimming lane.

The III flow cytometry: it is active with combining of VEGFR-2 natural antigen to adopt flow cytometer (FACS) to analyze anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment.Concrete grammar is, respectively with 2 * 10 ⁶Individual HUVEC and NIH3T3 cell are with the PBS flushing that contains 4 ℃ of the aseptic precoolings after half an hour of 0.1% Paraformaldehyde 96 PBS fixed cell three times, use anti-VEGFR-2 antibody chimeric Fab antibody fragment respectively, 60 μ g/ml and two different concns of 120 μ g/ml are hatched 4 ℃ altogether and are spent the night, PBS gives a baby a bath on the third day after its birth anti-all over back adding goat-anti human Fab rhodamine fluorescence two, the lucifuge incubated at room after 2 hours PBS give a baby a bath on the third day after its birth time, it is resuspended to contain 0.1% Paraformaldehyde 96 PBS, 400 μ l, on FACS, carry out immunofluorescence analysis and measure average fluorescent strength, with only add two anti-be background, the anti-VEGFR-2 antibody chimeric Fab antibody fragment and the HUVEC combination rate of 60 μ g/ml and two different concns of 120 μ g/ml are respectively 34.46% and 35.16% as a result; The anti-VEGFR-2 antibody chimeric Fab antibody fragment and the NIH3T3 cell combination rate of same concentration are respectively 14.06% and 16.26%.

Above result shows that anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment can combine with VEGFR-2 antigen, and antibody character and specificity that anti-VEGFR-2 antibody that this chimeric Fab fragment has kept former preparation possesses are described.

Described anti-VEGFR-2 antibody chimeric Fab antibody fragment is characterized in that: utilize the chimeric Fab gene fragment of above-mentioned acquisition, can carry out recombinant expressed, preparation in prokaryotic cell prokaryocyte, yeast cell, eukaryotic cell.

Described anti-VEGFR-2 antibody chimeric Fab antibody fragment is used for relevant vascular diseases, particularly the tumor research of high expression level VEGFR-2.

Described anti-VEGFR-2 antibody chimeric Fab antibody fragment is in the diagnostic reagent of preparation anti-VEGFR-2, the application in the curative drug.

The advantage that the present invention compared with prior art has:

Fast development along with modern biotechnology, modern biochemistry and Protocols in Molecular Biologies such as language function genomics, proteomics, information biology, in conjunction with technology such as genetically engineered, protein engineering, cell engineerings, climaxes appear repeatedly to make biotech drug research and development.Therapeutic antibodies is the medicine based on the preparation of the antibody engineering technology of cell engineering and genetic engineering technique, with its high specificity, safe and effective and become the focus of current biotech drug research and development.The anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment of preparation, show analogue antigen character and the specificity that has kept mouse source property anti-VEGFR-2 antibody by test, be used for relevant vascular diseases, the tumor research of high expression level VEGFR-2 particularly can be used for preparing diagnostic reagent, the curative drug of anti-VEGFR-2.

Use gene recombination technology that this mouse resource monoclonal antibody is transformed the preparation chimeric Fab antibody fragment, the antibody fragment of preparation has following advantage:

(1) greatly reduces the mouse derived component of anti-VEGFR-2 antibody, help further carrying out in vivo test.

(2) keep antibody character and specificity that the mouse endogenous antibody possesses, can potentially be used for the target diagnosis and treatment of relevant vascular diseases (tumour that comprises some high expression level VEGFR-2).

(3) prokaryotic expression carrier is easy to make up, but great expression is produced fast and is convenient to purifying.

(4) help preparing the humanization modified of chimeric full molecular antibody or antibody, the mouse derived components that further reduces in the anti-VEGFR-2 antibody is studied with the target diagnosis and treatment of carrying out relevant vascular diseases (tumour that comprises some high expression level VEGFR-2).

Description of drawings

The invention will be further described below with reference to accompanying drawing:

Fig. 1 is the total RNA electrophoresis result of monoclonal anti VEGFR-2 monoclonal antibody hybridoma cell, shows that the RNA extracting is better, does not degrade.

Fig. 2 is monoclonal anti VEGFR-2 antibody heavy chain variable region and variable region of light chain pcr amplification product electrophoresis result, M: nucleic acid standard molecular weight (Takara DL2000); The VL gene fragment of 1:336bp; The VH gene fragment of 2:351bp.

Fig. 3: with pComb3XTT is the human IgG of template amplification ₁Constant region of light chain and CH1 fragment electrophoresis result, M: nucleic acid standard molecular weight (Takara DL2000); The C kappa gene fragment of 1:321bp; The CH of 2:393bp ₁Gene fragment.

Fig. 4: the nucleic acid electrophoresis result of chimeric Fd section and L chain amplified production, M: nucleic acid standard molecular weight (Takara DL2000); The L gene fragment of 1:657bp; The Fd gene fragment of 2:744bp.

Fig. 5: the nucleic acid electrophoresis result of chimeric Fab amplified production, M: nucleic acid standard molecular weight (Takara DL2000); 1: the Fab gene fragment of about 1.5kb.

Fig. 6: with the western-blot result of the goat-anti human Fab antibody of mark HRP, antigen is that ultrasonic supernatant of ultrasound precipitation, thalline and culture supernatant: the M of chimeric Fab bacterium and contrast bacterium TOP10F ' is Fermentas, #SM0641,1-3 are respectively ultrasonic supernatant of ultrasound precipitation, thalline and the culture supernatant of chimeric Fab bacterium; 4-6 is respectively ultrasonic supernatant of ultrasound precipitation, thalline and the culture supernatant of contrast bacterium TOP10F '.

Fig. 7: chimeric Fab purifying protein SDS-PAGE electrophoresis result, M is protein standard (Fermentas, #SM0441), 1-6 is respectively the elutriant that contains 50mM, 100mM, 200mM, 300mM, 400mM, 500mM imidazoles, and wherein 5 is the chimeric Fab fragment of purifying in the elutriant of 400mM imidazoles.

Fig. 8: with commercialization recombinant human VEGF R-2 albumen is antigen, and anti-VEGFR-2 antibody chimeric Fab antibody fragment is an anti-ELISA result.

Fig. 9: with HUVEC, NIH3T3 total protein of cell is natural antigen, and the anti-VEGFR-2 antibody chimeric Fab molecule is anti-western-blot (DAB colour developing) result, and M is albumen marker; 1 is the HUVEC total protein of cell, and 2 is the NIH3T3 total protein of cell.

Figure 10: with anti-VEGFR-2 antibody chimeric Fab antibody fragment is one anti-, with the FACS result of HUVEC and NIH3T3 cell response, with do not add one anti-only add two anti-be background: 1 is background; 2,3 is anti-VEGFR-2 antibody Fab molecule and the figure that combines of HUVEC; 4,5 is anti-VEGFR-2 antibody Fab molecule and the figure that combines of NIH3T3 cell.

Embodiment

The preparation method of embodiment 1, anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment:

1) segmental amplification of antibody variable gene and checking:

Mouse source property anti-VEGFR-2 antibody hybridoma 8H1 is cultured to logarithmic phase, Trizol-chloroform-isopropanol method extracting cell total rna; Dried cell total rna is dissolved in the 10 μ l water, gets 1 μ l and do the 10g/l agarose gel electrophoresis, occur 3 band (see figure 1)s, 28s, 18s and 5s behind the electrophoresis.Better and the less degradation of extracting of RNA is described, its content is about 0.1g/l.The RNA that gets 9 μ l carries out reverse transcription, is template with the mRNA among total RNA, with oligodT ₁₅Be primer, post transcription cloning obtains strand cDNA.

Design 19 VH upstream primers and 17 V κ upstream primers, 4 VH downstream primers and 3 V κ downstream primer primers:

V κ 5 ' upstream primer:

Vκ-1

5’-GGG CCC AGG CGG CCG AGC TCG AYA TCC AGC TGA CTC AGC C-3’

Vκ-2

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCW CCC AGT C-3’

Vκ-3

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGM TMA CTC AGT C-3’

Vκ-4

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGY TRACAC AGT C-3’

Vκ-5

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TRA TGA CMC AGT C-3’

Vκ-6

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTM AGA TRA MCC AGT C-3’

Vκ-7

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTC AGA TGA YDC AGT C-3’

Vκ-8

5’-GGG CCC AGG CGG CCG AGC TCG AYA TYC AGA TGA CAC AGA C-3’

Vκ-9

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCA WCC AGT C-3’

V κ-10

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG WGC TSA CCC AAT C-3’

V κ-11

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTS TRA TGA CCC ART C-3’

Vκ-12

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTK TGA TGA CCC ARA C-3’

Vκ-13

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CBC AGK C-3’

Vκ-14

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TAA CYC AGG A-3’

Vκ-15

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CCC AGW T-3’

Vκ-16

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CAC AAC C-3’

Vκ-17

5’-GGG CCC AGG CGG CCG AGC TCG AYA TTT TGC TGA CTC AGT C-3’

V κ 3 ' downstream primer

VκR1

5’-AGA TGG TGC AGC CAC AGT TCG TTT KAT TTC CAG YTT GGT CCC-3’

VκR2

5’-AGA TGG TGC AGC CAC AGT TCG TTT TAT TTC CAA CTT TGT CCC-3’

VκR3

5’-AGA TGG TGC AGC CAC AGT TCG TTT CAG CTC CAG CTT GGT CCC-3’

VH5 ' upstream primer

VH1

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR MAG CTT CAG GAG TC-3’

VH2

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTB CAG CTB CAG CAG TC-3’

VH3

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG CTG AAG SAS TC-3’

VH4

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAR CTG CAA CAR TC-3’

VH5

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAG CTB CAG CAR TC-3’

VH6

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAR CTG CAG CAG TC-3’

VH7

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAC GTG AAG CAG TC-3’

VH8

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAS STG GTG GAA TC-3’

VH9

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AWG YTG GTG GAG TC-3’

VH10

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG SKG GTG GAG TC-3’

VH11

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAM CTG GTG GAG TC-3’

VH12

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG ATG GAR TC-3’

VH13

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAR CTT GTT GAG TC-3’

VH14

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR AAG CTT CTC GAG TC-3’

VH15

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAR STT GAG GAG TC-3’

VH16

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTT ACT CTR AAA GWG TST G-3’

VH17

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAA CTV CAG CAR CC-3’

VH18

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAC TTG GAA GTG TC-3’

VH19

5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG GTC ATC GAG TC-3’

VH3 ' downstream primer

VH R1

5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CGT GGT-3’

VH R2

5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC TGT GAG AGT GGT-3’

VH R3

5’-CGA TGG GCC CTT GGT GGA GGC TGC AGA GAC AGT GAC CAG AGT-3’

VH R4

5’-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC TGA GGT-3’

With V κ 5 ' upstream primer, V κ 3 ' downstream primer, VH5 ' upstream primer, VH3 ' downstream primer each molten be final concentration 100pmol/ul, then all with 1: 1 volume ratio mixing, VH, VL gene increase respectively, amplification condition be 95 ℃ 4 minutes, 95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; Last 72 ℃ were extended 10 minutes, and electrophoresis result is shown the band of VH gene 351bp, the band (see figure 2) of VL gene 336bp, the gene fragment that reclaim, purifying increases is connected to the pMD-18T carrier, transformed into escherichia coli Top10F ', the order-checking back obtains light chain, weight chain variabl area sequence, and sequence is as follows:

Wherein the nucleotides sequence of VH is classified as:

TAC TGG GGT CAA GGA ACC ACG GTC ACC GTC TCC TCA

The nucleotides sequence of VL is classified as:

GGG ACC AAG CTG GAG CTG AAA

2) the segmental amplification of chimeric Fab:

Design the V of amplification single-chain antibody respectively according to the correct sequence of above-mentioned order-checking _H, V _LArticle 4, oligonucleotide primer, the VH that increases respectively, VL fragment, the upstream primer of variable region of light chain (VL) is VLF:5 '-G GGCCCAGGCGGCCGAGCTCGACATTGTGATGA CACAGTC-3 ', downstream primer be VLR:5 '-

TTTCAGCTCCAGCTTGGTCCC-3 ', holding the enzyme of having introduced SfiI to cut recognition site at 5 of VLF ' is the underscore partial sequence, 5 of VLR ' end has been introduced and human IgG ₁C κ upstream primer 5 ' 21 bases of end complementary be the italicized item sequence, be beneficial to the VL and the human IgG of mouse source property anti-VEGFR-2 antibody ₁The segmental overlapping pcr amplification of C κ; The upstream primer of variable region of heavy chain (VH) be VHF:5 '-

CTCGAGGTGAAGCTGGTGGAGTC-3 ', downstream primer be VHR:5 '-

(1) human IgG ₁The CH of antibody ₁Amplification with C κ section:

With plasmid pComb3XTT is template, and human IgG increases respectively ₁The C κ and the CH of antibody ₁Nucleotide fragments; The upstream primer of C κ section is C κ F:5 '-CGAACTGTGGCTGCACCATCTGTC-3 ', and downstream primer is C κ R:5 '-GGCCATGGCTGGTTGGGCAGC-3 '; CH ₁Upstream primer be CH ₁F:5 ' GCCTCCACCAAGGGCCCATCGGTC-3 ', downstream primer are CH ₁R:AGAAGCGTAGTCCGGAACGTC-3 '.Amplification condition be 95 ℃ 4 minutes; 95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes, and agarose gel electrophoresis amplifies the band (see figure 3) of about 393bp, 321bp respectively, and glue recovery purifying amplified band is dissolved in the deionized water, and-20 ℃ frozen standby;

(2) chimeric L chain, the segmental amplification of Fd fragment gene:

VL and human IgG with mouse source property anti-VEGFR-2 antibody ₁The pcr amplification product of the C κ of antibody is a template, carry out the overlapping extension PCR chimeric L chain that increases with upstream primer LF:5 '-GGGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ' and downstream primer LR:5 '-GGCCATGGCTGGTTGGGCAGC-3 ', amplification condition is 95 ℃, 4 minutes; 95 ℃, 30 seconds, 56 ℃, 30 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃, 10 extended 10 minutes.Agarose gel electrophoresis amplifies the band of about 657bp, and glue reclaims amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby.

VH and human IgG with mouse source property anti-VEGFR-2 antibody ₁The CH of antibody ₁Pcr amplification product be template, carry out the overlapping extension PCR chimeric Fd fragment (see figure 4) that increases with upstream primers F dF:5 '-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGGTGGAGTC-3 ' and downstream primer FdR:5 '-AGAAGCGTAGTCCGGAACGTC-3 ', amplification condition is 95 ℃, 4 minutes; 95 ℃, 30 seconds, 56 ℃, 30 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃, 10 extended 10 minutes.Agarose gel electrophoresis amplifies the band (see figure 4) of about 744bp, and glue reclaims amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby.

(3) amplification of chimeric Fab gene fragment:

With the chimeric Fd section of amplification acquisition and the glue recovery purified product of L chain nucleotide sequence is template, carry out overlapping extension PCR amplification with upstream primers F abF:5 '-GGGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ' and downstream primer FabR:5 '-AGAAGCGTAGTCCGGAACGTC-3 ', during amplification, do not add earlier primer, 95 ℃ 4 minutes; 94 ℃ 30 seconds, 45 ℃ 30 seconds, 72 ℃ 90 seconds, 8 circulations; Then add behind FabF, the FabR primer 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 90 seconds, 22 circulations; 72 ℃ were extended 10 minutes, and agarose gel electrophoresis amplifies the band of about 1.5kb, sees Fig. 5, and glue reclaims the purifying amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby.

3) chimeric Fab construction of prokaryotic expression vector and evaluation:

Adopt the pComb3XSS plasmid, the segmental PCR glue of plasmid and Fab reclaims product and cuts 12-16h with the SfiI restriction enzyme at 50 ℃ of enzymes, and glue reclaims the big fragment of plasmid that enzyme is cut behind the electrophoresis, is dissolved in the deionized water.Get the enzyme of pComb3XSS, Fab amplified production and cut product, in same centrifuge tube, connect 12-16h for 16 ℃ with the T4 ligase enzyme by 1: 4 mol ratio mixing.

The chimeric Fab fragment of L chain that the expression of recombinant plasmid that makes up is chimeric and the reorganization of Fd section, 22 amino acid of leader peptide sequences pelB translation between the two connect, leading peptide pelB was sheared when the protein fragments of translation was secreted to bacterium pericentral siphon chamber, and chimeric L chain is connected by interchain disulfide bond with the Fd section.The aminoacid sequence of chimeric L chain is:

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys ter Phe ter

The aminoacid sequence of chimeric Fd section is:

His His His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser ter

The bacterium liquid that will contain correct recombinant plasmid changed in the 2ml SB solution by 1: 100, and the penbritin working concentration is 100 μ g/ml, and the glucose final concentration is 4%, 37 ℃ and shakes bacterium and spend the night; Centrifugal 15 minutes of the bacterium 10000g that spends the night, abandoning supernatant, then use 2ml SB solution resuspended, with the bacterium liquid after resuspended by changing at 1: 100 in the SB solution that 2ml contains 100 μ g/ml penbritins, cultivate about OD600=1.0 for 37 ℃, adding isopropyl-(IPTG) is 1mmol/L to final concentration, sucrose final concentration to 4%, 23 ℃ of shaking culture 20h.Centrifugal collection thalline, handle culture supernatant, the ultrasonic supernatant of thalline and ultrasound precipitation respectively, carrying out SDS-PAGE and western-blot detects, the chimeric Fab fragment all has expression in culture supernatant, the ultrasonic supernatant of thalline and ultrasound precipitation as a result, wherein soluble proteins mainly is in the ultrasonic supernatant of thalline, chimeric Fd segment molecule amount is about 29kDa, and chimeric L chain molecular weight is about 24kDa, and the middle no target protein band (see figure 6) of contrast bacterium TOP10F '.

4) purifying of expressing protein

Bacterium liquid 10000g is centrifugal 15 minutes behind a large amount of abduction deliverings of bacterium, abandons culture supernatant, adds the 20mM phosphate balance damping fluid (containing 0.5M/L NaCl, the 10mM imidazoles) of original bacteria liquid 1/10 volume in the precipitation, and bacterium is resuspended; Then bacterium liquid is carried out ultrasonicly, surpass 10 seconds, stopped 10 seconds, surpass altogether 90 times, then 4 ℃ of 12000rpm centrifugal 30 minutes, abandon precipitation, and ultrasonic supernatant carries out purifying with 5ml Histrap HP pillar then with 0.22 μ m membrane filtration.Earlier wash pillar with the speed of 2.5ml/min, then use the level pad balance pillar of at least 10 times of column volumes, then with sample on the speed of 2ml/min with water with 5 times of column volumes; With level pad balance nickel post to baseline, wash pillar with the damping fluid that contains 50mM, 100mM, 200mM, 300mM, 400mM, 500mM imidazoles of 5 times of column volumes respectively, and the elutriant of collection respective concentration imidazoles, carry out the 12%SDS-PAGE electrophoresis, observe protein purification situation (see figure 7).The result contains the Fab albumen of seeing purifying in the elutriant of 400mM imidazoles, carries out desalination with the millpore ultrafiltration pillar of 10KD and concentrates, and PBS washes 3 times.

The activity identification of embodiment 2 chimeric Fab antibody fragments:

The I euzymelinked immunosorbent assay (ELISA): with containing the 0.1M carbonate buffer solution, the coating buffer of pH9.6 dilutes recombinant human VEGF R-2 albumen, and this albumen is available from U.S. R﹠amp; D company, production code member 357-KD-050.By 96 hole elisa plates, every hole adds 100 μ l to recombinant human VEGF R-2 albumen with the concentration bag of 2 μ g/ml, and 4 ℃ are spent the night; After the PBST washing,, hatch 2h for 37 ℃ with 5% skimmed milk-lavation buffer solution sealing; After the PBST washing 5 times, add 100 μ l anti-VEGFR-2 antibody chimeric Fab antibody fragments in each hole, with 200 μ g/ml initial concentrations, 11 concentration gradient dilutions, 4 ℃ are spent the night; Goat-anti human Fab second antibody 100 μ l/ holes with dilution in 1: 5000 join in the hole, hatch 1h for 37 ℃; Peroxidase substrate colour developing liquid 100 μ l/ holes, with 2M sulfuric acid stopped reaction, last machine testing colorimetric adopts dual wavelength 450nm/630nm to room temperature after following 15 minutes.Chimeric Fab antibody fragment can play antigen-antibody ELISA reaction with recombinant human VEGF R-2 albumen as a result, sees Fig. 8.

HUVEC, NIH3T3 total protein of cell are carried out the 10%SDS-PAGE electrophoresis and electricity forwards on the nitrocellulose membrane, with confidential reference items antibody β-actin of the anti-VEGFR-2 antibody chimeric Fab antibody fragment of this film and 50 μ g/ml and dilution in 1: 2000 simultaneously 37 ℃ hatch 2h, the PBST washing room is every 5 minutes 4 times, goat-anti human Fab two resists 37 ℃ and hatches 1h, the DAB result that develops the color, one thick bright wisp band, three filaments of sun bands appear in HUVEC total protein swimming lane from the top down, respectively successively corresponding to VEGFR-2 albumen three band 230kDa, 200kDa, 150kDa and confidential reference items β-actin band; Confidential reference items β-actin band of 43kDa then only appears in NIH3T3 total protein of cell swimming lane, sees Fig. 9.

III flow cytometry: adopt flow cytometry analysis anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment active with combining of VEGFR-2 natural antigen.Concrete grammar is, respectively with 2 * 10 ⁶Individual HUVEC and NIH3T3 cell are with the PBS flushing that contains 4 ℃ of the aseptic precoolings after half an hour of 0.1% Paraformaldehyde 96 PBS fixed cell three times, use anti-VEGFR-2 antibody chimeric Fab antibody fragment respectively, 60 μ g/ml and two different concns of 120 μ g/ml are hatched 4 ℃ altogether and are spent the night, PBS gives a baby a bath on the third day after its birth anti-all over back adding goat-anti human Fab rhodamine fluorescence two, the lucifuge incubated at room after 2 hours PBS give a baby a bath on the third day after its birth time, it is resuspended to contain 0.1% Paraformaldehyde 96 PBS, 400 μ l, on flow cytometer, carry out immunofluorescence analysis and measure average fluorescent strength, with only add two anti-be background, the anti-VEGFR-2 antibody chimeric Fab antibody fragment and the HUVEC combination rate of 60 μ g/ml and two different concns of 120 μ g/ml are respectively 34.46% and 35.16% as a result; The anti-VEGFR-2 antibody chimeric Fab antibody fragment and the NIH3T3 cell combination rate of same concentration are respectively 14.06% and 16.26%, see Figure 10.

Above result shows that anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment can combine with VEGFR-2 antigen, illustrates that this chimeric Fab fragment has kept antibody character and specificity that former anti-VEGFR-2 antibody possesses.

Anti-VEGFR-2 antibody chimeric Fab antibody fragment Nucleotide and protein sequence table

＜110〉Chinese People's Liberation Army Medical Research Institute Of Nanjing Military Region, Nanjing Medical University

＜120〉anti-VEGFR-2 antibody chimeric Fab antibody fragment and preparation method thereof, application

<160>3

<210>1

<211>1483

<212>DNA

＜213〉artificial sequence

<220>

<221>mis-feature

<222><1>…<12>

＜223〉the SfiI restriction enzyme site of connection chimeric Fab antibody fragment gene and carrier pComb3XSS

<220>

<221>mis-feature

<222><13>…<18>

＜223〉carrier pComb3XSS goes up the SacI restriction enzyme site

<220>

<221>V_region

<222><19>…<354>

＜223〉mouse source property anti-VEGFR-2 antibody chain variable region gene fragment sequence

<220>

<221>C_region

<222><355>…<675>

＜223〉people's IgG antibody 1 constant region of light chain C kappa gene fragment sequence

<220>

<221>mis-feature

<222><676>…<678>

＜223〉terminator codon of light chain gene fragment translation

<220>

<221>mis-feature

<222><679>…<708>

＜223〉nucleotide sequence of chimeric Fab fragment light chain and the overlapping extension of Fd gene fragment contains ribosome bind site.

<220>

<221>mis-feature

<222><670>…<674>

＜223〉carrier pComb3XSS goes up the XbaI enzyme cutting site

<220>

<221>mis-feature

<222><709>…<774>

＜223〉the pelB signal peptide sequence that increases for the Fd solubility expression that makes among the anti-VEGFR-2 antibody Fab will be in protein excretion

Be sheared during to the pericentral siphon chamber.

<220>

<221>mis-feature

<222><775>…<780>

＜223〉carrier pComb3XSS goes up the XhoI restriction enzyme site

<220>

<221>V_region

<222><781>…<1131>

＜223〉mouse source property anti-VEGFR-2 antibody heavy chain variable region gene fragment sequence

<220>

<221>C_region

<222><1132>…<1446>

＜223〉people's IgG antibody 1 CH CH1 gene fragment order

<220>

<221>mis-feature

<222><1447>…<1452>

＜223〉carrier pComb3XSS goes up the SpeI restriction enzyme site

<220>

<221>mis-feature

<222><1453>…<1464>

<220>

<221>mis-feature

<222><1465>…<1521>

＜223〉carrier pComb3XSS goes up sequence, contains the His label

<220>

<221>mis-feature

<222><1522>…<1524>

＜223〉the succsinic acid terminator on the carrier pComb3XSS

<400>1

GCC AGA GCG GCC GAG CTC GAC ATT GTG ATG ACA CAG TCT GCA TTC TCC AAT CCA GTC ACT 60

Glu Leu Asp Ile Val Met Thr G1n Ser Ala Phe Ser Asn Pro Val Thr

1 5 10 15

CTT GGA ACA TCA GCT TCC ATC TCC TGC AGG TCT AGT AAG AGT CTC CTA CAT AGT AAT GGC 120

Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly

20 25 30 35

ATC ACT TAT TTG TAT TGG TAT CTG CAG AAG CCA GGC CAG TCT CCT CAG CTC CTG ATT TAT 180

Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr

40 45 50 55

CAG ATG TCC AAC CTT GCC TCA GGA GTC CCA GAC AGG TTC AGT AGC AGT GGG TCA GGA ACT 240

Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr

60 65 70 75

GAT TTC ACA CTG AGA ATC AGC AGA GTG GAG GCT GAG GAT GTG GGT GTT TAT TAC TGT GCT 300

Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala

80 85 90 95

CAA AAT CTA GAA CTT CCT CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGA ACT 360

Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr

100 105 110 115

GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA ACT 420

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

120 125 130 135

GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG 480

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

140 145 150 155

GTG GAT AAC GCC CTC CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG 540

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

160 165 170 175

GAC AGC ACC TAC AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC 600

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

180 185 190 195

AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TTG CCC GTC ACA AAG AGC TTC 660

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

200 205 210 215

AAC AGG GGA GAG TGT TAG TTC TAG ATA ATT AAT TAG GAG GAA TTT AAA ATG AAA TAC CTA 720

Asn Arg Gly Glu Cys ter Phe ter

220 224

TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCC ATG GCC CTC GAG 780

GTG AAG CTG GTG GAG TCA GGA CCT GGC CTA GTG CAG CCC TCA CAG AGC CTG TCC ATC ACC 840

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

1 5 10 15 20

TGC ACA GTC TCT GGT TTC TCA TTA ACT AGC CAT GGT GTA CAC TGG GTT CGC CAG TCT CCA 900

Cys Thr Val Ser Gly Phe Ser Leu Thr Ser His Gly Val His Trp Val Arg Gln Ser Pro

25 30 35 40

GGA AAG GGT CTG GAG TGG CTG GGA GTG ATA TGG AGT GGT GGA AGC ACA GAC TAT AAT GCA 960

Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala

45 50 55 60

GCT TTC ATA TCC AGA CTG AGC ATC AGC AAG GAC AAT TCC AAG AGC CAA GTT TTC TTT AAA 1020

Ala Phe Ile Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys

65 70 75 80

ATG AAC AGT CTG CAA GCT AAT GAC ACA GCC ATA TAT TAC TGT GCC AGA AAT AGG AGG GGG 1080

Met Asn Ser Leu Gln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn Arg Arg Gly

85 90 95 100

TAC TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC ACG GTC ACC GTC TCC TCA GCC TCC ACC 1140

Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

105 110 115 120

AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG 1200

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

125 130 135 140

GCC CTG GGC TGC CTG GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA 1260

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

145 150 155 160

GGC GCC CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC 1320

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175 180

TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATC TGC 1380

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

185 190 195 200

AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCC AAA TCT TGT 1440

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

205 210 215 220

GAC AAA ACT AGT GGC CAG GCC GGC CAG CAC CAT CAC CAT CAC CAT GGC GCA TAC CCG TAC 1500

Asp Lys Thr Ser Gly Gln Ala Gly Gln His His His His His His Gly Ala Tyr Pro Tyr

225 230 235 240

GAC GTT CCG GAC TAC GCT TCT TAG 1524

Asp Val Pro Asp Tyr Ala Ser ter

245 248

<210>2

<211>224

<212>PRT

＜213〉artificial sequence

<220>

＜223〉chimeric light chain of anti-VEGFR-2 antibody (L section), the i.e. variable region of light chain of anti-VEGFR-2 antibody and human IgG1's light chain

The fusion rotein that constant region C κ reorganization back forms, 224 amino acid of total length.

<400>2

Glu Leu Asp Ile Val Met Thr Gln Ser Ala Phe Ser Asn Pro Val

1 5 10 15

Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser

20 25 30

Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln

35 40 45

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn

50 55 60

Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly

65 70 75

Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val

80 85 90

Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe

95 100 105

Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

110 115 120

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

125 130 135

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

140 145 150

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

155 160 165

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

170 175 180

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

185 190 195

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Leu

200 205 210

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys ter Phe ter

215 220 224

<210>3

<211>248

<212>PRT

＜213〉artificial sequence

<220>

＜223〉chimeric heavy chain of anti-VEGFR-2 antibody (Fd section), the i.e. variable region of heavy chain of anti-VEGFR-2 antibody and human IgG1's heavy chain

The fusion rotein that constant region CH1 reorganization back forms, 248 amino acid of total length.

<400>3

Val Lys Leu Val Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser

20 25 30

His Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Leu Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala

50 55 60

Ala Phe Ile Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser

65 70 75

Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ala Asn Asp Thr Ala

80 85 90

Ile Tyr Tyr Cys Ala Arg Asn Arg Arg Gly Tyr Tyr Ala Met Asp

95 100 105

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

110 115 120

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

125 130 135

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

140 145 150

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

155 160 165

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

170 175 180

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

185 190 195

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

200 205 210

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr Ser Gly

215 220 225

Gln Ala Gly Gln His His His His His His Gly Ala Tyr Pro Tyr

230 235 240

Asp Val Pro Asp Tyr Ala Ser ter

245 248

Claims

1. anti-VEGFR-2 antibody chimeric Fab fragment, the i.e. heavy chain of mouse source property anti-VEGFR-2 antibody and variable region of light chain and human IgG ₁Heavy chain part constant region CH ₁Reach the fusion rotein that constant region of light chain C κ recombinates and forms; The variable region of light chain and the human IgG of mouse source property anti-VEGFR-2 antibody ₁The chimeric L chain of constant region of light chain C κ reorganization and the variable region of heavy chain VH and the human IgG of mouse source property anti-VEGFR-2 antibody ₁CH ₁The chimeric Fd section of reorganization connects by leader sequence pelB in the middle of both, and leader sequence pelB was sheared when chimeric Fd section, L chain were secreted into bacterium pericentral siphon chamber, and both connect and compose chimeric Fab antibody fragment by interchain disulfide bond;

The aminoacid sequence of chimeric L chain is:

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys ter

The aminoacid sequence of chimeric Fd section is:

His His His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser ter。

2. the preparation method of the chimeric Fab antibody fragment described in the claim 1, this antibody fragment is to utilize the genetic engineering technique preparation, concrete grammar is as follows:

1) segmental amplification of antibody variable gene and checking:

Mouse source property anti-VEGFR-2 antibody hybridoma 8H1, its deposit number is CGMCC No.3013, is cultured to logarithmic phase, Trizol-chloroform-isopropanol method extracting cell total rna; With the mRNA among total RNA is template, with oligodT ₁₅Be primer, post transcription cloning obtains strand cDNA;

The variable region of heavy chain (VH) of design mouse source property anti-VEGFR-2 antibody, the amplimer of variable region of light chain (VL), the VH that increases respectively, VL fragment, the upstream primer of variable region of light chain (VL) is VLF:5 '-G GGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ', downstream primer be VLR:5 '- AGATGGTGCAGCCACAGTTCGTTTCAGCTCCAGCTTGGTCCC-3 ', holding the enzyme of having introduced SfiI to cut recognition site at 5 of VLF ' is the underscore partial sequence, 5 of VLR ' end has been introduced and human IgG ₁C κ upstream primer 5 ' 21 bases of end complementary be the italicized item sequence, be beneficial to the VL and the human IgG of mouse source property anti-VEGFR-2 antibody ₁The segmental overlapping pcr amplification of C κ; The upstream primer of variable region of heavy chain (VH) is VHF: 5 '- GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGGTGGAGTC-3 ', downstream primer be VHR:5 '- CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCGTGGT-3 ' has introduced and human IgG at 5 of VHF ' end ₁21 base sequences of C κ downstream primer complementary be the italicized item sequence, 5 of VHR ' end has been introduced and human IgG ₁CH ₁21 base sequences of upstream primer complementary, i.e. italicized item is beneficial to the VH and the human IgG of anti-VEGFR-2 antibody ₁CH ₁Segmental overlapping pcr amplification;

CDNA with post transcription cloning is a template respectively, with the VH gene fragment of primer VHF, VHR amplification mouse source property anti-VEGFR-2 antibody, with the VL gene fragment of primer VLF, VLR amplification mouse source property anti-VEGFR-2 antibody; Amplification condition be 95 ℃ 4 minutes; 95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes; Agarose gel electrophoresis, glue reclaim purifying amplification gene fragment, and VH, VL PCR glue recovery purified product carry out TA with the pMD-18T carrier respectively and be connected; Connect product transformed into escherichia coli XL1-Blue, coating contains the LB flat board of 100 μ g/ml penbritins, 30 μ g/ml tsiklomitsins, puts 37 ℃ of 12-16h; Next day is picking transformed bacteria and empty plasmid conversion contrast bacterium at random, 37 ℃ are shaken bacterium and with the upstream and downstream special primer of VH, VL gene fragment bacterium liquid are carried out PCR respectively after 5 hours and identify, contain the bacterial strain that inserts VH gene fragment plasmid and amplify a band about 351bp, contain the bacterial strain that inserts VL gene fragment plasmid and amplify a band about 336bp; The bacterium liquid that bacterium liquid PCR checking amplifies the correct band of size send biotech firm to check order;

Wherein the nucleotides sequence of VH is classified as:

TAC TGG GGT CAA GGA ACC ACG GTC ACC GTC TCC TCA

The nucleotides sequence of VL is classified as:

GAG CTC GAC ATT GTG ATG ACA CAG TCT GCA TTC TCC AAT CCA GTC ACT CTT GGA ACA TCA GCT

TCC ATC TCC TGC AGG TCT AGT AAG AGT CTC CTA CAT AGT AAT GGC ATC ACT TAT TTG TAT TGG

TAT CTG CAG AAG CCA GGC CAG TCT CCT CAG CTC CTG ATT TAT CAG ATG TCC AAC CTT GCC TCA

GGA GTC CCA GAC AGG TTC AGT AGC AGT GGG TCA GGA ACT GAT TTC ACA CTG AGA ATC AGC AGA

GTG GAG GCT GAG GAT GTG GGT GTT TAT TAC TGT GCT CAA AAT CTA GAA CTT CCT CTC ACG TTC

GGT GCT GGG ACC AAG CTG GAG CTG AAA

2) the segmental amplification of chimeric Fab:

(1) human IgG ₁The CH of antibody ₁Amplification with C κ section:

With plasmid pComb3XTT is template, and human IgG increases respectively ₁The C κ and the CH of antibody ₁Nucleotide fragments; The upstream primer of C κ section is C κ F:5 '-CGAACTGTGGCTGCACCATCTGTC-3 ', and downstream primer is C κ R:5 '-GGCCATGGCTGGTTGGGCAGC-3 '; CH ₁Upstream primer be CH ₁F:5 '-GCCTCCACCAAGGGCCCATCGGTC-3 ', downstream primer are CH ₁R:AGAAGCGTAGTCCGGAACGTC-3 '; Amplification condition be 95 ℃ 4 minutes; 95 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes, and agarose gel electrophoresis amplifies the band of about 393bp, 321bp respectively, and glue recovery purifying amplified band is dissolved in the deionized water, and-20 ℃ frozen standby;

(2) chimeric L chain, the segmental amplification of Fd fragment gene:

VL and human IgG with mouse source property anti-VEGFR-2 antibody ₁The pcr amplification product of the C κ of antibody is a template, carry out the overlapping extension PCR chimeric L chain that increases with upstream primer LF:5 '-GGGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ' and downstream primer LR:5 '-GGCCATGGCTGGTTGGGCAGC-3 ', amplification condition is 95 ℃, 4 minutes; 95 ℃, 30 seconds, 56 ℃, 30 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes; Agarose gel electrophoresis amplifies the band of about 657bp, and glue reclaims amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby;

VH and human IgG with mouse source property anti-VEGFR-2 antibody ₁The CH of antibody ₁Pcr amplification product be template, carry out the overlapping extension PGR chimeric Fd fragment that increases with upstream primers F dF:5 '-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGGTGGAGTC-3 ' and downstream primer FdR:5 '-AGAAGCGTAGTCCGGAACGTC-3 ', amplification condition is 95 ℃, 4 minutes; 95 ℃, 30 seconds, 56 ℃, 30 seconds, 72 ℃, 30 seconds, 30 circulations; 72 ℃ were extended 10 minutes; Agarose gel electrophoresis amplifies the band of about 744bp, and glue reclaims amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby;

(3) amplification of chimeric Fab gene fragment:

With the chimeric Fd section of amplification acquisition and the glue recovery purified product of L chain nucleotide sequence is template, carry out overlapping extension PCR amplification with upstream primers F abF:5 '-GGGCCCAGGCGGCCGAGCTCGACATTGTGATGACACAGTC-3 ' and downstream primer FabR:5 '-AGAAGCGTAGTCCGGAACGTC-3 ', during amplification, do not add earlier primer, 95 ℃ 4 minutes; 94 ℃ 30 seconds, 45 ℃ 30 seconds, 72 ℃ 90 seconds, 8 circulations; Then add behind FabF, the FabR primer 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 90 seconds, 22 circulations; 72 ℃ were extended 10 minutes, and agarose gel electrophoresis amplifies the band of about 1.5kb, and glue reclaims the purifying amplified band, is dissolved in the deionized water, and-20 ℃ frozen standby;

3) chimeric Fab construction of prokaryotic expression vector and evaluation:

Adopt plasmid pComb3XSS, the segmental PCR glue of plasmid and Fab reclaims product and cuts 12-16h with the SfiI restriction enzyme at 50 ℃ of enzymes, and glue reclaims the big fragment of plasmid that enzyme is cut behind the electrophoresis, is dissolved in the deionized water; Get the enzyme of pComb3XSS, Fab amplified production and cut product, in same centrifuge tube, connect 12-16h for 16 ℃ with the T4 ligase enzyme by 1: 4 mol ratio mixing;

To connect product transformed competence colibacillus intestinal bacteria TOP10F ', coating contains the LB flat board of 100 μ g/ml penbritins, puts 37 ℃ of 12-16h; Next day, picking transformed bacteria and empty plasmid transformed the contrast bacterium at random, and 37 ℃ were shaken bacterium after 5 hours, identified bacterium liquid being carried out pcr amplification with the special primer of Fab respectively, and the bacterial strain that contains insertion Fab fragment plasmid amplifies the band about a treaty 1.5kb; The bacterium liquid that bacterium liquid PCR checking amplifies the correct band of size send biotech firm to check order, dna sequence analysis confirms to contain the chimeric Fab fragment of structure in recombinant plasmid, sequence is correct, wherein horizontal line part A TG AAA TAC CTA TTG CCTACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCC ATG GCC is leader sequence pelB, TAG is chimeric Fd chain, L chain translation stop codon, GCC AGA GCG GCC, GGC CAG GCC GGC is for connecting the SfiI restriction enzyme site of chimeric Fab antibody fragment gene and carrier pComb3XSS, GAG CTC is the SacI restriction enzyme site of carrier pComb3XSS, CAG CAC CAT CAC CAT CAC CAT GGC GCA TAC CCG TAC GAC GTT CCG GAC TAC GCT TCT contains the His label for carrier pComb3XSS goes up sequence, ACT AGT is the SpeI restriction enzyme site, CTAGA is the XbaI enzyme cutting site, and CTC GAG is the XhoI restriction enzyme site;

GCC AGA GCG GCC GAG CTC GAC ATT GTG ATG ACA CAG TCT GCA TTC TCC AAT CCA GTC ACT CTT

GGA ACA TCA GCT TCC ATC TCC TGC AGG TCT AGT AAG AGT CTC CTA CAT AGT AAT GGC ATC ACT

TAT TTG TAT TGG TAT CTG CAG AAG CCA GGC CAG TCT CCT CAG CTC CTG ATT TAT CAG ATG TCC

AAC CTT GCC TCA GGA GTC CCA GAC AGG TTC AGT AGC AGT GGG TCA GGA ACT GAT TTC ACA CTG

AGA ATC AGC AGA GTG GAG GCT GAG GAT GTG GGT GTT TAT TAC TGT GCT CAA AAT CTA GAA CTT

CCT CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA 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 AGC TTG CCC GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT TAG TT C TAG ATA ATT AAT

TAG GAG GAA TTT AAA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT

GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG GTG GAG TCA GGA CCT GGC CTA GTG CAG CCC

TCA CAG AGC CTG TCC ATC ACC TGC ACA GTC TCT GGT TTC TCA TTA ACT AGC CAT GGT GTA CAC

TGG GTT CGC CAG TCT CCA GGA AAG GGT CTG GAG TGG CTG GGA GTG ATA TGG AGT GGT GGA AGC

ACA GAC TAT AAT GCA GCT TTC ATA TCC AGA CTG AGC ATC AGC AAG GAC AAT TCC AAG AGC CAA

GTT TTC TTT AAA ATG AAC AGT CTG CAA GCT AAT GAC ACA GCC ATA TAT TAC TGT GCC AGA AAT

AGG AGG GGG TAC TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC ACG GTC ACC GTC TCC TCA GCC

TCC ACC AAG 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 GGC ACC CAG ACC TAC ATC TGC AAC GTG

AAT CAC AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCC AAA TCT TGT GAC AAA ACT

AGT GGC CAG GCC GGC CAG CAC CAT CAC CAT CAC CAT GGC GCA TAC CCG TAC GAC GTT CCG GAC

TAC GCT TCT TAG

The chimeric Fab fragment of L chain that the expression of recombinant plasmid that makes up is chimeric and the reorganization of Fd section, 22 amino acid of leader peptide sequences pelB translation between the two connect, leading peptide pelB was sheared when the protein fragments of translation was secreted to bacterium pericentral siphon chamber, and chimeric L chain is connected by interchain disulfide bond with the Fd section;

Bacterium liquid 10000g is centrifugal 15 minutes behind a large amount of abduction deliverings of bacterium, abandons culture supernatant, and add original bacteria liquid 1/10 volume in the precipitation and contain 0.5M/L NaCl, the 20mM phosphate balance damping fluid of 10mM imidazoles, bacterium is resuspended; Then bacterium liquid is carried out ultrasonicly, surpass 10 seconds, stopped 10 seconds, surpass altogether 90 times, then 4 ℃ of 12000rpm centrifugal 30 minutes, abandon precipitation, and ultrasonic supernatant carries out purifying with 5ml Histrap HP pillar then with 0.22 μ m membrane filtration; Earlier wash pillar with the speed of 2.5ml/min, then use the level pad balance pillar of at least 10 times of column volumes, then with sample on the speed of 2ml/min with water with 5 times of column volumes; With level pad balance nickel post to baseline, wash pillar with the damping fluid that contains 50mM, 100mM, 200mM, 300mM, 400mM, 500mM imidazoles of 5 times of column volumes respectively, and the elutriant of collection respective concentration imidazoles, carry out the 12%SDS-PAGE electrophoresis, observe the protein purification situation; The result contains the Fab albumen of seeing purifying in the elutriant of 400mM imidazoles, carries out desalination with the millpore ultrafiltration pillar of 10KD and concentrates, and PBS washes 3 times, promptly obtains the chimeric Fab antibody fragment of purifying;

5) activity identification of chimeric Fab antibody fragment:

The I euzymelinked immunosorbent assay (ELISA)

With containing the 0.1M carbonate buffer solution, the coating buffer of pH9.6 dilution recombinant human VEGF R-2 albumen, by 96 hole elisa plates, every hole adds 100 μ l with the concentration bag of 2 μ g/ml, and 4 ℃ are spent the night; After the PBST washing,, hatch 2h for 37 ℃ with 5% skimmed milk-lavation buffer solution sealing; After the PBST washing 5 times, add 100 μ l anti-VEGFR-2 antibody chimeric Fab antibody fragments in each hole, with 200 μ g/ml initial concentrations, 11 concentration gradient dilutions, 4 ℃ are spent the night; Goat-anti human Fab second antibody 100 μ l/ holes with dilution in 1: 5000 join in the hole, hatch 1h for 37 ℃; Peroxidase substrate colour developing liquid 100 μ l/ holes, with 2M sulfuric acid stopped reaction, last machine testing colorimetric adopts dual wavelength 450nm/630nm to room temperature after following 15 minutes; Chimeric Fab antibody fragment can play antigen-antibody ELISA reaction with recombinant human VEGF R-2 albumen as a result;

The II immunoblotting

People's navel endothelial venule cell HUVEC of high expression level VEGFR-2, l cell NIH3T3 total protein low or that do not have a VEGF expression R-2 are natural antigen; Cell is cultured to 1 * 10 respectively ⁷, remove substratum under 4 ℃ of conditions and clean with PBS, with the abundant lysing cell of 500 μ l RIPA lysates, 10000g got supernatant and total protein of cell in centrifugal 5 minutes, measured the concentration of each total protein of cell; With each total protein of cell concentration make after the 1mg/ml equal concentrations packing frozen-20 ℃ standby;

HUVEC, NIH3T3 total protein of cell are carried out the 10%SDS-PAGE electrophoresis and electricity forwards on the nitrocellulose membrane, with confidential reference items antibody β-actin of the anti-VEGFR-2 antibody chimeric Fab antibody fragment of this film and 50 μ g/ml and dilution in 1: 2000 simultaneously 37 ℃ hatch 2h, the PBST washing room is every 5 minutes 4 times, goat-anti human Fab two resists 37 ℃ and hatches 1h, the DAB result that develops the color, one thick bright wisp band, three filaments of sun bands appear in HUVEC total protein swimming lane from the top down, respectively successively corresponding to VEGFR-2 albumen three band 230kDa, 200kDa, 150kDa and confidential reference items β-actin band; Confidential reference items β-actin band of 43kDa then only appears in NIH3T3 total protein of cell swimming lane;

The III flow cytometry

Adopt flow cytometry analysis anti-VEGFR-2 antibody people mouse chimeric Fab antibody fragment active with combining of VEGFR-2 natural antigen; Concrete grammar is, respectively with 2 * 10 ⁶Individual HUVEC and NIH3T3 cell are with the PBS flushing that contains 4 ℃ of the aseptic precoolings after half an hour of 0.1% Paraformaldehyde 96 PBS fixed cell three times, use anti-VEGFR-2 antibody chimeric Fab antibody fragment respectively, 60 μ g/ml and two different concns of 120 μ g/ml are hatched 4 ℃ altogether and are spent the night, PBS gives a baby a bath on the third day after its birth anti-all over back adding goat-anti human Fab rhodamine fluorescence two, the lucifuge incubated at room after 2 hours PBS give a baby a bath on the third day after its birth time, it is resuspended to contain 0.1% Paraformaldehyde 96 PBS, 400 μ l, on flow cytometer, carry out immunofluorescence analysis and measure average fluorescent strength, with only add two anti-be background, the anti-VEGFR-2 antibody chimeric Fab antibody fragment and the HUVEC combination rate of 60 μ g/ml and two different concns of 120 μ g/ml are respectively 34.46% and 35.16% as a result; The anti-VEGFR-2 antibody chimeric Fab antibody fragment and the NIH3T3 cell combination rate of same concentration are respectively 14.06% and 16.26%;

Above result shows that anti-VEGFR-2 antibody chimeric Fab antibody fragment can combine with VEGFR-2 antigen, and antibody character and specificity that anti-VEGFR-2 antibody that this chimeric Fab fragment has kept former preparation possesses are described.

3. according to the anti-VEGFR-2 antibody chimeric Fab antibody fragment of the described preparation method of claim 2 preparation.

4. the chimeric Fab antibody fragment described in the claim 1 is in the diagnostic reagent of preparation anti-VEGFR-2, the application in the curative drug.