CN105524166A - Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof - Google Patents
Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof Download PDFInfo
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- CN105524166A CN105524166A CN201410508253.XA CN201410508253A CN105524166A CN 105524166 A CN105524166 A CN 105524166A CN 201410508253 A CN201410508253 A CN 201410508253A CN 105524166 A CN105524166 A CN 105524166A
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Landscapes
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Abstract
The present invention provides a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells, and applications thereof. According to the present invention, with the monoclonal antibody, the cervical cancer biomarker HPV18E7 protein in the tumor cells can be detected in a highly specific manner so as to distinguish between the cancerous cervical epithelial cells and the abnormal cervical epithelial cells or non-cancerous cervical epithelial cells and provide basis for the accurate diagnose of the cancer caused by the HPV infection, such that the treatment can be timely taken, the cancer occurrence or cancer spreading can be prevented, and the suffering of patients can be alleviated.
Description
Technical field
The invention belongs to biological diagnosis and field of medicaments, specifically, the present invention relates to the monoclonal antibody and application thereof that identify the positive epithelium of cervix uteri cancer cells of HPV18.
Background technology
Cervical cancer is the common cancer of female reproductive system, occupies female malignant second.1949, first Sttauss observed human papillomavirus (humanpapillomavirus, HPV) particle under Electronic Speculum in wart body leach liquor.HaraldZurHausen in 1976 propose HPV may be spread through sex intercourse carcinogenic factor after, HPV infects the heat subject becoming tumour virus etiological study with the research of Cervical Cancer.Large quantity research finds, human papillomavirus HPV is the arch-criminal causing cervical cancer, can also cause other tumour multiple, comprise reproductive tract, mammary gland, digestive tube and respiratory cancer.The propagation of HPV in recent years in population of China is growing on and on, and the research work of the cancerous precaution of the Forbidden City neck, treatment is extremely important.
Most of women can infect sexual organ HPV in life at it, and whole world sexual organ HPV infection rate is up to 10.4%.Most of HPV to infect in 1-2 can remove by self immune system, and the HPV infection of sustainable existence will develop into high-grade cervical intraepithelial neoplasia (cin) (cervicalintraepithelialneoplasia, CIN) damage, as CIN2 and CIN3, develop into cervical cancer even further.According to statistics, the low cervical lesions of about 20% will change high injury into, if treated not in time, wherein 30% will transfer to further for malignant tumour.Therefore, early diagnosis and prevention HPV infect is the important breakthrough mouth reducing the relative disease mortality ratio such as cervical cancer and reduce treatment of human cervical cancer spending.What current cervical carcinoma screening mainly adopted is Pap smear inspection, detects the cytological appearance of cervical exfoliated cell.Morphocytology detect there is certain subjectivity, film-making difficulty, batch in and batch between poor repeatability, there is higher false positive and false negative rate, therefore low to early-stage cancer detection sensitivity, rate of missed diagnosis is high, positive rate is only 30% ~ 50%; Some area, Pap smear inspection replace by liquid based cytology (LBC), LBC is semi-automatic or Full automatic specimen process new technology, can automatic analysis detect sample and can provide remaining cell sample supply other HPV infection analysis.Another kind uses Molecular Detection widely clinically---HPVDNA detect (as HC2) can helper detection high-risk HPV virus existence.Detect sensitive although HPVDNA detects than morphocytology, cannot distinguish HPV is transient infections or persistent infection, and the sensitivity of detection method and specificity are all necessary in malignancy of tumor evolution process.Because it can accurately not reflect that therefore the generation of cervical cancer can not be used for detecting cancer (U.S. is consistent with the cervical carcinoma screening guide that China 2012 will upgrade the end of the year respectively to point out " be not recommended in the crowd of any age bracket be used alone HPV detect carry out examination ").
HPV be a kind of have species specificity addicted to epithelium virus, belong to the small DNA virus of double-strand closed loop, comprise about 8000 base pairs.Comprising 8 early stage open reading frames (E1-E8), 2 late period single open reading frame and 1 non-coding Chang Kong district.In early days in open reading frame, E6 and E7 gene pairs Growth of Cells stimulates the most important.The research such as Ziegent (2003) has confirmed that HPVE6, E7 gene has cell transformation function, it is potential oncogene, coding HPVE6, E7 albumen is cancer protein, in vitro can transformed mouse epithelial cell, also can make human epithelial cell generation immortalization, and the continuous expression of HPVE6, E7 albumen is that maintenance cultured cell in vitro immortalization is necessary.Therefore, early expression albumen E6 and E7 of high-risk HPV plays an important role in the generation of cervical cancer.In development of cancer, virus DNA integrates enters in human cel gene group, the disappearance controlled along with E6 and E7 protein expression, continuous expression E6 and E7 albumen in the epithelial cell of high-grade cervical atypical hyperplasia and Patients with Cervical Cancer.E7 is tumor antigenicity albumen, and has stronger antigenicity, can make tumour cancer suppressor gene pRb inactivation, finally cause unregulated cell growth, cause cellular immortalization and canceration occurs.This makes E7 can be used as a tumor markers of high-grade cervical damage and cervical cancer detection.
Current clinical immunization groupization detects HPV and infects mainly employing HPVL1 and some other auxiliary biological marker, as p16INK4A, Ki67, (ValentinaF, RenzoB, the SerenaB such as hTERT, Deng, DetectionofHPVE7Oncoviralproteinincervicallesionsbyanewa ntibody.ApplimmunohistochemMolMorphol, 2013,21 (4): 341-350).Clinical HPV detects does not have suitable antibody to mainly contain three reasons: 1, HPV albumen expression amount in clinical tissue or cell sample is lower, and the antibody of degree of needs high-affinity detects; 2, HPV virus can not be survived in laboratory culture under existing dard tissue culture techniques; Itself there is immunosuppression in 3, E7 albumen, makes to adopt E7 protein immune animal can not obtain good immune response, makes the antibody prepared often there is cross reaction with other HPV albumen in addition and do not have specificity to E7 albumen.
Therefore, need to provide a kind of easy, objective cancer (particularly cervical cancer) of infecting to detect high-risk HPV of method.
Summary of the invention
The object of the present invention is to provide a kind of monoclonal antibody and the application thereof that can identify the positive epithelium of cervix uteri cancer cells of HPV18.
In a first aspect of the present invention, provide a kind of variable region of heavy chain of antibody, described variable region of heavy chain comprises following three complementary determining region CDR:
CDR1 shown in SEQIDNO.:4,
CDR2 shown in SEQIDNO.:6, and
CDR3 shown in SEQIDNO.:8.
In another preference, described variable region of heavy chain has the aminoacid sequence shown in SEQIDNO.:10.
A second aspect of the present invention, provides a kind of heavy chain of antibody, and described heavy chain has variable region of heavy chain as described in the first aspect of the invention, and
CH.
In another preference, described CH behaviour source or mouse source.
A third aspect of the present invention, provides a kind of variable region of light chain of antibody, and described variable region of light chain has the complementary determining region CDR being selected from lower group:
CDR1' shown in SEQIDNO.:14,
CDR2' shown in SEQIDNO.:16, and
CDR3' shown in SEQIDNO.:18.
In another preference, described variable region of light chain has the aminoacid sequence shown in SEQIDNO.:20.
A fourth aspect of the present invention, provides a kind of light chain of antibody, and described light chain has the variable region of light chain as described in third aspect present invention, and
Constant region of light chain.
In another preference, described constant region of light chain behaviour source or mouse source.
A fifth aspect of the present invention, provides a kind of antibody, and described antibody has:
(1) variable region of heavy chain as described in the first aspect of the invention; And/or
(2) variable region of light chain as described in third aspect present invention.
In another preference, described antibody has: heavy chain as described in respect of the second aspect of the invention; And/or the light chain as described in fourth aspect present invention.
In another preference, described antibody is the antibody of the anti-HPV of specificity; Preferably, described antibody is the antibody of the anti-HPV18 of specificity; More preferably, described antibody is the antibody of the anti-HPV18E7 albumen of specificity.
In another preference, described antibody comprises: single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody (as human mouse chimeric antibody), mouse source antibody or humanized antibody.
In another preference, described antibody has following characteristic:
(1) with HPV18E7 protein affinity≤2.67nM; With
(2) not with HPV16E7 protein binding.
Described " HPV18E7 albumen " can be wild-type HPV18E7 albumen, also can be the derived protein of wild-type HPV18E7 albumen.Described " HPV16E7 albumen " can be wild-type HPV16E7 albumen, also can be the derived protein of wild-type HPV16E7 albumen.
In another preference, described antibody is specificity can distinguish the monoclonal antibody of HPV18E7 albumen and HPV16E7 albumen.
In another preference, described antibody is not combined with other HPV hypotype or lower with the avidity of other HPV hypotype.
In another preference, described antibody also has following characteristic:
(3) can combine the protein-specific of the 44-58 amino acids comprising HPV18E7.
In another preference, described antibody is IgG1 type antibody.
A sixth aspect of the present invention, provides a kind of recombinant protein, and described recombinant protein has:
(i) variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention, the variable region of light chain 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; And
(ii) optional assistance expression and/or the sequence label of purifying.
In another preference, described sequence label comprises 6His label.
In another preference, the described anti-HPV of recombinant protein specificity; Preferably, the anti-HPV18 of specificity; More preferably, the anti-HPV18E7 albumen of specificity.
A seventh aspect of the present invention, provides a kind of polynucleotide, and its coding is selected from the polypeptide of lower group:
(1) variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention, the variable region of light chain 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; Or
(2) recombinant protein as described in sixth aspect present invention.
In another preference, described polynucleotide have SEQIDNO.:3,5,7,9,13,15, the sequence shown in 17 or 19.
A eighth aspect of the present invention, provides a kind of carrier, and it contains the polynucleotide described in seventh aspect present invention of the present invention.
In another preference, described carrier comprises: bacterial plasmid, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral as adenovirus, retrovirus or other carriers.
A ninth aspect of the present invention, provides a kind of genetically engineered host cell, and it contains in carrier described in eighth aspect present invention or genome the polynucleotide be integrated with described in seventh aspect present invention.
A tenth aspect of the present invention, provides a kind of immune conjugate, and this immune conjugate contains:
(a) variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention, the variable region of light chain 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; With
B () is selected from the coupling moiety of lower group: detectable, medicine, toxin, cytokine, radionuclide or enzyme.
In another preference, described conjugate is selected from: fluorescence or luminous marker, radioactively labelled substance, MRI (nuclear magnetic resonance) or CT (CT technology) contrast medium, maybe can produce the enzyme that can detect product, radionuclide, biotoxin, cytokine (as IL-2 etc.), antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/nanometer rod, virion, liposome, magnetic nanosphere, pro-drug activation enzymes (such as, DT-diaphorase (DTD) or xenyl lytic enzyme-sample protein (BPHL)), chemotherapeutics (such as, cis-platinum) or any type of nano particle etc.
A eleventh aspect of the present invention, provide a kind of pharmaceutical composition, it is characterized in that, it contains:
(i) variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention, the variable region of light chain 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 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 for the preparation of the medicine for the treatment of tumour, and described tumour is selected from lower group: cancer of the stomach, liver cancer, leukemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, mammary cancer, large bowel cancer, prostate cancer, cervical cancer, adrenal tumor or tumor of bladder.
A twelveth aspect of the present invention, provide the purposes of variable region of heavy chain as described in the first aspect of the invention, heavy chain as described in respect of the second aspect of the invention, the variable region of light chain 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 immune conjugate as described in tenth aspect present invention, it is characterized in that, for the preparation of medicament, reagent, check-out console or test kit;
Described reagent, check-out console or test kit are used for:
(1) HPV18E7 albumen in sample is detected; And/or
(2) endogenic HPV18E7 albumen in tumour cell is detected; And/or
(3) tumour cell of expressing HPV18E7 albumen is detected; And/or
(4) type of HPV is differentiated;
Described medicament is used for the treatment of or prevents to express the tumour of HPV18E7 albumen.
In another preference, containing HPV18E7 albumen in described sample.
In another preference, described tumour comprises: the tumour of urogenital system, small cell lung cancer, melanoma or H/N tumors, cancer of the stomach, liver cancer, leukemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, mammary cancer, large bowel cancer, prostate cancer or adrenal tumor.
In another preference, described " tumour of urogenital system " comprising: cervical cancer, bladder cancer, carcinoma of endometrium or penile cancer.
In another preference, described reagent comprises the immune particulate of chip, coated antibody.
A thirteenth aspect of the present invention, provide a kind of method detecting HPVE7 albumen in sample, described method comprises step:
(1) by the antibody contacts described in sample and fifth aspect present invention;
(2) detect whether form antigen-antibody complex, wherein form mixture and just represent in sample to there is HPVE7 albumen.
In another preference, detected by ELISA method in step (2).
In another preference, described HPVE7 albumen comprises HPV18E7 albumen.
In another preference, in step (1), by sample and two kinds of antibody contacts for HPVE7 albumen, and detected by ELISA method in step (2), described two kinds is the antibody described in fifth aspect present invention at least one in the antibody of HPVE7 albumen.
In another preference, described " antigen-antibody complex " is " first antibody-antigen-second antibody " ternary complex, wherein, described first antibody is the antibody described in fifth aspect present invention, and different in conjunction with epi-position in conjunction with epi-position and described first antibody of described second antibody.
In another preference, described second antibody is the monoclonal antibody that hybridoma cell strain CGMCCNO.5714 produces.
In another preference, described in step (1), after the antibody contacts described in sample and fifth aspect present invention, in reaction system, add the 3rd antibody of anti-described first antibody again, and detect the formation of " antigen-first antibody-three antibody " mixture in step (2).
In another preference, with detectable label on described first antibody, described second antibody or described 3rd antibody.
In another preference, described detectable label is biotin labeling, colloid gold label, horseradish peroxidase-labeled, radioisotope labeling, fluorescein-labelled.
In another preference, described sample comprises: human or animal tissues sample, tumor resection sample, cast-off cells sample.
In another preference, described method is used for the object of nondiagnostic.
A fourteenth aspect of the present invention, provides a kind of check-out console, and described check-out console comprises substrate (back up pad) and test strip, and described test strip contains the antibody described in fifth aspect present invention or the immune conjugate described in sixth aspect present invention.
In another preference, described test strip is also containing antigen point sample district.
In another preference, described test strip overlaps successively form by filtering sample paper, chromatographic material, nitrocellulose filter and thieving paper.
A fifteenth aspect of the present invention, provides a kind of test kit, and described test kit comprises:
(1) first container, containing the antibody described in fifth aspect present invention in described first container; And/or
(2) second container, resists containing two of the antibody described in anti-fifth aspect present invention in described second container; And/or
(3) the 3rd containers, containing cell cracking agent in described 3rd container;
Or,
Described test kit contains the check-out console described in the present invention the tenth four sides.
In another preference, the antibody in described first container is with detectable label.
In another preference, the antibody in described second container is with detectable label.
A sixteenth aspect of the present invention, provide a kind of preparation method preparing recombinant polypeptide, the method comprises:
A () under conditions suitable for the expression, cultivates the host cell described in ninth aspect present invention;
B () isolates recombinant polypeptide from culture, described recombinant polypeptide is the antibody described in fifth aspect present invention or the recombinant protein described in sixth aspect present invention.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 is HPV18E7 monoclonal antibody MAB-MAB-F2 antigen-binding specificity ELISA detected result, and specific experiment details is shown in embodiment 1, and 2.2 trifles: MAB-F2 can be special in conjunction with HPV18E7 recombinant protein with HPV16E7 recombinant protein without combination.
Fig. 2 is HPV18E7 monoclonal antibody conjugated antigen epitope amino acid sequence region ELISA detected result, and specific experiment details is shown in embodiment 1,2.3 trifles: Fig. 2 A is HPV18E7 aminoacid sequence schematic diagram, and Fig. 2 B is ELISA detected result figure.MAB-F2 can in conjunction with His-HPV18E7 recombinant protein and polypeptide HPV18E7-1 and HPV18E7-2, and not Binding peptide His-HPV16E7 recombinant protein.
Fig. 3 is HPV18E7 monoclonal antibody protein binding affinity assays ELISA detected result figure, specific experiment details is shown in embodiment 1,2.4 trifles: His-HPV18E7 bag is respectively 8 μ g/ml, 2 μ g/ml, 0.5 μ g/ml, 0.125 μ g/ml by concentration, MAB-F2 is hatched concentration and is done doubling dilution from 8 μ g/ml, totally seven gradients, substitute into K
aff=(n-1)/2 (n [Ab ']-[Ab]), calculate six K values altogether, average, and finally calculate MAB-F2 protein level avidity is 2.67nM.
Fig. 4 is the result figure adopting HPV18E7 monoclonal antibody MAB-F2 Hela cellular endogenous HPV18E7 to be carried out to IP-WB detection, and specific experiment details is shown in embodiment 1, and 2.5 trifles: mIgG is that negative control can not be combined by the HPV18E7 in tumor cell lysis liquid.MAbMAB-F2 can be combined by the HPV18E7 in tumor cell lysis liquid, therefore has obvious band at 15KDa place.Wherein the band of about 50KDa and 25KDa is the heavy chain and the light chain that use antibody in IP process.
Fig. 5 is HPV18E7 monoclonal antibody immunocytochemical stain detected result figure, specific experiment details is shown in embodiment 1,2.6 trifles: A is C-33A cell dyeing result, B is Hela cell dyeing result, C is CaSki cell dyeing result, the strong staining reaction that monoclonal antibody MAB-F2 and Hela cell have, and with C-33A cell and CaSki cell dye-free.
Fig. 6 is the immunocytochemical stain result figure that the cervical cancer cell lines C-33A that fixes of different stationary liquid and Hela cell adopt the dyeing of HPV18E7 monoclonal antibody specific MAB-F2; Specific experiment details is shown in embodiment 2; C-33A cell and HPV18E7 monoclonal antibody MAB-F2 are without immune response; After two kinds of cytomixis, HPV18E7 monoclonal antibody MAB-F2 makes to account for the Hela look (red arrow place) of total cellular score 10% and negative cells C-33A dye-free; Suspend fixing 100% Hela cell and HPV18E7 monoclonal antibody MAB-F2 have strong immune response.4% paraformaldehyde and LBC stationary liquid on the specificity of HPV18E7 monoclonal antibody MAB-F2 in ICC detects without impact.
Embodiment
The present inventor, by extensive and deep research, through screening in a large number, unexpectedly obtains a kind of anti-HPVE7 monoclonal antibody MAB-F2, experimental result shows, should for the monoclonal antibody of HPVE7 albumen, and specificity is high, avidity is strong, significantly can distinguish the HPV18E7 albumen in cell.This monoclonal antibody can combine with HPV18E7 albumen specifically, but with HPV16E7 albumen without binding activities, therefore this antibody can not only be used for detecting HPV18E7 albumen, can also be used for differentiating different HPV types.Present invention also offers the method detecting and/or identify HPV18E7 albumen, the method good stability, detection sensitivity is high.Present invention also offers the test kit and check-out console that comprise above-mentioned antibody.
Particularly, the present invention adopts restructuring GST-HPV18E7 fusion protein immunization mouse, uses another kind of fusion rotein His-HPV18E7 as selective mechanisms antigen.Filter out Positive hybridoma clones strain, all can secretory antibody IgG, and all can in conjunction with recombinant protein His-HPV18E7, in specific binding HPVE7 fusion rotein in ELISA detects.
Except ELISA is in conjunction with the qualification of recombinant protein antigen, positive monoclonal antibody further across antigen in conjunction with epitope analysis, Affinitybindingcharacterization, WesternBlot, immunoprecipitation, immunocytochemical stain (ImmunocytochemistryICC), tissue slice immunostaining (ImmunohistochemistryIHC) method is identified.By above qualification test, 1 strain antibody clone strain MAB-F2 have passed survey requirement, shows protein molecular level, cell levels, organizes the function of horizontal specific binding high-risk HPV E7 cancer protein.
For filtering out the antibody reagent having Clinical Laboratory and be worth, this monoclonal antibody is again further across the detection experiment for cervical cancer cell lines.Finally obtaining a strain can the monoclonal antibody of the positive cervical epithelial cells of specific recognition HPV18E7, specificity based on this antibody develops can distinguish epithelium of cervix uteri cancer cells and Normocellular method, these methods are enough to identify those early cervical carcinomas patient with regard to sensitivity, are enough to distinguish normal and malignant cell with regard to specificity.
Of the present invention one preferred embodiment in, the aminoacid sequence of described HPV16E7 albumen is as follows:
HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE
DEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP(SEQIDNO.:1)。
Of the present invention one preferred embodiment in, the aminoacid sequence of described HPV18E7 albumen is as follows:
MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQIDNO.:2)。
As used herein, term " antibody " or " immunoglobulin (Ig) " have about 150000 of same structure feature daltonian different four glycan albumen, and it is made up of the heavy chain (H) that two identical light chains (L) are identical with two.Every bar light chain is connected with heavy chain by a covalent disulfide bonds, and the disulfide linkage number difference between the heavy chain of different Immunoglobulin Isotype.The intrachain disulfide bond at every bar heavy chain and light chain also regular interval.There is variable region (VH) one end of every bar heavy chain, is thereafter multiple constant region.There is variable region (VL) one end of every bar light chain, and the other end has constant region; The constant region of light chain is relative with first of heavy chain constant region, and the variable region of light chain is relative with the variable region of heavy chain.Special amino-acid residue forms interface between light chain and the variable region of heavy chain.
As used herein, term " variable " represents that some part of variable region in antibody is different in sequence, which forms various specific antibodies to the combination of its specific antigen and specificity.But mutability is not evenly distributed in whole antibody variable region.It concentrates in light chain and variable region of heavy chain in three fragments be called in complementary determining region (CDR) or hypervariable region.Part comparatively conservative in variable region is called framework region (FR).Each self-contained four FR districts in the variable region of native heavy and light chain, they in beta sheet configuration, are connected by three CDR forming shack haply, in some cases can forming section β-pleated sheet structure structure.CDR in every bar chain is closely close together by FR district and together form the antigen-binding site (see Kabat etc., NIHPubl.No.91-3242, volume I, 647-669 page (1991)) of antibody with the CDR of another chain.Constant region does not participate in the combination of antibody and antigen directly, but they show different effector functions, such as, participate in the cytotoxicity depending on antibody of antibody.
" light chain " of vertebrate antibodies (immunoglobulin (Ig)) can be classified as the class in visibly different two classes (being called κ and λ) according to the aminoacid sequence of its constant region.According to the aminoacid sequence of its CH, immunoglobulin (Ig) can be divided into different kinds.Mainly contain 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can be divided into subclass (isotype) further, as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.CH corresponding to inhomogeneity immunoglobulin (Ig) is called α, δ, ε, γ and μ.The subunit structure of inhomogeneity immunoglobulin (Ig) and 3-d modelling are known by those skilled in the art.
As used herein, term " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from the colony that a class is substantially homogeneous, and the single antibody namely comprised in this colony is identical, except the sudden change of the natural generation that may exist except minority.Monoclonal antibody is with high specificity for single antigen site.And different from conventional polyclonal antibody preparation (normally having the different antibodies for different determinant), each monoclonal antibody is for the single determinant on antigen.Except their specificity, the benefit of monoclonal antibody is also that they are cultivated by hybridoma and synthesizes, and can not be polluted by other immunoglobulin (Ig).Modifier " mono-clonal " illustrates the characteristic of antibody, is to obtain from substantially homogeneous antibody population, and this should not be construed as needing to produce antibody with any special methods.
The present invention also comprises the monoclonal antibody of the corresponding aminoacid sequence with described anti-HPV18E7 protein monoclonal antibody, has the monoclonal antibody of described anti-HPV18E7 protein monoclonal antibody variable region chain, and has other protein of these chains or protein conjugate and fusion expressed product.Particularly, the present invention includes and have containing hypervariable region (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (i.e. immune conjugate and fusion expressed product), as long as this hypervariable region is identical with the hypervariable region of heavy chain with light chain of the present invention or at least 90% homology, preferably at least 95% homology.
As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: that medicine, toxin, cytokine (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule are combined with described HPV16E7 protein monoclonal antibody or its fragment and the conjugate that formed.The present invention also comprises the cell surface marker thing or antigen that are combined with described anti-HPV18E7 protein monoclonal antibody or its fragment.
The present invention not only comprises complete monoclonal antibody, also comprises and has immunocompetent antibody fragment, as Fab or (Fab')
2fragment; Heavy chain of antibody; Light chain of antibody.
As used herein, term " variable region of heavy chain " and " V
h" be used interchangeably.
As used herein, term " variable region " and " complementary determining region (complementaritydeterminingregion, CDR) " are used interchangeably.
Of the present invention one preferred embodiment in, the variable region of heavy chain of described antibody comprises following three complementary determining region CDR:
CDR1, its aminoacid sequence is GFTFNRYD (SEQIDNO.:4), and its coding nucleotide sequence is, ggattcactttcaatagatatgac (SEQIDNO.:3);
CDR2, its aminoacid sequence is IRNDGSKT (SEQIDNO.:6), and its coding nucleotide sequence is, atcagaaatgatggtagtaagact (SEQIDNO.:5);
CDR3, its aminoacid sequence is TRDSYGTVFAY (SEQIDNO.:8), and its coding nucleotide sequence is, acaagggactcctatggtaccgtgtttgcttac (SEQIDNO.:7).
In another preference, the aminoacid sequence of described variable region of heavy chain is:
LVQSGGGSVKPGGSLKLSCAASGFTFNRYDMSWFRQSPEKRLEWVAEIRNDGSKTHYSDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCTRDSYGTVFAYWGQGTLVTVSA(SEQIDNO.:10)；
Its coding nucleotide sequence is:
Ctggtgcagtcagggggaggctcagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcaatagatatgacatgtcttggtttcgccagtctccagagaagaggctggagtgggtcgcagagatcagaaatgatggtagtaagactcactattcagacactgtgacgggccgattcaccatctccagagacaatgccaagaacaccctgtacctggaaatgagcagtctgaggtctgaggacacggccatgtattactgtacaagggactcctatggtaccgtgtttgcttactggggccaagggactctggtcactgtctctgcag(SEQIDNO.:9)。
Of the present invention one preferred embodiment in, the heavy chain of described antibody comprises above-mentioned variable region of heavy chain and CH, and described CH can be mouse source or people source.
As used herein, term " variable region of light chain " and " VL " are used interchangeably.
Of the present invention one preferred embodiment in, according to the variable region of light chain of antibody of the present invention, there is the complementary determining region CDR being selected from lower group:
CDR1', its aminoacid sequence is QSLLYSNGKTY (SEQIDNO.:14), and its coding nucleotide sequence is, cagagcctcttatatagtaatggaaaaacctat (SEQIDNO.:13);
CDR2', its aminoacid sequence is LVS (SEQIDNO.:16), and its coding nucleotide sequence is, ctggtgtct (SEQIDNO.:15)
CDR3', its aminoacid sequence is VQGTHFPQT (SEQIDNO.:18), and its coding nucleotide sequence is, gtgcaaggtacacattttcctcagacg (SEQIDNO.:17)
In another preference, the aminoacid sequence of described variable region of light chain is:
VMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPQTFGGGTKLEIK(SEQIDNO.:20)，
Its coding nucleotide sequence is:
gtcatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagtcaagtcagagcctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaatcagcagagtggaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctcagacgttcggtggaggcaccaagctggaaatcaaac(SEQIDNO.:19)。
Of the present invention one preferred embodiment in, the light chain of described antibody comprises above-mentioned variable region of light chain and constant region of light chain, and described constant region of light chain can be mouse source or people source.
In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " are used interchangeably, all refer to the antibody of specific binding HPV18E7 albumen, such as, there is albumen or the polypeptide of variable region of heavy chain (aminoacid sequence as SEQIDNO.:10) and/or variable region of light chain (aminoacid sequence as SEQIDNO.:20).They can contain or not contain initial methionine.
In another preference, described antibody is mouse or the people mouse chimeric mAb of anti-HPV18E7 albumen, and its CH and/or constant region of light chain can be humanized CH or constant region of light chain.More preferably, described humanized CH or constant region of light chain are CH or the constant region of light chain of human IgG1, IgG2 etc.
Present invention also offers other protein or fusion expressed product with antibody of the present invention.Particularly, the present invention includes and have containing the heavy chain of variable region and any protein of light chain or protein conjugate and fusion expressed product (i.e. immune conjugate and fusion expressed product), as long as this variable region is with the heavy chain of antibody of the present invention and the variable region of light chain is identical or at least 90% homology, preferably at least 95% homology.
Generally, the antigenic binding property of antibody can be described by 3 the specific regions being positioned at heavy chain and variable region of light chain, be called Variable Area (CDR), this is intersegmentally divided into 4 frame areas (FR), the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texture, and the β-pleated sheet structure formed by FR is therebetween close to each other on space structure, and the CDR on the CDR on heavy chain and corresponding light chain constitutes the antigen binding site of antibody.FR or the CDR region that has been which Amino acid profile can be determined by the aminoacid sequence of antibody more of the same type.
The heavy chain of antibody of the present invention and/or the variable region of light chain interesting especially because relate to conjugated antigen at least partly in them.Therefore, the present invention includes those and there is the band monoclonal antibody light chain of CDR and the molecule of variable region of heavy chain, as long as the homology that its CDR and the CDR identified have more than 90% (preferably more than 95%, best more than 98%) herein.
The present invention not only comprises complete monoclonal antibody, also comprises the fusion rotein that the fragment or antibody with immunocompetent antibody and other sequences are formed.Therefore, the present invention also comprises the fragment of described antibody, derivative and analogue.
As used herein, term " fragment ", " derivative " and " analogue " refer to the polypeptide substantially keeping biological function that antibody of the present invention is identical or activity.Polypeptide fragment of the present invention, derivative or analogue can be the polypeptide that (i) has one or more conservative or non-conservative amino acid residue (preferred conservative amino acid) and be substituted, and the amino-acid residue of such replacement can may not be and encoded by genetic code, or (ii) has the polypeptide of substituted radical in one or more amino-acid residue, or (iii) mature polypeptide and another compound (such as extend the compound of polypeptide transformation period, such as polyoxyethylene glycol) merge the polypeptide formed, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide formed (as leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying or proprotein sequence, or the fusion rotein to be formed with 6His label).According to instruction herein, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
Antibody of the present invention refers to polypeptide that have HPV18E7 protein binding activity, that comprise above-mentioned CDR district.This term also comprise have with antibody identical function of the present invention, the variant form of the polypeptide that comprises above-mentioned CDR district.These variant forms comprise (but being not limited to): one or morely (be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally within 20, within being preferably 10, within being more preferably 5) amino acid.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal and/or N-terminal the function that or several amino acid also can not change protein usually.This term also comprises active fragments and the reactive derivative of antibody of the present invention.
The variant form of this polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, the albumen coded by DNA can hybridized with the coding DNA of antibody of the present invention under high or low stringency condition and the polypeptide utilizing the antiserum(antisera) of anti-antibody of the present invention to obtain or albumen.
Present invention also offers other polypeptide, as comprised the fusion rotein of people's antibody or its fragment.Except the polypeptide of almost total length, present invention includes the fragment of antibody of the present invention.Usually, this fragment have antibody of the present invention at least about 50 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
In the present invention, " conservative variant of antibody of the present invention " refers to compared with the aminoacid sequence of antibody of the present invention, has 10 at the most, preferably at the most 8, more preferably at the most 5, best at the most 3 amino acid replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out amino acid replacement according to Table A and produce.
Table A
Present invention also offers the polynucleotide molecule of encoding such antibodies or its fragment or its fusion rotein.Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can the varient of or degeneracy identical with the coding region sequence shown in SEQIDNO.:3,5,7,9,13,15,17,19.As used herein, " varient of degeneracy " refer in the present invention coding there is the aminoacid sequence identical with polypeptide of the present invention, but with the differentiated nucleotide sequence of coding region sequence shown in SEQIDNO.:3,5,7,9,13,15,17,19.
The polynucleotide of mature polypeptide of the present invention of encoding comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional coding sequence; The encoding sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide comprising encoding such peptides, also can be the polynucleotide also comprising additional code and/or non-coding sequence.
The invention still further relates to and above-mentioned sequence hybridization and have at least 50% between two sequences, preferably at least 70%, the more preferably polynucleotide of at least 80% homogeny.The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions.In the present invention, " stringent condition " refers to: (1) compared with the hybridization under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, 0.1%SDS, 60 DEG C; Or be added with denaturing agent during (2) hybridization, and as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) homogeny only between two sequences, at least more than 90%, is just hybridized when being more preferably more than 95%.Further, the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in SEQIDNO.:10 and/or SEQIDNO.:20.
The Nucleotide full length sequence of antibody of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.Feasible method synthesizes a relevant sequence, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.In addition, also the encoding sequence of heavy chain and expression label (as 6His) can be merged, form fusion rotein.
Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.The biomolecules that the form that biomolecules (nucleic acid, albumen etc.) involved in the present invention comprises being separated exists.
At present, the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof) can be obtained completely by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA molecular (or as carrier) as known in the art and cell.In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.
The invention still further relates to the carrier comprising above-mentioned suitable DNA sequence dna and suitable promotor or control sequence.These carriers may be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell is as yeast; The insect cell of fruit bat S2 or Sf9; The zooblast etc. of CHO, COS7,293 cells.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods as microinjection, electroporation, liposome packaging etc.
The transformant obtained can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.
Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cytolemma.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Antibody of the present invention can be used alone, and also can modify built up section or the coupling of part or any these materials above with detectable (for diagnostic purpose), therapeutical agent, PK (protein kinase).
Detectable for diagnostic purpose includes but not limited to: fluorescence or luminous marker, radioactively labelled substance, MRI (nuclear magnetic resonance) or CT (CT technology) contrast medium, maybe can produce the enzyme that can detect product.
Can include but not limited to the therapeutical agent of antibodies of the present invention or coupling: 1. radionuclide (Koppe etc., 2005, metastasis of cancer comment (Cancermetastasisreviews) 24,539); 2. biological poison (Chaudhary etc., 1989, nature (Nature) 339,394; Epel etc., 2002, Cancer Immunol and immunotherapy (CancerImmunologyandImmunotherapy) 51,565); 3. cytokine is as (Gillies etc., 1992, institute of NAS periodical (PNAS) 89,1428 such as IL-2; Card etc., 2004, Cancer Immunol and immunotherapy (CancerImmunologyandImmunotherapy) 53,345; Halin etc., 2003, cancer research (CancerResearch) 63,3202); 4. gold nano grain/nanometer rod (Lapotko etc., 2005, cancer communication (Cancerletters) 239,36; Huang etc., 2006, U.S. chemical institute magazine (JournaloftheAmericanChemicalSociety) 128,2115); 5. virion (Peng etc., 2004, gene therapy (Genetherapy) 11,1234); 6. liposome (Mamot etc., 2005, cancer research (Cancerresearch) 65,11631); 7. magnetic nanosphere; 8. pro-drug activation enzymes (such as, DT-diaphorase (DTD) or xenyl lytic enzyme-sample protein (BPHL)); 10. chemotherapeutics (such as, 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 above-mentioned antibody or its active fragments or its fusion rotein, and pharmaceutically acceptable carrier.Usually, but these materials are formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can with being formulated the character of material and illness to be treated and changing to some extent.The pharmaceutical composition prepared can carry out administration by conventional route, comprising (but being not limited to): knurl is interior, intraperitoneal, intravenously or topical.
Pharmaceutical composition of the present invention can be directly used in conjunction with HPV16E7 protein molecular, thus can be used for prevention and therapy tumour.In addition, also can use other treatment agent simultaneously.
Pharmaceutical composition of the present invention contains safe and effective amount (as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) of the present invention and pharmaceutically acceptable carrier or vehicle.This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.Pharmaceutical composition such as injection, solution should aseptically manufacture.The dosage of activeconstituents is treatment significant quantity, such as every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use together with other treatment agent.
When making pharmaceutical composition, that the immune conjugate of safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and be in most of the cases no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Hybridoma cell strain
Present invention also offers the hybridoma cell strain can produced and the present invention is directed to HPVE7 protein monoclonal antibody; Preferably, the invention provides the hybridoma cell strain for HPV18E7 protein monoclonal antibody of high-titer.
After obtaining the hybridoma producing HPVE7 protein monoclonal antibody of the present invention, those skilled in the art can utilize this hybridoma cell strain Dispersal risk easily.In addition, those skilled in the art also can know the structure (variable region of heavy chain of such as antibody and variable region of light chain) of antibody of the present invention easily, then prepare monoclonal antibody of the present invention by recombination method.
The preparation of monoclonal antibody
Antibody of the present invention can be prepared by the various technology that those skilled in that art are known.Such as, antigen of the present invention, can be applied to animal to induce the generation of monoclonal antibody.For monoclonal antibody, hybridoma technology can be utilized prepare (see people such as Kohler, Nature256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, InMonoclonalAntibodiesandTCellHybridomas, Elsevier, N.Y., 1981) or available recombinant DNA method (U.S. Patent number 4,816,567) preparation.
Representational myeloma cell is effective integration, support that the stable high level of antibody produces by the antibody produced cell selected, and those myeloma cells responsive to substratum (HAT medium matrix), comprise myeloma cell strain, the myeloma cell strain of such as muroid, the myeloma cell strain comprised derived from MOPC-21 and MPC-11 mouse tumor (can purchased from SalkInstituteCellDistributionCenter, San Diego, California, the U.S.) and SP-2, NZ0 or X63-Ag8-653 cell (can purchased from AmericanTypeCultureCollection, Luo Keweier, Maryland, the U.S.).Human myeloma and mouse-human heteromyeloma's cell strain have also been described for generation of human monoclonal antibodies [Kozbor, J.Immunol., 133:3001 (1984); Brodeur etc., the production technology of monoclonal antibody and application (MonoclonalAntibodiesProductionTechniquesandApplications), 51-63 page (MarcelDekker, Inc., New York, 1987)].
Growth of Hybridoma Cell is detected to the generation with required specific monoclonal antibody in substratum analysis wherein, as, by external binding analysis such as, Enzyme Linked Immunoadsorbent Assay (ELISA) or radioimmunoassay (RIA).The position of expressing the cell of antibody can be detected with FACS.Then, hybridoma clone can be formed subclone (subcloned) by limiting dilution procedures, and by standard method growth (Goding, monoclonal antibody (MonoclonalAntibodies): principle and putting into practice (PrinciplesandPractice), AcademicPress (1986) 59-103 page).The substratum be applicable to used to reach this purpose comprises, such as, DMEM or RPMI-1640 substratum.In addition, hybridoma can grow as ascitic tumor in animal body.
The monoclonal antibody of being secreted by subclone is suitably separated by conventional immunoglobulin purification technique from substratum, ascites or serum, these purifying process are such as, Protein A-agarose method (proteinA-Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The invention provides a kind of monoclonal antibody for HPVE7 albumen, particularly for the monoclonal antibody of HPV16E7 albumen.In a preferred scheme of the present invention, monoclonal antibody adopts cultivates hybridoma method preparation.Get the supernatant liquor of Hybridoma Cell Culture, slightly propose IgG through saturated ammonium sulphate method, then by the antibody slightly carried through affinity column (ProteinG-Sephrose) purifying.
In a preferred scheme of the present invention, monoclonal antibody adopts the method preparation of Balb/C mouse ascites manufacture order clonal antibody.Be inoculated in the mouse peritoneal of sensitization by about hybridoma, in 2-4 week, visible belly obviously swells.Extract ascites, after saturated ammonium sulphate method is slightly carried, then by the antibody slightly carried through affinity column (ProteinG-Sephrose) purifying.
The immunoglobulin (Ig) of mark
In a preference of the present invention, described immunoglobulin (Ig) is with detectable.More preferably, described marker is selected from lower group: colloid gold label thing, colored labels or fluorescent marker.
Colloid gold label can adopt method known to those skilled in the art to carry out.In a preferred scheme of the present invention, the monoclonal antibody colloid gold label of HPV18E7 albumen, obtains the monoclonal antibody of colloid gold label.
HPV18E7 protein monoclonal antibody of the present invention has good specificity, and very high tires.
Check-out console and material thereof
The check-out console material that check-out console of the present invention can adopt this area conventional, adopts conventional check-out console preparation method to make.
The present invention detects the plate for detecting immunity of HPV18E7 albumen, comprises the back up pad of test strip and support test strip, as adopted PVC polyester offset plate etc.; Described test strip overlaps successively form by filtering sample paper, chromatographic material, nitrocellulose filter and thieving paper, and overlapping part can adopt conventional method, as adhesive tape etc. is fixedly connected with; Wherein: the HPVE7 protein monoclonal antibody of the pre-coated colloid gold label of chromatographic material or coloured label or polyclonal antibody, preferably by the HPVE7 protein monoclonal antibody of colloid gold label, nitrocellulose filter adsorbs detection line and nature controlling line;
In a preferred scheme: on chromatographic material, the HPVE7 protein monoclonal antibody of pre-coated colloid gold label adopts concentration to be that the HPVE7 protein monoclonal antibody solution of 0.5-1.5mg/ml colloid gold label carries out pre-coated, and package amount is 50 μ l/cm
2; Preferred concentration is 0.5 or 1.5mg/ml, 50 μ l/cm
2;
Detection method and result judge
Keep flat check-out console, sample is dropped on filter sample paper, in sample about 120 μ l, 3 ~ 5min, observe tomographic results.Fringe position according to occurring carrys out judged result.
Negative: obvious colour band all appears in quality control region, detection zone, shows for feminine gender;
Positive: only to occur obvious colour band in quality control region, and in detection zone without colour band, show for the positive;
Invalid: quality control region, detection zone is without any colour band or do not occur colour band in quality control region and occur colour band in detection zone, show detection method mistake or check-out console rotten or lost efficacy, again should exchange check-out console for and detect.
Method and sample
The present invention relates to for the method for the pattern detection cervical cancer of cell and/or histolysis.The method step is roughly as follows: obtain cell and/or tissue samples; Sample is dissolved in media as well; Detect the level of HPV cancer protein in the sample of described dissolving.The sample that the inventive method uses can be any sample comprising cell be present in cell-preservation liquid, as what use in liquid basal cell detection method.
The present invention may be used for the detection that HPV infects HPV cancer protein in associated cancer, wherein HPV infects the tumour of the urogenital systeies such as relevant cancer such as cervical cancer, bladder cancer, carcinoma of endometrium, penile cancer, the preliminary stage of small cell lung cancer, melanoma and H/N tumors and these cancers.
According to the present invention, use HPV cancer protein molecule marker can support or even replace cytology and/or Histologic Examination Method.In special case, protein molecular mark can be used as diagnostic tool and not need further based on the support of the Morphology observation of cell.When not supporting based on cellular informatics, only in protein molecular level, be limited to case to the diagnostic method of cancer, wherein the level of mark or mark should have specificity to the situation that will detect.Biomarker of the present invention is HPV cancer protein, and this biomarker derives from virus, because the mark characteristic that in tissue, virus exists can not occur in the people's tissue do not infected, therefore can complete in sample solution the detection that HPV infects.
The sample (sample) adopted in the present invention comprises cell, tissue samples and biopsy specimen.The term " biopsy " that the present invention uses should comprise the biopsy of all kinds well known by persons skilled in the art.Therefore the excision sample that the biopsy used in the present invention can comprise such as tumour, the tissue samples prepared by puncture or the needle puncture biopsy of endoscopic procedures or organ.
The sample used in the present invention can comprise cell or tissue sample that is fixing or that preserve.Cell or tissue sample can such as be stored in the sample collection of standard, storage or conveying medium, and the known business of such as those skilled in the art can obtain preserves medium (formalin, Cytyc " PreservCyt " or TripathImaging " Cytorich " etc.).Suitable cell preserving medium can comprise one or more mixture for preserving cellular component being selected from alcohol, aldehyde, ketone, acid, metal ion or mercury, ether etc.Alcohol comprises methyl alcohol, ethanol, (just or different) propyl alcohol, (just, XOR uncle) butanols or high side chain or unbranched alcohol.Aldehyde comprises formaldehyde, acetaldehyde, glutaraldehyde etc.Also the ketone of such as acetone can be used.The acid used in the sample medium of standard comprises the mineral acid of organic acid (acetic acid, trichoroacetic acid(TCA), Whitfield's ointment and picric acid) or such as chromic acid.Metal such as silver, copper, chromium, mercury, osmium and uranium can be comprised in the sample solution of standard.The such as salts solution of uranyl acetate, two potassiumchromates, ammonium sulfate etc. can be the component of preserving medium.
In addition, the sample of lysis is carried out after acquisition can being used in method disclosed herein immediately.Sample carries out cracking after obtaining immediately, and morphologic information is lost in the process, and the protein molecular information of sample is saved.Sample can directly be transferred to the solution containing suitable washing agent and preservatives from the body of individuality.In cracking medium, use suitable reagent, raw-material molecular components can be preserved, and do not degrade.Such as by using enzyme inhibitors but enzyme activity minimum degradation.Therefore, the solution of the detection sample in this cracking medium can show the protein molecular characteristic detecting sample when dissolving.
According to the present invention, sample can be dissolved in any suitable cracking medium.This cracking medium can be such as urea, methane amide, the aqueous solution of washing agent, such as teepol is (as SDS, N-dodecanol creatine sodium, sodium deoxycholate, alkylaryl sulfonate, long-chain (aliphatics) alcohol sulfate, olefin sulphates and sulfonate, alpha-olefin vitriol and sulfonate, vitriol monoglyceride, sulfuric acid ether, thio succinate, chain alkyl sulfonate, phosphoric acid ester, the different thiosulphate of alkyl, sucrose ester), cationic detergent (as, cetyltrimethylammonium chloride), non-ionic octoxynol detergent (as, polysorbas20, NonidetP-40, TritonX-100, NP-40, lgepalCA-630, N-octyl group-glycoside) or both sexes washing agent (as, CHAPS, 3-dodecyl-dimethyl amine-propane-1-sulfonic acid, dodecanol dimethylamine oxide) and/or hydroxide bases, such as sodium hydroxide or potassium hydroxide.Usual any suitable liquid can be used as the solvent of cracking medium of the present invention.Liquid can be organic or inorganic, and can be neat liquid, and liquid mixture or the liquid containing substance solution also can containing other materials to strengthen the character of solvent.In embodiments of the invention, the formula of cracking medium is 50mMTris-HCl, 150mMNaCl, 1%NP-40,0.5% Sodium desoxycholate, 0.1%SDS.
One or more reagent preventing component degradation in starting material can be contained further for the cracking medium dissolving sample according to the present invention.This component such as comprises enzyme inhibitors, such as proteinase inhibitor.In embodiments of the invention, sample By Direct Pyrolysis.Proteinase inhibitor such as can comprise serpin, cystatin, aspartic protease inhibitor, inhibitors of metalloproteinase, acid protease inhibitor, alkaline protease inhibitor or calpastatin.In embodiments of the invention, what proteinase inhibitor adopted is Pepstatin (pepstatin), Leupeptin (bright inhibiting peptide), (aprotinin) and 100 μ g/mlPMSF (phenylmethylsulfonyl fluoride).
Test kit
Present invention also offers the test kit that one refers to containing antibody of the present invention (or its fragment) or check-out console of the present invention, in a preference of the present invention, described test kit also comprises container, working instructions, buffer reagent etc.
The present invention is designed for the detection kit detecting HPV cancer protein level further, this test kit comprises the antibody identifying HPV cancer protein, for dissolving the cracking medium of sample, the common reagent needed for detection and damping fluid, as various damping fluid, certification mark, detection substrate etc.Described antibody is anti-HPVE7 antibody preferably, is more preferably anti-HPV16E7 antibody.This detection kit can be in-vitro diagnosis device.
The present invention designs and develops the test kit for infecting correlation circumstance diagnostic assessment to the HPV from solution sample further, this test kit can detect the HPV cancer protein be present in sample solution, and the cell-preservation liquid wherein preserving sample can be the cell-preservation liquid in such as liquid basal cell detection method.By cytolysis in suitable cracking medium, and be used to develop at the detection kit detected based on the infection of the HPV to the sample from the dissolving related neoplasms on acellular analysis foundation and in-vitro diagnosis device.
An object of the present invention is to provide a kind of method detecting HPV18E7 protein expression, and described method can be used for detecting the detection that HPV infects associated cancer particularly cervical cancer.
The present inventor etc. have made the monoclonal antibody MAB-F2 for human papillomavirus HPV18E7 protein and have studied its reactivity, the antigen of this monoclonal antibody is between amino acid 44-58 position in conjunction with epitope peptide, comprising aminoacid sequence VNHQHLPARRAEPQR.This anti-HPV18E7 monoclonal antibody MAB-F2 is used to carry out immunocytochemical stain to the Human cervical cancer cell lines CaSki of cervical cancer cell lines C-33A and expression HPV16E7 not expressing HPV and the human cervical carcinoma cell lines Hela of expression HPV18E7, found that: MAB-F2 and HPV18E7 positive cell Hela shows strong staining reaction, with HPV16E7 positive cell CaSki and the C-33A cell response not expressing HPV albumen.
And then the anti-HPV18E7 monoclonal antibody MAB-F2 made by the present inventor etc. use carries out immunocytochemical stain to the cervical cancer tumer line that 4% paraformaldehyde or LBC stationary liquid fix a few days.The expression of HPV18E7 is not detected in the human cervical carcinoma cell lines C-33A not containing HPVDNA, when after Hela cell and C-33A cell in proportion (1:9) mixing, when positive tumor cell ratio is low to moderate 10%, by detecting the HPV18E7 of inside tumor cells, MAB-F2 also can detect the existence of positive tumor cell specifically, and two kinds of stationary liquids on the judgement of positive and negative findings without impact.According to this discovery result, the present inventor completes the present invention.
That is, method of the present invention detects the method for tumor marker, it is characterized in that: the step comprising the HPV18E7 detected in sample.
In the method for the invention, described detection sample is preferentially the section of culture maybe this tissue of cast-off cells, maybe this tissue gathered from a corpse or other object for laboratory examination and chemical testing, also can be the suspension cell prepared by the culture organizing maybe this tissue gathered from a corpse or other object for laboratory examination and chemical testing.In addition, described cell preferably cervical exfoliated cell.
In method of the present invention, a described corpse or other object for laboratory examination and chemical testing preferably likely suffers from the patient of cervical lesions, also can be the patient that pathology has occurred uterine neck.
Described HPV18E7 is preferably HPV18E7 protein or its fragment.When this situation, the step of the HPV18E7 described in detection preferably uses the immunocytochemical stain analysis of HPV18E7.The anti-HPV18E7 antibody used is anti-HPV18E7 monoclonal antibody preferably.
The reliability index occurred as the pernicious or pre-malignant cells that HPV18 is correlated with from the albumen of HPV18E7 oncogene expression that described method immunodetection adopts.One of aspect that the present invention is the most useful is infecting the application in the diagnosis of relevant any epithelial cell exception to cervical cancer, squamous cell and gland cancer and to carcinogenic HPV18, and shown carcinogenic HPV18 infects and comprises Koilocytosis; Hyperkeratosis; Comprise illness precancer of intraepithelial neoplasia formation or intraepithelial lesions; Height dysplasia; Go or malignant cancer with infecting.Except cervical cancer, to the detection of HPV18E7 to the tumour detecting the urogenital systeies such as bladder cancer, carcinoma of endometrium, penile cancer, small cell lung cancer, melanoma and H/N tumors are also useful.
Another object of the present invention provides a kind of detection kit by method of the present invention.This test kit can be diagnostic kit or research kit.
Test kit of the present invention is the test kit detecting tumor marker, it is characterized in that having anti-HPV18E7 monoclonal antibody.Test kit of the present invention is preferentially also have to detect required common reagent and damping fluid, as various damping fluid, certification mark, detection substrate etc.Described antibody is anti-HPV18E7 antibody preferably, be more preferably anti-HPV18E7 monoclonal antibody, the little mouse-anti HPV18E7 monoclonal antibody especially preferably produced by hybridoma MAB-F2 or the monoclonal antibody of the binding activities equal with this little mouse-anti HPV18E7 monoclonal antibody tool.
The invention provides a kind of method and distinguish the tumour cell that HPV18E7 infects and the tumour cell not containing HPVDNA by the HPV18E7 albumen detecting cellular endogenous.This method still can be detected accurately based on when accounting for 10% of total cellular score when HPV18E7 positive tumor cell, and after cell is fixing in clinical extensive employing LBC stationary liquid, still can accurately detect positive cell, thus can cancer develop make diagnosis in early days, in time treatment foundation is provided.
Further, the present invention also provides a kind of detection kit adopting this detection method to be formed.
Major advantage of the present invention is:
(1) antibody for HPV18E7 albumen provided by the invention, specificity is high, and avidity by force, and can be prepared in a large number, monoclonal anti weight easily controls.
(2) antibody for HPV18E7 albumen provided by the invention can specificly combine with HPV18E7 albumen, but not with HPV16E7 protein binding, therefore this antibody can not only be used for detecting HPVE7 albumen, can also be used for differentiating different HPV types.
(3) provided by the invention detection in the method for HPVE7 albumen uses antibody provided by the invention, good stability, and detection sensitivity is high, reaches 2.67nM with the binding affinity of HPV18E7 albumen.
(4) monoclonal antibody provided by the invention and detection method, is applicable to the early diagnosis of associated cancer and large-scale patient's examination, and can be used for monitoring recurrence patient.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1
1. human papillomavirus HPV18E7 monoclonal antibody preparation
1.1 animal immune
Get 6-8 week female BAl BIc/C mouse, immunogen is GST-HPV18E7 albumen, and immune programme for children is in table 1.Carry out mouse docking blood sampling before each immunity, adopt Hi
s-HPV18E7 is detected mice serum as detectable antigens bag by indirect elisa method and tires.Until immune serum tire titre reach maximum and no longer raise time extracting spleen cell merge.
Table 1 mouse immune program
1.2 cytogamy and cultivation
The splenocyte and the myeloma cell SP2/0 that collect immune mouse according to a conventional method merge, and merge ratio splenocyte: SP2/0=5:1, fused cell is sub-packed in 96 well culture plates, is placed in 37 DEG C, and in 5%CO2 constant incubator, selectivity is cultivated.Liquid is entirely changed 3 times with HAT substratum after merging; When hybridoma covers with a field of microscope, carry out ELISA detection.
The screening of 1.3 positive hybridomas
During detection, employing indirect ELISA screens: antigen selects His-HPV18E7 fusion rotein, 2 μ g/ml wrapper sheets, 4 DEG C are spent the night, after PBST washing pats dry, add 5% skim-milk to close, 37 DEG C of effect 2h or 4 DEG C spend the night, after PBST washing pats dry, add hybridoma supematant and the positive (P) is set, negative (N) and blank (blank directly adds ELIAS secondary antibody), 37 DEG C of reaction 1h, add anti-(SigmaA2554) (1:10000) 37 DEG C of sheep anti mouse-HRP two after PBST washing pats dry again and react 45 ~ 60min, PBST washing pats dry rear TMB and develops the color and 2MH
2sO
4stop.
ELISA the selection result with OD value be greater than 0.6 for going out positive hole, and to recheck.After twice continuous detecting is the positive, this positive porocyte is increased, in time frozen and subclone.
The foundation of 1.4 hybridoma cell lines
For the hole that 2 screenings are all the positive, adopt limiting dilution assay to carry out subclone, get one piece of 96 porocyte culture plate, in every hole, add 150 μ lHAT nutrient solutions; To needing the positive hole of subclone to blow and beat suspension gently, drawing 100 μ l cell suspensions and being added in 96 porocyte plates, doubling dilution from the first hole.Kong Zhongyue is chosen in cell counting to be had the hole of 100 cells and joins in the loading slot containing 6mlHAT nutrient solution, is added in the cell plate of paving feeder layer, adds first three columns by 100 μ l/ holes; Add HAT substratum 3ml again, 100 μ l/ holes are added in the cell plate of paving feeder layer, three row in adding; Add HAT substratum 5ml again, 100 μ l/ holes are added in the cell plate of paving feeder layer, six row after adding.When the positive rate calculating every block plate reaches 100%, stable cell line can be obtained.Transfer in Tissue Culture Plate, enlarged culturing is also frozen in a large number.
A strain positive hybridoma cell strain MAB-F2 is obtained after screening.
A large amount of productions (ascites preparation) of 1.5 monoclonal antibodies
Mass propgation hybridoma (MAB-F2), 8-10 week age BALB/C female mice use whiteruss sensitization in advance, then abdominal injection 1x10
6hybridoma/only, within about about 10 days, obviously expand to a certain extent until mouse web portion, extract ascites with No. 9 syringe needles.The ascites His-HPV18E7 bag of results is detected titer of ascites by ELISA and is reached 1:10, more than 000.Ascites fluid through remove scleroproein and saltout process after, with ProteinG affinity column chromatography method purifying.Collect protein peak effluent liquid, ultraviolet spectrophotometer OD260 is used with after phosphate buffered saline buffer (PBS) dialysis, 280 to measure antibody protein concentration be 0.7-1.5mg/ml, and indirect ELISA detected result shows: tiring at more than 1:10000 of the monoclonal antibody of purifying.
2. the qualification of monoclonal antibody
The qualification of 2.1 monoclonal antibody Ig subclass:
Purified monoclonal antibody adopts PBS1:10000 dilution, and operate according to the hypotype identification kit specification sheets of Sigma company, monoclonal antibody MAB-F2 is IgG1.
2.2ELISA detects anti-HPV18E7 monoclonal antibody specificity and cross reaction:
Antigen selects His-HPV18E7 (HPV18E7 aminoacid sequence SEQIDNO.:2MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNH QHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCA SQQ) and His-HPV16E7 (HPV16E7 aminoacid sequence SEQIDNO.:1HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEP DRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP) fusion rotein respectively, 2 μ g/ml wrapper sheets, 4 DEG C are spent the night, after PBST washing pats dry, add 5% skim-milk to close, 37 DEG C of effect 2h or 4 DEG C spend the night, after PBST washing pats dry, add anti-HPV18E7 monoclonal antibody MAB-F2 (1 μ g/ml), 37 DEG C of reaction 1h, add anti-(SigmaA2554) (1:10000) 37 DEG C of sheep anti mouse-HRP two after PBST washing pats dry again and react 45 ~ 60min, PBST washing pats dry rear TMB and develops the color and 2MH
2sO
4stop, OD450nm place reading.The results are shown in Figure 1:MAB-F2 only can specific in conjunction with HPV18E7 recombinant protein with HPV16E7 without combination.
The antigen of 2.3Anti-HPV18E7 monoclonal antibody is in conjunction with epitope analysis
The qualification epitope amino acid sequence region of monoclonal antibody in HPV18E7 antigen protein.ELISA method is adopted to identify: antigen selects polypeptide or recombinant protein, wherein polypeptide is respectively HPV18E7-1 (aminoacid sequence SEQIDNO.:11SDSEEENDEIDGVNHQHLPARRAEPQRH), HPV18E7-2 (aminoacid sequence SEQIDNO.:12IDGVNHQHLPARRAEPQR), recombinant protein is His-HPV18E7 and His-HPV16E7, proteantigen 4 μ g/ml wrapper sheet, polypeptide antigen 2 μ g/ml wrapper sheet, 4 DEG C are spent the night, after PBST washing pats dry, add 5% skim-milk to close, 37 DEG C of effect 2h or 4 DEG C spend the night, after PBST washing pats dry, add anti-HPV18E7 monoclonal antibody MAB-F2 (1 μ g/ml), 37 DEG C of reaction 1h, add anti-(SigmaA2554) (1:10000) 37 DEG C of sheep anti mouse-HRP two after PBST washing pats dry again and react 45 ~ 60min, PBST washing pats dry rear TMB and develops the color and 2MH
2sO
4stop, OD450nm place reading.The results are shown in Figure 2: because MAB-F2 can Binding peptide HPV18E7-1 and HPV18E7-2, therefore initial guess MAB-F2 and HPV18E7's is 41-55 amino acids in conjunction with epi-position, and due to MAB-F2 only can specific binding HPV18E7 recombinant protein and not in conjunction with HPV16E7 recombinant protein, and 41-43 amino acids exists in two kinds of recombinant proteins simultaneously, therefore MAB-F2 and HPV18E7 binding amino acid sequence is between VNHQHLPARRAEPQR, i.e. between the 44-58 amino acids of HPV18E7.
2.4Anti-HPV18E7 monoclonal antibody protein binding affinity assays
Monoclonal antibody protein measures the method set up with reference to people (J.DavidBeatty, BarbaraG.BeattyandWilliamG.Vlahos1987) such as Beatty in conjunction with affinity costant.His-HPV18E7 albumen is with 8 μ g/ml, 2 μ g/ml, 0.5 μ g/ml, 0.125 μ g/ml concentration bag quilt, the ratio of four concentration is 1:4:16:64, and adding primary antibodie is MAB-F2 antibody, and concentration starts to do doubling dilution from 8 μ g/ml, totally seven gradients, carry out ELISA detection.Value will be obtained and substitute into formula K
aff=(n-1)/2 (n [Ab ']-[Ab]) calculate affinity costant, in formula, [Ab] and [Ab '] represents respectively when antigen concentration is Ag and Ag ', produce the antibody concentration of half light absorption value, namely get antibody concentration corresponding to curve maximum OD value half (i.e. OD50%) place by graphing method.N=Ag/Ag ' in formula.Then compare between two, as n=4,3 K values can be obtained, during n=16, obtain 2 K values, 1 K value during n=64, can be obtained, obtain the mean of 6 K values as net result.Result is as shown in Figure 3: calculate that MAB-F2 and His-HPV18E7 recombinant protein combines avidity 2.67nM.
2.5IP-WB detects monoclonal antibody in conjunction with the endogenous HPV18E7 of tumour cell:
Get 5x10
5the cervical cancer cell lines Hela cell of the HPV18E7 positive of bibliographical information adds 0.5ml and contains 1%TritonX-100,1mMEDTA, 50mMNaCl, 0.1 (m/v) SDS, 1%Sodiumdeoxycholate, 1mMPMSF, 2 μ g/mlAprotinin, the cell pyrolysis liquid of 1 μ g/mlLeupeptin and 1 μ g/mlPepstain, wherein all proteinase inhibitor are adding before use.Cell is placed on ice after cracking 30min, and 12500rpm, 4 DEG C of centrifugal 10min collect supernatant, are target protein extract.
In lysis supernatant, add 2 μ g monoclonal antibody MAB-F2 or mIgG respectively, hatch two hours for 4 DEG C, then add 15 μ lproteinA/Gbeads4 DEG C overnight incubation.Second day, adopt after not washing beads twice containing the cell pyrolysis liquid of proteinase inhibitor, add 1xSDS loading buffer, carry out immune-blotting method, adopt NC film, 100V voltage, transferring film 35min.After 2h closed by 5% skim-milk, add MAB-F2 or the mIgG albumen of 5% skim-milk dilution, antibody working concentration is 2 μ g/ml, 4 DEG C of overnight incubation, after adopting the TBS containing 0.1%Tween to wash 4 times, add Anti-MouseIgG (Fcspecific)-peroxidaseantibody (SigmaA2554) (1:1000), incubated at room 1h, DAB (Wuhan doctor moral SA2024), colour developing, result as shown in Figure 4, is 15KDa place at arrow indication molecular weight, the HPV16E7 albumen of the identification Hela cellular endogenous that MAB-F2 antibody is special.
2.6 immunocytochemical stains detect the expression of the HPV18E7 in cervical cancer tumer line:
Express the cervical cancer cell lines Hela cell of HPV18E7 albumen, express the cervical cancer cell lines CaSki cell of HPV16E7 albumen and do not carry out immunocytochemical stain test containing the cervical cancer cell lines C-33A cell monoclonal antibody MAB-F2 of HPVDNA.Specific experiment method is as follows:
CaSki, Hela, C-33A cell to plant on the cover glass of poly-L-Lysine process 37 DEG C respectively, 5%CO
2cultivate 24h, cover glass is taken out employing 4% paraformaldehyde stationary liquid room temperature and fix 30min; Add TBS washing lotion and wash 5min, dry; The TBS damping fluid room temperature placement 15min added containing 0.3%TritonX-100 makes cell membrane penetration; Add TBS washing lotion and wash 5min, dry; Add containing 1%H to make Endogenous peroxidase inactivation
2o
2tBS damping fluid room temperature treatment 5min, dry; Add TBS washing lotion and wash 5min, dry; Add 10%FBS/TBS confining liquid close spend the night, dry; Add mAbanti-HPV18E7MAB-F2 (5 μ g/ml) 37 DEG C and hatch 2h; Add TBS/0.1%Tween washing lotion and wash 5 times, each 5min, dry; Add Anti-MouseIgG (Fcspecific)-peroxidaseantibody (SigmaA2554) (1:1000) two anti-37 DEG C and hatch 1h; Add TBS/0.1%Tween washing lotion and wash 5 times, each 5min, dry; Add DAB nitrite ion (Beijing Zhong Shan Golden Bridge ZLI-9017), room temperature reaction 10min, distilled water wash termination reaction.Basis of microscopic observation result record.
The results are shown in Figure 5, detection display under microscope: HPV18E7 monoclonal antibody MAB-F2 only has strong immunochemistry staining reaction with the cervical cancer cell lines Hela expressing HPV18E7 albumen, and with the CaSki cell of expressing HPV16E7 and the cervical cancer cell lines C-33A that do not express HPV albumen without, staining reaction.Consistent with ELISA detected result before, namely the HPV18E7 of MAB-F2 and cellular endogenous has strong binding ability, the HPV18E7 albumen of identification cellular endogenous that namely can be special.
Embodiment 2
Immunocytochemical stain detects HPV18E7 monoclonal antibody MAB-F2 in the specificity in fixing cervical cancer tumer line that suspends:
Express the cervical cancer cell lines Hela cell of HPV18E7 albumen and after different cell stationary liquid is fixing, do not adopt monoclonal antibody MAB-F2 to carry out immunocytochemical stain test containing the cervical cancer cell lines C-33A cell of HPVDNA.Specific experiment method is as follows:
Collect C-33A cell and Hela cell respectively, 4% paraformaldehyde or LBC stationary liquid is adopted to fix a few days, get and add up to 50000 cells, wherein cervical cancer cell Hela cell mixes with certain proportion with negative control C-33A cell, and the ratio of Hela cell and C-33A cell number is 1:9,1:19,1:49,1:99,1:499,1:999.On the cover glass that mixing is placed on poly-L-Lysine process, room temperature is air-dry, makes cell be fixed on cover glass; Add TBS washing lotion and wash 5min, dry; The TBS damping fluid room temperature placement 15min added containing 0.3%TritonX-100 makes cell membrane penetration; Add TBS washing lotion and wash 5min, dry; Add containing 1%H to make Endogenous peroxidase inactivation
2o
2tBS damping fluid room temperature treatment 5min, dry; Add TBS washing lotion and wash 5min, dry; Add 10%FBS/TBS confining liquid close spend the night, dry; Add mAbanti-HPV16E7MAb-F2 (2 μ g/ml) 37 DEG C and hatch 2h; Add TBS/0.1%Tween washing lotion and wash 5 times, each 5min, dry; Add Anti-MouseIgG (Fcspecific)-peroxidaseantibody (SigmaA2554) (1:1000) two anti-37 DEG C and hatch 1h; Add TBS/0.1%Tween washing lotion and wash 5 times, each 5min, dry; Add DAB nitrite ion (Beijing Zhong Shan Golden Bridge ZLI-9017), room temperature reaction 10min, distilled water wash termination reaction; Dye 2min to add haematoxylin dyeing liquid (green skies C0107), and rinse in leaching tap water and remove unnecessary staining fluid, about 10min, distilled water washs one time (several seconds) again, basis of microscopic observation result record.
Detection display under microscope: HPV18E7 monoclonal antibody specific MAB-F2 only has strong immunochemistry staining reaction with the cervical cancer cell lines Hela expressing HPV18E7 albumen, and with not containing the cervical cancer cell lines C-33A of HPVDNA without immunology staining reaction.After Hela cell mixes with C-33A cell number 1:9, MAB-F2 can specifically in conjunction with Hela cell with C-33A cell without combination, MAB-F2 negative and positive cell in still can distinguishing accurately when namely positive tumor cell accounts for total cell count 10%.In adopting 4% paraformaldehyde or LBC stationary liquid fixed cell to detect ICC in addition, the judgement of feminine gender and positive staining is without impact.
Therefore, result shows that fixed number is in the future in advance when tumour cell adopts 4% paraformaldehyde or LBC stationary liquid, HPV18E7 monoclonal antibody MAB-F2 still specificly can detect the HPV18E7 albumen of inside tumor cells, and namely the stationary liquid process cell a few days does not produce as dyeing weakens or the impact of background enhanced immunocytochemical stain result.In addition when HPV18E7 cell proportion is low to moderate 10%, target cell during MAB-F2 antibody still can detect accurately, illustrates that this detection method has good specificity and sensitivity.Owing to using LBC detection technique widely clinically at present, remaining cell sample can be provided for other HPV infection analysis, and in this embodiment, adopt LBC fixed number still can accurately detect the cervical cancer cell that HPV18E7 is relevant, therefore the method can develop the detection of the associated malignancies caused for clinical HPV18E7 persistent infection further in the future.
Discuss:
The present inventor adopts aforesaid method to prepare HPV18E7 monoclonal antibody, screens 1000 polyclonal cellulars, and be sieved to 8 strain specificitys altogether good, with the cell strain of HPV16E7 albumen no cross reaction, after subclone, 5 strains are turned out cloudy, and 3 strains are still positive.Finally, only have monoclonal antibody MAB-F2 to detect and Immuncytochemical detection by IP-WB, clinical detection application has very large value.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (16)
1. a variable region of heavy chain for antibody, is characterized in that, described variable region of heavy chain comprises following three complementary determining region CDR:
CDR1 shown in SEQIDNO.:4,
CDR2 shown in SEQIDNO.:6, and
CDR3 shown in SEQIDNO.:8;
Preferably, described variable region of heavy chain has the aminoacid sequence shown in SEQIDNO.:10.
2. a heavy chain for antibody, is characterized in that, described heavy chain has variable region of heavy chain as claimed in claim 1 and CH.
3. a variable region of light chain for antibody, is characterized in that, described variable region of light chain has the complementary determining region CDR being selected from lower group:
CDR1' shown in SEQIDNO.:14,
CDR2' shown in SEQIDNO.:16, and
CDR3' shown in SEQIDNO.:18;
Preferably, described variable region of light chain has the aminoacid sequence shown in SEQIDNO.:20.
4. a light chain for antibody, is characterized in that, described light chain has variable region of light chain as claimed in claim 3 and constant region of light chain.
5. an antibody, is characterized in that, described antibody has: variable region of heavy chain as claimed in claim 1; And/or variable region of light chain as claimed in claim 3;
Or described antibody has: heavy chain as claimed in claim 2; And/or light chain as claimed in claim 4.
6. a recombinant protein, is characterized in that, described recombinant protein has:
(i) variable region of heavy chain as claimed in claim 1, heavy chain as claimed in claim 2, variable region of light chain as claimed in claim 3, light chain as claimed in claim 4 or antibody as claimed in claim 5; And
(ii) optional assistance expression and/or the sequence label of purifying.
7. polynucleotide, is characterized in that, its coding is selected from the polypeptide of lower group:
(1) variable region of heavy chain as claimed in claim 1, heavy chain as claimed in claim 2, variable region of light chain as claimed in claim 3, 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 carrier, is characterized in that, it contains the polynucleotide described in the claims in the present invention 7.
9. a genetically engineered host cell, is characterized in that, it contains in carrier according to claim 8 or genome and is integrated with polynucleotide according to claim 7.
10. an immune conjugate, is characterized in that, this immune conjugate contains:
(a) variable region of heavy chain as claimed in claim 1, heavy chain as claimed in claim 2, variable region of light chain as claimed in claim 3, light chain as claimed in claim 4 or antibody as claimed in claim 5; With
B () is selected from the coupling moiety of lower group: detectable, medicine, toxin, cytokine, radionuclide or enzyme.
11. a pharmaceutical composition, it is characterized in that, it contains:
(i) variable region of heavy chain as claimed in claim 1, heavy chain as claimed in claim 2, variable region of light chain as claimed in claim 3, light chain as claimed in claim 4 or antibody as claimed in claim 5, recombinant protein as claimed in claim 6 or immune conjugate as claimed in claim 10; And
(ii) pharmaceutically acceptable carrier.
The purposes of 12. variable region of heavy chain as claimed in claim 1, as claimed in claim 2 heavy chain, variable region of light chain as claimed in claim 3, light chain as claimed in claim 4 or antibody as claimed in claim 5, as claimed in claim 6 recombinant protein or as claimed in claim 10 immune conjugate, it is characterized in that, for the preparation of medicament, reagent, check-out console or test kit;
Described reagent, check-out console or test kit are used for:
(1) HPV18E7 albumen in sample is detected; And/or
(2) endogenic HPV18E7 albumen in tumour cell is detected; And/or
(3) tumour cell of expressing HPV18E7 albumen is detected; And/or
(4) type of HPV is differentiated;
Described medicament is used for the treatment of or prevents to express the tumour of HPV18E7 albumen.
13. 1 kinds of methods detecting HPVE7 albumen in sample, it is characterized in that, described method comprises step:
(1) by sample and antibody contacts according to claim 5;
(2) detect whether form antigen-antibody complex, wherein form mixture and just represent in sample to there is HPVE7 albumen.
14. 1 kinds of check-out consoles, is characterized in that, described check-out console comprises substrate (back up pad) and test strip, and described test strip contains antibody according to claim 5 or immune conjugate according to claim 10.
15. 1 kinds of test kits, is characterized in that, described test kit comprises:
(1) first container, containing antibody according to claim 5 in described first container; And/or
(2) second container, resists containing two of anti-antibody according to claim 5 in described second container; And/or
(3) the 3rd containers, containing cell cracking agent in described 3rd container;
Or,
Described test kit contains check-out console according to claim 14.
16. 1 kinds of preparation methods preparing recombinant polypeptide, the method comprises:
A () under conditions suitable for the expression, cultivates host cell according to claim 9;
B () isolates recombinant polypeptide from culture, described recombinant polypeptide is antibody according to claim 5 or recombinant protein according to claim 6.
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