CN109021097A - A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45 - Google Patents

Abstract

The present invention provides a kind of monoclonal antibody and its application for identifying HPV18 and/or HPV45, the antibody can detect HPV18 the and/or HPV45 E7 carcinogenic protein that epithelium of cervix uteri cancer (preceding) attenuates in born of the same parents with high specificity, it is normal so as to obviously distinguish (preceding) variation of the epithelium of cervix uteri cancer as caused by HPV18 and/or HPV45 persistent infection, effective auxiliary detection information is provided for doctor's Accurate Diagnosis relevant case, improve cervical carcinoma detection and early intervention, fail to pinpoint a disease in diagnosis and the clinical excessive diagnosis and treatment of the related cervical carcinogenesis of reduction, reduce and avoid unnecessary vaginoscopy.

Description

A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45

Technical field

The invention belongs to biological diagnosis and field of medicaments, specifically, the present invention relates to identifications by human papilloma virus (HPV) (preceding) the change tumour cell of cancer caused by 18 and/or 45 persistent infections attenuates the Dan Ke of born of the same parents including people's epithelium of cervix uteri cancer (preceding) Grand antibody and its immunohistochemical staining application.

Background technique

The last diagnostic of cervical carcinoma (preceding) lesion is using pathological tissue cytomorphology as goldstandard.Cervical lesions grade , be not divided into Cervical intraepitheliaI neoplasia (cervical intraepithelial neoplasia, CIN) (CIN1, CIN2 and CIN3), carcinoma in situ and infiltrating carcinoma.Wherein minuent intraepithelial neoplasia (cin) CIN1 is the performance of HPV acute infection, and lesion recession switchs to just The probability often organized is very high.Height intraepithelial neoplasia (cin) CIN3 has the significant risk for progressing to cancer, therefore clinical treatment is bright Really.Clinically there is dispute to the processing of CIN2 at present, because Precise Diagnosis is had any problem, suitable therapeutic scheme is not known yet. Observer has largely difference to CIN2 diagnosis, even to the interpretation of CIN2, there is also bright in the trained stringent U.S. Show inconsistent.CIN2 is not only a kind of independent intermediate lesion, it appears that is even more the mixing of low level and high-level lesion, and leads to Histology is crossed to be difficult simply to distinguish this.Therefore low level and high-level lesion how are distinguished, Precise Diagnosis uterine neck is disliked Venereal disease becomes, and provides more accurately information for clinical intervention and treatment, becomes urgent problem to be solved in clinical uterine neck diagnosis and treatment.

In all tumours of the mankind, cervical carcinoma is the current cause of disease uniquely specific tumour, while being also that can uniquely prevent Tumour.It is the result of human papilloma virus (human papillomavirus, HPV) persistent infection.Development of cancer Middle HPV DNA is integrated into human cel gene group, and transformant starts continuous expression HPV E6 and E7 albumen into the cell.The two Carcinogenic protein is the major reason for leading to cervical carcinoma, these small molecular proteins contain 158 and 98 amino acid respectively, has zinc finger Structure (tryptophan-X-X- tryptophan), the vicious transformation of this structure and cell is closely related.HPV E6 albumen passes through E6-AP The mediation of (E6associated protein), in conjunction with and the cancer suppressor protein p53 that degrades.HPV E7 albumen and cancer suppressor protein pRb are tied It closes, so that pRb is discharged nuclear factor E2F, cause cell hyperproliferation, cell cycle regulating is out of control, leads to the immortalization of cell And promoting the generation of cervical carcinoma, this makes the tumor markers that E7 can be used as high-grade cervical damage and cervical carcinoma detects.

HPV is a kind of small DNA virus, the characteristic with very strong thermophilic scaly epithelium, the main substrate for invading scaly epithelium Confluent monolayer cells and metaplasia cell positioned at uterine neck zone of transformation.What is be currently known has a type more than 100, according to carcinogenicity, is divided into high-risk Two class of type HPV and low risk HPV.High-risk HPV relevant to human tumor has forties kinds, wherein common mainly includes The types such as HPV18, HPV45.The infection rate of HPV18 occupies second in cervical squamous cell carcinoma, but in adenocarcinoma of the uterine cervix based on HPV18 The infection type (47.4%) wanted.Therefore HPV18 be main distribution in cervix cancer high-risk HPV types not one of.

Those skilled in the art are dedicated to developing a kind of detection method and skill independent of pathological tissue cytomorphology Art, to detect the cervical carcinoma as caused by HPV18 and/or HPV45, especially high-grade cervical intraepithelial neoplasia (cin).One side area simultaneously Divide the cell development of non-malignant lesions abnormal, on the other hand can also prompt the Early pathological changes of uterine cervix with height canceration risk, be Clinic diagnosis provides more objective, accurately diagnosis scheme.

Summary of the invention

The monoclonal antibody and its application that the purpose of the present invention is to provide a kind of by identifying HPV18 and/or HPV45.

In the first aspect of the present invention, provide a kind of heavy chain variable region of antibody, the heavy chain variable region include with Lower three complementary determining region CDR:

CDR1 shown in SEQ ID NO.:4,

CDR2 shown in SEQ ID NO.:6, and

CDR3 shown in SEQ ID NO.:8.

In another preferred example, the heavy chain variable region has amino acid sequence shown in SEQ ID NO.:10.

The second aspect of the present invention, provides a kind of heavy chain of antibody, and the heavy chain has such as first aspect present invention The heavy chain variable region and heavy chain constant region.

In another preferred example, the heavy chain constant region is source of people, source of mouse, rabbit source or chimeric antibody.

The third aspect of the present invention provides a kind of light chain variable region of antibody, and the light chain variable region, which has, to be selected from down The complementary determining region CDR of group:

CDR1' shown in SEQ ID NO.:12,

CDR2' shown in SEQ ID NO.:14, and

CDR3' shown in SEQ ID NO.:16.

In another preferred example, the light chain variable region has amino acid sequence shown in SEQ ID NO.:18.

The fourth aspect of the present invention, provides a kind of light chain of antibody, and the light chain has such as third aspect present invention The light chain variable region and constant region of light chain.

In another preferred example, the constant region of light chain is source of people, source of mouse, rabbit source or chimeric antibody.

The fifth aspect of the present invention, provides a kind of antibody, and the antibody includes

(1) heavy chain variable region as described in the first aspect of the invention；And/or

(2) light chain variable region as described in the third aspect of the present invention.

In another preferred example, the antibody includes heavy chain as described in respect of the second aspect of the invention；And/or such as the present invention Light chain described in fourth aspect.

In another preferred example, the antibody is the antibody of the anti-HPV of specificity；Preferably, the antibody is specificity The antibody of anti-HPV18 and HPV45；It is highly preferred that the antibody is the antibody of specificity anti-HPV18 E7 and HPV45 E7 albumen.

In another preferred example, the antibody includes: single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody (such as people rabbit chimeric antibody), source of mouse antibody, rabbit source antibody or humanized antibody.

In another preferred example, described " HPV18 E7 albumen " can be wild type HPV18 E7 albumen, or wild The derived protein of raw type HPV18 E7 albumen." the HPV45 E7 albumen " can be wild type HPV45 E7 albumen, can also be with For the derived protein of wild type HPV45 E7 albumen.

In another preferred example, the antibody is the list that can specifically bind HPV18 E7 albumen and HPV45 E7 albumen Clonal antibody.

In another preferred example, affinity of the antibody not in conjunction with other HPV types or with other HPV hypotypes compared with It is low.

In another preferred example, the antibody also has characteristics that

It (3) can be in conjunction with the protein-specific of the space conformation of HPV18 E7 and HPV45 E7.

The sixth aspect of the present invention, provides a kind of recombinant protein, and the recombinant protein includes

(i) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention Antibody；And

(ii) sequence label of optional assistance expression and/or purifying.

In another preferred example, the sequence label includes 6 × His label.

In another preferred example, the anti-HPV of recombinant protein specificity；Preferably, the anti-HPV18 and HPV45 of specificity； It is highly preferred that the anti-HPV18 E7 of specificity and HPV45 E7 albumen.

The seventh aspect of the present invention provides a kind of polynucleotides, it encodes polypeptide selected from the group below:

(1) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention Antibody；Or

(2) recombinant protein as described in sixth aspect present invention.

In another preferred example, the polynucleotides have SEQ ID NO.:3,5,7,9,11,13,15 or 17, institute The sequence shown.

The eighth aspect of the present invention provides a kind of carrier, it contains polynucleotides described in seventh aspect present invention.

In another preferred example, the carrier include: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, Mammalian cell virus such as adenovirus, retrovirus or other carriers.

The ninth aspect of the present invention provides a kind of genetically engineered host cell, it contains eighth aspect present invention Polynucleotides described in seventh aspect present invention are integrated in the carrier or genome.

The tenth aspect of the present invention provides a kind of immune conjugate, which contains:

(a) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention Antibody；With

(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or Enzyme.

In another preferred example, the conjugate is selected from: (magnetic is total by fluorescence or luminous marker, radioactively labelled substance, MRI Vibration imaging) CT (x-ray tomography of electronic computer) contrast agent or can generate detectable product enzyme, radiation Property nucleic, biotoxin, cell factor (such as IL-2), antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/receive Rice stick, virion, liposome, magnetic nanosphere, pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or biphenyl base hydrolase- Sample protein (BPHL)), chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

The eleventh aspect of the present invention provides a kind of pharmaceutical composition, which is characterized in that it contains:

(i) as described in the first aspect of the invention heavy chain variable region, heavy chain as described in respect of the second aspect of the invention, such as this hair Light chain variable region described in the bright third aspect, light chain as described in the fourth aspect of the present invention or as described in fifth aspect present invention Antibody, the recombinant protein as described in sixth aspect present invention or the immune conjugate as described in tenth aspect present invention；And

(ii) pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition is injection type.

In another preferred example, the pharmaceutical composition is used to prepare the drug for the treatment of tumour, and the tumour is selected from The following group: gastric cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, large intestine Cancer, cervical carcinoma, carcinoma of endometrium, carcinoma of penis, adrenal tumor or tumor of bladder.

The twelveth aspect of the present invention provides heavy chain variable region as described in the first aspect of the invention, such as present invention the Two aspect described in heavy chain, light chain variable region as described in the third aspect of the present invention, light chain as described in the fourth aspect of the present invention, Or the antibody as described in fifth aspect present invention, the recombinant protein as described in sixth aspect present invention or the tenth side such as of the invention The purposes of immune conjugate described in face, which is characterized in that be used to prepare medicament, reagent, detection plate or kit；

The reagent, detection plate or kit are used for:

(1) HPV18 and/or HPV45 E7 albumen in test sample；And/or

(2) endogenic HPV18 and/or HPV45 E7 albumen in tumour cell is detected；And/or

(3) tumour cell of detection expression HPV18 and/or HPV45 E7 albumen；And/or

(4) identify the type of HPV；

The medicament is used to treat or prevent the tumour of expression HPV18 and/or HPV45 E7 albumen.

In another preferred example, HPV18 and/or HPV45 E7 albumen is contained in the sample.

In another preferred example, the tumour includes: the tumour of urogenital system, the tumour of respiratory system, digestion The tumour of road system, comprising: cervical carcinoma, carcinoma of endometrium, carcinoma of penis, Small Cell Lung Cancer, melanoma or H/N tumors, stomach Cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer or kidney Upper adenoncus tumor.

In another preferred example, described " tumour of urogenital system " includes: cervical carcinoma, bladder cancer, carcinoma of endometrium Or carcinoma of penis.

In another preferred example, the reagent includes the immune particle of chip, coated antibody.

The thirteenth aspect of the present invention provides a kind of method of HPV E7 albumen in test sample, the method includes Step:

(1) sample is contacted with antibody described in fifth aspect present invention；

(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are HPV E7 eggs in sample It is white.

In another preferred example, it is detected in step (2) by ELISA method.

In another preferred example, the HPV E7 albumen includes HPV18 and/or HPV45 E7 albumen.

In another preferred example, in step (1), sample is contacted with two kinds of antibody for HPV E7 albumen, and Detected in step (2) by ELISA method, it is described two at least one of antibody of HPV E7 albumen be this hair Antibody described in bright 5th aspect.

In another preferred example, described " antigen-antibody complex " is that " first antibody-antigen-secondary antibody " ternary is multiple Close object, wherein the first antibody is antibody described in fifth aspect present invention, and the combination epitope of the secondary antibody with The combination epitope of the first antibody is different.

In another preferred example, described in step (1), sample is contacted with antibody described in fifth aspect present invention Afterwards, the third antibody for resisting the first antibody is added in the reaction system, and " antigen-the first is anti-for detection in step (2) The formation of body-third antibody " compound.

In another preferred example, with detectable mark on the first antibody, the secondary antibody or the third antibody Note.

In another preferred example, the detectable label is biotin labeling, colloid gold label, horseradish peroxidase mark Note, radioisotope labeling, fluorescein label.

In another preferred example, the sample includes: human or animal tissues sample, tumor resection sample, cast-off cells sample Product.

In another preferred example, the method is used for nondiagnostic purpose.

The fourteenth aspect of the present invention, provides a kind of detection plate, and the detection plate includes substrate (support plate) and surveys Strip, the test-strips contain immunoconjugates described in antibody described in fifth aspect present invention or sixth aspect present invention Object.

In another preferred example, the test-strips also contain antigen point sample area.

In another preferred example, the test-strips by filter sample paper, chromatographic material, nitrocellulose filter and blotting paper successively Overlap joint composition.

The fifteenth aspect of the present invention provides a kind of kit, includes: in the kit

(1) the first container contains antibody described in fifth aspect present invention in the first container；And/or

(2) second container, the secondary antibody containing antibody described in anti-fifth aspect present invention in the second container；And/or

(3) third container contains cell cracking agent in the third container；

Alternatively,

The kit contains detection plate described in the tenth four sides of the invention.

In another preferred example, the antibody in the first container has detectable label.

In another preferred example, the antibody in the second container has detectable label.

The sixteenth aspect of the present invention provides a kind of preparation method of preparation and reorganization polypeptide, and this method includes:

(a) under conditions suitable for the expression, host cell described in ninth aspect present invention is cultivated；

(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in fifth aspect present invention Or recombinant protein described in sixth aspect present invention.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 is the potency result figure for exempting from serum with immunogen protein ELISA detection three.It as a result is inspection with His-HPV18 E7 When surveying antigen, the mice serum potency highest of number #F reaches fusion standard.

Fig. 2 is the nine plants of monoclonal cell culture supernatant activity and intersect ELISA testing result that screening obtains.As a result have six Strain Colony Culture supernatant MAB-01,06,07,11,13,14 can specifically bind His-HPV18E7 recombinant protein, and and His Label unrelated protein does not combine.Its excess-three strain MAB-03,04,10 in addition in conjunction with His-HPV18 E7 recombinant protein, while with His label unrelated protein also has stronger combination.

Fig. 3 is HPV18 E7 mouse monoclonal antibody MAB-14 activity ELISA testing result.As a result MAB-14 can be high affine The combination His-HPV18 E7 recombinant protein of power.

Fig. 4 is antibody subtype detection, and as a result MAB-14 antibody subtype is IgG1, kappa.

Fig. 5 is that mouse monoclonal antibody MAB-14 intersects ELISA testing result.As a result MAB-14 not only high-affinity is special Property combination recombinant protein His-HPV18 E7, and the specific binding His-HPV45 E7 of high-affinity.While and His- HPV16 E7, His-HPV31 E7, His-HPV52 E7, His-HPV58 E7 are without apparent cross reaction.

Fig. 6 A and Fig. 6 B are the heavy chain of monoclonal antibody Immunoglobulin IgG, light chain variable region CDR gene order, and specific experiment is thin Section is shown in 1,2.3 trifle of embodiment.Fig. 6 A shows the heavy chain variable region CDR gene order of MAB-14 monoclonal antibody Immunoglobulin IgG. Fig. 6 B shows the light chain variable region CDR gene order of MAB-14 monoclonal antibody Immunoglobulin IgG.

Fig. 7 A, Fig. 7 B and Fig. 7 C are HPV18 E7 mouse monoclonal antibody immunohistochemical staining testing result figure.Fig. 7 A For the tissue section strain result of MAB-06；Fig. 7 B is the tissue section strain result of MAB-13；Fig. 7 C is the tissue of MAB-14 It is sliced coloration result.As a result MAB-14 shows strong dyeing, and interstitial areas clean background by tumour cell to tumour cell.MAB- 14 are substantially better than other strain antibodies on specificity.

Specific embodiment

The present inventor successfully obtains the anti-His-HPV18 E7 of one plant of specificity by largely screening by in-depth study Monoclonal antibody MAB-14, the experimental results showed that, should for His-HPV18 E7 albumen monoclonal antibody, affinity is strong, Recombinant protein His-HPV18 E7 can be specifically combined, and in combination with recombinant protein His-HPV45 E7.Therefore the antibody It is used not only for detection 18 E7 albumen of high-risk HPV, additionally it is possible to for detecting 45 E7 albumen of high-risk HPV.Further grind Study carefully and shows that the antibody can also be used in formalin and fix, the detection of the clinical cervical tissue sample slice of paraffin embedding.The present invention The method for additionally providing detection and/or identifying HPV18/45 E7 albumen, this method stability is good, and detection sensitivity is high.This hair The bright kit and detection plate additionally provided comprising above-mentioned antibody.

Specifically, the present invention is using recombination His-HPV18 E7 fusion protein immunization BALB/C mice, and uses His- HPV18 E7 recombinant protein obtains positive monoclonal hybridoma cell strain as screening antigen, screening.And with His unrelated protein into Row secondary screening, it is final to obtain 6 plants of clone strains that specifically bind His-HPV18 E7 recombinant protein.

To filter out the antibody reagent with clinical examination value in histological changes of uterine cervix identification, by 6 plants of monoclonal cells Strain carries out mouse hydroperitoneum antibody of RGDV production purifying, and carry out further clinical verification: formalin is fixed, the cervical carcinoma group of paraffin embedding The detection of slice is knitted, it is final to obtain one plant of monoclonal antibody for capableing of specific recognition E7 protein positive cervical carcinoma paraffin section MAB-14.In order to explore the crossing instances of the antibody Yu other high-risk type HPV, His-HPV16,31,45,52,58 E7 are used Recombinant protein carries out ELISA detection to MAB-14, as a result, it has been found that MAB-14 monoclonal antibody not only know by the specific of high-affinity Other HPV18 E7, while the specific recognition HPV45 E7 of high-affinity.Specificity based on the antibody, which develops, to be identified The method of the canceration of people's epithelium of cervix uteri or precancerous lesion as caused by HPV18 and/or HPV45, this method can be used in auxiliary and face The bed morphologic diagnosis of uterine neck pathology histocyte.

In a preferred embodiment of the invention, the amino acid sequence of the HPV18 E7 albumen is as follows:

MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVE SSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQ ID NO.:1)。

In a preferred embodiment of the invention, the amino acid sequence of the HPV45 E7 albumen is as follows:

MHGPRATLQEIVLHLEPQNELDPVDLLCYEQLSESEEENDEADGVSHAQLPARRAEPQRHKILCVCCKCDGRIDLTV ESSADDLRTLQQLFLSTLSFVCPWCATNQ(SEQ ID NO.:2)。

As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical structure feature Different four glycan albumen is made of two identical light chains (L) and two identical heavy chains (H).Every light chain is total by one Valence disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Each heavy chain and The intrachain disulfide bond at light chain also regular interval.One end of each heavy chain has variable region (VH), is followed by multiple constant regions.Every One end of light chain has variable region (VL), and the other end has constant region；The constant region of light chain is opposite with first constant region of heavy chain, gently The variable region of chain and the variable region of heavy chain are opposite.Special amino acid residue forms boundary between the variable regions of the light chain and the heavy chain Face.

As used herein, " variable " the certain parts for indicating variable region in antibody of term are different in sequence, its shape Combination and specificity at various specific antibodies to its specific antigen.However, changeability and being unevenly distributed over entire anti- In body variable region.It concentrates in light chain and heavy chain variable region three segments being known as in complementary determining region (CDR) or hypervariable region In.More conservative part is known as framework region (FR) in variable region.Four FR are respectively contained in the variable region of native heavy and light chain Area, they are generally in beta sheet configuration, are connected by three CDR of formation connection ring, can form part β folding in some cases Stack structure.CDR in every chain is by the area FR firmly against the antigen for together forming antibody together and with the CDR of another chain Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody Cellular toxicity.

" light chain " of vertebrate antibodies (immunoglobulin) can be classified as obviously not according to the amino acid sequence of its constant region One kind in same two classes (referred to as κ and λ).According to the amino acid sequence of its heavy chain constant region, immunoglobulin can be divided into not Same type.Mainly there are 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them can also be further separated into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Light chain constant corresponding to different immunoglobulin like protein is distinguished It is also known as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of different immunoglobulin like protein are known to those skilled in the art 's.

As used herein, term " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from a kind of substantially uniform group, i.e., The single antibody for including in the group be it is identical, in addition to a small number of mutation that may be present naturally occurred.Monoclonal antibody is high Specifically it is directed to single antigen site.Moreover, (usually having for different determinants from conventional polyclonal antibody preparation Different antibodies) it is different, each monoclonal antibody is for the single determinant on antigen.Other than their specificity, herein Rabbit monoclonal antibodies be that overall length rabbit monoclonal antibodies base is constructed by the method for molecular biosciences after screening by phage library Because of expression vector, which is transferred to eukaryotic expression system, cell conditioned medium is collected after culture and obtains, it will not be by other immune balls Protein contamination.Modifier " monoclonal " illustrates the characteristic of antibody, is obtained from substantially uniform antibody population, this should not be by It is construed to need to produce antibody with any specific process.

The invention also includes the lists of the corresponding amino acid sequence with the anti-HPV18/45 E7 protein monoclonal antibody Clonal antibody, the monoclonal antibody with the anti-HPV18/45 E7 protein monoclonal antibody variable region chain, and there is this Other protein or protein conjugate and fusion expressed product of a little chains.Specifically, the present invention includes having containing hypervariable region (mutually Mend determine area, CDR) light chain and heavy chain any protein or protein conjugate and fusion expressed product (i.e. immunoconjugates Object and fusion expressed product), as long as the hypervariable region is identical as the hypervariable region of light chain of the invention and heavy chain or at least 90% is homologous Property, preferably at least 95% homology.

As it is known by the man skilled in the art, immune conjugate and fusion expressed product include: drug, toxin, cell factor (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and the HPV16 E7 protein monoclonal antibody or Its segment in conjunction with and the conjugate of formation.The invention also includes with the anti-HPV16 E7 protein monoclonal antibody or its piece The cell surface marker object or antigen that section combines.

The present invention not only includes complete monoclonal antibody, further includes having immunocompetent antibody fragment, such as Fab or (Fab')₂Segment；Heavy chain of antibody；Antibody light chain.

As used herein, term " heavy chain variable region " and " V_H" be used interchangeably.

As used herein, term " variable region " and " complementary determining region (complementarity determining Region, CDR) " it is used interchangeably.

In a preferred embodiment of the invention, the heavy chain variable region of the antibody includes that following three complementations are determined Determine area CDR:

CDR1, amino acid sequence are GFTFNRYD (SEQ ID NO.:4), and coding nucleotide sequence is ggattcactttcaatagatatgac(SEQ ID NO.:3)；

CDR2, amino acid sequence are IRNDGSKT (SEQ ID NO.:6), and coding nucleotide sequence is atcagaaatgatggtagtaagact(SEQ ID NO.:5)；

CDR3, amino acid sequence are TRDSYGTVFAY (SEQ ID NO.:8), and coding nucleotide sequence is acaagggactcctatggtaccgtgtttgcttac(SEQ ID NO.:7)。

In another preferred example, the amino acid sequence of the heavy chain variable region are as follows:

QLELVQSGGGSVKPGGSLKLSCAASGFTFNRYDMSWFRQSPEKRLEWVAEIRNDGSKTHYSDTVTGRFTISRDNAKN TLYLEMSSLRSEDTAMYYCTR(SEQ ID NO.:10)；

Its coding nucleotide sequence are as follows:

cagctcgagctggtgcagtcagggggaggctcagtgaagcctggagggtccctgaaactctcctgtgcagcctctgg attcactttcaatagatatgacatgtcttggtttcgccagtctccagagaagaggctggagtgggtcgcagagatca gaaatgatggtagtaagactcactattcagacactgtgacgggccgattcaccatctccagagacaatgccaagaac accctgtacctggaaatgagcagtctgaggtctgaggacacggccatgtattactgtacaagg(SEQ ID NO.: 9)。

In a preferred embodiment of the invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain Constant region, the heavy chain constant region can be source of mouse, source of people or rabbit source.

As used herein, term " light chain variable region " and " V_L" be used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of antibody according to the present invention has and is selected from The complementary determining region CDR of the following group:

CDR1', amino acid sequence are QSLLYSNGKTY (SEQ ID NO.:12), and coding nucleotide sequence is cagagcctcttatatagtaatggaaaaacctat(SEQ ID NO.:11)；

CDR2', amino acid sequence are LVS (SEQ ID NO.:14), coding nucleotide sequence ctggtgtct (SEQ ID NO.:13)

CDR3', amino acid sequence are VQGTHFPQT (SEQ ID NO.:16), and coding nucleotide sequence is gtgcaaggtacacattttcctcagacg(SEQ ID NO.:15)

In another preferred example, the amino acid sequence of the light chain variable region are as follows:

ELVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDLGVYYCVQGTHFP (SEQ ID NO.:18),

Its coding nucleotide sequence are as follows:

gagctcgtcatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagtcaag tcagagcctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgcc taatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttaca ctgaaaatcagcagagtggaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcct(SEQ ID NO.:17)。

In a preferred embodiment of the invention, the light chain of the antibody includes above-mentioned light chain variable region and light chain Constant region, the constant region of light chain can be source of mouse, source of people or rabbit source.

In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all Refer to the antibody of specific binding HPV18/45 E7 albumen, such as with heavy chain variable region (such as amino acid of SEQ ID NO.:10 Sequence) and/or light chain variable region (such as amino acid sequence of SEQ ID NO.:18) albumen or polypeptide.They can contain or not Containing initial methionine.

In another preferred example, the antibody is mouse or the people's mouse chimeric mAb of anti-HPV18/45 E7 albumen, Its heavy chain constant region and/or constant region of light chain can be the heavy chain constant region or constant region of light chain of humanization.It is highly preferred that institute The heavy chain constant region or constant region of light chain for the humanization stated are the heavy chain constant region or constant region of light chain of human IgG1, IgG2 etc..

The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention It is (i.e. immune even including any protein or protein conjugate and fusion expressed product with heavy chain and light chain containing variable region Join object and fusion expressed product), as long as the variable region is identical as the variable region of the heavy chain of antibody of the present invention and light chain or at least 90% homology, preferably at least 95% homology.

Generally, the antigenic binding property of antibody can be described by being located at 3 specific regions of heavy chain and light chain variable region, The section is partitioned into 4 frame areas (FR) by referred to as Variable Area (CDR), and the amino acid sequence of 4 FR is relatively conservative, Association reaction is not participated in directly.These CDR form cyclic structure, the β-pleated sheet formed by FR therebetween phase on space structure Mutually close, the CDR on CDR and corresponding light chain on heavy chain constitutes the antigen binding site of antibody.It can be by comparing similar The amino acid sequence of the antibody of type determines be which Amino acid profile FR or CDR region domain.

The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because at least partly relating in them And combine antigen.Therefore, the present invention includes those molecules with the monoclonal antibody light chain with CDR and heavy chain variable region, only Want its CDR and CDR that identifies herein have 90% or more (preferably 95% or more, most preferably 98% or more) homology.

The present invention not only includes complete monoclonal antibody, further include have immunocompetent antibody segment or antibody with The fusion protein that other sequences are formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.

As used herein, term " segment ", " derivative " refer to that be kept substantially antibody of the present invention identical with " analog " Biological function or active polypeptide.Polypeptide fragment of the invention, derivative or the like, which can be (i), one or more Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino Sour residue, which can be, may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second Glycol) fusion is formed by polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (as before Lead sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or merge egg with what 6His label was formed It is white).According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.

Antibody of the present invention refers to polypeptide with HPV18/45 E7 protein binding activity, including above-mentioned CDR region.The term It further include the variant form with polypeptide with antibody identical function of the present invention, comprising above-mentioned CDR region.These variant form packets It includes (but being not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10 It is a) missing, insertion and/or the substitution of amino acid, and in C-terminal and/or N-terminal addition one or several (usually 20 Within, more preferably it is within 5 within preferably 10) amino acid.For example, in the art, with similar performance or similar Amino acid when being replaced, do not usually change the function of protein.For another example, one is added in C-terminal and/or N-terminal Or several amino acid will not generally also change the function of protein.The term further includes the active fragment and activity of antibody of the present invention Derivative.

The variant form of the polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induction Mutant, the encoded albumen of DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency, with And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.

The present invention also provides other polypeptides, such as the fusion protein comprising human antibody or its segment.In addition to almost overall length Outside polypeptide, the invention also includes the segments of antibody of the present invention.In general, the segment has at least about 50 companies of antibody of the present invention Continue amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about 100 continuous amino acids.

In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention, There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on Table A and generate.

Table A

The present invention also provides encoding such antibodies or the polynucleotide molecules of its segment or its fusion protein.Of the invention Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with Coding region sequence shown in SEQ ID NO.:3,5,7,9,11,13,15,17 is identical or the variant of degeneracy.Such as this paper institute With " variant of degeneracy " refers to that coding has amino acid sequence identical with polypeptide of the invention, but and SEQ in the present invention The differentiated nucleic acid sequence of coding region sequence shown in ID NO.:3,5,7,9,11,13,15,17.

The polynucleotides for encoding mature polypeptide of the invention include: the coded sequence of an encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume Code sequence.

The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include The polynucleotides of additional code and/or non-coding sequence.

The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90% with On, more preferably 95% or more when, just hybridizes.Further, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.:10 And/or mature polypeptide shown in SEQ ID NO.:20 has identical biological function and activity.

The nucleotide full length sequence of antibody of the invention or its segment can usually use PCR amplification method, recombination method or artificial Synthetic method obtains.A kind of feasible method is that manually synthetic method synthesizes related sequence, especially fragment length When shorter.In general, being then attached the very long segment of available sequence again by first synthesizing multiple small fragments.In addition, may be used also The coded sequence of heavy chain and expression label (such as 6His) are fused together, fusion protein is formed.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method. Biomolecule (nucleic acid, albumen etc.) according to the present invention includes existing biomolecule in a separate form.

At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.

The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This A little carriers can be used for converting host cell appropriate, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell；Fungal cell's such as yeast；The insect cell of drosophila S2 or Sf9；CHO, COS7, zooblast of 293 cells etc..

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is using MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Antibody of the invention can be used alone, can also be with detectable marker (for diagnostic purpose), therapeutic agent, PK (egg White kinases) combination of modified part or any the above substance combines or coupling.

Detectable marker for diagnostic purposes includes but is not limited to: fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.

Can in conjunction with antibody of the present invention or coupling therapeutic agent include but is not limited to: 1. radionuclides (Koppe etc., 2005, (Cancer metastasis reviews) 24,539 is commented in metastasis of cancer)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. gold nano (Lapotko etc., 2005, cancer communicates (Cancer letters) 239,36 to particle/nanometer rods；Huang etc., 2006, it is Americanized Association magazine (Journal of the American Chemical Society) 128,2115)；5. virion (Peng Deng 2004, gene therapy (Gene therapy) 11,1234)；6. liposome (Mamot etc., 2005, cancer research (Cancer Research) 65,11631)；7. magnetic nanosphere；8. pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or xenyl hydrolysis Enzyme-sample protein (BPHL))；10. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

The present invention also provides a kind of compositions.In preference, the composition is pharmaceutical composition, it contains The antibody stated or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, these substances can be prepared In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is usually about 5-8, and preferably pH is about 6-8, although pH value can be varied with the property and illness to be treated for being formulated substance.Prepared pharmaceutical composition It can be administered by conventional route, including (but being not limited to): tumor is interior, peritonaeum is interior, intravenous or local administration.

Pharmaceutical composition of the invention can be directly used for combining HPV16 E7 protein molecular, thus can be used for preventing and treating Tumour.In addition, also can be used simultaneously other therapeutic agents.

Pharmaceutical composition of the invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more Good ground 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) and pharmaceutically acceptable carrier or tax of the present invention Shape agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Drug system Agent should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain There are glucose and the aqueous solution of other adjuvants to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile Under the conditions of manufacture.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg of about 1 microgram/kg body weight-daily Weight.In addition, polypeptide of the invention can be also used together with other therapeutic agents.

It is that the immune conjugate of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety Effective quantity typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg weight, preferably The ground dosage is about 1 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration route, disease The factors such as people's health status, within the scope of these are all skilled practitioners technical ability.

The preparation of monoclonal antibody

Antibody of the invention can be prepared by various technologies known to those skilled in the art.For example, this hair Bright antigen can be applied to animal to induce the generation of monoclonal antibody.For monoclonal antibody, can using hybridoma technology come Preparation is (see Kohler et al., Nature 256；495,1975；Kohler et al., Eur.J.Immunol.6:511,1976； Kohler et al., Eur.J.Immunol.6:292,1976；Hammerling et al., In Monoclonal Antibodies And T Cell Hybridomas, Elsevier, N.Y., 1981), display technique of bacteriophage or the recombinant DNA method (U.S. can be used The patent No. 4,816,567) preparation.

Representative myeloma cell is effective integration, the stabilization Gao Shui for supporting by the antibody produced cell of selection antibody It shows no increases in output and gives birth to and to those of culture medium (HAT medium matrix) sensitivity myeloma cell, including myeloma cell strain, such as mouse The myeloma cell strain of class (is purchased from Salk including the myeloma cell strain derived from MOPC-21 and MPC-11 mouse tumor Institute Cell Distribution Center, Santiago, California, the U.S.) and SP-2, NZ0 or X63-Ag8-653 cell (is purchased from American Type Culture Collection, Luo Keweier, Maryland, the U.S.). Human myeloma and mouse-human heteromyeloma's cell strain be also described for generate human monoclonal antibodies [Kozbor, J.Immunol., (1984) 133:3001；Brodeur etc., the production technology and application (Monoclonal of monoclonal antibody Antibodies Production Techniques and Applications), 51-63 pages (Marcel Dekker, Inc., New York, 1987)].

Growth of Hybridoma Cell is analyzed in culture medium therein to detect and have the monoclonal of required specificity anti- The generation of body, e.g., by external binding analysis for example, Enzyme Linked Immunoadsorbent Assay (ELISA) or radiommunoassay (RIA). The position for expressing the cell of antibody can be detected with FACS.Then, hybridoma clone can be formed by limiting dilution procedures It is subcloned (subcloned), and grows (Goding, monoclonal antibody (Monoclonal by standard method Antibodies): principle and practicing (Principles and Practice), Academic Press (1986) 59-103 Page).The suitable culture medium used to reach this purpose includes, for example, DMEM or RPMI-1640 culture medium.In addition, Hybridoma can be grown as ascites tumor in animal body.

Pass through conventional immunoglobulin purification from culture medium, ascites or serum by the monoclonal antibody of subclone secretion Technique is suitably separated, these purifying process are for example, Protein A-agarose method (protein A-Sepharose), hydroxyl Base apatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Display technique of bacteriophage is a screening technique, and allogenic polypeptide or albumen are merged table with the capsid protein of bacteriophage It reaches, fusion protein is shown on the surface of virion, and the DNA for encoding the fusant is then located in virion, to make big It establishes between amount polypeptide and its DNA encoding sequence and directly contacts, make various target molecules (antibody, enzyme, cell surface receptor etc.) Polypeptide ligand Rapid identification is able to by elutriation.

The present invention provides a kind of monoclonal antibodies for HPV E7 albumen, especially for HPV18/45 E7 albumen Monoclonal antibody.In of the invention one preferred scheme, monoclonal antibody is using culture hybridoma method preparation. The supernatant for taking Hybridoma Cell Culture is purified through affinity column (Protein A/G-Sephrose).

In the preferred scheme of of the invention one, monoclonal antibody is using Balb/C mouse ascites production monoclonal antibody Method preparation.Hybridoma is inoculated into the mouse peritoneal of sensitization, ascites is extracted, through affinity column (Protein A/ G-Sephrose it) is purified.

In the preferred scheme of of the invention one, monoclonal antibody sets up eukaryotic expression system, wink using recombinant DNA method Turn HEK293 cell to express antibody, then by antibody of the secretion in culture medium through affinity column (Protein A/G- Sephrose it) is purified.

Method and sample

The present invention relates to for the method in tissue samples detection cervical carcinoma.This method step approximately as: obtain tissue Sample；Detect the level of HPV cancer protein in the sample.Sample used in the method for the present invention is that clinical pathology is commonly used Formalin fix, the cervical tissue of paraffin embedding slice.

The present invention can be used for the detection of HPV cancer protein in HPV infection associated cancer, wherein the relevant cancer of HPV infection Such as the tumour of the urogenital systems such as cervical carcinoma, bladder cancer, carcinoma of endometrium, carcinoma of penis, Small Cell Lung Cancer, melanoma with And the preliminary stage of H/N tumors and these cancers.

According to the present invention, Histologic Examination Method can be supported or even replaced using HPV cancer protein molecular labeling.In spy In different case, protein molecular label is used as diagnostic tool without the branch of the Morphology observation further based on cell It holds.Biomarker of the present invention is HPV cancer protein, and the biomarker is from virus, because virus exists in tissue Label characteristic do not occur at be uninfected by people tissue in, therefore to the detection of the sample of HPV infection have specificity.

Sample (sample) employed in the present invention includes tissue samples and biopsy specimen.Terminology used in the present invention is " living Inspection " should include the biopsy of all kinds well known by persons skilled in the art.Therefore biopsy used in the present invention may include example As tumour excision sample, pass through tissue samples prepared by the puncture of endoscopic procedures or organ or needle puncture biopsy.

Sample used in the present invention may include fixed or preservation tissue samples.Tissue samples can be protected for example There are can commercially obtain in the sample collection of standard, storage or conveying medium, such as known to those skilled in the art It saves medium (formalin, Cytyc " PreservCyt " or Tripath Imaging " Cytorich " etc.).It is suitable to save Medium may include it is one or more selected from alcohol, aldehyde, ketone, acid, metal ion or mercury, ether etc. for saving the mixed of cellular component Close object.Alcohol includes methanol, ethyl alcohol, (just or different) propyl alcohol, (just, exclusive or uncle) butanol or high branch or unbranched alcohol.Aldehyde includes Formaldehyde, acetaldehyde, glutaraldehyde etc..Also the ketone of such as acetone can be used.The acid used in the sample medium of standard includes The inorganic acid of machine acid (acetic acid, trichloroacetic acid, salicylic acid and picric acid) or such as chromic acid.May include in the sample solution of standard Metal such as silver, copper, chromium, mercury, osmium and uranium.The salting liquid of uranyl acetate, two potassium chromates, ammonium sulfate etc. can be preservation and be situated between The component of matter.

Kit

The present invention also provides a kind of reagents for containing only antibody of the invention (or its segment) or detection plate of the invention Box, in a preference of the invention, the kit further includes container, operation instructions, buffer etc..

The present invention is further designed for the detection kit of detection high-risk HPV E7 cancer protein, which includes knowing The antibody of other high-risk HPV E7 cancer protein, detects required common reagent and buffer, such as various buffers, enzyme-linked tag Secondary antibody, detection label, detection substrate etc..The antibody is preferably anti-HPV E7 antibody, more preferably anti-HPV18 and/or HPV45 E7 monoclonal antibody.The detection kit can be in-vitro diagnosis device.

The present invention is further designed and developed for the HPV infection correlation circumstance diagnostic assessment from cervical biopsy sample Kit, the kit can detecte the high-risk HPV E7 cancer protein being present in sample.Tissue is carried out formalin to consolidate It is fixed, paraffin embedding processing, and it is related to be used to develop the HPV infection to sample on the basis of based on acellular morphological analysis The detection kit and in-vitro diagnosis device that tumour is detected.

It is and described one of the objects of the present invention is to provide a kind of method for detecting HPV18/45 E7 protein expression Method can be used for detecting the detection of HPV infection associated cancer especially cervical carcinoma.

The present inventor etc. has made the monoclonal antibody for human papilloma virus HPV18/45 E7 protein MAB-14 simultaneously studies its reactivity.HPV18 and/or HPV45 is infected using this anti-HPV18/45 E7 monoclonal antibody MAB-14 Positive cervical cancer tissues sample carries out immunohistochemical staining, as a result, it has been found that MAB-14 has strong dyeing anti-in tumour cell It answers, while interstitial cell and reactive cellular context are clean by tumour cell.

According to the discovery as a result, the present inventor completes the present invention.

That is, method of the invention is the method for detecting tumor marker, it is characterised in that: including in detection sample HPV18/45 E7 the step of.

In the method for the invention, the detection sample is preferentially that epithelium of cervix uteri damage is possible to the trouble for suffering from cervical lesions Person is also possible to the patient that uterine neck has occurred and that lesion.

The HPV18/45 E7 is preferably HPV18/45 E7 protein or its segment.In this situation, detect described The step of HPV18/45 E7, preferably uses the immunohistochemical staining of HPV18/45 E7 to analyze.Used anti-HPV18/ 45 E7 antibody are preferably anti-HPV18/45 E7 monoclonal antibody.

Albumen from HPV18/45 E7 oncogene expression used by the method immune detection is as HPV18/45 phase The reliability index that the pernicious or pre-malignant cells closed occurs.The most useful aspect of the present invention first is that cervix cancer, squamous Epithelial cell damage and gland cancer and application in the diagnosis of relevant any epithelial cell exception is infected to carcinogenic HPV18/45, institute The carcinogenic HPV18/45 infection shown includes Koilocytosis；Hyperkeratosis；It is formed including intraepithelial neoplasia or intraepithelial lesions Precancer illness；Height depauperation；With infectivity or malignant cancer.In addition to cervical carcinoma, to the detection of HPV18/45 E7 to inspection The tumour of the urogenital systems such as bladder cancer, carcinoma of endometrium, carcinoma of penis is surveyed, Small Cell Lung Cancer, melanoma and neck are swollen Tumor is also useful.

Another object of the present invention provides a kind of detection kit by means of the present invention.The kit, which can be, examines Disconnected kit or research kit.

Kit of the invention is the kit to detect tumor marker, it is characterised in that has anti-HPV18/45 E7 monoclonal antibody.Kit of the invention is had to detect required common reagent and buffer, such as various slow Fliud flushing, the secondary antibody of enzyme-linked tag, detection label, detection substrate etc..The antibody is preferably anti-HPV18/45 E7 antibody, more Preferably anti-HPV18/45 E7 monoclonal antibody obtains particularly preferably by recombinant DNA technology and is used for eukaryotic expression system Anti- HPV18/45 E7 monoclonal antibody gene recombinant expression carrier.The anti-HPV18/45 E7 mouse generated by eukaryotic expression system Monoclonal antibody or and the anti-HPV18/45 E7 mouse monoclonal antibody have the active monoclonal antibody of same combination.

The present invention provides a kind of methods by the HPV18/45 E7 albumen of detection cellular endogenous, and it is non-malignant to distinguish uterine neck The tumour cell of lesion, and the neutral formalin being clinically widely used is fixed, in the histotomy of paraffin embedding With use, so as to make diagnosis early stage cancer develops, foundation is provided for treatment in time.

Further, detection kit is formed by using the detection method the present invention also provides a kind of.

Main advantages of the present invention are:

(1) antibody provided by the invention for HPV18/45 E7 albumen, specificity is high, and affinity is strong, and can be big Amount preparation, monoclonal antibody quality are easy to control.

(2) antibody provided by the invention for HPV18/45 E7 albumen be capable of specificity with HPV18/45 E7 albumen It is combined, therefore the antibody is used not only for detection HPV18 E7 albumen, additionally it is possible to for detecting HPV45 E7 albumen.

(3) antibody provided by the invention is used in the method for detection HPV E7 albumen provided by the invention, stability is good, inspection It is high to survey sensitivity.

(4) monoclonal antibody and detection method provided by the invention, suitable for the early diagnosis of associated cancer, and can be used In monitoring recurrence patient.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1

The preparation of 1 human papilloma virus HPV18 E7 monoclonal antibody

1.1 animal immune

Five 4-6 weeks female BAl BIc/C mices are taken, immunogene is His-HPV18 E7 albumen, and immune programme is shown in Table 1.Every time Mouse docking blood sampling is carried out before immune, it is indirect as detection antigen coat using His-HPV18 E7 recombinant protein (5 μ g/ml) ELISA method detection mice serum potency, to immune serum potency titre > 1:10, extracting spleen cell is merged when 000.Knot Fruit #F mice serum potency highest, reaches fusion standard (Fig. 1), #F mouse is taken to be merged.

1 mouse immune program of table

1.2 cell fusions and culture

The splenocyte for collecting immune mouse according to a conventional method is merged with myeloma cell SP2/0, and fusion ratio spleen is thin Born of the same parents: SP2/0=5:1, fused cell are sub-packed in 96 well culture plates, are placed in 37 DEG C, selectively cultivate in 5%CO2 constant incubator. It is changed full liquid 3 times after fusion with HAT culture medium；When hybridoma covers with a field of microscope, ELISA detection is carried out.

The screening of 1.3 positive hybridomas

When detection, screened using indirect ELISA: antigen selects immunogen protein mixed liquor, 5 μ g/ml and 1 μ g/ml difference Wrapper sheet, 4 DEG C overnight, after PBST washing pats dry, the closing of 5% skimmed milk power is added, 37 DEG C act on 2h or 4 DEG C overnight, PBST washing After patting dry, hybridoma supematant is added and positive (P), negative (N) and blank control (blank is directly added into ELIAS secondary antibody) are set, 37 DEG C reaction 1h, PBST washing pat dry after add 37 DEG C of sheep anti mouse-HRP secondary antibody (Sigma A2554) (1:10,000) reaction 45 ~60min, PBST washing pat dry rear TMB colour developing and 2M H₂SO₄It terminates.

It is positive hole that ELISA the selection result, which is greater than 1 with OD value, and is rechecked.By continuous test positive twice Afterwards, by the positive hole, cell is expanded, and is frozen and is subcloned in time.

1.4 hybridomas subclone

All it is positive hole for 2 screenings, is subcloned using limiting dilution assay, takes one piece of 96 hole cell culture 150 μ l HAT culture solutions are added in every hole in plate；Suspension is gently blown and beaten to the positive hole that need to be subcloned, draws 100 μ l cell suspensions It is added in 96 porocyte plates, the doubling dilution since the first hole.There are about the hole of 100 cells and additions in cell count selection hole Into the loading slot containing 6ml HAT culture solution, it is added to by 100 holes μ l/ in the cell plates of paving feeder layer, adds first three columns；It mends again Add HAT culture medium 3ml, 100 holes μ l/ are added in the cell plates of paving feeder layer, add in three column；Add HAT culture medium 5ml again, 100 The hole μ l/ is added in the cell plates of paving feeder layer, six column after adding.It is available steady when calculating the positive rate of every block of plate and reaching 100% Determine cell strain.Obtain nine plants of stabilization monoclonal antibody strain MAB-01 for combining immunogen proteins altogether, 03,04,06,07,10,11, 13,14.And secondary screening is carried out with His-HPV18 E7, His label unrelated protein respectively to this nine plants of monoclonal antibody strains, as a result take Monoclonal antibody the strain MAB-01,06,07,11,13,14 (Fig. 2) of His-HPV18 E7 albumen are specifically bound to six plants.Transfer Into tissue culture plate, expands and cultivate and freeze.

The production (ascites preparation) of 1.5 monoclonal antibodies

The BALB/C female mice of mass propgation hybridoma, 8-10 week old uses atoleine sensitization in advance, then abdomen Chamber injects 1x10⁶Hybridoma/only, it observes after a week, after mouse web portion obviously expands to a certain extent, with No. 9 syringe needles Extract ascites.The ascites of harvest His-HPV18 E7, which is coated with, reaches 1:10,000 or more by ELISA detection titer of ascites.Abdomen Aqueous is after removal fibrin processing, with the purifying of Protein A/G affinity column chromatography method.Protein peak efflux is collected, phosphorus is used Ultraviolet specrophotometer OD260,280 measurement antibody protein concentration, indirect ELISA detection are used after phthalate buffer (PBS) dialysis Antibody titer.As a result MAB-14 monoclonal antibody shows the immune response (Fig. 3) of high-affinity to His-HPV18 E7 recombinant protein.

2. the identification of monoclonal antibody

The identification of 2.1 monoclonal antibody Ig subclass:

Purified monoclonal antibody is diluted using PBS 1:10 000, is operated according to the subtype identification kit specification of Sigma company, MAB-14 monoclonal antibody is IgG1, kappa (Fig. 4).

2.2ELISA detects monoclonal antibody cross reaction:

Antigen selects His-HPV16 E7 (HPV16 E7 amino acid sequence MHGDTPTLHE respectively YMLDLQPETTDLYCYEQLSDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTL GIVCPICSQKP, SEQ ID NO.19), His-HPV18 E7 (SEQ ID NO.1), His-HPV31 E7MRGETPTLQDYVLDLQPEATDLYCYEQLPDSSDEEDVIDSPAGQAKPDTSNYNIVTFCCQCESTLRLCVQSTQVD IRILQELLMGSFGIVCPNCSTRL, SEQ ID NO.20), His-HPV45 E7 (SEQ ID NO.2), His-HPV52 E7 MRGDKATIKDYILDLQPETTDLHCYEQLGDSSDEEDTDGVDRPDGQAEQATSNYYIVTYCHSCDSTLRLCIHSTATD LRTLQQMLLGTLQVVCPGCARL, SEQ ID NO.21), His-HPV58 E7 MRGNNPTLREYILDLHPEPTDLFCYEQLCDSSDEDEIGLDGPDGQAQPATANYYIVTCCYTCGTTVRLCINSTTTDV RTLQQLLMGTCTIVCPSCAQQ, SEQ ID NO.22) fusion protein, 2 μ g/ml wrapper sheets, 4 DEG C are overnight, and PBST washing pats dry Afterwards, the closing of 5% skimmed milk power is added, overnight, after PBST washing pats dry, monoclonal antibody MAB-14 (1 μ g/ is added in 37 DEG C of 2h or 4 DEG C of effects Ml), 37 DEG C of reaction 1h, PBST washings add 37 DEG C of sheep anti mouse-HRP secondary antibody (Sigma A2554) (1:10 000) after patting dry 45~60min is reacted, PBST washing pats dry rear TMB colour developing and 2M H2SO4 is terminated, and reads at OD450nm.As a result see Fig. 5: The specific binding recombinant protein His-HPV18 E7 of MAB-14 monoclonal antibody not only high-affinity, but also the specificity knot of high-affinity Close His-HPV45 E7.Simultaneously with other high-risk type E7 albumen without apparent cross reaction.

2.3 Cloning Human Immunoglobulin Genes clone and sequencing

2.3.1 hybridoma Total RNAs extraction

Hybridoma total serum IgE is extracted referring to Invitrogen " TRIZOL Reagent " operation manual.

2.3.2 reverse transcription and antibody cloning sequencing

It is carried out referring to Fermantas " RevertAid First Strand cDNA Synthesis Kit " specification anti- Transcription, and the end of synthesis 5 ' and 3 ' primer is designed, it is template amplification antibody gene with obtained cDNA, reaction condition:

94 DEG C, 1min；94 DEG C, 30s；55 DEG C, 30s；72 DEG C, 1min (30 cycle)；72 DEG C, 5min.PCR is recycled to produce It is connected to after object band on pMD-19T carrier, and converts TOP10 competent cell, be applied to IPTG, the LB of X-gal is flat Plate carries out blue hickie screening and culturing (37 DEG C, overnight).Picking white single colonie is inoculated into 2ml LB culture medium (Amp+), 37 DEG C of trainings Extracting bacterium solution DNA sends to sequencing after supporting overnight, as a result sees Fig. 6.

The HPV18/45 E7 albumen in cervical cancer tissues detects in 2.4 monoclonal antibody immunity histochemical staining methods

Respectively with mouse monoclonal antibody MAB-01,06,07,11,13,14 for primary antibody to uterine neck normal tissue and HPV18 and/or The positive cervical cancer tissues of HPV45 infection carry out immunohistochemical staining.Method is as follows: pathology paraffin section is first immersed in In dimethylbenzene twice, every time 10 minutes；Then be successively immersed in 100% ethyl alcohol, 95% ethyl alcohol, 90% ethyl alcohol, 80% ethyl alcohol, In 70% ethyl alcohol, each 5 minutes, last deionization washing was twice；It is molten that histotomy is put into the 0.01M sodium citrate buffering boiled In liquid (pH6.0), high temperature and pressure 2 minutes；Room temperature is 30 minutes cooling, and TBST is washed three times, and 5 minutes every time；In order to make endogenous peroxide 3%H is added in compound enzyme inactivation₂O₂Room temperature is handled 10 minutes；TBST is washed 3 times, every time 5 minutes；It is added containing 10% calf serum TBST room temperature is closed 20 minutes；Confining liquid is discarded, above-mentioned 6 strain antibody is separately added into, it is small to be incubated at room temperature 1 by concentration 15ug/ml When；Anti-Mouse/Rabbit IgG-HRP (Dako K5007) is added dropwise after sufficiently washing, is incubated at room temperature 30 minutes；Through filling 2-5 minutes (microscopically observation) of DAB (Dako) colour developing, flowing water after washing is divided to rinse 5 minutes；Haematoxylin redyes 1 minute, originally Water is rinsed well and returns indigo plant；Substep is dehydrated, and successively 70% ethyl alcohol, 80% ethyl alcohol, 95% ethyl alcohol, dehydrated alcohol respectively impregnate 5 minutes； Last xylene soak twice, 5 minutes every time；Neutral gum mounting again；Last micro- sem observation is taken pictures.

As a result see Fig. 7: mouse monoclonal antibody MAB-14 has apparent dark-brown to dye in cervical cancer tissues cancerous tumor cell, and dyeing is fixed Positioned at cytoplasm, and organize interstitial cells and reactive cellular context clean；Mouse monoclonal antibody MAB-13, MAB-06 are in cervical cancer tissues There is apparent dark-brown to dye in cancerous tumor cell, while there is also obvious palm fibres for interstitial cell and reactive cell in the tissue Color background coloration；MAB-01,07,11 in cervical cancer tissues without brown colouring.In conclusion only source of mouse monoclonal antibody MAB- 14 can be special identification cervical cancer tissues in HPV18/45 E7 albumen to make cervical carcinogenesis cell be in specific stain, And dyeing is positioned in cytoplasm.

It discusses:

The present inventor prepares HPV18 E7 source of mouse monoclonal antibody using the above method, by hybridoma supematant in HPV E7 And immunohistochemistry application of preliminary screening and ascites the production antibody purification of label unrelated protein on cervical tissue has screened nine One plant of affinity height is finally taken in strain antibody strain, can high-affinity specific binding His-HPV18 E7, and it is high simultaneously affine The monoclonal antibody MAB-14 of the specific binding His-HPV45 E7 recombinant protein of power, and can identify HPV18/ in cervical carcinogenesis tissue 45 E7 carcinogenic proteins.The study show that MAB-14 monoclonal antibody can specifically bind HPV18 in cervical carcinogenesis cell and/or HPV45 E7 carcinogenic protein is amplified and is catalyzed by two antinoise signals the colour developing of DAB chromogen substrate, makes the evil in cervical cancer tissues slice Property cancerous tumor cell cytoplasm specificity brown colouring is presented, make that cancerous tumor cell is intuitive, objectively labeled comes out.

Uterine neck is finally progressed to the time that cervical carcinoma generally requires 10 years or so, entire cervical lesions by HPV persistent infection Progression is divided into Cervical intraepitheliaI neoplasia (CIN), carcinoma in situ and infiltrating carcinoma.Cervical intraepitheliaI neoplasia (CIN) is according to lesion journey Degree is divided into low lesion (CINI), moderate lesion (CINII) and height lesion (CINIII) again, and cervical carcinoma early intervention is most Good treatment window is CINII and CINIII.Clinic cervical tissue pathological diagnosis goldstandard is still Cytomorphology at present, because The accuracy of this pathological diagnosis result is often depending on the experience and level of pathologist, especially to CINII rank sample In diagnosis, even there are still obvious inconsistencies for the interpretation of CINII sample in the trained stringent U.S..Chinese pathology Doctor lacks, and experienced pathologist is even more fewer and fewer, therefore compels to be essential to the diagnosis of CIN especially CINII sample More effective supplementary means is wanted, it can objectively adjuvant clinical diagnosis.In China, lead to the main type of the HPV infection of cervical squamous cell carcinoma It is still HPV16 and HPV18, accounts for 85% or so of HPV infection type in cervical squamous cell carcinoma.And HPV16 E7, HPV18 E7 albumen Amino acid identity is only 42%, therefore the other antibody of single type is difficult to cover and detects clinically most cases.This hair Bright offer immunohistochemical staining antibody reagent and method, can be in specific marker Cervical intraepitheliaI neoplasia cell and cancerous tumor cell HPV18 E7 carcinogenic protein, prompt HPV18 infection tissue cervical carcinogenesis high risk, reduction is failed to pinpoint a disease in diagnosis and excessively diagnosis and treatment all There is certain clinical application significance.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Aituojin Biological Medicine (Suzhou) Co., Ltd.

<120>a kind of monoclonal antibody and its application for identifying HPV18 and/or HPV45

<130> P2017-0439

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 105

<212> PRT

<213> HPV18 E7

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Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu

1 5 10 15

Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu Ser

20 25 30

Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His

35 40 45

Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met

50 55 60

Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala

65 70 75 80

Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe

85 90 95

Val Cys Pro Trp Cys Ala Ser Gln Gln

100 105

<210> 2

<211> 106

<212> PRT

<213> HPV45 E7

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Met His Gly Pro Arg Ala Thr Leu Gln Glu Ile Val Leu His Leu Glu

1 5 10 15

Pro Gln Asn Glu Leu Asp Pro Val Asp Leu Leu Cys Tyr Glu Gln Leu

20 25 30

Ser Glu Ser Glu Glu Glu Asn Asp Glu Ala Asp Gly Val Ser His Ala

35 40 45

Gln Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Lys Ile Leu Cys

50 55 60

Val Cys Cys Lys Cys Asp Gly Arg Ile Asp Leu Thr Val Glu Ser Ser

65 70 75 80

Ala Asp Asp Leu Arg Thr Leu Gln Gln Leu Phe Leu Ser Thr Leu Ser

85 90 95

Phe Val Cys Pro Trp Cys Ala Thr Asn Gln

100 105

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<211> 24

<212> DNA

<213>artificial sequence

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ggattcactt tcaatagata tgac 24

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Gly Phe Thr Phe Asn Arg Tyr Asp

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<212> DNA

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atcagaaatg atggtagtaa gact 24

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Ile Arg Asn Asp Gly Ser Lys Thr

1 5

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<211> 33

<212> DNA

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acaagggact cctatggtac cgtgtttgct tac 33

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Thr Arg Asp Ser Tyr Gly Thr Val Phe Ala Tyr

1 5 10

<210> 9

<211> 294

<212> DNA

<213>artificial sequence

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cagctcgagc tggtgcagtc agggggaggc tcagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcaat agatatgaca tgtcttggtt tcgccagtct 120

ccagagaaga ggctggagtg ggtcgcagag atcagaaatg atggtagtaa gactcactat 180

tcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctggaaatga gcagtctgag gtctgaggac acggccatgt attactgtac aagg 294

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<213>artificial sequence

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Gln Leu Glu Leu Val Gln Ser Gly Gly Gly Ser Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr

20 25 30

Asp Met Ser Trp Phe Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Asn Asp Gly Ser Lys Thr His Tyr Ser Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg

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<211> 33

<212> DNA

<213>artificial sequence

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cagagcctct tatatagtaa tggaaaaacc tat 33

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Gln Ser Leu Leu Tyr Ser Asn Gly Lys Thr Tyr

1 5 10

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<212> DNA

<213>artificial sequence

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ctggtgtct 9

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Leu Val Ser

1

<210> 15

<211> 27

<212> DNA

<213>artificial sequence

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gtgcaaggta cacattttcc tcagacg 27

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<211> 9

<212> PRT

<213>artificial sequence

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Val Gln Gly Thr His Phe Pro Gln Thr

1 5

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<211> 300

<212> DNA

<213>artificial sequence

<400> 17

gagctcgtca tgacccagac tccactcact ttgtcggtta ccattggaca accagcctct 60

atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaattgg 120

ttattacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggaa cagattttac actgaaaatc 240

agcagagtgg aggctgagga tttgggagtt tattactgcg tgcaaggtac acattttcct 300

<210> 18

<211> 100

<212> PRT

<213>artificial sequence

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Glu Leu Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Gly

85 90 95

Thr His Phe Pro

100

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<211> 98

<212> PRT

<213> HPV16 E7

<400> 19

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Ser Asp Ser Ser

20 25 30

Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp

35 40 45

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr

50 55 60

Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu

65 70 75 80

Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln

85 90 95

Lys Pro

<210> 20

<211> 98

<212> PRT

<213>artificial sequence

<400> 20

Met Arg Gly Glu Thr Pro Thr Leu Gln Asp Tyr Val Leu Asp Leu Gln

1 5 10 15

Pro Glu Ala Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Pro Asp Ser Ser

20 25 30

Asp Glu Glu Asp Val Ile Asp Ser Pro Ala Gly Gln Ala Lys Pro Asp

35 40 45

Thr Ser Asn Tyr Asn Ile Val Thr Phe Cys Cys Gln Cys Glu Ser Thr

50 55 60

Leu Arg Leu Cys Val Gln Ser Thr Gln Val Asp Ile Arg Ile Leu Gln

65 70 75 80

Glu Leu Leu Met Gly Ser Phe Gly Ile Val Cys Pro Asn Cys Ser Thr

85 90 95

Arg Leu

<210> 21

<211> 99

<212> PRT

<213>artificial sequence

<400> 21

Met Arg Gly Asp Lys Ala Thr Ile Lys Asp Tyr Ile Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu His Cys Tyr Glu Gln Leu Gly Asp Ser Ser

20 25 30

Asp Glu Glu Asp Thr Asp Gly Val Asp Arg Pro Asp Gly Gln Ala Glu

35 40 45

Gln Ala Thr Ser Asn Tyr Tyr Ile Val Thr Tyr Cys His Ser Cys Asp

50 55 60

Ser Thr Leu Arg Leu Cys Ile His Ser Thr Ala Thr Asp Leu Arg Thr

65 70 75 80

Leu Gln Gln Met Leu Leu Gly Thr Leu Gln Val Val Cys Pro Gly Cys

85 90 95

Ala Arg Leu

<210> 22

<211> 98

<212> PRT

<213>artificial sequence

<400> 22

Met Arg Gly Asn Asn Pro Thr Leu Arg Glu Tyr Ile Leu Asp Leu His

1 5 10 15

Pro Glu Pro Thr Asp Leu Phe Cys Tyr Glu Gln Leu Cys Asp Ser Ser

20 25 30

Asp Glu Asp Glu Ile Gly Leu Asp Gly Pro Asp Gly Gln Ala Gln Pro

35 40 45

Ala Thr Ala Asn Tyr Tyr Ile Val Thr Cys Cys Tyr Thr Cys Gly Thr

50 55 60

Thr Val Arg Leu Cys Ile Asn Ser Thr Thr Thr Asp Val Arg Thr Leu

65 70 75 80

Gln Gln Leu Leu Met Gly Thr Cys Thr Ile Val Cys Pro Ser Cys Ala

85 90 95

Gln Gln

Claims (16)

1. a kind of heavy chain variable region of antibody, which is characterized in that the heavy chain variable region includes following three complementary determining regions CDR:

CDR1 shown in SEQ ID NO.:4,

CDR2 shown in SEQ ID NO.:6, and

CDR3 shown in SEQ ID NO.:8；

Preferably, the heavy chain variable region has amino acid sequence shown in SEQ ID NO.:10.

2. a kind of heavy chain of antibody, which is characterized in that the heavy chain has heavy chain variable region as described in claim 1, and Heavy chain constant region.

3. a kind of light chain variable region of antibody, which is characterized in that the light chain variable region has complementary determining region selected from the group below CDR:

CDR1' shown in SEQ ID NO.:12,

CDR2' shown in SEQ ID NO.:14, and

CDR3' shown in SEQ ID NO.:16；

Preferably, the light chain variable region has amino acid sequence shown in SEQ ID NO.:18.

4. a kind of light chain of antibody, which is characterized in that the light chain has light chain variable region as claimed in claim 3, and Constant region of light chain.

5. a kind of antibody, which is characterized in that the antibody includes

(1) heavy chain variable region as described in claim 1；And/or

(2) light chain variable region as claimed in claim 3；

Preferably, the antibody includes heavy chain as claimed in claim 2；And/or light chain as claimed in claim 4.

6. a kind of recombinant protein, which is characterized in that the recombinant protein includes

(i) heavy chain variable region as described in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；And

(ii) sequence label of optional assistance expression and/or purifying.

7. a kind of polynucleotides, which is characterized in that it encodes polypeptide selected from the group below:

(1) heavy chain variable region as described in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；Or

(2) recombinant protein as claimed in claim 6.

8. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 7.

9. a kind of genetically engineered host cell, which is characterized in that it contains in carrier or genome according to any one of claims 8 It is integrated with polynucleotides as claimed in claim 7.

10. a kind of immune conjugate, which is characterized in that the immune conjugate contains:

(a) heavy chain variable region as described in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5；With

(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or enzyme.

11. a kind of pharmaceutical composition, which is characterized in that it contains:

(i) heavy chain variable region as described in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, light chain as claimed in claim 4 or antibody as claimed in claim 5, recombination as claimed in claim 6 Albumen or immune conjugate as claimed in claim 10；And

(ii) pharmaceutically acceptable carrier.

12. heavy chain variable region as described in claim 1, heavy chain as claimed in claim 2, as claimed in claim 3 light Chain variable region, antibody as claimed in claim 5, recombinates egg at light chain as claimed in claim 4 as claimed in claim 6 The purposes of immune conjugate white or as claimed in claim 10, which is characterized in that be used to prepare medicament, reagent, detection plate or Kit；

The reagent, detection plate or kit are used for:

(1) HPV18 and/or HPV45E7 albumen in test sample；And/or

(2) endogenic HPV18 and/or HPV45E7 albumen in tumour cell is detected；And/or

(3) tumour cell of detection expression HPV18 and/or HPV45E7 albumen；And/or

(4) identify the type of HPV；

The medicament is used to treat or prevent the tumour of expression HPV18 and/or HPV45E7 albumen.

13. a kind of method of HPV E7 albumen in test sample, which is characterized in that the method includes the steps:

(1) sample is contacted with the antibody described in claim 5；

(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are HPV E7 albumen in sample.

14. a kind of detection plate, which is characterized in that the detection plate includes substrate and test-strips, and the test-strips, which contain, has the right Benefit require 5 described in antibody or immune conjugate as claimed in claim 6.

15. a kind of kit, which is characterized in that include: in the kit

(1) the first container contains the antibody described in claim 5 in the first container；And/or

(2) second container, the secondary antibody containing antibody described in anti-claim 5 in the second container；And/or

(3) third container contains cell cracking agent in the third container；

Alternatively,

The kit contains detection plate described in claim 14.

16. a kind of preparation method of preparation and reorganization polypeptide, which is characterized in that this method includes:

(a) under conditions suitable for the expression, host cell as claimed in claim 9 is cultivated；

(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in claim 5 or claim Recombinant protein described in 6.