CN103772501A - VEGF (Vascular Endothelial Growth Factor) antibody as well as preparation method and application thereof - Google Patents
VEGF (Vascular Endothelial Growth Factor) antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a VEGF (Vascular Endothelial Growth Factor) single-domain antibody as well as a fusion antibody and a polymer antibody of the VEGF antibody. The invention also discloses a nucleotide sequence for encoding the antibodies, an expression vector containing the nucleotide sequence, a recombinant bacterium containing the nucleotide sequence as well as a preparation method and applications of the antibodies. The VEGF antibody is low in molecular weight, high in plasticity and wide in application prospect.
Description
Technical field
The present invention relates to new VEGF antibody and its production and use.
Background technology
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a kind of homodimer glycoprotein, has the effects such as the division of the vascular endothelial cell of promotion, propagation and induction of vascular formation in human body.VEGF can, by promoting that vasculogenesis causes tumour Fast Growth, be an important tumour medicine target.
VEGF antibody can be combined with VEGF and be suppressed its physiological vigor, and then suppresses the Fast Growth of tumour, and in addition, VEGF antibody can also be treated agedness yellow spot degenerative disease.Conventional antibody has whole antibody, Fab, single-chain antibody etc.At present, VEGF antibody is mostly whole antibody, as, rhuMAb-VEGF (bevacizumab, Avastin), HuMV833 monoclonal antibody, VEGF-Trap antibody, also have small part single-chain antibody, as the single-chain antibody of rhuMAb-VEGF.These antibody drug molecular weight are large, are difficult for through vessel wall, cause local drug concentration on the low side, and drug effect is limited; Also because molecular weight is larger, there is the defect that immunogenicity is high simultaneously.
VH single domain antibody is the small molecule antibody occurring in recent years, it only contains an antibody-like of the variable region of heavy chain (VH) of whole antibody, and its molecular weight is approximately 1/10 of whole antibody, and immunogenicity is low, penetrance is good, is conducive to improve the drug level of target area.But, because single domain antibody lacks variable region of light chain, being difficult to form antigen binding domain, ubiquity is without antigen binding capacity or the extremely low problem of binding ability.
Summary of the invention
The problem existing for solving existing VEGF antibody, the invention provides new VEGF antibody and its production and use.
The invention provides VEGF single domain antibody, its aminoacid sequence is as shown in SEQ ID NO:1～SEQ ID NO:6 any one.
Single domain antibody, refers to the antibody that only has complete antibody heavy variable region.
Single domain antibody shown in SEQ ID NO:1～SEQ ID NO:6 is called after: Vh-VEGF (Vh), Vh-VEGF/Y31(Y31 respectively), Vh-VEGF/Q61(Q61), Vh-VEGF/YDA(Vh-YDA), Vh-VEGF/30(30), Vh-VEGF/94(94), their aminoacid sequence is as follows:
Vh（SEQ?ID?NO.1）：
MEVQLEQSGGGLVQPGGSLRLSCAASGFTFTNYGMNWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGS; Molecular weight is 14.46kD.
Y31（SEQ?ID?NO.2）：
MEVQLEQSGGGLVQPGGSLRLSCAASGFTFTYYGMNWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGS; Molecular weight is 14.51kD.
Q61（SEQ?ID?NO.3）：
MEVQLEQSGGGLVQPGGSLRLSCAASGFTFTYYGMNWVRQAPGKGLEWVGWINAYT GEPTYAQDFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGS; Molecular weight is 14.56kD.
Vh-YDA（SEQ?ID?NO.4）:
MEVQLEQSGGGLVQPGGSLRLSCAASGYTDTYYGMGWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLE; Molecular weight is 14.178kD.
30（SEQ?ID?NO.5）:
MEVQLEQSGGGLVQPGGSLRLSCAASGYTDTNYGMGWVRRAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPFLYCNSEWYFD VWGQGTLVTVSSLVGGGS; Molecular weight is 14.41kD.
94（SEQ?ID?NO.6）:
MEVQLEQSGGGLVQPGGSLRLSCVASGYTDTNYGMGWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPNFCWCSRWYFD VWGQGTLVTVSSLVGGGS; Molecular weight is 14.45kD.
The present invention also provides VEGF fusion antibody, and it is the fusion antibody being formed by connecting by connection peptides by aforementioned single domain antibody, or connects at the C-terminal of aforementioned single domain antibody the fusion antibody that Fc fragment forms.
Connection peptides is a peptide species, and its aminoacid sequence is (GGGGS)
n, wherein, n can be 1～10.Preferably, the aminoacid sequence of described connection peptides is: GGGGSGGGGSGGGGS.
Preferably, the aminoacid sequence of the fusion antibody that described connection peptides is formed by connecting as shown in SEQ ID NO:7, called after: Vh-double.
The aminoacid sequence (SEQ ID NO:7) of Vh-double is:
MEVQLEQSGGGLVQPGGSLRLSCAASGFTFTNYGMNWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGGSGGGGSGGGGSEVQLEQSGGGLVQPGGSLRLSCAASGF TFTNYGMNWVRQAPGKGLEWVGWINAYTGEPTYAADFKRRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARYPHYYGSSHWYFDVWGQGTLVTVSSLE; Molecular weight is 29.20kD.
The aminoacid sequence of the fusion antibody that preferably, the described connection of the C-terminal at single domain antibody Fc fragment forms is as shown in SEQ ID NO:10, by its called after Vh-Fc.
The aminoacid sequence of Vh-Fc is:
MEVQLEQSGGGLVQPGGSLRLSCAASGFTFTNYGMNWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGSEVQLEQSGGGLVQPGGSLRLSCAASGFTFTNYGMNWVR QAPGKGLEWVGWINAYTGEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARYPHYYGSSHWYFDVWGQGTLVTVSSLEGGGSPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEVTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGS; Molecular weight is 54.8kD.
The present invention also provides VEGF tripolymer antibody, and monomer whose is to be formed by connecting by aforementioned single domain antibody and isoleucine zipper.
Tripolymer antibody, refers to the antibody being formed by three monomer polymerizations.Some protein molecule can aggregate into polymer, and the repeating unit in polymer is called monomer.
Wherein, it is curling that isoleucine zipper is made up of the amino acid stretching--α-helixstructure, the 7th amino acid in every 7 amino acid is Isoleucine, Isoleucine is hydrophobic amino acid, is arranged in a side of alpha-helix, and all charged amino-acid residues are arranged in opposite side, in the time that 3 single domain antibodies that are connected with isoleucine zipper are arranged in parallel, between Isoleucine, interact, form " slide fastener " spline structure, and then make to form tripolymer.Therefore the albumen, connecting with isoleucine zipper can be assembled formation tripolymer.
The aminoacid sequence of described isoleucine zipper is:
QRMKQIEDKIEEILSKIYHIENEIARIKKLVGER。
Preferably, the aminoacid sequence of described tripolymer antibody is as shown in SEQ ID NO:8 or 9.
VEGF polymer antibody shown in SEQ ID NO:8, SEQ ID NO:9 is called after: Vh-trime and Vh-YDA-trime respectively.
The aminoacid sequence (SEQ ID NO:8) of the monomer of Vh-trime is:
MEVQLEQSGGGLVQPGGSLRLSCAASGFTFTNYGMNWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGGSGGGGSGGGGSQRMKQIEDKIEEILSKIYHIENEIARI KKLVGERGGGS; Molecular weight is 19.48kD; Vh-trime antibody is the tripolymer antibody being formed by aforementioned monomer polymerization.
The aminoacid sequence (SEQ ID NO:9) of the monomer of Vh-YDA-trime is:
MEVQLEQSGGGLVQPGGSLRLSCAASGFTDTNYGMNWVRQAPGKGLEWVGWINAYT GEPTYAADFKRRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYPHYYGSSHWYFD VWGQGTLVTVSSLEGGGGSGGGGSGGGGSQRMKQIEDKIEEILSKIYHIENEIARI KKLVGERGGGS; Molecular weight is 19.508kD.
Vh-YDA-trime antibody is the tripolymer antibody being formed by aforementioned monomer polymerization.
The present invention further provides the nucleotide sequence of the above-mentioned antibody of encoding.
Preferably, above-mentioned sequence is as shown in SEQ ID NO:11～SEQ ID NO:20:
Nucleotide sequence shown in SEQ ID NO:11 (encoding antibody Vh) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGTGGCGGTAGC；
Nucleotide sequence shown in SEQ ID NO:12 (encoding antibody Y31) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACTATTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGTGGCGGTAGC；
Nucleotide sequence shown in SEQ ID NO:13 (encoding antibody Q61) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCTACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGCAGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGAACACTGGTCACCGTCTCTTCACTCGTGGGTGGCGGTAGC；
Nucleotide sequence shown in SEQ ID NO:14 (encoding antibody Vh-YDA) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAG；
Nucleotide sequence shown in SEQ ID NO:15 (encoding antibody 30) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTTTCCTGTGCGGCCTCTGGATACACCGACACCAACTATGGCATGGGCTGGGTCCGCCGGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGTTCCTCTACTGCAACAGCGAGTGGTATTTTGATGTGTGGGGCCAGGGAACACTGGTCACCGTCTCTTCACTCGTGGGTGGCGGTAGC；
Nucleotide sequence shown in SEQ ID NO:16 (encoding antibody 94) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGTGGCCTCTGGATACACCGACACCAACTATGGCATGGGCTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGAACTTCTGCTGGTGCAGCCGGTGGTATTTTGATGTGTGGGGCCAGGGAACACTGGTCACCGTCTCTTCACTCGTGGGTGGCGGTAGC；
Nucleotide sequence shown in SEQ ID NO:17 (encoding antibody Vh-double) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGTGGTGGCGGTTCCGGCGGTGGTGGCTCTGGCGGTGGCGGATCGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAG；
Nucleotide sequence shown in SEQ ID NO:18 (monomer of encoding antibody Vh-trime) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGCGCATGAAACAGATTGAAGATAAAATTGAAGAAATTCTGAGCAAAATTTATCATATTGAAAACGAAATTGCGCGCATTAAAAAACTGGTGGGCGAACGCGGCGGCGGCAGC；
Nucleotide sequence shown in SEQ ID NO:19 (monomer of encoding antibody Vh-YDA-trime) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGCGCATGAAACAGATTGAAGATAAAATTGAAGAAATTCTGAGCAAAATTTATCATATTGAAAACGAAATTGCGCGCATTAAAAAACTGGTGGGCGAACGCGGCGGCGGCAGC。
Nucleotide sequence shown in SEQ ID NO:20 (encoding antibody Vh-Fc) is:
ATGGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGTGGCGGTAGCGAAGTGCAGCTGGAGCAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTACCAACTATGGCATGAACTGGGTCCGCCAGGCTCCGGGCAAAGGCCTGGAATGGGTGGGCTGGATTAACGCCTATACCGGCGAACCGACCTATGCGGCGGATTTTAAACGTCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATATCCGCATTATTATGGCAGCAGCCATTGGTATTTTGATGTGTGGGGCCAGGGCACCCTGGTTACCGTGAGTTCACTCGAGGGTGGCGGTAGCCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGGTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGTGGCGGTAGC。
The present invention also provides the expression vector that contains above-mentioned nucleotide sequence.
Preferably, above-mentioned expression vector is restructuring pET22b plasmid.
The present invention further provides the recombinant bacterium that contains above-mentioned expression vector.
Preferably, described recombinant bacterium is BL21(DE3) intestinal bacteria.
The preparation method who the present invention further provides above-mentioned VEGF antibody, comprises following steps:
(1) get aforementioned recombinant bacterium, be inoculated into and contain 0.5～1.5%(w/v) in the LB substratum or YT substratum of glucose, 50～150mg/L penbritin, cultivate 10～15h, obtain bacterium liquid for 34～40 ℃;
(2) according to 0.5～1.5%(v/v) inoculum size, step (1) bacterium liquid is seeded in the LB substratum or YT substratum that contains 50～150mg/L penbritin, 34～40 ℃ are cultured to OD
600=0.6～0.8;
(3) add IPTG to final concentration be 0.2～0.4mmol/L, under the condition of 20～30 ℃, inducing culture 3～7 hours;
(4) collect culture, separation and purification, obtains VEGF antibody.
LB substratum: contain Tryptones 10g, yeast extract 5g and sodium-chlor 10g in every 1L solution, all the other are water.
YT substratum: contain Tryptones 16g, yeast extract 10g and sodium-chlor 4g in every 1L solution, all the other are water.
Preferably, in step (1), in described substratum, the concentration of glucose is 1%(w/v), the concentration of penbritin is 100mg/L; The temperature of described cultivation is 37 ℃; Described incubation time is 12h; In step (2), in described substratum, the concentration of penbritin is 100mg/L, and the temperature of described cultivation is 37 ℃; In step (3), the final concentration of IPTG is 0.3mmol/L; The temperature of inducing culture is 25 ℃, and the time is 5 hours.
The present invention also provides the purposes of above-mentioned VEGF antibody in the medicine of preparing antitumor drug or treatment agedness yellow spot degenerative disease, and wherein, described agedness yellow spot degenerative disease is moist agedness yellow spot degenerative disease.
The present invention is by the optimization to nucleotide sequence, successfully prepare highly active VEGF single domain antibody Vh, Y31, Q61, Vh-YDA, 30,94, the affinity costant of itself and VEGF is 0.669～2.042, with positive drug Avastin single-chain antibody (affine Changshu is 1.001) quite or higher, meanwhile, their molecular weight is little, can effectively penetrate tumour, immunogenicity is low, and safety is a kind of safe and effective antitumor drug; The present invention has also prepared has highly active VEFG fusion antibody and tripolymer antibody, and their affinity costant is 1.102～2.655, and for clinical antitumor drug provides a kind of new selection, application prospect is good.
Obviously,, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
The Western-Blot result of the soluble proteins that Fig. 1 comprises Vh, Vh-double, Vh-trime or Vh-Fc;
The ELISA detected result of the soluble proteins that Fig. 2 comprises Vh, Vh-double, Vh-trime or Vh-Fc;
The ELISA detected result of the Vh of Fig. 3 purifying;
The ELISA detected result of the Vh-double of Fig. 4 purifying;
The ELISA detected result of the Vh-trime of Fig. 5 purifying;
The ELISA detected result of the Vh-Fc of Fig. 6 purifying;
Western-Blot and the ELISA detected result of the single-chain antibody of the Y31 antibody of Fig. 7 purifying, Q61 antibody, Avastin;
Fig. 8 comprise Y31 antibody, Q61 antibody, Avastin the Western-Blot detected result of soluble proteins;
The ELISA detected result of 94 antibody, 30 antibody, Vh-YDA antibody and the tripolymer antibody Vh-YDA-trime thereof of Fig. 9 purifying.
Embodiment
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following embodiment.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
The preparation of embodiment 1 VEGF antibody of the present invention and the detection of affinity thereof
In the present embodiment, prepared shown in the single domain antibody Vh shown in SEQ ID NO:1 and fusion rotein Vh-double(SEQ ID NO:7 thereof), shown in tripolymer Vh-trime(SEQ ID NO:8), shown in syzygy Vh-Fc(SEQ ID NO:9), and its molecular weight, affinity etc. have been done to detection.
1, the preparation of antibody
The structure of 1.1 plasmids
Nucleotide sequence C-end shown in SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:20 adds CGU or the CAC nucleotide sequence of 6 repetitions separately, also the label that has added 6 Histidine compositions after the albumen of expressing separately, is abbreviated as (His)
6label, so that the detection to expression product, each nucleotide sequence carries out full gene by Jiangsu Jin Wei intelligence biotech company and synthesizes, and uses EcoR I and Nco I enzyme to cut, and is connected into respectively pET22b carrier.
The conversion of 1.2 plasmids and expression engineering bacteria build
Electricity turns: get 50 μ l electricity conversion competent cell BL21(DE3), the plasmid that adds 1 μ l step 1.1 to build.Mixture is proceeded in the electric revolving cup of precooling, leave standstill 2-5min on ice.It is 1800V/cm that electricity turns condition, electric shock 4-5 millisecond.The electricity thing of changing the line of production adds in 250 μ l SOC liquid nutrient mediums, and 37 ℃, 200r/min recovery 1 hour.After getting appropriate recovery, cell is coated the LB solid medium containing 50mg/L penbritin.Be inverted overnight incubation for 37 ℃.
The evaluation of 1.3 positive colony bacterium
Random 20 mono-clonals of picking in 1ml LB substratum (containing 50mg/L penbritin), 37 ℃, 280r/min incubated overnight.Next day, get 1 μ l bacterium liquid and do two primer PCRs, and reference numeral.Bacterium colony PCR reaction system is: 1 μ l incubated overnight bacterium liquid is template, 2 × Taq PCR Master Mix5 μ l, and the each 0.25 μ l of the positive antisense primer of T7terminator/B and T7forward/F, deionized water is supplemented to 10 μ l.Reaction conditions: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 54 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 20 circulations; 72 ℃ are extended 5min again.Bacterium colony PCR product is identified through 1% agarose gel electrophoresis, the glycerine guarantor bacterium that PCR positive bacteria is 10% with final concentration.
1.4 clone's mycetocyte cultivations and protein expression, the extraction of albumen, purifying
The positive of identifying through PCR is cultivated inoculation and is contained in the LB substratum (containing 100mg/L penbritin) of 1% glucose in 5ml, and 37 ℃, 220r/min incubated overnight.Incubated overnight bacterium liquid is seeded to 30ml LB(containing 100mg/L penbritin by 1%) in substratum, 37 ℃, 220r/min shaking culture to OD600 be 0.6～0.8 o'clock, add IPTG to final concentration be 0.3mmol/L, 25 ℃, 200r/min inducing culture, after 5 hours, is measured and is stopped OD600.Culturing process is set up negative control bacterial strain, is the engineering bacteria containing empty expression vector, and the method for its all cultivations, induction and sample preparation is identical with positive colony bacterial strain.
Total protein detects: get the bacterium liquid of equal densities, the centrifugal 1min of 12000r/min, discards supernatant.Resuspended with 40 μ l deionized waters after washing of precipitate, add 10 μ l5 × SDS albumen sample-loading buffers, 94 ℃ of heating 5min, the centrifugal 1min of 12000r/min, gets supernatant 2 μ l and carries out the analysis of 15%SDS-PAGE electrophoresis detection.
1.4.1 the extracting of soluble proteins: by bacterium liquid 5000rpm, the centrifugal 10min of room temperature, collects thalline.Add extraction agent Bugbuster extracting soluble proteins by 5mL/g bacterium, thalline and the vibration of bugbuster reagent are mixed.Mixed solution is placed in to decolorization swinging table room temperature jolting 20min, 4 ℃, 13,000rpm, centrifugal 20min, collects supernatant.
1.4.2 the purifying of antibody
The cell precipitation of positive colony is after ultrasonic disruption, and 4 ℃, 12600rpm centrifuging and taking supernatant, adopts GE Ni Sepharose fast flow filler antibody purification.Use buffer A(NaH
2pO
450mM, NaCl0.3M, Imidazole5mM, glycerine 10%; PH7.4) balance and washing pillar, use buffer B(NaH
2pO
450mM, NaCl0.3M, Imidazole150mM, glycerine 10%; PH7.4) wash-out is collected purification of samples, and sample solution is placed 4 ℃ of preservations.
2, the detection of the performance such as Antibody avidity
Adopt respectively and detect with the following method soluble proteins extract or purification of samples:
2.1Western Blot method detects: get soluble proteins extract or purification of samples 40ul and add 10 μ l5 × SDS albumen sample-loading buffers, 94 ℃ of heating 5min.Negative control sample is processed in the same way.Loading 20 μ l carry out after SDS-PAGE electrophoresis, and methyl alcohol activation pvdf membrane 15s, is soaked in transfering buffering liquid, and is cut into gel size together with filter paper, together with folding with gel in order, and 130mA, 60min transferring film.2%BSA sealing 1h after transferring film, with the PBST damping fluid washing containing 0.05%Tween-20 3 times, the PBST solution of 1%BSA is pressed 1:8000 dilution proportion anti-His HRP antibody coupling matter, hatches 1h.PBST damping fluid washing 3 times, the colour developing of tmb substrate colouring reagents.
2.2 affinities detect: first detect hole with the antigen coated ELISA of VEGF165 that 100 μ l concentration are 1ug/ μ l, 4 ℃ are spent the night.PBS damping fluid is washed plate 3 times, with the PBST solution sealing containing 2%BSA, hatches 2h for 37 ℃.PBST damping fluid is washed plate 3 times, adds soluble antibody or the purification of samples of 100 μ l Bugbuster extractings in antigen coated hole, and antibody is all pressed 2 poly-dilution method dilutions, does 3 multiple holes (n=3), averages.The soluble proteins extract (Bugbuster extract) of preparing with negative control thalline is as feminine gender.Incubated at room 1h.With washing plate 3 times containing PBST lavation buffer solution, then add the anti-His HRP antibody coupling matter (1:6000) of 100 μ l containing the PBST dilution of 1%BSA, incubated at room 1h.With PBST lavation buffer solution washing orifice plate 3-5 time, in hole, add 100 μ L TMB development reagent, color development at room temperature 5min, add 50 μ l1M HCl color development stopping reactions, ELISA microplate reader is in 450nm wavelength readings, wherein the value of negative control hole is as system background value deduction, and the 450nm light absorption value after use deduction does makes affine graphic representation to extension rate.
3, result:
(1) Vh: as shown in the Western-blot experimental result of Fig. 1 a, in the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed, do not contain histidine-tagged albumen, engineering bacteria of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:11) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 16kD, adds (His) with protein shown in SEQ ID:1 (protein of SEQ ID NO:11 coding)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein Vh.
As the ELISA result of Fig. 2 a shows, the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed is without VEGF antigen binding capacity, and recombinant bacterial strain of the present invention is expressed with histidine-tagged target protein, there is very strong VEGF antigen binding capacity, illustrate that target protein Vh of the present invention has very strong VEGF antigen binding capacity.
As the ELISA result of Fig. 3 shows, the affinity costant of target protein Vh of the present invention and VEGF antigen is 2.042, further illustrates target protein Vh of the present invention and has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention expresses and has obtained the protein Vh shown in SEQ ID:1, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
(2) Vh-double: as shown in the Western-blot experimental result of Fig. 1 b, in the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed, do not contain histidine-tagged albumen, engineering bacteria of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:17) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 30kD, adds (His) with protein shown in SEQ ID:7 (protein of SEQ ID NO:17 coding)
6label, connection peptides (GGGGS)
3molecular weight consistent, illustrate that the present invention successfully expresses to have obtained target protein.
As the ELISA result of Fig. 2 b shows, the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed is without VEGF antigen binding capacity, and recombinant bacterial strain of the present invention is expressed with histidine-tagged target protein, there is very strong VEGF antigen binding capacity, illustrate that target protein Vh-double of the present invention has very strong VEGF antigen binding capacity.
As the ELISA result of Fig. 4 shows, the affinity costant of target protein Vh-double of the present invention and VEGF antigen is 1.102, further illustrates target protein Vh-double of the present invention and has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention expresses and has obtained the protein Vh-double shown in SEQ ID:7, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
(3) Vh-trime: as shown in the Western-blot experimental result of Fig. 1 c, in the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed, do not contain histidine-tagged albumen, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:18) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 22kd, with protein monomers shown in SEQ ID:8 (protein of SEQ ID NO:18 coding), add (His)
6the molecular weight of label, isoleucine zipper is consistent.Under Western-blot experiment condition of the present invention, Vh-trime tripolymer antibody can depolymerization, thereby Western-blot detects monomer, shows that the present invention successfully expresses to have obtained target protein Vh-trime.
As the ELISA result of Fig. 2 c shows, the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed is without VEGF antigen binding capacity, and engineering bacterium expression of the present invention with histidine-tagged target protein, there is very strong VEGF antigen binding capacity, illustrate that target protein Vh-trime of the present invention has very strong VEGF antigen binding capacity.
As the ELISA result of Fig. 5 shows, the affinity costant of target protein Vh-trime of the present invention and VEGF antigen is 2.655, further illustrates target protein Vh-trime of the present invention and has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention expresses and has obtained the tripolymer antibody Vh-trime of monomer as shown in SEQ ID:8, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
(4) Vh-Fc: as shown in the Western-blot experimental result of Fig. 1 d, in the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed, do not contain histidine-tagged albumen, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:20) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 22kd, with protein shown in SEQ ID:10 (protein of SEQ ID NO:20 coding), add (His)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein Vh-Fc.
As the ELISA result of Fig. 2 d shows, the soluble proteins that negative control bacterial strain (containing the recombinant bacterium of empty plasmid) is expressed is without VEGF antigen binding capacity, and engineering bacterium expression of the present invention with histidine-tagged target protein, there is very strong VEGF antigen binding capacity, illustrate that target protein Vh-Fc of the present invention has very strong VEGF antigen binding capacity.
As the ELISA result of Fig. 6 shows, it is 2.081 that target protein Vh-Fc of the present invention has with the affinity costant of VEGF antigen, further illustrates target protein Vh-Fc of the present invention and has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein Vh-Fc shown in SEQ ID:10, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
To sum up, successful expression of the present invention has obtained Vh, Vh-double, Vh-trime, Vh-Fc antibody, and they all have very strong VEGF antigen binding capacity.
The preparation of embodiment 2 VEGF antibody of the present invention and the detection of affinity
The antibody that the present embodiment relates to is respectively: the single domain antibody Vh-YDA shown in SEQ ID NO:4 and tripolymer antibody Vh-YDA-trime thereof; Single domain antibody Y31, Q61,30,94 shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 difference, using Avastin single-chain antibody (scFv) as positive control antibody, (Avastin single-chain antibody doubly puts forth energy to wait chief editor according to Shen, " recombinant antibodies ", Science Press, 2005, the method that p246-p250 records built).
1, the preparation of antibody of the present invention
The structure of 1.1 plasmids
Nucleotide sequence C-end shown in SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:16 adds CGU or the CAC nucleotide sequence of 6 repetitions separately, also the label that has added 6 Histidine compositions after the albumen of expressing separately, is abbreviated as (His)
6label, so that the detection to expression product, each nucleotide sequence carries out full gene by Jiangsu Jin Wei intelligence biotech company and synthesizes, and uses EcoR I and Nco I enzyme to cut, and is connected into respectively pET22b carrier.
The conversion of 1.2 plasmids and expression engineering bacteria build
Make electricity consumption conversion system that connection product is transformed into competent cell BL21(DE3), step of converting is with in embodiment 1 1.2, spread plate incubated overnight.
The evaluation of 1.3 positive colony bacterium
In the 2YT substratum (containing 50mg/L penbritin) that random 30 mono-clonals of picking contain 1% glucose in 1ml, 37 ℃, 280r/min incubated overnight.Next day, get 1 μ l bacterium liquid and do two primer PCRs, and reference numeral.Bacterium colony PCR reaction system is identified through 1% agarose gel electrophoresis with 1.3 bacterium colony PCR products in embodiment 1, the glycerine guarantor bacterium that PCR positive bacteria is 10% with final concentration.
1.4 clone's mycetocytes are cultivated and protein expression
The positive of identifying through PCR is cultivated inoculation and is contained in the YT substratum (containing 100mg/L penbritin) of 1% glucose in 5ml, and 37 ℃, 220r/min incubated overnight.Incubated overnight bacterium liquid is seeded to 30mlYT(containing 100mg/L penbritin by 1%) in substratum, 37 ℃, 220r/min shaking culture to OD600 be 0.6～0.8 o'clock, add IPTG to final concentration be 0.3mmol/L, 25 ℃, 200r/min inducing culture, after 3 hours, is measured and is stopped OD600.Culturing process is set up negative control bacterial strain, is the engineering bacteria containing empty expression vector, and the method for its all cultivations, induction and sample preparation is identical with positive colony bacterial strain.
The extraction of 1.5 soluble proteinss, separation and purification
With 1.4.1,1.4.2 step in embodiment 1.
2, detect
Western Blot detection and parent and ELISA detect, with 2.1 and 2.2 steps in embodiment 1.
3, result
（1）Y31
As shown in the Western-blot experimental result of Fig. 7, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:12) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 16kD, with protein shown in SEQ ID:2 (protein of SEQ ID NO:12 coding), add (His)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein.
As shown in the ELISA result of Fig. 7, the object of the invention protein Y 31 is 0.896 with the affinity costant of VEGF antigen, suitable with the affinity costant (1.001) of positive drug Avastin single-chain antibody, illustrates that it has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein Y31 shown in SEQ ID:2, and it has very strong VEGF antigen binding capacity, suitable with the binding ability of positive drug Avastin single-chain antibody, is a kind of highly active VEGF antibody.
（2）Q61
As shown in the Western-blot experimental result of Fig. 7, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:13) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 16kD, with protein shown in SEQ ID:3 (protein of SEQ ID NO:13 coding), add (His)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein.
As the ELISA result of Fig. 7 shows, the object of the invention PQ 61, with the affinity costant of VEGF antigen be 0.669, there is very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein Q61 shown in SEQ ID:3, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
（3）Vh-YDA
As shown in the Western-blot experimental result of Fig. 8, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:14) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 15kD, with protein shown in SEQ ID:4 (protein of SEQ ID NO:14 coding), add (His)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein Vh-YDA.
As shown in the ELISA result of Fig. 9, engineering bacteria target protein Vh-YDA of the present invention, the affinity costant of itself and VEGF antigen is 1.6, has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein Vh-YDA shown in SEQ ID:4, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
（4）Vh-YDA-trime
As shown in the Western-blot experimental result of Fig. 8, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:19) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 22kd, with protein monomers shown in SEQ ID:9 (protein of SEQ ID NO:19 coding), add (His)
6the molecular weight of label, isoleucine zipper is consistent.Under Western-blot experiment condition of the present invention, Vh-YDA-trime tripolymer antibody can depolymerization, thereby Western-blot detects monomer, shows that the present invention successfully expresses to have obtained target protein Vh-YDA-trime.
As shown in the ELISA result of Fig. 9, this target protein Vh-YDA-trime, the affinity costant of itself and VEGF antigen is 1.884, has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein Vh-YDA-trime shown in SEQ ID:9, and it has very strong VEGF antigen binding capacity, suitable with the binding ability of positive drug Avastin single-chain antibody, is a kind of highly active VEGF antibody.
(5)30
As shown in the Western-blot experimental result of Fig. 8, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:15) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 15kD, with protein shown in SEQ ID:5 (protein of SEQ ID NO:15 coding), add (His)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein 30.
As shown in the ELISA result of Fig. 9, the object of the invention albumen 30 is 1.502 with the affinity costant of VEGF antigen, has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein 30 shown in SEQ ID:5, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
(6)94
As shown in the Western-blot experimental result of Fig. 8, recombinant bacterial strain of the present invention (containing the positive plasmid that comprises nucleotide sequence shown in SEQ ID NO:16) is expressed and has been obtained containing histidine-tagged albumen, its molecular weight is 15kD, with protein shown in SEQ ID:6 (protein of SEQ ID NO:16 coding), add (His)
6the molecular weight of label is consistent, illustrates that the present invention successfully expresses to have obtained target protein 94.
As shown in the ELISA result of Fig. 9, engineering bacteria of the present invention (containing comprising the positive plasmid of nucleotide sequence shown in SEQ ID NO:16) express with histidine-tagged target protein 94, the affinity costant of itself and VEGF antigen is 1.424, has very strong VEGF antigen binding capacity.
Experimental result explanation, the present invention has expressed the protein 94 shown in SEQ ID:6, and it has very strong VEGF antigen binding capacity, is a kind of highly active VEGF antibody.
To sum up, successful expression of the present invention has obtained Vh-YDA, Vh-YDA-trime, Y31, Q61,94,30 antibody, and they all have very strong VEGF antigen binding capacity.
The present invention adopts engineered method, VEGF single domain antibody Vh, Y31, Q61, Vh-YDA, 30,94 are successfully prepared, the avidity of they and VEGF is high, and affinity costant is 0.669～2.042, with positive drug Avastin single-chain antibody (affinity costant is 1.001) quite or higher, simultaneously, molecular weight is little, can effectively penetrate tumour, and immunogenicity is low, safety is a kind of safe and effective antitumor drug; The present invention has also prepared the tripolymer antibody of fusion antibody, Vh antibody and the Vh-YDA antibody of Vh antibody, and the avidity of they and VEGF is high, and affinity costant is 1.102～2.655, can be used for preparing antitumor drug, and potential applicability in clinical practice is good.
Claims (10)
1.VEGF single domain antibody, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO:1～SEQ ID NO:6 any one.
2.VEGF fusion antibody, is characterized in that: it is the fusion antibody being formed by connecting by connection peptides by single domain antibody claimed in claim 1, or connects at the C-terminal of single domain antibody described in claim 1 fusion antibody that Fc fragment forms;
Preferably, the aminoacid sequence of described fusion antibody is as shown in SEQ ID NO:10.
3. fusion antibody according to claim 2, is characterized in that: the aminoacid sequence of described connection peptides is: GGGGSGGGGSGGGGS;
Preferably, described fusion antibody is as shown in SEQ ID NO:7.
4.VEGF tripolymer antibody, is characterized in that: monomer whose is to be formed by connecting by single domain antibody described in claim 1 and isoleucine zipper.
5. tripolymer antibody according to claim 4, is characterized in that: in described monomer, the aminoacid sequence of isoleucine zipper is QRMKQIEDKIEEILSKIYHIENEIARIKKLVGER;
Preferably, the aminoacid sequence of described monomer is as shown in SEQ ID NO:8 or 9.
6. the nucleotide sequence of antibody described in coding claim 1～5 any one;
Preferably, its sequence is as shown in SEQ ID NO:11～SEQ ID NO:20.
7. an expression vector that contains nucleotide sequence described in claim 6;
Preferably, described expression vector is restructuring pET22b plasmid.
8. a recombinant bacterium that contains expression vector described in claim 7;
Preferably, described recombinant bacterium is BL21(DE3) intestinal bacteria.
9. the preparation method of antibody described in claim 1～5 any one, is characterized in that: comprise following steps:
(1) get the recombinant bacterium described in claim 8, be inoculated into and contain 0.5～1.5%(w/v) in the LB substratum or YT substratum of glucose, 50～150mg/L penbritin, cultivate 10～15h, obtain bacterium liquid for 34～40 ℃;
(2) according to 0.5～1.5%(v/v) inoculum size, step (1) gained bacterium liquid is seeded in the LB substratum or YT substratum that contains 50～150mg/L penbritin, 34～40 ℃ are cultured to OD
600=0.6～0.8;
(3) add IPTG to final concentration be 0.2～0.4mmol/L, under the condition of 20～30 ℃, inducing culture 3～7 hours;
(4) collect culture, separation and purification, obtains VEGF antibody.
Preferably, in step (1), in described substratum, the concentration of glucose is 1%(w/v), the concentration of penbritin is 100mg/L; The temperature of described cultivation is 37 ℃; Described incubation time is 12h;
In step (2), in described substratum, the concentration of penbritin is 100mg/L, and the temperature of described cultivation is 37 ℃;
In step (3), the final concentration of IPTG is 0.3mmol/L; The temperature of inducing culture is 25 ℃, and the time is 5 hours.
Described in claim 1～5 any one antibody in the purposes of preparing in antitumor drug or treatment agedness yellow spot degenerative disease medicine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820245A (en) * | 2015-01-06 | 2016-08-03 | 珠海亿胜生物制药有限公司 | Anti-VEGF antibody |
| CN105820244A (en) * | 2015-01-06 | 2016-08-03 | 珠海亿胜生物制药有限公司 | Anti-VEGF antibody |
| CN106146657A (en) * | 2016-07-12 | 2016-11-23 | 晋明（天津）生物医药技术有限公司 | Wide spectrum recombinant antibody fragment combining influenza virus A and preparation method and application |
| CN107636013A (en) * | 2015-07-31 | 2018-01-26 | 苏州康宁杰瑞生物科技有限公司 | For programmed death ligand (PD L1) single domain antibody and its derived protein |
| US12024568B2 (en) | 2012-09-13 | 2024-07-02 | Cornell University | Treatment of brain cancers using central nervous system mediated gene transfer of monoclonal antibodies |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101663319A (en) * | 2007-02-21 | 2010-03-03 | 埃博灵克斯股份有限公司 | Be used for the treatment of at the aminoacid sequence of vascular endothelial growth factor and the polypeptide that comprises it and be characterised in that excessive and/or pathologic vessels takes place or the illness and the disease of neovascularization |
-
2014
- 2014-01-27 CN CN201410039879.0A patent/CN103772501A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101663319A (en) * | 2007-02-21 | 2010-03-03 | 埃博灵克斯股份有限公司 | Be used for the treatment of at the aminoacid sequence of vascular endothelial growth factor and the polypeptide that comprises it and be characterised in that excessive and/or pathologic vessels takes place or the illness and the disease of neovascularization |
Non-Patent Citations (3)
| Title |
|---|
| MAHDI BEHDANI ET AL: "Generation and characterization of a functional nanobody against the vascular endothelial growth factor receptor-2;angiogenesis cell receptor", 《MOLECULAR IMMUNOLOGY》 * |
| 刘珩等: "一种具有人VEGF结合活性的人源单一重链可变区的克隆与表达", 《生物工程学报》 * |
| 潘欣等: "单域重链抗体的分子特征", 《生物技术通讯》 * |
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