CN102321175A - Nano-antibody or polypeptide aiming at breast cancer Her2/new - Google Patents
Nano-antibody or polypeptide aiming at breast cancer Her2/new Download PDFInfo
- Publication number
- CN102321175A CN102321175A CN201110280031A CN201110280031A CN102321175A CN 102321175 A CN102321175 A CN 102321175A CN 201110280031 A CN201110280031 A CN 201110280031A CN 201110280031 A CN201110280031 A CN 201110280031A CN 102321175 A CN102321175 A CN 102321175A
- Authority
- CN
- China
- Prior art keywords
- gly
- ser
- antibody
- her2
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a polypeptide or a nano-antibody (or a single domain antibody fragment) aiming at a breast cancer over-expressed epidermal growth factor receptor 2 (Her2/new), and the invention also relates to nucleic acids coding the nano-antibody and the polypeptide, a method for preparing the nano-antibody and the polypeptide, and a host cell which expresses or can express the nano-antibody; the nano-antibody can be highly expressed in escherichia coli, and can be used to prepare enzyme immunoassay kits for quantitative detection of breast cancer patient blood and body fluid samples with breast cancer over-expressed epidermal growth factor receptor 2 (Her2/new).
Description
Technical field
The invention belongs to biomedicine or biological pharmacy technical field; Be particularly related to polypeptide and nano antibody (or the title single domain antibody fragment of mammary cancer being crossed expression epidermal growth factor acceptor 2 (Her2/new); Single domain antibody fragment); Invention has also related to the nucleic acid of encode said nano antibody and polypeptide, prepares the method for said nano antibody and polypeptide, expresses the host cell that maybe can express said nano antibody; This nano antibody can efficiently express in intestinal bacteria, can be applied to prepare detection by quantitative mammary cancer and cross the breast cancer patient's blood of expression epidermal growth factor acceptor 2 (Her2/new) and the Enzymoimmune reagent kit of humoral sample etc.
Background technology
Estimate recently that according to IARC of the World Health Organization annual global New Development women with breast cancer case reaches 1,200,000, account for 22.7% of women's malignant tumour morbidity, have every year 500000 women to die from mammary cancer approximately.It is that Her2/new crosses expression that 1/3-1/4 is arranged in the patient with breast cancer approximately; Can use monoclonal antibody medicine-Trastuzumab (Herceptin) to carry out efficacious therapy (Charles L. Vogel; Efficacy and Safety of Trastuzumab as a Single Agent in First-Line Treatment of HER2-Overexpressing Metastatic Breast Cancer, J Clin Oncol 20:719-726.).Cross the expression patient for the specific detection that mammary cancer Her2 is expressed and carry out individuation, target property efficacious therapy; At first patient's biopsy is carried out qualitative, the semiquantitative determination of Her2; Domestic method: " immunohistochemistry (IHC); fluorescence in situ hybridization (FISH); and chromogenic in situ hybridization (CISHH) method (reference is seen: 1.Press MF; Hung G, Godolphin W, Slamon DJ.Sensitivity of HER-2/neu antibodies in archival tissue samples:potential source of error in immunohistochemical studies of oncogene expression.Cancer Res.1994; 54:2771-2777.2.Press MF; Slamon DJ; Flom KJ, et al.Evaluation of HER-2/neu gene amplification and overexpression:comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens.J Clin Oncol.2002; 20:3095-3105.3.Schnitt SJ, Jacobs TW.Current status of HER2testing:caught between a rock and a hard place.Am J Clin Pathol.2001; 116:806-810.4.Foster CS, Gosden CM, Ke Y.HER2/neu expression in cancer:the pathologist as diagnostician or prophet? Hum Pathol.2003; 34:635-638.5.Bartlett J, Mallon E, Cooke T.The clinical evaluation of HER-2status:which test to use? J Pathol.2003; 199:411-417.); Utilization enzyme-labeled immunity method (ELISA) detects free Her2 extracellular region protein polypeptide among the patients serum of mammary cancer Her2 expression now; Express guidance that the patient carries out individuation, magnetic target therapy and therapeutic evaluation (Ramon Colomer, Sagrario Montero, the Ana Lluch after the treatment to reach mammary cancer Her2 crossed; Et al.Circulating HER2Extracellular Domain and Resistance to Chemotherapy in Advanced Breast Cancer; Clinical Cancer Research, 2000,6:2356-2364).
Nano antibody technology, be the biomedical science man on the basis of traditional antibody, the notion of utilization Protocols in Molecular Biology combining nano particle science, and the up-to-date and minimum antibody molecule of research and development.At first by Belgian scientist in camel blood, find (C.Hamers-Casterman, Naturally occurring antibodies devoid of light chains, Nature, 1993,363:446-448).Cross the polypeptide and the nano antibody of expression epidermal growth factor acceptor 2 (Her2/new) to mammary cancer; Be utilization nano antibody technology, the extracellular region polypeptide with the Her2 of recombination screens the nano antibody gene pool; And obtain to the specific nano antibody of Her2 (Technical Reference: Tanha J; Dubuc G, Selection by phage display of llama conventional V (H) fragments with heavy chain antibody V (H) H properties, J Immunol Methods.2002; 263 (1-2): 97-109) (Gueorguieva D; Li S, Identification of single-domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis; FASEB J.2006,20 (14): 2636-8).Polypeptide and the nano antibody of crossing expression epidermal growth factor acceptor 2 (Her2/new) to mammary cancer will have broad application prospects for detection and the guiding treatment of crossing expression epidermal growth factor acceptor 2 (Her2/new) patient to mammary cancer.
Summary of the invention
Technical problem
The purpose of this invention is to provide polypeptide and the nano antibody of crossing expression epidermal growth factor acceptor 2 (Her2/new) to mammary cancer; Provide encoding sequence and this nano antibody of this nano antibody preparing simultaneously, detect the application that the patient with breast cancer crosses the ELISA test kit of expressing epidermal growth factor acceptor 2.
Technical scheme
To the nano antibody of Her2/new, comprise framework region and complementary determining region; , wherein said framework region is for being respectively FR1-FR4, and the aminoacid sequence of the FR1-FR4 of framework region is specially:
FR1:QVKLVESGGGLVQPGGSLRLSCAASGSGFSQVKLVESGGGLVQPGGSLRLSC AASGFTFD sees SEQ NO.1;
FR2:WYRQTPGNRREWVWYRQTPGNRREWV sees SEQ NO.2;
FR3:RFAISRDNAKTTVYLQMNSLKPEDTAVYYCRFTISRDNTKNMLYLQMNSLKA EDTGLYYC sees SEQ NO.3;
FR4; WGQGTQVTVSSWGQGTQVTVSS sees SEQ NO.4;
, it is characterized in that complementary determining region is CDR1-CDR3, the aminoacid sequence of complementary determining region CDR1-CDR3 is specially:
CDR1 is PNVMG, sees SEQ NO.5 or DYAMS, sees SEQ NO.6;
CDR2 is AAANKYGTTTYADSVKG, sees SEQ NO.7 or SAISWNGGSTYYAESMKG, sees SEQ NO.8;
CDR3 is AASTATNWDYHY, sees SEQ NO.9 or VAPWKF, sees SEQ NO.10.
As the described nano antibody of a kind of optimal way is pH2G4 or pH2F1; Wherein the aminoacid sequence of pH2G4 does, sees SEQ NO.11:
QVKLVESGGGLVQPGGSLRLSCAASGSGFS
PNVMGWYRQTPGNRREWV
AAANKYGTTTYADSVKG
RFAISRDNAKTTVYLQMNSLKPEDTAVYYC
AASTATNWDYHYWGQGTQVTVSS；
The aminoacid sequence of pH2F1 is to see SEQ NO.12:
QVKLVESGGGLVQPGGSLRLSCAASGFTFD
DYAMSWVRQAPGKGLEWV
SAISWNGGSTYYAESMK
GRFTISRDNTKNMLYLQMNSLKAEDTGLYYC
VAPWKFWGQGTQVTVSS。
A kind of polypeptide to Her2/new is characterized in that polypeptide comprises described CDR1-CDR3 and FR1-FR4, and amino acid sequence of polypeptide has the aminoacid sequence similarity at least 80% with CDR1-CDR3 and FR1-FR4.
Have and the aminoacid sequence similarity at least 90%. preferred at least 95% of CDR1-CDR3 and FR1-FR4 or above sequence identity mutually as a kind of optimal way amino acid sequence of polypeptide.
Has the aminoacid sequence similarity 95% with CDR1-CDR3 and FR1-FR4 as a kind of optimal way amino acid sequence of polypeptide.
Be described further
The nucleotide sequence of pH2G4,354, SEQ NO.13:
CAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTG
TGCAGCCTCTGGAAGCGGCTTCAGTCCCAATGTGATGGGCTGGTACCGCCAAACTCCAGGGAACC
GGCGCGAGTGGGTCGCGGCAGCTAACAAATATGGTACGACTACCTATGCAGACTCCGTGAAGGGC
CGATTCGCCATCTCCAGAGACAACGCCAAGACTACGGTGTATCTCCAAATGAACAGCCTGAAACC
GGAGGACACGGCCGTCTATTACTGCGCCGCAAGTACGGCAACGAATTGGGACTATCACTACTGGG
GCCAGGGGACCCAGGTCACCGTCTCCTCA
PH2G4 (proteinic aminoacid sequence, 118):
QVKLVESGGGLVQPGGSLRLSCAASGSGFS
PNVMGWYRQTPGNRREWV
AAANKYGTTTYADSVKG
RFAISRDNAKTTVYLQMNSLKPEDTAVYYC
AASTATNWDYHYWGQGTQVTVSS
PH2F1 (nucleotide sequence, 339), SEQ NO.14:
CAGGTAAAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACTTTTGATGATTATGCCATGAGCTGGGTCCGACAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCAGCTATTAGCTGGAATGGTGGTAGCACATACTATGCAGAATCCATGAAG
GGCCGATTCACCATCTCCAGAGACAATACCAAGAACATGCTGTATCTGCAAATGAACAGCCTGAA
AGCTGAGGACACGGGTCTGTATTACTGTGTGGCACCGTGGAAATTCTGGGGCCAGGGGACCCAGG
TCACCGTCTCCTCA
PH2F1 (proteinic aminoacid sequence, 113):
QVKLVESGGGLVQPGGSLRLSCAASGFTFD
DYAMSWVRQAPGKGLEWV
SAISWNGGSTYYAESMK
GRFTISRDNTKNMLYLQMNSLKAEDTGLYYC
VAPWKFWGQGTQVTVSS
The proteinic aminoacid sequence of pH2G4 and pH2F1: the left side begins to be aminoterminal that the right side ends up being carboxyl terminal; It is in proper order: the sequence of not having setting-out down is respectively a framework region, FR1～FR4; What following setting-out was arranged is that antigen combines complementary district, CDR1～CDR3.
Principle introduction of the present invention:
The present invention relates to make up Her2 extracellular region polypeptide recombination clone's pET28a expression vector, obtained to have Her2 extracellular region (Her2ECD) the high purifying protein of His label, with the Ni that can high-affinity combines to have the His label protein
+Magnetic bead combines; The correct space structure of displayed polypeptides with the non-immune nano antibody gene of the antigen selection of this form storehouse (nano antibody phage display gene pool), is crossed the specific nano antibody of expression epidermal growth factor acceptor 2 (Her2/new) (or title single domain antibody fragment and obtained to be directed against mammary cancer; Single domain antibody fragment); This gene is connected reorganization with expression vector, made up the nano antibody strain that can in intestinal bacteria, efficiently express (pH2G4, pH2F1).Shake a bottle small-scale production in the laboratory, through Ni
+The resin gel affinitive layer purification can obtain that the SDS-PAGE electrophoresis is pure to reach 95%, the nano antibody of about 100 mg/litre bacterial culturess.Utilize the capture antibody of nano antibody pH2G4, utilize biotin labeled nano antibody pH2F1 or goat-anti Her2 polyclonal antibody, detect the level that clever Her2 sensitivity can reach 1ng/ml as detecting antibody as Her2.
Wherein framework region is that FR1-FR4 is the conventional framework of VHH of camel heavy chain antibody,
Beneficial effect:
The present invention relates to make up Her2 extracellular region polypeptide recombination clone's pET28a expression vector; Acquisition has the high purifying protein of Her2 extracellular region (Her2ECD) of His label; And have the Ni+ magnetic bead and combine; The correct space structure of displayed polypeptides with the non-immune nano antibody gene of the antigen selection of this form storehouse (deriving from non-immune camel nano antibody phage display gene pool), is crossed the specific nano antibody of expression epidermal growth factor acceptor 2 (Her2/new) (or title single domain antibody fragment and obtained to be directed against mammary cancer; Single domain antibody fragment); This gene is connected reorganization with expression vector, made up the nano antibody strain that can in intestinal bacteria, efficiently express (pH2G4, pH2F1).Shake a bottle small-scale production in the laboratory, through Ni
+The resin gel affinitive layer purification can obtain the SDS-PAGE electrophoresis purity and reach 95%, the nano antibody of about 100 mg/litre bacterial culturess.Utilize the capture antibody of nano antibody pH2G4, utilize biotin labeled nano antibody pH2F1 or goat-anti Her2 polyclonal antibody, detect the level that Her2 sensitivity can reach 1ng/ml as detecting antibody as Her2.
Description of drawings
The Her2ECD albumen of Fig. 1 for expressing is through Ni
+The resin gel affinitive layer purification, the electrophorogram of SDS-PAGE, shown in the result, E1～E7 is that 250mM imidazoles wash-out goes out the Her2ECD purifying protein, M is standard molecular weight albumen Marker.The purity of Her2ECD can reach more than 95%.
The pH2G4 albumen of Fig. 2 for expressing is through Ni
+The resin gel affinitive layer purification, shown in the SDS-PAGE result, W1～5 are the imidazoles flush away foreign protein part of 100mM; E1～E7 is that 250mM imidazoles wash-out goes out the pH2G4 purifying protein, and M is standard molecular weight albumen Marker.The purity of pH2G4 can reach more than 90%.
Fig. 3 is pH2G4, and the electrophorogram behind the pH2F1 purifying, SDS-PAGE show that its purity can reach more than 95%, and molecular weight is about 15kd.
Fig. 4 is shown that by filter the high pressure liquid phase analysis experiment through molecular sieve polydextran gel (superG75) the time warp that the elution peak of pH2G4 occurs is compared with standard molecular weight albumen Marker, and the molecular weight of its peak value should be 15kd.
Fig. 5 is not when adding biotinylated pH2G4 in Fig. 5 a dyeing system, and cell dyeing is the green of background, and Fig. 5 b with the Her2 receptors bind of cell surface, tangible stained positive occurs and reacts when adding biotinylated pH2G4.
Fig. 6 is shown in the result, and under the coated antibody of same concentrations and the condition that detects antibody, the antibody sandwich elisa plate is placed 37 degree, and to spend night be good to its result than placing 4.
Fig. 7 is shown in the ELISA orthogonal experiments, and capture antibody encapsulates concentration in 5～10 mcg/ml, is best effort concentration, and the optimum detection working concentration of biotin labeling antibody is 1～5 mcg/ml;
Fig. 8 is for shown in the result of the sensitivity that detects Her2 and typical curve, and when Her2 concentration dilution scope during from 2 microgram to 1 nanogram(ng)s, it is measured concentration and becomes better linearity to concern;
Fig. 9 is the pH2G4-pSJF2 plasmid map;
Figure 10 is the plasmid map of pH2Fi-pSJF2;
Figure 11 is the gene electrophorogram of nano antibody, and wherein M is depicted as the molecule Marker of 100bp, and PCR product band is about 500bp, uses the QIAgenPCRkit purifying, and with restriction endonuclease sfiI (New Negland Biolabs), 50 degree act on 3～5 hours.。
Five, embodiment
Below in conjunction with specific embodiment, further set forth the present invention.
Embodiment 1:
Make up the gene clone of Her2 extracellular region (Her2ECD) polypeptide, wherein Her2ECD is the recombinant protein of band His label:
(upstream primer GGAATTCatgaagctgcggctccctgc sees SEQ NO.17 and downstream primer CGGGATCCgcggttggtgtctatcagtgt to design a pair of primer; See SEQ NO.18); CDNA with Her2 is a template, and 480 Nucleotide of pcr amplification Her2ECD are connected in the pET28a carrier then; Through gene sequencing, to confirm the exactness of the purpose clone gene sequence that obtained.(2) in e. coli bl21, efficiently express Her2ECD, and through Ni
+The resin gel affinitive layer purification can obtain the SDS-PAGE electrophoresis purity and reach the Her2ECD purifying protein (see figure 1) more than 95%.
The nucleotide sequence of Her2, see SEQ NO.15:
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCATGAAGCTGCGGCTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGC
Wherein the condition of pcr amplification is: with the PCR test kit of buying (Beijing Quanshijin Biotechnology Co., Ltd); Make an experiment to specifications; In 50 microlitre reaction volumes: template 1 microlitre (50ng); Each 1 microlitre of upstream and downstream primer (10uM), 2 times of super mixed solution 25 microlitres of test kit, water is supplied 50 microlitre TVs.Amplification condition: 94 ℃, 4 minutes; At 94 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃, totally 30 circulations under 1 minute condition; Then at 72 ℃, 5 minutes.Detect the PCR product that obtains 500bp through agarose electrophoresis.
The aminoacid sequence of Her2ECD, see SEQ NO.16:
MGSS
SSGLVPRGSHMASMTGGQQMGRGSEF
MKLRLPASPETHLDMLRHLYQGCQVVQGNL ELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPV TGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNR。
Wherein the black matrix italic is the position of His; Underscore is the aminoacid sequence of HerECD.
Embodiment 2:
Cross the screening process of the specific nano antibody of expression epidermal growth factor acceptor 2 (Her2/new) to mammary cancer:
(1) in 1.5 milliliters centrifuge tube; At first add the empty carrier band His label polypeptide (the empty carrier band His label polypeptide of described pET28a vector expression is that professor Sun Jian of University Of Tianjin is so kind as to give) of the pET28a vector expression of 50 microgram purifying, add 100 microlitre phage (5x10
11The non-immune camel nano antibody phage display gene pool of tfu adds the Ni of 50 microlitres
+Magnetic bead adds 2% skimmed milk 0.01M phosphate buffered saline buffer (2%MPBS) of 500 microlitres again, and pH7.0 is placed on the rotary drum of adjustable speed, rotates kind gently 60 times/minute, at room temperature 1 hour.
(2) with 1.5 milliliters of centrifuge tubes of reaction mixture in above-mentioned (1), be placed on the pipe support of magnetic force, through magnetic force absorption in 1 minute, Ni
+Magnetic bead firmly is adsorbed on the pipe end and leans on a side of magnetic force frame (this step is to remove in the non-immune camel nano antibody phage display gene pool empty carrier band His label polypeptide bonded polypeptide and the Ni with the pET28a vector expression
+The non-characteristic bonded nano antibody of magnetic bead), supernatant is transferred in new 1.5 milliliters of centrifuge tubes.
(3) supernatant in (2) (adsorbed later phage) lining adds the Her2ECD purifying protein of 50 micrograms, is placed on the rotary drum of adjustable speed, rotates kind gently 60 times/minute, at room temperature combines 2 hours.
(4) in 1.5 milliliters of reaction tubess of (3), add the 50 microlitre Ni that sealed through 2%MPBS
+Magnetic bead is placed on the rotary drum of adjustable speed, rotates kind gently 60 times/minute, at room temperature combines 30 minutes.
(5) with 1.5 milliliters of reaction tubess in (4), be placed on the pipe support of magnetic force, through magnetic force absorption in 1 minute, inhale and remove supernatant, respectively wash Ni with 1 milliliter 0.05～0.1% polysorbas20 (T) PBS (PBST) and PBS
+Magnetic bead 10 times is to wash unconjugated phage off.
(6) will dissociate down with Her2ECD purifying protein specificity bonded phage with TEA (triethylamine (7.18M)), and infect the e. coli tg1 that is in logarithmic phase, generation and purifying phage are used for the screening of next round.Identical screening process repeats 3～4 and takes turns.
The construction process of wherein non-immune camel nano antibody phage display gene pool; Reference is seen (Tanha J; Dubuc G; Selection by phage display of llama conventional V (H) fragments with heavy chain antibody V (H) H properties, J Immunol Methods.2002,263 (1-2): 97-109)
1.cDNA synthetic: pick up from the camel in zoological park and the alpaca peripheral blood of Shanxi agricultural university; Extract lymphocyte; Use the QIAgenRNA purification kit, purifying RNA carries out cDNA with Amersham Biosciences First-Sttrand cDNA Synthesis Kit and synthesizes.
2. camel heavy chain pcr amplification for the first time: design 2 upstream primers: LT1-5 ' CGC CAT CAA GGTACC AGT TGA 3 ' sees that SEQ NO.19 and LT2--5 ' GGG GTA CCT GTC ATC CAC GGA CCA GCT GA 3 ' see SEQ NO.20; Article 2, downstream primer: LB1-5 ' GCC CAG CCG GCC ATG GCC SMK GTG CAG CTG GTG GAK TCT GGG GGA 3 ' sees that SEQ NO.21 and LB2--5 ' GCC CAG CCG GCC ATG GCC CAG GTA AAG CTG GAG GAG TCT GGG GGA 3 ' see SEQ NO.22.The condition of pcr amplification is: 94 ℃ 3 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds., did 30 circulations in 1 minute for 72 ℃; 72 ℃ were being increased 7 minutes then.
3. use 1% agarose electrophoresis, detect the PCR product, and will be greater than 500bp, the product band less than 800bp excises from agarose, and reclaims kit with QIAgen glue, reclaims the PCR product.
4. PCR prepares product for making up gene pool for the second time: design upstream primer gKF--5 ' CAT GTG TAG ACT CGC GGC CCA GCC GGC CAT GGC C3 ' sees that SEQ NO.23 and gKB--5CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCT GG 3 ' see SEQ NO.24.The condition of pcr amplification is: 94 ℃ 3 minutes; 94 ℃ 30 seconds, 55 ℃ 30 seconds., did 30 circulations in 1 minute for 72 ℃; 72 ℃ were being increased 7 minutes then.
5. use 1% agarose electrophoresis, detect the PCR product, obtain the about 500bp of gene of nano antibody, Figure 11 as follows this moment:
6. phage vector pHEN6 (transforming through pHEN1), with restriction endonuclease sfiI (New Negland Biolabs), 50 spend, and act on 3～5 hours.
7. nano antibody gene VHH links reaction with the pHEN6 carrier:
Reaction mixture: T4 ligase enzyme (invitrogen) (high density 5u/ul) 1ul
VHH PCR product 3mmol
pHEN6vector 1mmol
Ligase enzyme damping fluid 4ul
Water is mended 20ul
16 degree, link is spent the night.
8. bacterium transforms: get ligation thing 1ul, 2ul joins in the e. coli tg1 competence, after changeing with some transfer appearance (BioRad company) electricity, is coated on the LB aminobenzylpenicillin flat board, and 32 spend night, counting bacterial clone number.
9. according to the lab scale conversion results, when transformation efficiency reaches 10
8During clone/ng carrier, do 30～50 electricity and transform, be non-immune camel phage display gene pool.The capacity of this gene pool is about 10
10The clone.
10. when carrying out gene pool when screening, the microbial culture with in above-mentioned 9 prepares phage by producing the phage program.
Embodiment 3:
The single positive colony of enzyme-linked immunoassay method (ELISA) screening Her2ECD specificity with phage:
(1) from after 3～4 take turns screening, the containing the Micro-Organism Culture Dish of phage of the foregoing description 2 (6) step, selects single bacterium colony and be inoculated in the 96 hole microbial culture plates overnight cultures; (2) its supernatant that contains phage is transferred in the elisa plate that the Her2ECD purifying protein encapsulates in advance, placed 1～2 hour in room temperature or 37 degree, (3) are with the unconjugated phage of 0.05%PBST flush away; (4) the phage-resistance antibody of adding horseradish peroxidase-labeled; Act on 1 hour, (5) TMB colour developing is on the ELISA appearance; At the 450nm wavelength, read absorption value (OD).(5) more than 2 times, be judged to the positive colony hole greater than control wells OD value when sample well OD value.(6) plasmid in the positive hole of pcr amplification or purifying and carry out gene sequencing.
According to the gene order of each clone strain, CDR1, CDR2, the strain that the CDR3 sequence is identical had been same clone strain both, and the different strain of its sequence both had been different clone strain.
Embodiment 4:
The structure of specific nano antibody expression plasmid:
The specific nano antibody gene that pcr amplification embodiment 3 is obtained; And acquisition has restriction enzyme BbsI and BamHI site PCR product; Handle PCR product and carrier (pSJF2 carrier) respectively with restriction enzyme BbsI and BamHI, connect reorganization through the T4 ligase enzyme, and the plasmid pH2G4 that acquisition can efficiently express in intestinal bacteria; PH2F1, and carry out gene sequencing to confirm the exactness of its sequence.
The amplification condition of PCR: with implementing row 1, primer: upstream primer TATGAAGACACCAGGCCCAGGTRMAGCTGGWGGAGTCT sees SEQ NO.25; Downstream primer gaagatctccggatccTGAGGAGACGGTGACCTGGGT sees SEQ NO.26.
PH2G4-pSJF2 sees that SEQ NO.27 and pH2F1-pSJF2 see that the plasmid map of SEQ NO.28 sees accompanying drawing 9,10.
Embodiment 5:
Nano antibody albumen is at expression in escherichia coli, purifying:
(1) with the embodiment 4 described plasmid pH2G4 that contain, the bacterial classification inoculation of pH2F1 is containing on the LB culture plate of aminobenzylpenicillin, and 37 spend night; (2) select single colony inoculation in 20 milliliters the LB nutrient solution that contains aminobenzylpenicillin, 37 degree, shaking table overnight cultures; (3) change kind in 1 liter of 2YT nutrient solution that contains aminobenzylpenicillin, 37 degree shaking tables are cultivated 240 rev/mins; Cultivate the OD value and reach at 0.6～1.0 o'clock, add 0.1～0.5M IPTG, continue overnight cultures.(4) centrifugal, receive bacterium, (5) add and melt bacterium enzymatic lysis bacterium, and are centrifugal, receive solubility nano antibody albumen in the supernatant.(6) prepare purity through Ni+ ion affinity chromatography high-pressure liquid phase and can reach the albumen more than 90%.PH2G4, per 1 liter of bacterial cultures albumen yield of pH2F1 is respectively 100 milligrams and 65 milligrams.The result sees Fig. 2, Fig. 3 and shown in Figure 4, and nano antibody pH2G4 and pH2F1 express the about 15kd of band His label protein, and through molecular sieve polydextran gel (superG75) experiment, the elution peak of pH2G4 shows that also its molecular weight is 15kd.
Embodiment 6:
Whether what evaluation was obtained can discern and combine natural Her2 to the Her2ECD nano antibody:
Utilize the pH2G4 of mark vitamin H that the breast cancer cell (SK-BR3) that can express the Her2 acceptor is dyeed, visible specific staining reaction, the result sees accompanying drawing 5.Shown in Fig. 5 a, when not adding biotinylated pH2G4 in the dyeing system, cell dyeing is the green of background, and Fig. 5 b with the Her2 receptors bind of cell surface, tangible stained positive reaction occurs when adding biotinylated pH2G4.
Embodiment 7:
Experimental study double-antibody sandwich enzyme-labeled immunity detection method:
(1) encapsulate the selection of condition: use 0.05M, the pH9.5 carbonate buffer solution is diluted to different concentration with the pH2G4 nano antibody, encapsulates elisa plate, places 4 respectively and spends night and 37 degree 1.5 hours, and its result shows, is good in 1.5 hours with 37 degree.The result sees Fig. 6; Shown in the result, under the coated antibody of same concentrations and the condition that detects antibody, the antibody sandwich elisa plate is placed 37 degree, and to spend night be good to its result than placing 4.
(2) the capture antibody optimum antibody encapsulates the preferred of concentration: capture antibody pH2G4 and detection antibody biotin labeled goat-anti Her2 polyclonal antibody IgG or pH2F1 are diluted to different working concentration; Detect Her2 concentration in 1 mcg/ml; OD450nm is under 1.0～1.5 condition; Carry out orthogonal test, optimizing best capture antibody sandwich concentration is 5～10 mcg/ml.See Fig. 7.
Fig. 7: shown in the ELISA orthogonal experiments, capture antibody encapsulates concentration in 5～10 mcg/ml, is best effort concentration, and the optimum detection working concentration of biotin labeling antibody is 1～5 mcg/ml.
(3) formulation of detection Her2 sensitivity and typical curve: encapsulate elisa plate with capture antibody pH2G4 working concentration 10 mcg/ml; Her2 is diluted to different concns; Add then and detect TPPA, the result shows that the sensitivity of its mensuration can reach 1～10 millimicro grams per milliliter (ng/ml).See Fig. 8.Accompanying drawing 8: detect shown in the result of sensitivity and typical curve of Her2, when Her2 concentration dilution scope during from 2 microgram to 1 nanogram(ng)s, it is measured concentration and becomes better linearity to concern.
(4) the double antibody sandwich method enzyme-labeled immunity detects confirming of the best optimum condition of Her2:
1) sodium carbonate buffer (pH=9.5,0.05M) dilution 10ug/ml pH2G4, the 100ul/ hole, 37 ℃ encapsulate 1.5h;
2) the PBST washing is 1 time, 4min;
3) 0.2% casein (0.2%CPBS) 250ul/ hole, 37 ℃ of sealing 1h;
4) the PBST washing is 1 time, 4min;
5) with Her2ECD as antigen, with 0.2%CPBS dilution, 100ul/ hole, 37 ℃ of 1h;
6) the PBST washing is 3 times, 4min;
7) be 2ug/ml with 0.2%CPBS dilution bio-IgG, 100ul/ hole, 37 ℃ of 1h;
8) the PBST washing is 3 times, 4min;
9) with 5000 times of 0.2%CPBS dilution avidin-HRP, 100ul/ hole, 37 ℃ of 1h;
10) the PBST washing is 3 times, 4min;
With quantitative tmb substrate color development at room temperature 10min.
SEQUENCE?LISTING
< 110>Tianjin Shengfa Nabio Tech. Co., Ltd.
< 120>to nano antibody or the polypeptide of mammary cancer Her2/new
<130>
<160> 28
<170> PatentIn?version?3.3
<210> 1
<211> 60
<212> PRT
<213> FR1
<400> 1
Gln?Val?Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Ser?Gly?Phe?Ser?Gln?Val
20 25 30
Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu
35 40 45
Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asp
50 55 60
<210> 2
<211> 26
<212> PRT
<213> FR2
<400> 2
Trp?Tyr?Arg?Gln?Thr?Pro?Gly?Asn?Arg?Arg?Glu?Trp?Val?Trp?Tyr?Arg
1 5 10 15
Gln?Thr?Pro?Gly?Asn?Arg?Arg?Glu?Trp?Val
20 25
<210> 3
<211> 60
<212> PRT
<213> FR3
<400> 3
Arg?Phe?Ala?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Thr?Thr?Val?Tyr?Leu?Gln
1 5 10 15
Met?Asn?Ser?Leu?Lys?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Arg?Phe
20 25 30
Thr?Ile?Ser?Arg?Asp?Asn?Thr?Lys?Asn?Met?Leu?Tyr?Leu?Gln?Met?Asn
35 40 45
Ser?Leu?Lys?Ala?Glu?Asp?Thr?Gly?Leu?Tyr?Tyr?Cys
50 55 60
<210> 4
<211> 22
<212> PRT
<213> FR4
<400> 4
Trp?Gly?Gln?Gly?Thr?Gln?Val?Thr?Val?Ser?Ser?Trp?Gly?Gln?Gly?Thr
1 5 10 15
Gln?Val?Thr?Val?Ser?Ser
20
<210> 5
<211> 5
<212> PRT
<213> CDR1
<400> 5
Pro?Asn?Val?Met?Gly
1 5
<210> 6
<211> 5
<212> PRT
<213> CDR1
<400> 6
Asp?Tyr?Ala?Met?Ser
1 5
<210> 7
<211> 17
<212> PRT
<213> CDR2
<400> 7
Ala?Ala?Ala?Asn?Lys?Tyr?Gly?Thr?Thr?Thr?Tyr?Ala?Asp?Ser?Val?Lys
1 5 10 15
Gly
<210> 8
<211> 18
<212> PRT
<213> CDR2
<400> 8
Ser?Ala?Ile?Ser?Trp?Asn?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Glu?Ser?Met
1 5 10 15
Lys?Gly
<210> 9
<211> 12
<212> PRT
<213> CDR3
<400> 9
Ala?Ala?Ser?Thr?Ala?Thr?Asn?Trp?Asp?Tyr?His?Tyr
1 5 10
<210> 10
<211> 6
<212> PRT
<213> CDR3
<400> 10
Val?Ala?Pro?Trp?Lys?Phe
1 5
<210> 11
<211> 118
<212> PRT
<213> pH2G4
<400> 11
Gln?Val?Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Ser?Gly?Phe?Ser?Pro?Asn
20 25 30
Val?Met?Gly?Trp?Tyr?Arg?Gln?Thr?Pro?Gly?Asn?Arg?Arg?Glu?Trp?Val
35 40 45
Ala?Ala?Ala?Asn?Lys?Tyr?Gly?Thr?Thr?Thr?Tyr?Ala?Asp?Ser?Val?Lys
50 55 60
Gly?Arg?Phe?Ala?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Thr?Thr?Val?Tyr?Leu
65 70 75 80
Gln?Met?Asn?Ser?Leu?Lys?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Ala?Ser?Thr?Ala?Thr?Asn?Trp?Asp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr
100 105 110
Gln?Val?Thr?Val?Ser?Ser
115
<210> 12
<211> 113
<212> PRT
<213> pH2F1
<400> 12
Gln?Val?Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asp?Asp?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Trp?Asn?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Glu?Ser?Met
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Thr?Lys?Asn?Met?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Gly?Leu?Tyr?Tyr?Cys
85 90 95
Val?Ala?Pro?Trp?Lys?Phe?Trp?Gly?Gln?Gly?Thr?Gln?Val?Thr?Val?Ser
100 105 110
Ser
<210> 13
<211> 354
<212> DNA
<213> pH2G4
<400> 13
caggtgaagc?tggtggagtc?tgggggaggc?ttggtgcagc?ctggggggtc?tctgagactc 60
tcctgtgcag?cctctggaag?cggcttcagt?cccaatgtga?tgggctggta?ccgccaaact 120
ccagggaacc?ggcgcgagtg?ggtcgcggca?gctaacaaat?atggtacgac?tacctatgca 180
gactccgtga?agggccgatt?cgccatctcc?agagacaacg?ccaagactac?ggtgtatctc 240
caaatgaaca?gcctgaaacc?ggaggacacg?gccgtctatt?actgcgccgc?aagtacggca 300
acgaattggg?actatcacta?ctggggccag?gggacccagg?tcaccgtctc?ctca 354
<210> 14
<211> 339
<212> DNA
<213> pH2F1
<400> 14
caggtaaagc?tggtggagtc?tgggggaggc?ttggtgcagc?ctggggggtc?tctgagactc 60
tcctgtgcag?cctctggatt?cacttttgat?gattatgcca?tgagctgggt?ccgacaggct 120
ccagggaagg?ggctggagtg?ggtctcagct?attagctgga?atggtggtag?cacatactat 180
gcagaatcca?tgaagggccg?attcaccatc?tccagagaca?ataccaagaa?catgctgtat 240
ctgcaaatga?acagcctgaa?agctgaggac?acgggtctgt?attactgtgt?ggcaccgtgg 300
aaattctggg?gccaggggac?ccaggtcacc?gtctcctca 339
<210> 15
<211> 582
<212> DNA
<213> Her2
<400> 15
atgggcagca?gccatcatca?tcatcatcac?agcagcggcc?tggtgccgcg?cggcagccat 60
atggctagca?tgactggtgg?acagcaaatg?ggtcgcggat?ccgaattcat?gaagctgcgg 120
ctccctgcca?gtcccgagac?ccacctggac?atgctccgcc?acctctacca?gggctgccag 180
gtggtgcagg?gaaacctgga?actcacctac?ctgcccacca?atgccagcct?gtccttcctg 240
caggatatcc?aggaggtgca?gggctacgtg?ctcatcgctc?acaaccaagt?gaggcaggtc 300
ccactgcaga?ggctgcggat?tgtgcgaggc?acccagctct?ttgaggacaa?ctatgccctg 360
gccgtgctag?acaatggaga?cccgctgaac?aataccaccc?ctgtcacagg?ggcctcccca 420
ggaggcctgc?gggagctgca?gcttcgaagc?ctcacagaga?tcttgaaagg?aggggtcttg 480
atccagcgga?acccccagct?ctgctaccag?gacacgattt?tgtggaagga?catcttccac 540
aagaacaacc?agctggctct?cacactgata?gacaccaacc?gc 582
<210> 16
<211> 194
<212> PRT
<213> Her2ECD
<400> 16
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
1 5 10 15
Arg?Gly?Ser?His?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln?Met?Gly?Arg
20 25 30
Gly?Ser?Glu?Phe?Met?Lys?Leu?Arg?Leu?Pro?Ala?Ser?Pro?Glu?Thr?His
35 40 45
Leu?Asp?Met?Leu?Arg?His?Leu?Tyr?Gln?Gly?Cys?Gln?Val?Val?Gln?Gly
50 55 60
Asn?Leu?Glu?Leu?Thr?Tyr?Leu?Pro?Thr?Asn?Ala?Ser?Leu?Ser?Phe?Leu
65 70 75 80
Gln?Asp?Ile?Gln?Glu?Val?Gln?Gly?Tyr?Val?Leu?Ile?Ala?His?Asn?Gln
85 90 95
Val?Arg?Gln?Val?Pro?Leu?Gln?Arg?Leu?Arg?Ile?Val?Arg?Gly?Thr?Gln
100 105 110
Leu?Phe?Glu?Asp?Asn?Tyr?Ala?Leu?Ala?Val?Leu?Asp?Asn?Gly?Asp?Pro
115 120 125
Leu?Asn?Asn?Thr?Thr?Pro?Val?Thr?Gly?Ala?Ser?Pro?Gly?Gly?Leu?Arg
130 135 140
Glu?Leu?Gln?Leu?Arg?Ser?Leu?Thr?Glu?Ile?Leu?Lys?Gly?Gly?Val?Leu
145 150 155 160
Ile?Gln?Arg?Asn?Pro?Gln?Leu?Cys?Tyr?Gln?Asp?Thr?Ile?Leu?Trp?Lys
165 170 175
Asp?Ile?Phe?His?Lys?Asn?Asn?Gln?Leu?Ala?Leu?Thr?Leu?Ile?Asp?Thr
180 185 190
Asn?Arg
<210> 17
<211> 27
<212> DNA
< 213>artificial sequence
<400> 17
ggaattcatg?aagctgcggc?tccctgc 27
<210> 18
<211> 29
<212> DNA
< 213>artificial sequence
<400> 18
cgggatccgc?ggttggtgtc?tatcagtgt 29
<210> 19
<211> 21
<212> DNA
<213> LT1
<400> 19
cgccatcaag?gtaccagttg?a 21
<210> 20
<211> 29
<212> DNA
<213> LT2
<400> 20
ggggtacctg?tcatccacgg?accagctga 29
<210> 21
<211> 45
<212> DNA
<213> LB1
<400> 21
gcccagccgg?ccatggccsm?kgtgcagctg?gtggaktctg?gggga 45
<210> 22
<211> 45
<212> DNA
<213> LB2
<400> 22
gcccagccgg?ccatggccca?ggtaaagctg?gaggagtctg?gggga 45
<210> 23
<211> 34
<212> DNA
<213> gKF
<400> 23
catgtgtaga?ctcgcggccc?agccggccat?ggcc 34
<210> 24
<211> 47
<212> DNA
<213> gKB
<400> 24
catgtgtaga?ttcctggccg?gcctggcctg?aggagacggt?gacctgg 47
<210> 25
<211> 38
<212> DNA
< 213>artificial sequence
<400> 25
tatgaagaca?ccaggcccag?gtrmagctgg?wggagtct 38
<210> 26
<211> 37
<212> DNA
< 213>artificial sequence
<400> 26
gaagatctcc?ggatcctgag?gagacggtga?cctgggt 37
<210> 27
<211> 167
<212> PRT
<213> pH2G4-pSJF2
<400> 27
Met?Lys?Thr?Ala?Ile?Ala?Ile?Ala?Val?Ala?Leu?Ala?Gly?Phe?Ala?Thr
1 5 10 15
Val?Ala?Gln?Ala?Gln?Val?Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val
20 25 30
Gln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Ser?Gly
35 40 45
Phe?Ser?Pro?Asn?Val?Met?Gly?Trp?Tyr?Arg?Gln?Thr?Pro?Gly?Asn?Arg
50 55 60
Arg?Glu?Trp?Val?Ala?Ala?Ala?Asn?Lys?Tyr?Gly?Thr?Thr?Thr?Tyr?Ala
65 70 75 80
Asp?Ser?Val?Lys?Gly?Arg?Phe?Ala?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Thr
85 90 95
Thr?Val?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Lys?Pro?Glu?Asp?Thr?Ala?Val
100 105 110
Tyr?Tyr?Cys?Ala?Ala?Ser?Thr?Ala?Thr?Asn?Trp?Asp?Tyr?His?Tyr?Trp
115 120 125
Gly?Gln?Gly?Thr?Gln?Val?Thr?Val?Ser?Ser?Gly?Ser?Glu?Gln?Lys?Leu
130 135 140
Ile?Ser?Glu?Glu?Asp?Leu?Asn?His?His?His?His?His?Lys?Leu?Gly?Thr
145 150 155 160
Gly?Arg?Arg?Phe?Thr?Thr?Ser
165
<210> 28
<211> 163
<212> PRT
<213> pH2F1-pSJF2
<400> 28
Met?Lys?Thr?Ala?Ile?Ala?Ile?Ala?Val?Ala?Leu?Ala?Gly?Phe?Ala?Thr
1 5 10 15
Val?Ala?Gln?Ala?Gln?Gln?Val?Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu
20 25 30
Val?Gln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe
35 40 45
Thr?Phe?Asp?Asp?Tyr?Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys
50 55 60
Gly?Leu?Glu?Trp?Val?Ser?Ala?Ile?Ser?Trp?Asn?Gly?Gly?Ser?Thr?Tyr
65 70 75 80
Tyr?Ala?Glu?Ser?Met?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Thr
85 90 95
Lys?Asn?Met?Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Lys?Ala?Glu?Asp?Thr
100 105 110
Gly?Leu?Tyr?Tyr?Cys?Val?Ala?Pro?Trp?Lys?Phe?Trp?Gly?Gln?Gly?Thr
115 120 125
Gln?Val?Thr?Val?Ser?Ser?Gly?Ser?Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu
130 135 140
Asp?Leu?Asn?His?His?His?His?His?Lys?Leu?Gly?Thr?Gly?Arg?Arg?Phe
145 150 155 160
Thr?Thr?Ser
Claims (5)
1. to the nano antibody of Her2/new, comprise framework region and complementary determining region; , wherein said framework region is for being respectively FR1-FR4, and the aminoacid sequence of the FR1-FR4 of framework region is specially:
FR1： QVKLVESGGGLVQPGGSLRLSCAASGSGFS
QVKLVESGGGLVQPGGSLRLSCAASGFTFD
FR2： WYRQTPGNRREWV
WYRQTPGNRREWV
FR3： RFAISRDNAKTTVYLQMNSLKPEDTAVYYC
RFTISRDNTKNMLYLQMNSLKAEDTGLYYC
FR4； WGQGTQVTVSS
WGQGTQVTVSS
, it is characterized in that complementary determining region is CDR1-CDR3, the aminoacid sequence of complementary determining region CDR1-CDR3 is specially:
CDR1 is PNVMG or DYAMS;
CDR2 is AAANKYGTTTYADSVKG or SAISWNGGSTYYAESMKG;
CDR3 is AASTATNWDYHY or VAPWKF.
2. the nano antibody to Her2/new according to claim 1 is characterized in that described nano antibody is pH2G4 or pH2F1; Wherein the aminoacid sequence of pH2G4 is:
QVKLVESGGGLVQPGGSLRLSCAASGSGFS
PNVMGWYRQTPGNRREWV
AAANKYGTTTYADSVKGRFAISRDNAKTTVYLQMNSLKPEDTAVYYC
AASTATNWDYHYWGQGTQVTVSS；
The aminoacid sequence of pH2F1 is:
QVKLVESGGGLVQPGGSLRLSCAASGFTFD
DYAMSWVRQAPGKGLEWV
SAISWNGGSTYYAESMKGRFTISRDNTKNMLYLQMNSLKAEDTGLYYC
VAPWKFWGQGTQVTVSS。
3. the polypeptide to Her2/new according to claim 1 is characterized in that polypeptide comprises described CDR1-CDR3 and FR1-FR4, and amino acid sequence of polypeptide has the aminoacid sequence similarity at least 80% with CDR1-CDR3 and FR1-FR4.
4. the polypeptide of Her2/new according to claim 2 is characterized in that amino acid sequence of polypeptide has and the aminoacid sequence similarity 90%. preferred 95% of CDR1-CDR3 and FR1-FR4 or above sequence identity mutually at least at least.
5. the polypeptide of Her2/new according to claim 2 is characterized in that amino acid sequence of polypeptide has and the aminoacid sequence similarity 95% of CDR1-CDR3 and FR1-FR4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110280031 CN102321175B (en) | 2011-09-21 | 2011-09-21 | Nano-antibody or polypeptide aiming at breast cancer Her2/new |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110280031 CN102321175B (en) | 2011-09-21 | 2011-09-21 | Nano-antibody or polypeptide aiming at breast cancer Her2/new |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102321175A true CN102321175A (en) | 2012-01-18 |
| CN102321175B CN102321175B (en) | 2013-08-07 |
Family
ID=45449030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110280031 Expired - Fee Related CN102321175B (en) | 2011-09-21 | 2011-09-21 | Nano-antibody or polypeptide aiming at breast cancer Her2/new |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102321175B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106866823A (en) * | 2017-03-08 | 2017-06-20 | 康众（北京）生物科技有限公司 | A kind of nano antibody of anti-Her2 |
| CN107236046A (en) * | 2017-05-15 | 2017-10-10 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | A kind of recombinant human endostatin fusion protein and its preparation method and application |
| CN109627319A (en) * | 2019-01-11 | 2019-04-16 | 北京协同创新研究院 | The heavy chain antibody of anti-HER-2 a kind of and its application |
| CN110452297A (en) * | 2019-09-03 | 2019-11-15 | 上海洛启生物医药技术有限公司 | Anti-VEGF single domain antibody and its application |
| CN110964113A (en) * | 2019-12-24 | 2020-04-07 | 北京纽安博生物技术有限公司 | Single-domain antibody for mediated immunoglobulin binding, bifunctional antibody constructed by same and application thereof |
| CN111825767A (en) * | 2020-07-31 | 2020-10-27 | 深圳市汉星生物科技有限责任公司 | Single-domain antibody of human epidermal growth factor receptor 2, detection kit and application thereof |
| CN114409787A (en) * | 2022-02-18 | 2022-04-29 | 南京英瀚斯生物科技有限公司 | Separation and purification method for HER3 nano antibody |
| WO2022127889A1 (en) * | 2020-12-18 | 2022-06-23 | 江苏先声药业有限公司 | Her2 antibody and application thereof |
| CN116162167A (en) * | 2021-11-25 | 2023-05-26 | 南京大学 | Double-targeting binding protein of human EGF receptor 2 and integrin and application thereof |
| CN116574186A (en) * | 2023-07-07 | 2023-08-11 | 云南大学 | Nanobody capable of specifically binding HER2 and application thereof |
| WO2024001844A1 (en) * | 2022-06-30 | 2024-01-04 | 复旦大学 | Method for preparing her2 nanobody and conjugate, and use thereof |
| CN120741857A (en) * | 2025-07-08 | 2025-10-03 | 浩铭医疗供应链管理(广东)有限公司 | HER2 detection kit and detection method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1350464A (en) * | 1999-04-09 | 2002-05-22 | 阿芳蒂制药股份有限公司 | Combination of taxotere and recombinant humanized HER2 monoclonal antibody for the treatment of cancer |
| CN1420128A (en) * | 2001-11-16 | 2003-05-28 | 上海中信国健药业有限公司 | Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof |
| CN101321784A (en) * | 2005-10-11 | 2008-12-10 | 埃博灵克斯股份有限公司 | Nanobodies TM and peptides against EGFR and IGF-IR |
| CN101721700A (en) * | 2008-10-15 | 2010-06-09 | 哈药集团生物工程有限公司 | Lyophilized preparation of anti-human Her2 antibody |
-
2011
- 2011-09-21 CN CN 201110280031 patent/CN102321175B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1350464A (en) * | 1999-04-09 | 2002-05-22 | 阿芳蒂制药股份有限公司 | Combination of taxotere and recombinant humanized HER2 monoclonal antibody for the treatment of cancer |
| CN1420128A (en) * | 2001-11-16 | 2003-05-28 | 上海中信国健药业有限公司 | Humanized anti-HER 2 monoclonal antibody, its preparation method and pharmaceutical composition thereof |
| CN101321784A (en) * | 2005-10-11 | 2008-12-10 | 埃博灵克斯股份有限公司 | Nanobodies TM and peptides against EGFR and IGF-IR |
| CN101721700A (en) * | 2008-10-15 | 2010-06-09 | 哈药集团生物工程有限公司 | Lyophilized preparation of anti-human Her2 antibody |
Non-Patent Citations (2)
| Title |
|---|
| JAMSHID TANHA 等: "Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH properties", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| 郑莉 等: "抗HER2/neu单克隆抗体的制备及其对乳腺癌细胞生长的抑制性", 《复旦学报（医学版）》 * |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106866823A (en) * | 2017-03-08 | 2017-06-20 | 康众（北京）生物科技有限公司 | A kind of nano antibody of anti-Her2 |
| CN107236046B (en) * | 2017-05-15 | 2021-05-07 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Recombinant human endostatin fusion protein and preparation method and application thereof |
| CN107236046A (en) * | 2017-05-15 | 2017-10-10 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | A kind of recombinant human endostatin fusion protein and its preparation method and application |
| CN109627319A (en) * | 2019-01-11 | 2019-04-16 | 北京协同创新研究院 | The heavy chain antibody of anti-HER-2 a kind of and its application |
| CN109627319B (en) * | 2019-01-11 | 2022-06-21 | 北京协同创新研究院 | anti-HER-2 heavy chain antibody and application thereof |
| CN110452297A (en) * | 2019-09-03 | 2019-11-15 | 上海洛启生物医药技术有限公司 | Anti-VEGF single domain antibody and its application |
| CN110452297B (en) * | 2019-09-03 | 2020-04-14 | 上海洛启生物医药技术有限公司 | Anti-VEGF single domain antibody and its application |
| CN110964113B (en) * | 2019-12-24 | 2021-09-03 | 北京纽安博生物技术有限公司 | Single-domain antibody for mediated immunoglobulin binding, bifunctional antibody constructed by same and application thereof |
| CN110964113A (en) * | 2019-12-24 | 2020-04-07 | 北京纽安博生物技术有限公司 | Single-domain antibody for mediated immunoglobulin binding, bifunctional antibody constructed by same and application thereof |
| CN111825767A (en) * | 2020-07-31 | 2020-10-27 | 深圳市汉星生物科技有限责任公司 | Single-domain antibody of human epidermal growth factor receptor 2, detection kit and application thereof |
| CN111825767B (en) * | 2020-07-31 | 2021-09-21 | 北京贝诗丹生物科技有限公司 | Single-domain antibody of human epidermal growth factor receptor 2, detection kit and application thereof |
| WO2022127889A1 (en) * | 2020-12-18 | 2022-06-23 | 江苏先声药业有限公司 | Her2 antibody and application thereof |
| CN116162167A (en) * | 2021-11-25 | 2023-05-26 | 南京大学 | Double-targeting binding protein of human EGF receptor 2 and integrin and application thereof |
| CN114409787A (en) * | 2022-02-18 | 2022-04-29 | 南京英瀚斯生物科技有限公司 | Separation and purification method for HER3 nano antibody |
| WO2024001844A1 (en) * | 2022-06-30 | 2024-01-04 | 复旦大学 | Method for preparing her2 nanobody and conjugate, and use thereof |
| CN116574186A (en) * | 2023-07-07 | 2023-08-11 | 云南大学 | Nanobody capable of specifically binding HER2 and application thereof |
| CN116574186B (en) * | 2023-07-07 | 2023-11-28 | 云南大学 | Nanobody capable of specifically binding HER2 and application thereof |
| CN120741857A (en) * | 2025-07-08 | 2025-10-03 | 浩铭医疗供应链管理(广东)有限公司 | HER2 detection kit and detection method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102321175B (en) | 2013-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102321175B (en) | Nano-antibody or polypeptide aiming at breast cancer Her2/new | |
| CN114539411B (en) | A ROR1 antibody or antigen-binding fragment thereof | |
| CN112028997B (en) | Anti-CEACAM5 Nanobody | |
| CN116769023B (en) | Mouse anti-Tasarella marneffei mannan protein hybridoma cell line, monoclonal antibodies and applications | |
| CN110577594B (en) | A kind of Staphylococcus aureus enterotoxin A nanobody A21, application and kit | |
| CN111793132A (en) | Monoclonal antibody of human procalcitonin and preparation method and application thereof | |
| CN111825767B (en) | Single-domain antibody of human epidermal growth factor receptor 2, detection kit and application thereof | |
| CN116199783B (en) | ALP monoclonal antibody and its application | |
| CN114106187A (en) | A specific shark single domain antibody targeting OGT and its preparation method and application | |
| CN108795880B (en) | Mouse hybridoma cell strain for generating human thymidine kinase 1(TK1) specific monoclonal antibody and application thereof | |
| CN106928363B (en) | A FAP Nanobody Nb12 | |
| CN110551218B (en) | A kind of GD2 nanobody and its application | |
| CN112625133A (en) | CDK2 nano antibody and application thereof | |
| CN106928364B (en) | A FAP Nanobody Nb26 | |
| CN112250765A (en) | Nano antibody aiming at HER2 and application thereof | |
| CN114591424B (en) | Specific antibody of new coronavirus S protein NTD region and preparation method and application thereof | |
| CN103059117A (en) | High throughput screening of important schistosoma japonicum antigens and application of antigens in schistosomiasis diagnosis | |
| CN106928358B (en) | A CD105 Nanobody Nb168 | |
| CN101550191A (en) | Malignant melanoma T cell nano antibody, coding sequence and application thereof | |
| CN110129277B (en) | Liver cancer diagnosis marker BRIX1 and application thereof | |
| CN106928365A (en) | A kind of FAP nano antibodies Nb36 | |
| CN110903359A (en) | Preparation of campylobacter jejuni recombinant protein and monoclonal antibody thereof | |
| CN117686722A (en) | S100A4 nano antibody and application thereof | |
| CN106928360B (en) | A CD105 Nanobody Nb68 | |
| CN116751299A (en) | anti-CD 30ki-1&4 antibody, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Li Shenghua Inventor after: Xu Jianbo Inventor before: Li Shenghua |
|
| COR | Change of bibliographic data | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160920 Address after: 300457 No. 220 Dongting Road, economic development zone, Binhai New Area, Tianjin Patentee after: Tianjin Shengfa NabioTech Co., Ltd. Patentee after: Xu Jianbo Address before: 300457 No. 220 Dongting Road, economic development zone, Binhai New Area, Tianjin Patentee before: Tianjin Shengfa NabioTech Co., Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130807 Termination date: 20170921 |