CN101864398B - Monoclonal antibody of anti-gonadotropin-releasing hormone receptor and application thereof - Google Patents
Monoclonal antibody of anti-gonadotropin-releasing hormone receptor and application thereof Download PDFInfo
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- CN101864398B CN101864398B CN2010101590351A CN201010159035A CN101864398B CN 101864398 B CN101864398 B CN 101864398B CN 2010101590351 A CN2010101590351 A CN 2010101590351A CN 201010159035 A CN201010159035 A CN 201010159035A CN 101864398 B CN101864398 B CN 101864398B
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Abstract
The present invention provides a mouse hybrid tumor cell strain (preserving number: ATCC GHR-106PTA-10252) and a monoclonal antibody (GHR-106) of anti-gonadotropin-releasing hormone receptor which is secreted from the mouse hybrid tumor cell strain. The antibody can perform effective inbibitional effect for the tumor cell through combining with the surface receptor of tumor cell. Then the antibody can be used for the diagnosis and treatment process of tumor and the application for preparing the antitumor drug.
Description
Technical field
The present invention relates to biological technical field, especially a kind of monoclonal antibody fragment of anti-gonadotropin-releasing hormone receptor and monoclonal antibody thereof and application.
Background technology
Gonadotropin releasing hormone (GnRH) originally is defined as the decapeptide hormone.Its function is the release that stimulates gonad-stimulating hormone.Through having the prepituitary gland cell-specific ground of GnRH acceptor to combine outward, discharge like gonad-stimulating hormone such as LH and FSH with the certain detail after birth.Follow-up study shows that GnRH and its acceptor are also many beyond hypophysis normally and in malignant cell or the tissue takes on the role, and the role of cell or tissue is understood as yet fully beyond hypophysis.Yet GnRH or its analogue are in the news to the antiproliferative effect of the cancer cells of different human body histoorgan.Many GnRH analogues connect cell toxicity medicament and report as the clinical study of anticarcinogen.In fact a part of cancer therapy drug is processed GnRH or its analogue and anticancer cytotoxin composition exactly, as: AN-152 or AN-207's is covalently bound.Another part then is as the medicine that suppresses tumor growth with GnRH analogue and antagonism property analogue thereof.Compare with general chemotherapeutics, these methods have manifested the effective and hypotoxicity of its treatment cancer.
Similar with GnRH, specificity has the shielding function of receptors to the monoclonal antibody of cell surface GnRH acceptor, stops the propagation activity of inside and outside cancer cells or changes reproductive function.The development that with antibody is the cancer therapy drug on basis has in recent years become a kind of optionally human body cancer treatment method (Pawson, A.J., Maudsley; S., Morgan, K.; Davidson, L., Naor; Z., and Millar, R.P.Inhibition of human Type I Gonadotropin-releasing hormonereceptor (GnRHR) function by expression of a human Type II GnRHRgene fragment.Endocrinol.2005; 146 (6): 2639-2649).
Yet the drug target on cancer cells surface should be clearly and is very abundant, and it is clear that the mechanism of action should be explained.The advantage of this method is: reduce spinoff, and keep treatment with antibody drug relative in circulation the long half-lift (5-20 days).Comprehensive these factors, anti-GnRH acceptor monoclonal antibody have the specificity and the certain effect that suppresses the cancer cell growth of target, and the ideal that will become neutralization or kill cancer cell is selected.
The preparation of anti-GnRH acceptor monoclonal antibody cell strain
Generally speaking the method for Kohler and Milstein just is enough to accomplish the required suitable antibody of preparation; Additive method is also arranged certainly, like (J.L., and Herzenberg.L.A. (1982) J.Immunlo.Meth.52:1.)
Combine the Mammals splenocyte of GnRH acceptor peptide (N1-29) immunity or peripheral blood lymphocyte to merge with hemocyanin through the myeloma cell, produce the hybridoma passage cell strain that to secrete desirable monoclonal antibody.The source that special needs to be pointed out is myeloma cell strain must be of the same race with antibody secreting cell.Had sophisticated myeloma cell strain to derive from small white mouse and big white mouse, these Mammalss can produce high-quality polyclonal antiserum and IgSC.Any successful passage cell strain can be as the secretory cell in source of the same race.Some infected antibody secreting cell also can go down to posterity in addition, like Epstein-Barr virus.These technology can be used as invention immunoglobulin,exocrine passage cell strain.
Under acvator existence conditions such as polyoxyethylene glycol, myeloma cell and antibody secreting cell merge becomes hybridoma.Now concrete standard and the method for publishing arranged, do not done statement at this.What the decision key of success was main is the selection and the propagating method of passage cell strain, and the population of selecting antibody secreting cell.The follow-up correct sensitization source of application that then depends on impels Mammals to produce these cells.
First-selected hybridoma preparation method, the splenocyte of separating immune mouse also merges with myeloma cell strain of the same race, as: NS-1 myeloma cell adds 50%PEG, and it is grown in specific substratum.Some other sophisticated myeloma cell strain has: HAT or the strain of AT sensitive cells, they can not contain the HAT substratum growth of xanthoglobulin, AMT and thymidine, perhaps can not contain the growth of azaserine and hypoxanthic AH substratum.
Therefore, have only the passage cell that merges with normal cell in culture medium selected, to survive; HAT that does not merge or AH sensitive cells be apoptosis, (finally still understanding apoptosis even if the normal cell that can not go down to posterity merges).Have only the passage cell of the fusion of cultivating to pass through screening and obtain ideal antibody.
Fused cell or the passage of additive method of cultivation in selected substratum need pass through screening.And the desired characteristics of screening cell is exactly can secretory antibody.There are many screening methods to adopt,, comprise the test of immune protein trace, EUSA and radioimmunoassay or the like as detecting substratum based on immunoreactive test to GnRH acceptor identification antibody.
Confirmed the cell strain that can secrete correct antibody, this antibody just can be separated through the purification technique of standard.The mark of antibody also has other standard technique in addition.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of monoclonal antibody or its fragment of anti-gonadotropin-releasing hormone receptor.
Another technical problem to be solved by this invention is to provide monoclonal antibody or its segmental application of above-mentioned anti-gonadotropin-releasing hormone receptor.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of mouse source hybridoma cell line; Its preserving number is ATCC GHR-106PTA-10252 (depositary institution: the biological article collecting center (No. 10801, American Type Culture Collection
Virginia, The United States Manassas, address town university main road, Virginia 20110-2209USA) of No. 10801 USSs in Virginia, The United States Manassas town university main road, preservation date on August 5th, 2009, deposit number GHR-106PTA-10252).
A kind of monoclonal antibody of anti-gonadotropin-releasing hormone receptor is named as GHR-106, is by above-mentioned mouse source hybridoma cell line excretory monoclonal antibody.
Preferably; The monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor; The variable region of light chain of said monoclonal antibody, heavy chain variable region gene and amino acid sequence coded thereof are the sequence shown in < 400>1, < 400>2, < 400>3 and < 400>4 in the sequence table, and promptly the protein sequence of the variable region of light chain of said monoclonal antibody is:
M D S Q A Q V L I L L L L W V S G T C G
D I V M S Q S P S S L A V S A G E K V T
M S C K S S Q S L L N S R T R K N Y L A
W Y Q Q K P G Q S P K L L I Y W A S T R
E S G V P D R F T G S G S G T D F T L T
I S S V Q A E D L A V Y Y C K Q S Y N L
Y T F G G G T K L E I K
The protein sequence of its variable region of heavy chain is:
Q V Q L K E S G P G L V A P S Q S L S I
T C T V S G F S L S R Y S V H W V R Q P
P G K G L E W L G M I W G G G S T D Y N
S A L K S R L S I S K D N S K S Q V F L
K M N S L Q T D D T A M Y Y C A R G N D
G Y Y S F A Y W G Q G T L V T V S S
Wherein the aminoacid sequence in three CDR districts of light chain is: CDR1 is K S S Q S L L N S R T RK N Y L A, and CDR2 is W A S T R E S, and CDR3 is K Q S Y N L Y T; The aminoacid sequence in three CDR districts of heavy chain is: CDR1 is R Y S V H, and CDR2 is M I W G G G S T D Y N S AL K S, and CDR3 is G N D G Y Y S F A Y.
Preferably, the monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor changes, lacks or add one or several amino acid to its aminoacid sequence, still keeps identical functions.
Preferably, the monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor, with the N1-29 amino acid fragment generation specific reaction of cell surface GnRH acceptor, with the separation constant of GnRH receptor response between 10
-7M and 10
-9Between the M.
Preferably, the monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor, with the GnRH acceptor combine competed by GnRH, the tumour cell that makes cultivation is because of turning down selectivity ribose body protein apoptosis.
Preferably, the monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor, its humanization form can be reacted with any cancer cells or the tissue of cell surface expression GnRH acceptor, suppresses tumor growth.
The monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor is as the application of preparation antitumor drug.
The monoclonal antibody of above-mentioned anti-gonadotropin-releasing hormone receptor can be considered the verivate of a kind of GnRH, has the effect of regulating the reproductive physiology function.
The MONOCLONAL ANTIBODIES SPECIFIC FOR method of above-mentioned anti-gonadotropin-releasing hormone receptor:
According to the anti-gonadotropin-releasing hormone receptor MONOCLONAL ANTIBODIES SPECIFIC FOR method of recording and narrating in the background technology, the cell strain of purifies and separates is gone down to posterity and selects:
At human body cell; The GnRH acceptor is the albuminoid membrane transfer protein of a kind of G; Monoclonal antibody only can be connected with the functional zone amino acid of GnRH acceptor in the zone, extracellular, in this case, and at the pairing N cardinal extremity of the palmitoyl oligopeptide amino-acid residue (1-29 of human body cell exterior domain GnRH acceptor; 182-193,195-206 and 293-306) combined with hemocyanin respectively.
These and the synthetic peptide of hemocyanin bonded are respectively as the antigenic substance immune mouse; Process secretion monoclonal antibody specific hybridoma; Through cytogamy repeatedly and screen many monoclonal antibodies; The final strain monoclonal antibody that produces, GHR-106, promptly immune and select it human body GnRH acceptor to be had the characteristic of high degree of specificity and affinity through hemocyanin binding peptide (N1-29).
The invention has the beneficial effects as follows:
The invention provides the monoclonal antibody GHR-106 of mouse source hybridoma cell line (preserving number is ATCC GHR-106PTA-10252) and excretory anti-gonadotropin-releasing hormone receptor thereof; Thereby said antibody can be expected to be used for the diagnosis of tumour and the application of therapeutic process and preparation antitumor drug then through with the tumor cell surface receptors bind tumour cell being played effective restraining effect.
Description of drawings
Fig. 1 (A) is: elisa immunoassay confirms the combining of polypeptide that GnRH acceptor N holds the 1-29 aminoacid sequence that contain of GHR106 monoclonal antibody and synthetic, increases with the increase of the GHR106 monoclonal anti scale of construction;
Fig. 1 (B) is: elisa immunoassay confirm GHR106 monoclonal antibody and OC-3-VGH tumour cell combine increase with the increase that GHR106 measures;
Fig. 2 (A) is: GnRH I is to GHR106 monoclonal antibody and OC-3-VGH tumour cell bonded restraining effect;
Fig. 2 (B) is: the polypeptide that contains GnRH acceptor N-end 1-29 aminoacid sequence of synthetic is to GHR106 monoclonal antibody and OC-3-VGH tumour cell bonded restraining effect;
Fig. 3 is: the programmed cell property apoptosis that the OC-3-VGH tumour cell of being cultivated is taken place after GHR-106 handles;
Fig. 4 is: the OC-3-VGH tumour cell of being cultivated is handled through the GHR-106 monoclonal antibody, can turn down the expression of the messenger RNA(mRNA) of four kinds of selected ribosomal proteins;
Fig. 5 is: detect at the expressed GnRH acceptor of different Staging of course of disease ovarian cancers through immunohistochemical staining;
Fig. 6 is: the nucleotide sequence of GHR106 monoclonal antibody and aminoacid sequence.
Preservation information
Classification noun: mouse source hybridoma cell line (Murine hybridoma)
Depositary institution's title: the biological article collecting center (ATCC) of USS
Depositary institution address: No. 10801, Virginia, The United States Manassas town university main road (Licensingand Services 10801 University Boulevard Manassas, Virginia 20110-2209USA)
Preservation date: on August 5th, 2009
Preserving number: GHR-106PTA-10252
Embodiment
Below in conjunction with specific embodiment technical scheme according to the invention is further described.
As shown in Figure 6, a kind of monoclonal antibody of anti-gonadotropin-releasing hormone receptor, the protein sequence of its variable region of light chain is:
M D S Q A Q V L I L L L L W V S G T C G
D I V M S Q S P S S L A V S A G E K V T
M S C K S S Q S L L N S R T R K N Y L A
W Y Q Q K P G Q S P K L L I Y W A S T R
E S G V P D R F T G S G S G T D F T L T
I S S V Q A E D L A V Y Y C K Q S Y N L
Y T F G G G T K L E I K
The protein sequence of its variable region of heavy chain is:
Q V Q L K E S G P G L V A P S Q S L S I
T C T V S G F S L S R Y S V H W V R Q P
P G K G L E W L G M I W G G G S T D Y N
S A L K S R L S I S K D N S K S Q V F L
K M N S L Q T D D T A M Y Y C A R G N D
G Y Y S F A Y W G Q G T L V T V S S
The concrete preparation method of the monoclonal antibody of said hybridoma cell line and excretory anti-gonadotropin-releasing hormone receptor thereof is following:
Corresponding to human GnRH acceptor n terminal amino acid sequence 1-29; 182-193; The polypeptide of the synthetic of 195-206 or 293-306 combines to process immunogenic substances (four kinds of neoantigens) with KLH (hemocyanin) respectively by ordinary method; 200 microlitres contain the synthetic antigenic phosphoric acid buffer of peptide-KLH of 100 micrograms, with equal-volume Freund's complete adjuvant mixing and emulsifying; After mixture is subcutaneously injected into interior .2 week of three BALB/C mice bodies and 4 weeks, injects the synthetic peptide-KLH antigen of same metering respectively once more, but use Freund's incomplete adjuvant instead. the antibody activity of immune mouse is measured by the ELISA method; Microwell plate encapsulates above four kinds of synthetic peptides respectively. and the goat anti-mouse IgG of alkali phosphatase enzyme mark is as SA; With the p-NPP colour developing, in the 405nm reading of data. mouse boosting cell that immunocompetence is higher and SP-2 spinal cord oncocyte merge with the preparation hybridoma, screen and identify prepared monoclonal antibody with the ELISA method; Screen and set up 15 strain hybridomas altogether; The antibody capable of its generation respectively with the GnRH receptors bind. the antibody that wherein immunocompetence is the highest is that the synthetic peptide that uses GnRH acceptor n terminal amino acid sequence 1-29 produces as immunogen, and called after GHR106 monoclonal antibody, the hypotype of GHR-106 are IgG1.
The characteristic of GHR-106 monoclonal antibody
Encapsulate microwell plate with GnRH acceptor N end 1-29 amino acid fragment and carry out the ELISA test, the separation constant between the monoclonal antibody GHR-106 of GnRH acceptor and anti-gonadotropin-releasing hormone receptor is between 10
-7M and 10
-9Between the M.The monoclonal antibody GHR-106 of the anti-gonadotropin-releasing hormone receptor of 100 microlitre different concns is added the reaction micropore; Outwell unreacted antibody 37 ℃ of reactions after 1 hour; The goat anti-mouse IgG that the reaction micropore adds 100 microlitre alkali phosphatase enzyme marks after cleaning once again is as SA; 37 ℃ were reacted 1 hour. after cleaning five times, the reaction micropore develops the color with p-NPP again, in the 405nm reading of data.Shown in Fig. 1 (A), the ELISA experiment shows that the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor is dose-dependently with the synthetic peptide that encapsulates (corresponding people GnRH acceptor N end 1-29 amino acid segment) and combines.
Encapsulate microwell plate with the OC-3-VGH ovarian cancer cell and carry out the ELISA test; All the other steps are the same; Shown in Fig. 1 (B), the ELISA experiment shows that the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor is dose-dependently with the OC-3-VGH cancer cells that encapsulates and combines.
It is thus clear that, the GHR-106 monoclonal antibody both can with the GnRH receptors bind, also can combine with tumour cell.
Embodiment 3
GnRH I or GnRH acceptor N end 1-29 amino acid fragment are to GHR106 monoclonal antibody and OC-3-VGH tumour cell bonded restraining effect.
The monoclonal antibody GHR-106 of 100 microlitres, 2 μ g/ml anti-gonadotropin-releasing hormone receptors is added the micropore that encapsulates the OC-3-VGH ovarian cancer cell, and experiment condition is with embodiment 2.Shown in Fig. 2 (A), its binding ability receives the inhibition of the GnRH I of different concns, and relative percentage signal and GnRH concentration are inversely proportional to.
The monoclonal antibody GHR-106 adding of 100 microlitres, 2 μ g/ml anti-gonadotropin-releasing hormone receptors is encapsulated the synthetic peptide micropore of (meeting people GnRH acceptor N end 1-29 amino acid fragment), and experiment condition is with embodiment 2.Shown in Fig. 2 (B), its binding ability receives the inhibition of the synthetic peptide (people GnRH acceptor N cardinal extremity 1-29 amino acid fragment) of different concns, and the concentration of relative percentage signal and synthetic peptide is inversely proportional to.Through carrying out immunoblot experiment, show that combining the protein band molecular weight of formation with the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor is 60KDa, is consistent with people GnRH acceptor with OC-3-VGH ovarian cancer cell extract.Test shows, be coated on the cancer cells of microwell plate, the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor and GnRH receptors bind, and suppressed combining of GnRH I and GnRH acceptor according to different content.This result has confirmed that the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor has high degree of specificity to human body GnRH acceptor, thereby promptly the GHR-106 monoclonal antibody has suppressed combining of GnRH and its acceptor through competitive and GnRH receptors bind.
Embodiment 4
The GHR-106 monoclonal antibody suppresses the result of treatment of growth of tumour cell
The GHR-106 monoclonal antibody is at external anti-proliferative effect to cancer cells
Handle the OC-3-VGH ovarian cancer cell with 1 μ g/ml GHR-106 (■) and 0.1 μ g/ml GnRH (), the per-cent of apoptosis increases, wherein the cancer cells culture condition: RPMI-1640 nutrient solution, 37 ℃, 5%CO2.The judgement that so-called cancer cell-apoptosis per-cent increases adds normal mouse IgG at the same time, confirms through the TUNEL test.As shown in Figure 3, when the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor concentration reached 10 μ g/ml, through cultivation in 24-72 hour, apoptosis appearred in cancer cells.
Monoclonal antibody GHR-106 (1ug/ml) (grey) and GnRH (0.1 μ g/ml) (black) with anti-gonadotropin-releasing hormone receptor cultivate 24 hours OC-3-VGH ovarian cancer cell together; As shown in Figure 4; The expression of the mRNA of selectivity ribose body protein (P0, P1, P2 and L37) descends, and the expression of the mRNA of GAPDH is used as internal control by 100%.The PCR product detects with 1.5% agar-agar gel electrophoresis; The monoclonal antibody GHR-106 and the GnRH that observe with anti-gonadotropin-releasing hormone receptor cultivate 24 hours cancer cells, and the selectivity ribose body protein of expressing mRNA comprises: descending appears in P0, P1, P2 and L37.
The primer that wherein is used for PCR is respectively:
P0:5 '-TTGTGTTCACCAAGGAGG-3 ' and 5 '-GTAGCCAATCTGCAGACAG-3 '
P1:5 '-CAAGGTGCTCGGTCCTTC-3 ' and 5 '-GAACATGTTATAAAAGAGG-3 '
P2:5 '-TCCGCCGCAGACGCCGC-3 ' and 5 '-TGCAGGGGAGCAGGAATT-3 '
L37:5 '-CAGAAGCGAGATGACGAAGG-3 ' and 5 '-CCAGAACATTTATTGCATGAC-3 '
GAPDH:5 '-GAAATCCCATCACCATCTTCC-3 ' and 5 '-CCAGGGGTCTTACTCCTTGG-3 '
PCR:94 ℃ of 40s, 50 ℃ of 40s, 72 ℃ of 1min (20-35 circulation)
See that from resulting result the monoclonal antibody GHR-106 of anti-gonadotropin-releasing hormone receptor on the cancer cells surface apoptosis that specificity combines the meeting priming cancer cell takes place with the GnRH acceptor.
In addition,, the result of reproductive function also can be occurred influencing, comprise the human body ovulation, fertility such as spermatogenesis regulatory function by the GnRH acceptor of Humanized antibody specific sealing for germinal tissue or organ; On the other hand, the humanized antibody medicine can show as long-acting GnRH analogy thing, can change the reproductive function of human body.
Embodiment 5
Immunohistochemical staining is found
Monoclonal antibody GHR-106 with anti-gonadotropin-releasing hormone receptor is directed against the GnRH acceptor; 39 examples are diagnosed as the difference specific immunity histochemical stain of ovarian cancer patients' ovary tissue enforcement by stages, and this experiment is undertaken by hospital of UBC.As shown in Figure 5, the positive staining rate is obviously relevant with the Staging of course of disease of ovarian cancer, and I phase case about 40% is positive, late period (III phase and IV phase) case positive rate then up to 73% to 100%; And normal ovarian tissue (13 example) is not all seen positive staining.Through experiment confirm the expressed GnRH acceptor of cancerous tissue follow the continuity and the development of the cancer course of disease, can constantly increase.
The above results be presented at make a definite diagnosis the ovarian cancer difference by stages patient's's (39 example) ovary tissue be at high proportion positive, and obviously increase with course of disease positive rate, it is negative that the ovary tissue that detects anovarism carninomatosis people in an identical manner all is dyeing.So the GHR-106 monoclonal antibody is expected to be used for and the identification of the marker of antibodies and the diagnosing tumor of pathological section.
Embodiment 6
The GnRH acceptor is in human cancer cell's expression
As shown in the table, in surpassing human cancer cell's strain of 90%, expression is arranged all through RT-PCR evidence GnRH receptor mRNA.This conclusion is clearly explained at all cancer cells, no matter be to betide which kind of histoorgan, in fact all can express the GnRH acceptor.
The primer that wherein is used for PCR is:
GnRHR1:5 '-CAGAAGAAAGAGAAAGGGAAAAAGC-3 ' and
5’-GATGAAAAGAGGGATGATGAAGAGG-3’；
Detect the JEG-3 of expressing GnRHR1 with RT-PCR
*: the power of signal is shown as ,+(by force), ± (weak) ,-(cannot see).
*: the fibroblast source and the embryo lung tissue of normal hyperplasia and propagation.
Can know through the foregoing description and to know that the GHR-106 monoclonal antibody can effectively competitively combine the GnRH acceptor, thereby has further confirmed the restraining effect of GHR-106 monoclonal antibody for tumour cell.
Above-mentioned detailed description of the monoclonal antibody fragment of this a kind of anti-gonadotropin-releasing hormone receptor and monoclonal antibody thereof and application being carried out with reference to embodiment; Be illustrative rather than determinate; Can enumerate out several embodiment according to institute's limited range; Therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Sequence table SEQ UENCE LISTING
< 110>Lee ， Ji Yu
< 120>a kind of monoclonal antibody of anti-gonadotropin-releasing hormone receptor and application
<130>
<150>US 61/239,704
<151>2009-09-03
<160>4
<170>PatentIn version 3.3
<210>1
<211>396
<212>DNA
<213>murine
<220>
<221>mRNA
<222>(1)..(396)
< 223>nucleotide sequence of the variable region of light chain of monoclonal antibody
<400>1
atggattcac aggcccaggt tcttatattg ctgctgctat gggtatctgg tacctgtggg 60
gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 120
atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 180
tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 240
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300
atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctt 360
tacacgttcg gaggggggac caagctggaa ataaaa 396
<210>2
<211>354
<212>DNA
<213>murine
<220>
<221>mRNA
<222>(1)..(354)
< 223>nucleotide sequence of the variable region of heavy chain of monoclonal antibody
<400>2
caggtgcagc tgaaggagtc aggacctggc ctggtggcac cctcacagag cctgtccatc 60
acatgcactg tctctgggtt ctcattatcc agatatagtg tacactgggt tcgccagcct 120
ccaggaaagg gcctggagtg gctgggaatg atatggggtg gtggaagcac agactataat 180
tcagctctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatgtact actgtgccag aggcaatgat 300
ggttactact cgtttgctta ctggggccaa gggactctgg tcactgtctc ttca 354
<210>3
<211>132
<212>PRT
<213>murine
<220>
<221>ACETYLATION
<222>(1)..(132)
< 223>aminoacid sequence of the variable region of light chain of monoclonal antibody
<400>3
Met Asp Ser Gln Ala Gln Val Leu Ile Leu Leu Leu Leu Trp Val Ser
1 5 10 15
Gly Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala
20 25 30
Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser
35 40 45
Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr
100 105 110
Tyr Cys Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys
115 120 125
Leu Glu Ile Lys
130
<210>4
<211>118
<212>PRT
<213>murine
<220>
<221>ACETYLATION
<222>(1)..(118)
< 223>aminoacid sequence of the variable region of heavy chain of monoclonal antibody
<400>4
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr
20 25 30
Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Met Ile Trp Gly Gly Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Arg Gly Asn Asp Gly Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
Claims (3)
1. mouse source hybridoma cell line, its preserving number is ATCC GHR-106 PTA-10252.
2. the monoclonal antibody of an anti-gonadotropin-releasing hormone receptor is characterized in that: be by the described mouse of claim 1 source hybridoma cell line excretory monoclonal antibody.
3. the application of the monoclonal antibody of the described anti-gonadotropin-releasing hormone receptor of claim 2 aspect the preparation ovarian cancer resistance medicament.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23970409P | 2009-09-03 | 2009-09-03 | |
| US61/239,704 | 2009-09-03 |
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| Publication Number | Publication Date |
|---|---|
| CN101864398A CN101864398A (en) | 2010-10-20 |
| CN101864398B true CN101864398B (en) | 2012-11-14 |
Family
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| CN2010101590351A Expired - Fee Related CN101864398B (en) | 2009-09-03 | 2010-03-24 | Monoclonal antibody of anti-gonadotropin-releasing hormone receptor and application thereof |
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| CN102174596B (en) * | 2011-03-22 | 2013-01-23 | 南京工业大学 | Method for producing butanol through staged sugar supplement anaerobic fermentation |
| CN106568979B (en) * | 2016-11-04 | 2019-01-08 | 中国水产科学研究院淡水渔业研究中心 | A kind of method and its application measuring knife long-tailed anchovy GnRH-R content |
| CN108484778B (en) * | 2017-03-31 | 2022-03-29 | 李吉祐 | Chimeric antigen receptor construct of humanized GHR106 monoclonal antibody, nucleic acid molecule and application |
| KR20200118092A (en) * | 2018-02-06 | 2020-10-14 | 밴쿠버 바이오테크 리미티드 | Use of GHR-106 monoclonal antibody as GnRH antagonist |
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| US5750366A (en) * | 1992-06-23 | 1998-05-12 | Mount Sinai School Of Medicine Of The City University Of New York | Cloning and expression of gonadotropin-releasing hormone receptor |
| WO2007144554A2 (en) * | 2006-06-16 | 2007-12-21 | Ardana Bioscience Limited | Gnrh (gonadotropin-releasing hormone) peptide variants |
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Patent Citations (2)
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|---|---|---|---|---|
| US5750366A (en) * | 1992-06-23 | 1998-05-12 | Mount Sinai School Of Medicine Of The City University Of New York | Cloning and expression of gonadotropin-releasing hormone receptor |
| WO2007144554A2 (en) * | 2006-06-16 | 2007-12-21 | Ardana Bioscience Limited | Gnrh (gonadotropin-releasing hormone) peptide variants |
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| CHI-YU GREGORY LEE 等.Immunoidentification of Gonadotropin Releasing Hormone Receptor in Human Sperm, Pituitary and Cancer Cells.《AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY》.2000,第44卷 * |
| CHI-YUGREGORYLEE等.ImmunoidentificationofGonadotropinReleasingHormoneReceptorinHumanSperm Pituitary and Cancer Cells.《AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY》.2000 |
| Gregory Lee · Bixia Ge.Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.《Cancer Immunol Immunother》.2010,第59卷 * |
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| CN101864398A (en) | 2010-10-20 |
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