CN107868127A - A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide - Google Patents
A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide Download PDFInfo
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- CN107868127A CN107868127A CN201610859309.5A CN201610859309A CN107868127A CN 107868127 A CN107868127 A CN 107868127A CN 201610859309 A CN201610859309 A CN 201610859309A CN 107868127 A CN107868127 A CN 107868127A
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- antibody
- light chain
- chain variable
- albumen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
Abstract
The invention provides a kind of monoclonal antibody for cervical carcinoma auxiliary diagnosis and its application, specifically, the invention provides one kind identification high-risk-type (HPV16, HPV18, HPV31, HPV33, HPV58 etc.) HPV positive cervical tissue rabbit resource monoclonal antibody and its application, the antibody can detect the biological marker HPVE7 albumen in tissue (including cervical carcinoma and cervical lesionses) with high specificity, become cervical epithelial tissue so as to distinguish the cervical carcinogenesis tissue related to HPV persistent infections and abnormal cervical or non-cancer, cervical carcinoma that can be caused by the high-risk HPV infection of Accurate Diagnosis, pathologist can give more accurate analysis result according to the expression of HPV E7 albumen to the state of an illness of patient.The rate of missed diagnosis of cervical lesionses can be effectively reduced, and improve injury and the wasting of resources that over-treatment is brought to patient.
Description
Technical field
The invention belongs to biomedicine field, and specifically the present invention relates to the list detected for cancer pathological section cancer protein
Clonal antibody and its application, more specifically the present invention relates to the detection of HPV E7 protein expressions in the cancer as caused by high-risk HPV.
Background technology
Cervical carcinoma is the common cancer of female reproductive system, occupies female malignant second, late cancer life in 5 years
Deposit that rate is low, worldwide there are higher morbidity and mortality.Zur Hansen propose HPV within 1976
(HPV) it is probably the carcinogenic factor that spreads through sex intercourse, and the relation between HPV and cervical carcinoma that begins one's study.At present, many epidemiology have been
Confirm that HPV is the arch-criminal for causing cervical carcinoma, a variety of other tumours, including genital tract, mammary gland, alimentary canal and breathing can also be caused
Road cancer.Therefore zur Hansen also obtained Nobel prize's soul in 2008.HPV is in recent years in population of China
Propagation be growing on and on, the Forbidden City neck cancer prevention, treatment research work it is extremely important.
80% women can infect HPV viruse in life at it, and most of HPV infections can be exempted from 1-2 by itself
Epidemic disease system is removed, and high-grade cervical intraepithelial neoplasia (cin) (cervical will be developed into by continuing existing HPV infection
Intraepithelial neoplasia, CIN) damage, such as CIN II and CIN III, or even further develops into cervical carcinoma.
According to statistics, about 20% low cervical lesions translate into high injury, if treated not in time, wherein 30% will be further
Switch to malignant tumour.The pathomorphism of most Middle and advanced cervical cancers is not it is clear that diagnosis is difficult.But for early stage
Cervix cancer, and its diagnosis of precancerous lesion are still the emphasis of research so far.Normal cervical epithelial → CIN → cervical carcinoma this
In one progression, it can all occur except there occurs the corresponding change, some gene structures, function etc. of histology, cytological appearance
Change, these genes can be as the molecular markers in this progress, so as to early detection, diagnosis CIN and cervical carcinoma.As Ki67,
p16INK4A, hTERT etc. increase with CIN ranks and express increase (Valentina F, Renzo B, Serena B, et al.,
Detection of HPV E7Oncoviral protein in cervical lesions by a new
antibody.Appl immunohistochem Mol Morphol,2013,21(4):341-350).Recent study shows,
Tumor suppressor gene p16INK4AIt is overexpressed in most of cervical carcinoma and precancerous lesion, it is believed that can be by p16INK4AAlbumen is as a kind of raw
Thing mark carries out the early screening of cervical carcinoma.
HPV persistent infections are to cause the main viral pathogenesis factor of canceration in epithelium of cervix uteri.In China, high-risk HPV 16,
18th, 31,33,52 and 58 be the several hypotypes of recall rate highest in Cervical squamous carcinoma.New closer studies have shown that p16INK4A
Overexpression and HPV persistent infections and cervical carcinoma closely related (Kalof AN, Cooper K., p16INK4a
immunoexpression:surrogate marker of high-risk HPV and high-grade cervical
intraepithelial neoplasia.Adv Anat Pathol.2006Jul；13(4):190-4.).Development of cancer
Middle viral DNA is integrated into human cel gene group, as the missing of E7 protein expressions control is by continuous expression Viral Carcinogenesis egg
White E7, make cell continue to break up and occur precancerous lesion, cause cell regulation and control out of control, immortalize.E7 albumen can preferentially with
Retinoblastoma cell cancer protein pRB is combined and is made its inactivation.Due to p16INK4ANegative-feedback regu- lation, HPV E7 be present with pRB quality inspections
Albumen be pRB inactivation after so that pRB is to p16INK4ANegative-feedback regu- lation action deprivation, so as to cause p16INK4AIn HPV infection
There is very high positive expression rate in positive cervical squamous cell carcinoma.In July, 2012, College of American Pathologists (CAP) and U.S.'s vagina
Mirror and the meeting of uterine neck pathology (ASCCP) guide point out that p16 can influence the mark of cell propagation as reflection HPV E6/E7, have
Enough evidences show to can be used in low level anus-genital tract Squamous cell lesions associated recommendation, it is proposed that use specific cloning number
(E6H4) p16INK4aWhether antibody has influence on the biomarker of cell cycle regulating as detection HPV infection.The clone number
It is global unique p16 for obtaining IVD certificationsINK4aAntibody.Roche Diagnistics CINtec histologies p16 (including p16 antibody E6H4) is
In May, 2014 in Discussion on Chinese Listed.Therefore, the high-grade cervical atypical hyperplasia caused by HPV infection and Patients with Cervical Cancer is upper
The E7 albumen of continuous expression in chrotoplast, is located at p16 in the pathogenesis of cervical carcinomaINK4aUpstream, itself also can conduct
High-grade cervical damages and a tumor markers of cervical carcinoma detection.
The conventional diagnostic method of cervical tissue is hematoxylin eosin staining method (H＆E) at present, although H＆E interpretations are current
The standard being classified to CIN, but it is vulnerable to Pathology Doctors ' subjective factor and epithelium of cervix uteri metaplasia, atrophy, the change repaired
Etc. the influence of factor, and then cause the poor repeatability of H＆E interpretations, diagnosis accuracy not high enough.Clinically there is an urgent need to it is more objective,
More accurately diagnostic criteria.Clinical HPV E7 detections mainly have two reasons currently without suitable antibody：1st, HPV albumen is facing
Expression quantity is relatively low, it is necessary to which the antibody of high-affinity is detected in bed tissue or cell sample；2, HPV viruse is in existing standard
It can not be survived under tissue culture technique in laboratory cultures；There is immunosupress in 3, E7 albumen so that exempted from using E7 albumen in itself
Epidemic disease animal can not obtain good immune response.In this case we provide a kind of HPV16 E7 albumen the grand antibody of list and
Detect the method that HPV E7 are overexpressed.The monoclonal antibody can be special the endogenous HPV16/18 E7 eggs of combination tumour cell
In vain.Diagnosing CIN I, CIN II and CIN III must judge according to H＆E form, by means of this method by detecting biology mark
Will thing E7 albumen can understand precancerous lesion exactly, improve the accuracy of CIN grade interpretations, it is ensured that patient be carried out accurate
Shunt to take appropriate follow-up, remedy measures.
The content of the invention
It is an object of the invention to provide a kind of grand antibody of list for auxiliary diagnosis before cervical carcinoma and its application.
The first aspect of the present invention, there is provided a kind of weight chain variable district of antibody, described weight chain variable district include following
Three complementary determining region CDR：
CDR1 shown in SEQ ID NO.4,
CDR2 shown in SEQ ID NO.6, and
CDR3 shown in SEQ ID NO.8.
In another preference, the weight chain variable district has the amino acid sequence shown in SEQ ID NO.10.
The second aspect of the present invention, there is provided a kind of heavy chain of antibody, described heavy chain have such as first aspect present invention
Described weight chain variable district, and
Heavy chain constant region.
In another preference, described heavy chain constant region behaviour source, mouse source or rabbit source.
The third aspect of the present invention, there is provided a kind of light chain variable district of antibody, the light chain variable district, which has, to be selected from down
The complementary determining region CDR of group：
CDR1' shown in SEQ ID NO.12,
CDR2' shown in SEQ ID NO.14, and
CDR3' shown in SEQ ID NO.16.
In another preference, described light chain variable district has the amino acid sequence shown in SEQ ID NO.18.
The fourth aspect of the present invention, there is provided a kind of light chain of antibody, described light chain have such as third aspect present invention
Described light chain variable district, and
Constant region of light chain.
In another preference, described constant region of light chain behaviour source, mouse source or rabbit source.
The fifth aspect of the present invention, there is provided a kind of antibody, the antibody have：
(1) weight chain variable district as described in the first aspect of the invention；And/or
(2) light chain variable district as described in third aspect present invention.
In another preference, the antibody has：Heavy chain as described in respect of the second aspect of the invention；And/or such as the present invention
Light chain described in fourth aspect.
In another preference, described antibody is the anti-HPV of specificity antibody；Preferably, the antibody is specificity
The antibody of anti-HPV E7 albumen；It is highly preferred that antibody of the antibody for the anti-HPV16 E7 albumen of specificity.It is it is preferred that described
Antibody also has the anti-HPV18 of specificity, HPV31, HPV33, HPV52, the activity of HPV58 E7 albumen.
In another preference, described antibody includes：Single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody
(such as people rabbit chimeric antibody), mouse source antibody, rabbit source antibody or humanized antibody.
In another preference, " the HPV16 E7 albumen " can be wild type HPV16 E7 albumen, or wild
The derived protein of raw type HPV16 E7 albumen." the HPV18 E7 albumen " can be wild type HPV18 E7 albumen, can also
For the derived protein of wild type HPV18 E7 albumen." the HPV31E7 albumen " can be wild type HPV31 E7 albumen, also may be used
Think the derived protein of wild type HPV31 E7 albumen." the HPV33 E7 albumen " can be wild type HPV33 E7 albumen,
Can also be the derived protein of wild type HPV33 E7 albumen, " the HPV52 E7 albumen " can be wild type HPV52 E7 eggs
In vain, or the derived protein of wild type HPV52 E7 albumen, " the HPV58 E7 albumen " can be wild type HPV58
E7 albumen, or the derived protein of wild type HPV58 E7 albumen.
In another preference, the antibody for can specifically bind HPV16 E7 albumen, HPV18 E7 albumen,
HPV31 E7 albumen, HPV33 E7 albumen, the monoclonal antibody of HPV52 E7 and HPV58 E7 albumen.
In another preference, the antibody do not combined with other HPV hypotypes or with the affinity of other HPV hypotypes compared with
It is low.
The sixth aspect of the present invention, there is provided a kind of recombinant protein, described recombinant protein have：
(i) weight chain variable district as described in the first aspect of the invention, such as heavy chain as described in respect of the second aspect of the invention, this hair
Light chain variable district described in the bright third aspect, the light chain as described in fourth aspect present invention or as described in fifth aspect present invention
Antibody；And
(ii) sequence label of optional assistance expression and/or purifying.
In another preference, described sequence label includes 6His labels.
In another preference, the anti-HPV of described recombinant protein specificity；Preferably, the anti-HPV E7 albumen of specificity；More
Preferably, the anti-HPV16 E7 albumen of specificity.It is preferred that the anti-high-risk HPV18, HPV31 of the recombinant protein also specificity,
HPV33, HPV52, HPV58 albumen.
The seventh aspect of the present invention, there is provided a kind of polynucleotides, it encodes the polypeptide being selected from the group：
(1) weight chain variable district as described in the first aspect of the invention, such as heavy chain as described in respect of the second aspect of the invention, this hair
Light chain variable district described in the bright third aspect, the light chain as described in fourth aspect present invention or as described in fifth aspect present invention
Antibody；Or
(2) recombinant protein as described in sixth aspect present invention.
In another preference, described polynucleotides have shown in SEQ ID NO.3,5,7,9,11,13,15 or 17
Sequence.
The eighth aspect of the present invention, there is provided a kind of carrier, it contains the multinuclear described in the aspect of invention the 7th
Thuja acid.
In another preference, described carrier includes：Bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus,
Mammalian cell virus such as adenovirus, retrovirus or other carriers.
The ninth aspect of the present invention, there is provided a kind of genetically engineered host cell, it contains eighth aspect present invention
The polynucleotides described in seventh aspect present invention are integrated with described carrier or genome.
The tenth aspect of the present invention, there is provided a kind of immune conjugate, the immune conjugate contain：
(a) weight chain variable district as described in the first aspect of the invention, such as heavy chain as described in respect of the second aspect of the invention, this hair
Light chain variable district described in the bright third aspect, the light chain as described in fourth aspect present invention or as described in fifth aspect present invention
Antibody；With
(b) coupling moiety being selected from the group：Detectable, medicine, toxin, cell factor, radionuclide or
Enzyme.
In another preference, the conjugate is selected from：(magnetic is common by fluorescence or luminous marker, radioactively labelled substance, MRI
Shake imaging) or CT (x-ray tomography of electronic computer) contrast agent or the enzyme of detectable product, radiation can be produced
Property nucleic, biotoxin, cell factor (such as IL-2), antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/receive
Rice rod, virion, liposome, magnetic nanosphere, pro-drug activation enzymes (for example, DT- diaphorases (DTD) or biphenyl base hydrolase-
Sample protein (BPHL)), chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
The eleventh aspect of the present invention, there is provided a kind of pharmaceutical composition, it contains：
(i) weight chain variable district as described in the first aspect of the invention, such as heavy chain as described in respect of the second aspect of the invention, this hair
Light chain variable district described in the bright third aspect, the light chain as described in fourth aspect present invention or as described in fifth aspect present invention
Antibody, the recombinant protein as described in sixth aspect present invention or the immune conjugate as described in tenth aspect present invention；And
(ii) pharmaceutically acceptable carrier.
In another preference, described pharmaceutical composition is injection type.
In another preference, described pharmaceutical composition is used for the medicine for preparing treatment tumour, and described tumour is selected from
The following group：Stomach cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, large intestine
Cancer, cervical carcinoma, carcinoma of endometrium, carcinoma of penis, adrenal tumor or tumor of bladder.
The twelveth aspect of the present invention, there is provided weight chain variable district as described in the first aspect of the invention, such as present invention the
Two aspect described in heavy chain, the light chain variable district as described in third aspect present invention, the light chain as described in fourth aspect present invention,
Or the antibody as described in fifth aspect present invention, the recombinant protein as described in sixth aspect present invention or the tenth side such as of the invention
The purposes of immune conjugate described in face, for preparing medicament, reagent, detection plate or kit；
The reagent, detection plate or kit are used for：
(1) HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58 in sample is detected
E7 albumen；And/or
(2) detect tumour cell in endogenic HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or
HPV52 and/or HPV58 E7 albumen；And/or
(3) detection expression HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58 E7
The tumour cell of albumen；
The medicament be used for treat or prevent expression HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or
The tumour of HPV52 and/or HPV58 E7 albumen.
In another preference, in the sample containing HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or
HPV52 and/or HPV58 E7 albumen.
In another preference, the tumour includes：The tumour of urogenital system, the tumour of respiratory system, digestion
The tumour of road system, including：Cervical carcinoma, carcinoma of endometrium, carcinoma of penis, ED-SCLC, melanoma or H/N tumors, stomach
Cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer or kidney
Upper adenoncus knurl.
In another preference, " tumour of urogenital system " includes：Cervical carcinoma, carcinoma of urinary bladder, carcinoma of endometrium
Or carcinoma of penis.
In another preference, described reagent includes chip, the immune particulate of coated antibody.
The thirteenth aspect of the present invention, there is provided a kind of method for detecting HPV E7 albumen in sample, methods described include
Step：
(1) sample is contacted with the antibody described in fifth aspect present invention；
(2) detect whether to form antigen-antibody complex, wherein forming compound means that in sample HPV E7 eggs be present
In vain.
In another preference, detected in step (2) by ELISA method.
In another preference, the HPV E7 albumen includes HPV16 and/or HPV18 and/or HPV31 and/or HPV33
And/or HPV52 and/or HPV58 E7 albumen.
In another preference, in step (1), sample is contacted with two kinds of antibody for HPV E7 albumen, and
Detected in step (2) by ELISA method, described two at least one of antibody of HPV E7 albumen is this hair
Antibody described in bright 5th aspect.
In another preference, " antigen-antibody complex " is that " first antibody-antigen-secondary antibody " ternary is multiple
Compound, wherein, the first antibody is the antibody described in fifth aspect present invention, and the combination epitope of the secondary antibody with
The combination epitope of the first antibody is different.
It is described in step (1) in another preference, sample is contacted with the antibody described in fifth aspect present invention
Afterwards, the 3rd antibody of the anti-first antibody is added in reaction system, and " antigen-the first is anti-for detection in step (2)
The formation of the antibody of body-the three " compound.
In another preference, with detectable mark on the first antibody, the secondary antibody or the 3rd antibody
Note.
In another preference, the detectable label is biotin labeling, colloid gold label, horseradish peroxidase mark
Note, radioisotope labeling, fluorescein mark.
In another preference, the sample includes：Human or animal tissues sample, tumor resection sample, cast-off cells sample
Product.
In another preference, methods described is used for nondiagnostic purpose.
The fourteenth aspect of the present invention, there is provided a kind of detection plate, described detection plate include substrate (supporting plate) and surveyed
Strip, described test-strips contain the antibody described in fifth aspect present invention or the immunoconjugates described in sixth aspect present invention
Thing.
In another preference, described test-strips also contain antigen point sample area.
In another preference, described test-strips by filter sample paper, chromatographic material, nitrocellulose filter and blotting paper successively
Overlap joint composition.
The fifteenth aspect of the present invention, there is provided a kind of kit, the kit include：
(1) first container, contain the antibody described in fifth aspect present invention in first container；And/or
(2) second container, the secondary antibody containing the antibody described in anti-fifth aspect present invention in the second container；And/or
(3) the 3rd containers, contain cell cracking agent in the 3rd container；
Or
The kit contains the detection plate described in the tenth four sides of the invention.
In another preference, the antibody in first container carries detectable label.
In another preference, the antibody in the second container carries detectable label.
The sixteenth aspect of the present invention, there is provided a kind of preparation method of Prepare restructuring polypeptide, this method include：
(a) under conditions suitable for the expression, the host cell described in ninth aspect present invention is cultivated；
(b) recombinant polypeptide is isolated from culture, described recombinant polypeptide is the antibody described in fifth aspect present invention
Or the recombinant protein described in sixth aspect present invention.
Brief description of the drawings
Fig. 1 is HPV16E7 single-chain antibodies (scFv) and protein binding ELISA testing result figures.
Fig. 2 is HPV16 E7 rabbit monoclonal antibodies antigen-binding specificity ELISA testing results.RAB-016, and RAB-
017 can only be combined with His-HPV16 E7, without being combined with His-HPV16 E7 and His unrelated proteins；RAB-139 can simultaneously with
His-HPV16 E7 and His-HPV18 E7 are combined, and are not combined with His unrelated proteins.
Fig. 3 is HPV16 E7 rabbit monoclonal antibodies potency ELISA testing results.As a result RAB-139 antibody titers are slightly better than
Other antibody, next to that RAB-016 and RAB-017.
Fig. 4 is cross reaction testing result of the HPV16 E7 rabbits monoclonal antibodies in different subtype HPV E7 albumen.RAB-016 energy
HPV hypotypes enough with reference to recombinant protein E7 are 16,31,33,35,52,58,6,11, and have and must hand over His unrelated proteins
Fork；RAB-017 can be 16,31,33 (weak), 52,58 (weak), 6,11 with reference to recombinant protein E7 HPV hypotypes；RAB-139 energy
HPV hypotypes enough with reference to recombinant protein E7 are 16,18,31,33,52,58.
Fig. 5 is that normal cervical tissues and cervical cancer tissues paraffin section use rabbit monoclonal antibody RAB-016, RAB-017 and RAB-
139 immunohistochemical staining result figure.Fig. 5 A are dyeing knots of the rabbit monoclonal antibody RAB-016 in normal structure and cervical cancer tissues
Fruit；Fig. 5 B are coloration results of the rabbit monoclonal antibody RAB-017 in normal structure and cervical cancer tissues；Fig. 5 C are the how anti-RAB-139 of rabbit
Coloration result in normal structure and cervical cancer tissues.
Fig. 6 is that rabbit monoclonal antibody RAB-139 is immunized in cervical tissue (chronic inflammation, CIN levels and cervical carcinoma) sample paraffin section
Histochemical staining partial results figure.Rabbit monoclonal antibody RAB-139 can not only detect the HPV E7 albumen in cervical carcinoma, can also detect
The HPV E7 albumen of CIN levels, can distinguish hyperplasia of prostate and neoplasm sample.Wherein Fig. 6 A are the coloration result of chronic inflammation,
Substantially without obvious dyeing.Fig. 6 B are CIN I coloration results, and left figure is CIN I level specific stains, and right figure is hyperplasia of prostate；Figure
6C is CIN II coloration results, and left figure is CIN II level specific stains, and right figure is hyperplasia of prostate；In Fig. 6 D, left figure CIN
III coloration result, right figure are the coloration result of cervical carcinoma.
Embodiment
The present inventor is by extensive and in-depth study, by a large amount of screenings, the final anti-high-risk HPVE7 rabbits source of one plant of acquisition
Monoclonal antibody RAB-139.Test result indicates that should be for the rabbit resource monoclonal antibody of high-risk HPV E7 albumen, specificity
Height, affinity is strong, has stronger affinity with the E7 albumen of high-risk HPV 16,18,31,33,58.Further study showed that should
Antibody can be additionally used in the detection of clinical paraffin section pathology sample.The invention provides detect and/or identify high-risk HPV E7
The method of albumen, this method stability is good, and detection sensitivity is high.Present invention also offers the kit for including above-mentioned antibody.
Specifically, the present invention uses His- using restructuring His-HPV16 E7 fusion protein immunization Japan large ear rabbits
HPV16 E7 and another His mark unrelated protein after rabbit anteserum reaches certain potency, to be drenched as selective mechanisms antigen
The bone-marrow-derived lymphocyte fawned on, for building bacteriophage master library.Specific antibody technology is prepared at this by phage display
Field is well known.The positive antibody strain Fv channel genes obtained will be screened into eukaryotic expression system, expression rabbit source total length resists
Body, it is in specific combination HPV E7 fusion proteins in ELISA detections.
Except the identification of ELISA combination recombinant protein antigens, positive monoclonal antibody is further across antigen binding epitope
Analysis, antigenic subtype cross reaction analysis, affinity combine identification, immunocytochemical stain method
(Immunocytochemistry ICC) and immunohistochemistry staining method (Immunohistochemistry IHC) identify.
By above qualification test, 1 strain antibody clone strain RAB-139 has passed through examination requirements, it is shown that protein molecular is horizontal, cellular water
The function of the gentle horizontal specific binding high-risk HPV E7 cancer proteins of tissue, it is most important to contain a variety of high-risk HPV hypotypes,
Add its application value.
In the preferred embodiment of the present invention, the amino acid sequence of the HPV16 E7 albumen is as follows：
HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRT
LEDLLMGTLGIVCPICSQKP(SEQ ID NO.:1)。
In the preferred embodiment of the present invention, the amino acid sequence of the HPV18 E7 albumen is as follows：
MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVE
SSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQ ID NO.:2)。
As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical architectural feature
Different four glycan albumen, it is made up of two identical light chains (L) and two identical heavy chains (H).Every light chain is common by one
Valency disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Every heavy chain and
The intrachain disulfide bond at light chain also regular interval.There is variable region (VH) one end of every heavy chain, is followed by multiple constant regions.Every
There is variable region (VL) one end of light chain, and the other end has constant region；The constant region of light chain is relative with the first of heavy chain constant region, gently
The variable region of chain is relative with the variable region of heavy chain.Special amino acid residue forms boundary between light chain and the variable region of heavy chain
Face.
As used herein, some parts of variable region are different in sequence in " variable " the expression antibody of term, its shape
The combination to its specific antigen and specificity into various specific antibodies.However, changeability and being unevenly distributed over whole anti-
In body variable region.It concentrates in light chain and weight chain variable district three fragments being referred to as in complementary determining region (CDR) or hypervariable region
In.More conservative part is referred to as framework region (FR) in variable region.Each self-contained four FR in the variable region of native heavy and light chain
Area, they are generally in beta sheet configuration, are connected by three CDR for forming connection ring, can form part β foldings in some cases
Stack structure.CDR in every chain is by FR areas firmly against the antigen that together form antibody together and with the CDR of another chain
Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly
With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody
Cellular toxicity.
" light chain " of vertebrate antibodies (immunoglobulin) can be classified as substantially not according to the amino acid sequence of its constant region
One kind in same two classes (being referred to as κ and λ).According to the amino acid sequence of its heavy chain constant region, immunoglobulin can be divided into not
Same species.Mainly there are 5 immunoglobulin like protein：IgA, IgD, IgE, IgG and IgM, some of them can also be further separated into subclass
(isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Distinguished corresponding to the light chain constant of different immunoglobulin like protein
It is also known as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of different immunoglobulin like protein are known to those skilled in the art
's.
As used herein, term " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from a kind of substantially uniform colony, i.e.,
The single antibody included in the colony is identical, in addition to a small number of mutation that may be present naturally occurred.Monoclonal antibody is high
Specifically it is directed to single antigen site.Moreover, (typically have for different determinants from conventional polyclonal antibody preparation
Different antibodies) it is different, each monoclonal antibody is for the single determinant on antigen.In addition to their specificity, herein
Rabbit monoclonal antibodies be that total length rabbit monoclonal antibodies base is built by the method for molecular biosciences after being screened by phage library
Because of expression vector, the carrier is transferred to eukaryotic expression system, cell conditioned medium is collected after culture and obtains, will not be by other immune balls
Protein contamination.Modifier " monoclonal " illustrates the characteristic of antibody, is obtained from substantially uniform antibody population, and this should not be by
It is construed to need to produce antibody with any specific process.
Present invention additionally comprises the list of the corresponding amino acid sequence with described anti-high-risk HPV E7 protein monoclonal antibodies
Clonal antibody, the monoclonal antibody with described anti-high-risk HPV E7 protein monoclonal antibodies variable region chain, and there is this
Other protein or protein conjugate and fusion expressed product of a little chains.Specifically, the present invention includes having containing hypervariable region (mutually
Mend determine area, CDR) light chain and heavy chain any protein or protein conjugate and fusion expressed product (i.e. immunoconjugates
Thing and fusion expressed product), as long as the hypervariable region is identical with the hypervariable region of light chain of the invention and heavy chain or at least 90% is homologous
Property, preferably at least 95% homology.
As it is known by the man skilled in the art, immune conjugate and fusion expressed product include：Medicine, toxin, cell factor
(cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and described HPV16 E7 protein monoclonal antibodies or
Its fragment with reference to and the conjugate of formation.Present invention additionally comprises with described anti-HPV16 E7 protein monoclonal antibodies or its piece
The cell surface marker thing or antigen that section combines.
The present invention not only includes complete monoclonal antibody, in addition to has immunocompetent antibody fragment, such as Fab or
(Fab')2Fragment；Heavy chain of antibody；Antibody light chain.
As used herein, term " weight chain variable district " and " VH" be used interchangeably.
Monoclonal antibody RAB-034 is sequenced using conventional method by the present invention, obtains its sequence information, sequence letter
Breath is described below.
As used herein, term " variable region " and " complementary determining region (complementarity determining
Region, CDR) " it is used interchangeably.
In the preferred embodiment of the present invention, the weight chain variable district of the antibody includes following three mutually
Mend and determine area CDR：
CDR1, its amino acid sequence are GFSLSSYT (SEQ ID NO.:4), its coding nucleotide sequence is,
ggattctccctcagtagctataca(SEQ ID NO.:3)；
CDR2, its amino acid sequence are ISTGDTT (SEQ ID NO.:6), its coding nucleotide sequence is,
attagtactggtgataccact(SEQ ID NO.:5)；
CDR3, its amino acid sequence are ARGYGKSSGYSGLNL (SEQ ID NO.:8), its coding nucleotide sequence is,
gcgagggggtatggtaaaagtagtggttattctggccttaacttg(SEQ ID NO.:7)。
In another preference, the amino acid sequence of the weight chain variable district is：
QSVEESGGDLVKPGASLTLTCKASGFSLSSYTMGWFRQAPGKGLEYIGAISTGDTTDYTNWAKGRFTISKTSSTTVA
LQMTSLTAADTATYFCARGYGKSSGYSGLNLWGPGTLVTVSS(SEQ ID NO.:10)；
Its coding nucleotide sequence is：
cagtcggtggaggagtccgggggagacctggtcaagcctggggcatccctgacactcacctgcaaagcctctggatt
ctccctcagtagctatacaatgggctggttccgccaggctccagggaaggggctggaatacatcggagccattagta
ctggtgataccactgactacacgaactgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggct
ctgcaaatgaccagtctgacagccgcggacacggccacctatttctgtgcgagggggtatggtaaaagtagtggtta
ttctggccttaacttgtggggcccaggtaccctggtcacagtgagctct(SEQ ID NO.:9)。
In the preferred embodiment of the present invention, the heavy chain of the antibody includes above-mentioned weight chain variable district and heavy chain
Constant region, the heavy chain constant region can be mouse source, people source or rabbit source.
As used herein, term " light chain variable district " and " VL" be used interchangeably.
In the preferred embodiment of the present invention, according to the light chain variable district of the antibody of the present invention, have and be selected from
The complementary determining region CDR of the following group：
CDR1', its amino acid sequence are ESVYSNNY (SEQ ID NO.:12),
Its coding nucleotide sequence is gagagcgtttatagtaacaactac (SEQ ID NO.:11)；
CDR2', its amino acid sequence are SAS (SEQ ID NO.:14),
Its coding nucleotide sequence is tctgcatcc (SEQ ID NO.:13)；
CDR3', its amino acid sequence are LGSYDCSSTDCFG (SEQ ID NO.:16),
Its coding nucleotide sequence is ctaggcagttatgattgtagtagtactgattgttttggt (SEQ ID
NO.:15)
In another preference, the amino acid sequence of described light chain variable district is：
DPVLTQTASPVSAAVGSTVTISCQSSESVYSNNYLSWFQQKPGQPPKQLIYSASSLASGVSSRFKGSGSGTQFTLTI
SDVQCDDAATYYCLGSYDCSSTDCFGFGGGTEVVVK(SEQ ID NO.:18),
Its coding nucleotide sequence is：
gaccctgtgctgacccagactgcatcgcccgtgtctgcagctgtgggaagcacagtcaccatcagttgccagtccag
tgagagcgtttatagtaacaactacttatcctggtttcagcagaaaccagggcagcctcccaagcaactgatctatt
ctgcatccagtctggcatctggggtctcatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatc
agcgacgtgcagtgtgacgatgctgccacttactactgtctaggcagttatgattgtagtagtactgattgttttgg
tttcggcggagggaccgaggtggtcgtcaaa(SEQ ID NO.:17)。
In the preferred embodiment of the present invention, the light chain of the antibody includes above-mentioned light chain variable district and light chain
Constant region, the constant region of light chain can be mouse source, people source or rabbit source.
In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all
Refer to the antibody of specific binding HPV16 E7 albumen, such as with weight chain variable district (such as SEQ ID NO.:10 amino acid sequence
Row) and/or light chain variable district (such as SEQ ID NO.:18 amino acid sequence) albumen or polypeptide.They can be with or without
Initial methionine.
In another preference, rabbit or people rabbit chimeric mAb of the described antibody for anti-HPV16 E7 albumen, it
Heavy chain constant region and/or constant region of light chain can be humanization heavy chain constant region or constant region of light chain.It is it is highly preferred that described
Humanization heavy chain constant region or heavy chain constant region or constant region of light chain that constant region of light chain is human IgG1, IgG2 etc..
Present invention also offers other protein or fusion expressed product with antibody of the present invention.Specifically, it is of the invention
It is (i.e. immune even including any protein or protein conjugate and fusion expressed product with the heavy chain containing variable region and light chain
Join thing and fusion expressed product), as long as the variable region is identical with the heavy chain of antibody of the present invention and the variable region of light chain or at least
90% homology, preferably at least 95% homology.
Typically, the antigenic binding property of antibody can by being described positioned at 3 specific regions of heavy chain and light chain variable district,
Referred to as Variable Area (CDR), by this it is intersegmental be divided into 4 frame areas (FR), 4 FR amino acid sequence is relatively conservative,
Association reaction is not participated in directly.These CDR form cyclic structure, the β-pleated sheet formed by FR therebetween phase on space structure
Mutually close, the CDR on CDR and corresponding light chain on heavy chain constitutes the antigen binding site of antibody.Can be by more similar
The amino acid sequence of the antibody of type determines be which Amino acid profile FR or CDR region domain.
The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because at least partly being related in them
And with reference to antigen.Therefore, the present invention, which includes those, has the monoclonal antibody light chain with CDR and the molecule of weight chain variable district, only
Want its CDR and the CDR identified herein that there is the homology of more than 90% (preferably more than 95%, most preferably more than 98%).
The present invention not only includes complete monoclonal antibody, the fragment or antibody that include there is immunocompetent antibody and
The fusion protein that other sequences are formed.Therefore, present invention additionally comprises the fragment of the antibody, derivative and analog.
As used herein, term " fragment ", " derivative " and " analog " refer to be kept substantially antibody of the present invention identical
Biological function or activity polypeptide.Polypeptide fragment, the derivative or the like of the present invention can be that (i) has one or more
Conservative or substituted non-conservative amino acid residue (preferably conservative amino acid) polypeptide, and such substituted amino
Sour residue can may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues
The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second
Glycol) the formed polypeptide of fusion, or the polypeptide that (iv) additional amino acid sequence is fused to this peptide sequence and formed is (as before
Lead sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or egg is merged with what 6His labels were formed
In vain).According to teaching herein, these fragments, derivative and analog belong to scope known to those skilled in the art.
Antibody of the present invention refers to the polypeptide of with HPV16 E7 protein binding activities including above-mentioned CDR region.The term also wraps
Including has the variant form of polypeptide with antibody identical function of the present invention, comprising above-mentioned CDR region.These variant forms include
(but being not limited to)：One or more (being usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10)
Missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal add one or several (be usually 20 with
It is interior, within preferably 10, more preferably within 5) amino acid.For example, in the art, with similar nature or similar
When amino acid is substituted, it will not generally change the function of protein.Again for example, C-terminal and/or N-terminal add one or
Several amino acid will not generally also change the function of protein.Active fragment and activity of the term also including antibody of the present invention spread out
Biology.
The variant form of the polypeptide includes：Homologous sequence, conservative variant, allelic variant, natural mutation, induction
Albumen coded by mutant, the DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of the high or low stringency, with
And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.
Present invention also offers other polypeptides, the fusion protein such as comprising human antibody or its fragment.Except almost total length
Outside polypeptide, present invention includes the fragment of antibody of the present invention.Generally, the fragment has at least about 50 companies of antibody of the present invention
Continue amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about
100 continuous amino acids.
In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention,
There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are similar or similar by property
Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on Table A and produced.
Table A
| Initial residue | Representational substitution | Preferable substitution |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Present invention also offers encoding such antibodies or the polynucleotide molecule of its fragment or its fusion protein.The present invention's
Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can
To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with
SEQ ID NO.:3rd, the coding region sequence shown in 5,7,9,13,15,17,19 is identical or the variant of degeneracy.Such as this paper institutes
With " variant of degeneracy " refers to that coding has the polypeptide identical amino acid sequence with the present invention, but and SEQ in the present invention
ID NO.:3rd, the differentiated nucleotide sequence of coding region sequence shown in 5,7,9,13,15,17,19.
Encoding the polynucleotides of the mature polypeptide of the present invention includes：The coded sequence of encoding mature polypeptide；Mature polypeptide
Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume
Code sequence.
Term " polynucleotides of coded polypeptide " can be included encoding the polynucleotides of this polypeptide or also include
The polynucleotides of additional code and/or non-coding sequence.
The invention further relates to having at least 50% between the hybridization of above-mentioned sequence and two sequences, preferably at least
70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more
The interfertile polynucleotides of nucleotides.In the present invention, " stringent condition " refers to：(1) compared with low ionic strength and higher temperature
Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl during (2) hybridization
Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or the phase same sex of (3) only between two sequences at least 90% with
On, just hybridize when more preferably more than 95%.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:10
And/or SEQ ID NO.:Mature polypeptide shown in 18 has identical biological function and activity.
The nucleotides full length sequence of the antibody of the present invention or its fragment can generally use PCR TRAPs, recombination method or artificial
The method of synthesis obtains.A kind of feasible method is the method that manually synthesizes to synthesize relevant sequence, especially fragment length
When shorter.Generally, by first synthesizing multiple small fragments, the very long fragment of sequence can be obtained by being then attached again.In addition, may be used also
The coded sequence of heavy chain and expression label (such as 6His) are merged, form fusion protein.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will
It is cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation isolated relevant sequence.
Biomolecule (nucleic acid, albumen etc.) involved in the present invention includes existing biomolecule in a separate form.
At present, it is already possible to obtain encoding albumen of the present invention (or its fragment, or its derivative by chemical synthesis completely
Thing) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and
In cell.It is introduced into addition, be able to will be also mutated by chemical synthesis in protein sequence of the present invention.
The invention further relates to include above-mentioned appropriate DNA sequence dna and the carrier of appropriate promoter or control sequence.This
A little carriers can be used for converting appropriate host cell, allow it to marking protein.
Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；It is or high
Deng eukaryotic, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；The bacterium of salmonella typhimurium
Cell；Fungal cell's such as yeast；Drosophila S2 or Sf9 insect cell；CHO, COS7,293 cells zooblast etc..
It can be carried out with recombinant DNA conversion host cell with routine techniques well known to those skilled in the art.When host is original
When core biology is such as Escherichia coli, can absorb DNA competent cell can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is to use MgCl2.If desired, conversion can also use the side of electroporation
Method is carried out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used
Host cell, culture medium used may be selected from various conventional mediums in culture.Under conditions of suitable for host cell growth
Cultivated.After host cell growth is to appropriate cell density, with suitable method (such as temperature transition or chemical induction)
The promoter of selection is induced, cell is further cultured for a period of time.
Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in the cell or on cell membrane.Such as
Fruit needs, can utilize its physics, chemical and other characteristic be separated by various separation methods and the albumen of purification of Recombinant.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine, use
Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), suction
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and other various liquid chromatography technologies and these methods.
The antibody of the present invention can be used alone, also can be with detectable (for diagnostic purpose), therapeutic agent, PK (eggs
White kinases) combination of modified part or any the above material combines or coupling.
Detectable for diagnostic purposes includes but is not limited to：Fluorescence or luminous marker, radioactively labelled substance,
MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can produce detectable product
Enzyme.
It can include but is not limited to the therapeutic agent of antibody binding of the present invention or coupling：1. radionuclide (Koppe etc.,
2005, metastasis of cancer comment (Cancer metastasis reviews) 24,539)；2. biology poison (Chaudhary etc., 1989,
Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and
Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, NAS's proceeding
(PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and
Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. gold nano
Particle/nanometer rods (Lapotko etc., 2005, cancer communication (Cancer letters) 239,36；Huang etc., 2006, it is Americanized
Association's magazine (Journal of the American Chemical Society) 128,2115)；5. virion (Peng
Deng, 2004, gene therapy (Gene therapy) 11,1234)；6. liposome (Mamot etc., 2005, cancer research (Cancer
Research) 65,11631)；7. magnetic nanosphere；8. pro-drug activation enzymes are (for example, DT- diaphorases (DTD) or xenyl hydrolysis
Enzyme-sample protein (BPHL))；10. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..
Present invention also offers a kind of composition.In preference, described composition is pharmaceutical composition, and it contains
The antibody stated or its active fragment or its fusion protein, and pharmaceutically acceptable carrier.Generally, these materials can be prepared
In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH ordinarily be about 5-8, and preferably pH is about
6-8, although pH value can be varied from the property and illness to be treated that are formulated material.The pharmaceutical composition prepared
It can be administered by conventional route, including (but being not limited to)：Knurl is interior, intraperitoneal, intravenous or part are administered.
The pharmaceutical composition of the present invention can be directly used for combining HPV16 E7 protein moleculars, thus can be used for preventing and treating
Tumour.In addition, other therapeutic agents can be also used simultaneously.
The pharmaceutical composition of the present invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more
Good ground 0.1-80wt%) above-mentioned monoclonal antibody (or its conjugate) of the invention and pharmaceutically acceptable carrier or tax
Shape agent.This kind of carrier includes (but being not limited to)：Salt solution, buffer solution, glucose, water, glycerine, ethanol, and combinations thereof.Medicine system
Agent should match with administering mode.The pharmaceutical composition of the present invention can be made into injection form, such as with physiological saline or contain
There are glucose and the aqueous solution of other assistant agents to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile
Under the conditions of manufacture.The dosage of active component is therapeutically effective amount, such as the mg/kg of about 1 microgram/kg body weight-about 5 daily
Body weight.In addition, the polypeptide of the present invention can be also used together with other therapeutic agents.
It is that the immune conjugate of safe and effective amount is applied to mammal during using pharmaceutical composition, the wherein safety
Effective dose typically at least about 10 micrograms/kg body weight, and 8 mg/kg body weight are in most cases no more than about, preferably
The ground dosage is the mg/kg body weight of about 10 micrograms/kg body weight-about 1.Certainly, specific dosage is also contemplated that method of administration, disease
The factors such as people's health status, within the scope of these are all skilled practitioners technical ability.
The preparation of monoclonal antibody
The antibody of the present invention can be prepared by various technologies known to a person skilled in the art.For example, this hair
Bright antigen, animal can be applied to induce the generation of monoclonal antibody.For monoclonal antibody, using hybridoma technology
(see Kohler et al., Nature 256；495,1975；Kohler et al., Eur.J.Immunol.6:511,1976；Kohler
Et al., Eur.J.Immunol.6:292,1976；Hammerling et al., In Monoclonal Antibodies and T
Cell Hybridomas, Elsevier, N.Y., 1981), display technique of bacteriophage or recombinant DNA method (U.S. Patent number can be used
4,816,567) prepare.
Display technique of bacteriophage is a triage techniques, and allogenic polypeptide or albumen are merged into table with the capsid protein of bacteriophage
Reach, fusion protein is illustrated in the surface of virion, and the DNA for encoding the fusant is then located in virion, so that greatly
Establish between amount polypeptide and its DNA encoding sequence and directly contact, make various target molecules (antibody, enzyme, cell surface receptor etc.)
Polypeptide ligand Rapid identification is able to by elutriation.
The invention provides a kind of monoclonal antibody for HPV E7 albumen, especially for high-risk HPV E7 albumen
Monoclonal antibody.In the preferable scheme of the present invention, monoclonal antibody is screened using display technique of bacteriophage,
Recombinant DNA method sets up eukaryotic expression system to express antibody, then will secrete antibody in culture medium through affinity column
(Protein A-Sephrose) is purified.
Method and sample
The present invention relates to the method for the pattern detection cervical carcinoma and Risk-warning for tissue.This method step is substantially such as
Under：Obtain tissue samples；Sample progress formalin is fixed, is prepared into paraffin section；Detect the sample in the paraffin section
High-risk HPV E7 cancer proteins are horizontal in this.
The present invention can be used for the detection of high-risk HPV E7 cancer proteins in high-risk HPV infection associated cancer, wherein high
The tumour of the related cancer urogenital system such as cervical carcinoma, carcinoma of urinary bladder, carcinoma of endometrium, carcinoma of penis of danger type HPV infection,
The preliminary stage of ED-SCLC, melanoma and H/N tumors and these cancers.
Sample (sample) employed in the present invention is tissue samples, and the paraffin section that formalin is fixed.
Kit
Present invention also offers a kind of reagent for referring to the antibody (or its fragment) containing the present invention or the detection plate of the present invention
Box, in the preference of the present invention, described kit also includes container, operation instructions etc..
Further the detection kit designed for detection high-risk HPV E7 cancer proteins, the kit include knowing the present invention
The antibody of other high-risk HPV E7 cancer proteins, detects required common reagent and buffer solution, such as various buffer solutions, enzyme-linked tag
Secondary antibody, detection mark, detection substrate etc..Described antibody is preferably anti-HPV E7 antibody, more preferably anti-HPV16 E7 Dan Ke
Grand antibody.The detection kit can be in-vitro diagnosis device.
The present invention is further designed and developed for the high-risk HPV infection correlation circumstance in cervical lesionses tissue samples
The kit of diagnostic assessment, the kit can detect the E7 cancer proteins of high-risk HPV 16 being present in sample, wherein sample
It can be paraffin section either frozen section.Section is made in cervical tissue, and is used for exploitation based on acellular
On the basis of morphological analysis the high-risk HPV infection conditions in sample are carried out with the detection kit and in-vitro diagnosis of immunology detection
Device.
An object of the present invention is to provide a kind of method for detecting high-risk HPV E7 protein expressions, and described
Method can be used for the detection of detection HPV infection associated cancer particularly cervical carcinoma.
The present inventor etc. has made the rabbit resource monoclonal for high-risk human mammilla papillomavirus (HPV) E7 protein
Antibody RAB-139, and study its application.The present inventor etc. uses made anti-high-risk HPV E7 rabbit monoclonal antibodies RAB-
The cervical tissue paraffin section that 139 pairs of formalin are fixed carries out immunohistochemical staining.By means of RAB-139 pairs of rabbit monoclonal antibody
The high-affinity and high specific of destination protein so that RAB-139 is not only able to high-risk in specific recognition cervical cancer tissues
Type HPVE7 albumen, additionally it is possible to identify the high-risk HPV E7 albumen in Cervical lesions.Therefore, the how anti-RAB-139 of rabbit both may be used
Detection for Middle and advanced cervical cancer, it can also be used to which detection has the possible Early pathological changes of uterine cervix of canceration, is clinical diagnosis and palace
The risk assessment of neck lesion growth provides reliable evidence.According to the discovery result, the present inventor completes the present invention.
That is, the method for the present invention is the method for detecting tumor marker, it is characterised in that：Including in detection sample
High-risk HPV E7 the step of.
In the method for the invention, the detection sample is preferentially the patient that epithelium of cervix uteri damage is possible to cervical lesionses,
Either uterine neck has occurred and that the patient of lesion.
The HPV16 E7 are preferably HPV16 E7 protein or its fragment.In this situation, described HPV16 is detected
The step of E7, is preferably to analyze using HPV16 E7 immunohistochemistry staining method.Used anti-HPV16 E7 antibody is preferred
It is anti-HPV16 E7 rabbit monoclonal antibodies.
Albumen from the HPV16 E7 oncogene expressions evil related as HPV16 used by methods described immune detection
Property or pre-malignant cells occur reliability index.One of most useful aspect of the present invention is thin to cervix cancer, scaly epithelium
Application in the diagnosis of cellular damage and gland cancer and any epithelial cell related to carcinogenic HPV16 infection exception；Shown is carcinogenic
HPV16 infection includes Koilocytosis；Hyperkeratosis；Precancer illness including intraepithelial neoplasia formation or intraepithelial lesions；
Height depauperation；With infectivity or malignant cancer.In addition to cervical carcinoma, HPV16 E7 detection is to detection carcinoma of urinary bladder, intrauterine
The tumour of the urogenital systems such as film cancer, carcinoma of penis, ED-SCLC, melanoma and H/N tumors are also useful.
Another object of the present invention provides a kind of detection kit by the method for the present invention.The kit can be examined
Disconnected kit or research kit.
The kit of the present invention is the kit for detecting tumor marker, it is characterised in that has anti-HPV16 E7 mono-
Clonal antibody.The kit of the present invention is preferentially also to have to detect required common reagent and buffer solution, such as various bufferings
Liquid, the secondary antibody of enzyme-linked tag, detection mark, detection substrate etc..Described antibody is preferably anti-HPV16 E7 antibody, more preferably
Anti- HPV16 E7 monoclonal antibodies, particularly preferably by phage display and recombinant DNA technology, obtain for eukaryotic expression system
The anti-HPV16 E7 monoclonal antibody gene recombinant expression carriers of system.The anti-HPV16 E7 rabbits Dan Ke as caused by eukaryotic expression system
Grand antibody or the monoclonal antibody for having equal binding activity with the anti-HPV16 E7 rabbit monoclonal antibodies.
The invention provides a kind of method HPV16 E7 albumen endogenous by detecting tissue, differentiation is free of the carcinogenic eggs of HPV
White tissue.And when tissue is fixed by clinical extensive neutral formalin, being made after paraffin section can accurately still examine
Oncogenic virus albumen is measured, so as to improve the accuracy of CIN grade interpretations, it is ensured that accurately shunting is carried out to patient to adopt
Take appropriate follow-up, remedy measures.
Further, the present invention also provides a kind of detection kit formed using the detection method.
Main advantages of the present invention are：
(1) antibody provided by the invention for HPV E7 albumen, specificity is high, and affinity is strong, and can largely make
It is standby, cheap.
(2) antibody provided by the invention for HPV16 E7 albumen can be specific with HPV16 E7 protein bindings,
Also can with the E7 protein bindings of the hypotype of HPV18,31,33,52,59, therefore the antibody can be used in HPV E7 albumen wide spectrum inspection
Survey.
(3) antibody provided by the invention being used in the method for detection HPV16 E7 albumen provided by the invention, stability is good,
Detection sensitivity is high.
(4) monoclonal antibody and detection method provided by the invention, suitable for risk before the early carcinomatous change of associated cancer
Early warning and middle and terminal cancer are made a definite diagnosis.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted detailed conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and
Number is calculated by weight.Experiment material and reagent used can obtain from commercially available channel unless otherwise instructed in following examples
.
Embodiment 1
Prepared by high-risk human mammilla papillomavirus 1. (HPV) E7 rabbits monoclonal antibody
The screening of 1.1 single-chain antibodies (scFv)
Using His-HPV16 E7 recombinant protein immune rabbits, with His-HPV16 E7 recombinant proteins and the uncorrelated eggs of His
It is white to carry out bioactivity.Separate rabbit bone-marrow-derived lymphocyte adaptive immune globulin gene.By a full set of variable region gene gram of B cell
It is grand to come out, it is assembled into phage antibody library.The phage antibody library of structure carries out elutriation using recombinant protein His-HPV16 E7.
By the enrichment of three-wheel elutriation；Determine phage titre；Plaque expands；DNA sequencing；The target that ELISA detections screen
Molecule binding peptide.Recombinant protein His-HPV16 E7 are selected in wherein ELISA detections, and set the moon with the uncorrelated albumen of His
Property control (N), Anti-6 × His antibody sets coating His antigen positives control (P), while sets blank control (envelope antigen
It is directly added into ELIAS secondary antibody).The μ g/ml wrapper sheets of His-HPV16 E7 0.1, the uncorrelated μ g/ml wrapper sheets of albumen 5 of His, 4 DEG C are overnight,
After PBST washings pat dry, the closing of 5% skimmed milk power is added, room temperature acts on 2h or 4 DEG C overnight, and after PBST washings pat dry, addition is treated
Survey the μ g/ml of antibody 5.37 DEG C of reaction 1h, PBST washings are separately added into anti-Flag-HRP secondary antibodies (Sigma A8592) again after patting dry
(1:10 000), Anti-6 × His (Abcam ab1187) (1:10 000), after 37 DEG C of reaction 60min, PBST washings pat dry
TMB is developed the color and 2M H2SO4 are terminated.ELISA the selection results react OD values greatly with antibody to be checked to recombinant protein His-HPV16E7
It is the positive in 2, and is rechecked.Screening obtains 8 plants of single-chain antibodies：ScFv001, scFv016, scFv017, scFv020,
ScFv023, scFv034, scFv133, scFv139.
The epitope amino acid sequence region of Preliminary Identification 8 plants of scFv combination.Identified using ELISA method：It is anti-
Original selects recombinant protein and polypeptide, and wherein recombinant protein is His-HPV16 E7；Polypeptide is respectively HPV16E7-1 (amino acid sequences
Arrange SEQ ID NO.:19PTLHEYMLDLQPETTDLYCYEQLNDSSEEE), HPV16E7-27 (amino acid sequence SEQ ID
NO.:20LNDSSEEEDEIDGPAGQAEPDRAH), HPV16 E7-5 (amino acid sequence SEQ ID NO.:
21KCDSTLRLCVQSTHVDIRTLE), His-Vac (amino acid sequence SEQ ID NO.:
22DEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGI V), His18E7-1 (amino acid sequences
Arrange SEQ ID NO.:23SDSEEENDEIDGVNHQHLPARRAEPQRH).And according to scFv and amino acid sequence binding ability
Difference is grouped to 8 plants of scFv.As a result Fig. 1 is seen：8 plants of scFv have must be specific, selection scFv016,
ScFv017 and scFv139 carries out the antibody producing of next step.
The production of 1.2 rabbit monoclonal antibodies
What the eucaryon vivoexpression technology of production rabbit monoclonal antibodies was well-known in the art.First according to rabbit antibody gene
Storehouse Fc sequences, with reference to the scFv antibody genes chosen above, total length rabbit monoclonal antibodies expression vector is built, builds three altogether
Individual carrier for expression of eukaryon.Expression vector collects culture supernatant by turning HEK293F cells liposome wink after 72 hours, and to training
Supernatant is supported to be purified through affinity column (Protein A-Sephrose).Three plants of rabbit monoclonal antibodies are obtained altogether, are respectively：
RAB-016, RAB-017 and RAB-139.The specificity of 1.3 rabbit monoclonal antibodies
During detection, using indirect ELISA method：Antigen selects His-HPV16 E7 and His-HPV18 E7 fusion proteins.
And antibody negative controls (N) to be checked are set with the uncorrelated albumen of His, Anti-6 × His antibody sets coating His antigen positives pair
According to (P), while blank control (envelope antigen is directly added into ELIAS secondary antibody) is set.His-HPV16 E7 and His-HPV18 E7 melt
The μ g/ml wrapper sheets of hop protein 0.5, the uncorrelated μ g/ml wrapper sheets of albumen 5 of His, 4 DEG C overnight, after PBST washings pat dry, adds 5% degreasing
Milk powder is closed, and 37 DEG C of effects 2 hours or 4 DEG C overnight, after PBST washings pat dry, add the μ g/ml of test antibodies 0.1,37 DEG C of reactions 1
Hour, PBST washings are separately added into goat-anti rabbit-HRP secondary antibodies (Sigma A0545) (1 again after patting dry:20 000), and Anti-6 ×
His(Abcam ab1187)(1:10 000), and 37 DEG C are reacted 1 hour, and PBST washings pat dry rear TMB colour developings and 2M H2SO4Terminate.
As a result Fig. 2 is seen：RAB-016, RAB-017, RAB-139 can be combined with His-HPV16 E7, without albumen knot uncorrelated to His
Close；Simultaneously RAB-139 and His-HPV18 E7 have it is weak intersect, and RAB-016 and RAB-017 can only combine His-HPV16 E7 weigh
Histone.
2. the identification of monoclonal antibody
2.1ELISA detects rabbit monoclonal antibodies potency
The μ g/ml wrapper sheets of fusion protein His-HPV16E7 0.5,4 DEG C overnight, after PBST washings pat dry, adds 5% defatted milk
Powder is closed, and 37 DEG C of effects 2 hours or 4 DEG C overnight, after PBST washings pat dry, add anti-HPV16E7 monoclonal antibodies RAB-016, RAB-
017, RAB-139, initial concentration is 1 μ g/ml, doubling dilution, totally 11 concentration gradients, and 37 DEG C are reacted 1 hour, and PBST washings are clapped
Goat-anti rabbit-HRP secondary antibodies (SigmaA0545) (1 are added after dry:20 000), and 37 DEG C are reacted 1 hour, after PBST washings pat dry
TMB develops the color and 2M H2SO4Terminate, reading at OD450nm.As a result Fig. 3 is seen：When using His-HPV16E7 for antigen, 3 plants anti-
Body has higher adhesion.
The intersection Subtype of 2.2 rabbit monoclonal antibodies
Identify cross reaction of the monoclonal antibody in different subtype HPV E7 albumen.Identified using ELISA method：
Antigen selects recombinant protein, and the HPV hypotypes of recombinant protein are respectively 16,18,31,33,35,45,52,58,6,11.Antigen with
0.5 μ g/ml wrapper sheets, 4 DEG C overnight, after PBST washings pat dry, adds the closing of 5% skimmed milk power, 37 DEG C of effects 2 hours or 4 DEG C of mistakes
At night, after PBST washings pat dry, anti-HPV16 E7 monoclonal antibodies RAB-016, RAB-017, RAB-139 (1 μ g/ml) are added, 37 DEG C anti-
Answer 1 hour, PBST washings add goat-anti rabbit-HRP secondary antibodies (SigmaA0545) (1 after patting dry:20 000), and 37 DEG C of reactions 1 are small
When, PBST washings pat dry rear TMB colour developings and 2M H2SO4Terminate, reading at OD450nm.As a result Fig. 4 is seen：RAB-016 can be combined
Recombinant protein E7 HPV hypotypes are 16,31,33,35,52,58,6,11, and have and must intersect with His unrelated proteins；RAB-
017 can be 16,31,33 (weak), 52,58 (weak), 6,11 with reference to recombinant protein E7 HPV hypotypes；RAB-139 can combine weight
Histone E7 HPV hypotypes are 16,18,31,33,52,58.
HPV16 E7 albumen in 2.3 rabbit resource monoclonal antibody immunohistochemistry staining methods detection cervical cancer tissues
It is respectively primary antibody to uterine neck normal structure and cervical cancer tissues using rabbit monoclonal antibody RAB-016, RAB-017 and RAB-139
Carry out immunohistochemical staining.Method is as follows：Pathology paraffin section is first immersed in dimethylbenzene twice, 10 minutes every time；
Then it is immersed in successively in 100% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, each 5 minutes, finally
PBST is washed three times；Histotomy is put into the 0.01M sodium citrates cushioning liquid (pH6.0) boiled, HTHP 2 minutes.
Room temperature is cooled down 30 minutes, and PBST is washed three times, 5 minutes every time；In order that Endogenous peroxidase inactivation adds and contains 3%H2O2PBS
Buffer solution room temperature treatment 10 minutes；PBST is washed 3 times, every time 5 minutes；Add the PBST room temperatures closing 20 containing 10% calf serum
Minute；Confining liquid is discarded, is separately added into 3 strain antibodies, concentration is RAB-016 (20ug/ml), RAB-017 (20ug/ml), RAB-
139 (0.1ug/ml), it is incubated at room temperature 1 hour；Anti-Mouse/Rabbit IgG-HRP (Dako are added dropwise after fully washing
K5007), it is incubated at room temperature 30 minutes；DAB (Dako K5007) colour developings 3-5 minutes (micro- Microscopic observation), stream after fully washing
Water rinses 5 minutes；Haematoxylin is redyed 1 minute, and running water is rinsed well, is immersed in running water 10 minutes；Substep is dehydrated, successively
70% ethanol, 80% ethanol, 95% ethanol, absolute ethyl alcohol respectively soak 5 minutes；Last xylene soak twice, 5 minutes every time；
Neutral gum mounting again；Last micro- sem observation is taken pictures.
As a result Fig. 5 is seen：Rabbit monoclonal antibody RAB-016 and RAB-017 equal dye-frees in normal cervical tissues and cervical cancer tissues；
Rabbit monoclonal antibody RAB-139, without specific staining, has obvious brown color to dye in normal cervical tissues in cervical cancer tissues, and contaminates
Color bits is in kytoplasm.In summary, in the identification cervical cancer tissues that only rabbit resource monoclonal antibody RAB-139 can be specifically
HPV16 E7 albumen is so that cervical carcinogenesis portion of tissue is in specific stain, and dyeing is positioned in kytoplasm, therefore selects
RAB-139 is for further study.
The rabbit resource monoclonal antibody RAB-139 of embodiment 2 is using immunohistochemistry staining method's detection cervical lesionses tissue
According to antibody screening result, it is further explored using rabbit source monoclonal antibody RAB-139 as primary antibody before cervix cancer is detected
Application in lesion sample.Cervical intraepitheliaI neoplasia (CIN) is the precancerous lesion of cervix cancer, including slight (the CIN I of cervix
Level), moderate (CIN II levels), severe atypical hyperplasia and carcinoma in situ (CIN III levels).Selection uterine neck chronic inflammation, CIN-I ,-
II ,-III pathology paraffin section and cervical carcinoma paraffin section, immunohistochemical staining detection, concrete operations are carried out using RAB-139
With reference to the trifle of embodiment 1,2.3.As a result：In uterine neck chronic inflammation sample, positive rate 16.7%；In CIN I samples, positive rate is
28.6%；In CIN II samples, positive rate 78.6%；In CINIII and cervical carcinoma sample, positive rate be 100% (table 1,
Clinical practice data of the rabbit monoclonal antibody RAB-139 in cervical tissue paraffin section).Sample dyeing situations at different levels are included, are had
Body is as shown in fig. 6, wherein Fig. 6 A are the coloration result of chronic inflammation, substantially without obvious dyeing.Fig. 6 B are CIN I coloration results, left
Figure is CIN I level specific stains, and right figure is hyperplasia of prostate；Fig. 6 C are CIN II coloration results, and left figure is that CIN II levels are special
Property dyeing, right figure is hyperplasia of prostate；In Fig. 6 D, left figure is CIN III coloration result, and right figure is the coloration result of cervical carcinoma.
The positive staining rate of each grade sample in the cervical tissue of table 1
| Sample type | Total sample number | Positive staining | Positive staining rate |
| Uterine neck chronic inflammation | 30 | 5 | 16.7% |
| CINI | 7 | 2 | 28.6% |
| CIN II | 14 | 11 | 78.6% |
| CIN III | 8 | 8 | 100% |
| Cervical carcinoma | 18 | 18 | 100% |
Discuss：
The present inventor prepares HPV16 E7 rabbit resource monoclonal antibodies using the above method, from three plants of rabbit single-chain antibody genes
Carry out the eukaryotic system expression of rabbit-anti full-length gene, and the total length rabbit monoclonal antibody of expression screened, finally select high sensitivity,
High specificity, the endogenous HPV16 E7 albumen of histotomy, while the monoclonal antibody RAB-139 that background is minimum can be identified.Originally grind
Study carefully and show that RAB-139 can effectively identify cervical tissue section sample by combining cervical carcinoma correlation high-risk HPV E7 albumen
Cancerous tumor cell in this.Further study showed that RAB-139 is not only able to identify that the virus protein in cervical cancer tissues can also be known
Virus protein in other precancerous lesions of uterine cervix tissue (such as CIN II-III levels).CIN includes all precancerous lesions and original position
Cancer, the pathologic process continuously developed in uterine neck carcinogenesis is reflected, i.e., by cervical dysplasia (light → in → weight) → in situ
A series of pathological changes of cancer → early invasive carcinoma.Though CIN is a lesion continuously developed, also there is a possibility that to reverse,
Whether cervical dysplasia, or carcinoma in situ have reverse possible, and simply carcinoma in situ is reversed and may very lacked.It is clinical for
Youth, there is the CIN I levels patient that fertility requires, extent of disease is small can be with follow-up observation, for CIN II levels patient using freezing
The local treatments such as laser；And for CIN III levels, based on surgery excision uterus, foreign countries have opinion to use local treatment for the country
Person.In clinical practice operation, the judgement for CIN II levels is often due to more dispute be present in the subjectivity of Pathology Doctors '.
Because the generation of high-risk HPV and cervical carcinoma is closely related, high-risk HPV sense can be detected in more than 90% cervical carcinoma sample
Dye, at least high-risk-type can be detected in uterine neck moderate lesion sample in CIN early stages by means of method provided by the invention
HPV, the index whether deteriorated in this, as cervical lesionses, operation whether can be carried out to CIN patient and shunted, for clinical doctor
Teacher provides patient's diagnosis and treatment auxiliary foundation, can both reduce the rate of missed diagnosis due to judging to bring to the subjectivity of tectology, again
Loss caused by over-treatment can be reduced.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (16)
1. a kind of weight chain variable district of antibody, it is characterised in that described weight chain variable district includes following three complementary determining regions
CDR：
CDR1 shown in SEQ ID NO.4,
CDR2 shown in SEQ ID NO.6, and
CDR3 shown in SEQ ID NO.8；
Preferably, the weight chain variable district has the amino acid sequence shown in SEQ ID NO.10.
A kind of 2. heavy chain of antibody, it is characterised in that described heavy chain has weight chain variable district as claimed in claim 1, and
Heavy chain constant region.
3. a kind of light chain variable district of antibody, it is characterised in that the light chain variable district has the complementary determining region being selected from the group
CDR：
CDR1' shown in SEQ ID NO.12,
CDR2' shown in SEQ ID NO.14, and
CDR3' shown in SEQ ID NO.16；
Preferably, described light chain variable district has the amino acid sequence shown in SEQ ID NO.18.
A kind of 4. light chain of antibody, it is characterised in that described light chain has light chain variable district as claimed in claim 3, and
Constant region of light chain.
5. a kind of antibody, it is characterised in that the antibody has：
(1) weight chain variable district as claimed in claim 1；And/or
(2) light chain variable district as claimed in claim 3；
Preferably, the antibody has：Heavy chain as claimed in claim 2；And/or the light chain described in claim 4.
6. a kind of recombinant protein, it is characterised in that described recombinant protein has：
(i) weight chain variable district as claimed in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light
Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；And
(ii) sequence label of optional assistance expression and/or purifying.
7. a kind of polynucleotides, it is characterised in that it encodes the polypeptide being selected from the group：
(1) weight chain variable district as claimed in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light
Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；Or
(2) recombinant protein as claimed in claim 6.
8. a kind of carrier, it is characterised in that it contains the polynucleotides described in claim 7.
9. a kind of genetically engineered host cell, it is characterised in that it contains in carrier or genome described in claim 8
It is integrated with the polynucleotides described in claim 7.
10. a kind of immune conjugate, it is characterised in that the immune conjugate contains：
(a) weight chain variable district as claimed in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light
Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；With
(b) coupling moiety being selected from the group：Detectable, medicine, toxin, cell factor, radionuclide or enzyme.
11. a kind of pharmaceutical composition, it is characterised in that it contains：
(i) weight chain variable district as claimed in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light
Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5, restructuring as claimed in claim 6
Albumen or immune conjugate as claimed in claim 10；And
(ii) pharmaceutically acceptable carrier.
It is 12. weight chain variable district as claimed in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light
Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5, restructuring as claimed in claim 6
The purposes of albumen or immune conjugate as claimed in claim 10, it is characterised in that for preparing medicament, reagent, detection plate
Or kit；
The reagent, detection plate or kit are used for：
(1) HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 eggs in sample are detected
In vain；And/or
(2) detect tumour cell in endogenic HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/
Or HPV58E7 albumen；And/or
(3) detection expression HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52 and/or HPV58E7 albumen
Tumour cell；
The medicament is used to treat or prevent expression HPV16 and/or HPV18 and/or HPV31 and/or HPV33 and/or HPV52
And/or the tumour of HPV58E7 albumen.
A kind of 13. method for detecting HPV E7 albumen in sample, it is characterised in that methods described includes step：
(1) sample is contacted with the antibody described in claim 5；
(2) detect whether to form antigen-antibody complex, wherein forming compound means that in sample HPV E7 albumen be present.
14. a kind of detection plate, it is characterised in that described detection plate includes substrate (supporting plate) and test-strips, described test
Bar contains the immune conjugate described in antibody or claim 10 described in claim 5.
15. a kind of kit, it is characterised in that the kit includes：
(1) first container, contain the antibody described in claim 5 in first container；And/or
(2) second container, the secondary antibody containing the antibody described in anti-claim 5 in the second container；And/or
(3) the 3rd containers, contain cell cracking agent in the 3rd container；
Or
The kit contains the detection plate described in claim 14.
16. a kind of preparation method of Prepare restructuring polypeptide, this method include：
(a) under conditions suitable for the expression, the host cell described in claim 9 is cultivated；
(b) recombinant polypeptide is isolated from culture, described recombinant polypeptide is the antibody or claim described in claim 5
Recombinant protein described in 6.
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| PCT/CN2017/095883 WO2018059117A1 (en) | 2016-09-28 | 2017-08-03 | Monoclonal antibody for detecting oncoprotein expression in pathologic tissue section |
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| CN110196332A (en) * | 2019-05-22 | 2019-09-03 | 艾托金生物医药（苏州）有限公司 | A kind of method and its application detecting more hypotype HPV E7 albumen |
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| CN108586607B (en) * | 2018-04-13 | 2020-09-11 | 郑州大学 | Preparation method and application of monoclonal antibody for resisting HPV16L1 protein |
| CN116199772B (en) * | 2022-11-16 | 2023-10-17 | 怡道生物科技(苏州)有限公司 | HPV31 type capsid protein L1 monoclonal antibody, preparation method and application |
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Application publication date: 20180403 |