CN109575130B - Monoclonal antibody for detecting HPV18E7 protein and preparation and application thereof - Google Patents

Monoclonal antibody for detecting HPV18E7 protein and preparation and application thereof Download PDF

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CN109575130B
CN109575130B CN201811465944.0A CN201811465944A CN109575130B CN 109575130 B CN109575130 B CN 109575130B CN 201811465944 A CN201811465944 A CN 201811465944A CN 109575130 B CN109575130 B CN 109575130B
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常小迦
施丽君
郑雅婷
时成龙
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Attogen Biomedical Suzhou Inc ltd
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Abstract

The invention belongs to the field of biological medicines, and particularly relates to a monoclonal antibody for detecting HPV18E7 protein, and preparation and application thereof. The monoclonal antibody which is high in specificity, strong in affinity, capable of stably expressing and good in repeatability is finally obtained, can specifically recognize HPV18E7 protein, and has the potential of being used for preparing medicines for preventing and treating cancers such as cervical cancer, penile cancer, perineum cancer, vaginal cancer, anal cancer, oropharyngeal cancer and the like.

Description

Monoclonal antibody for detecting HPV18E7 protein and preparation and application thereof
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a monoclonal antibody for detecting HPV18E7 protein, and preparation and application thereof.
Background
HPV is a small DNA virus with strong squamous epithelial character, mainly invading basal lamina cells of squamous epithelium and metaplastic cells located in cervical transformation zone. Currently, there are over one hundred types of HPV known, and the HPV types are classified into high-risk HPV and low-risk HPV according to carcinogenicity. High risk HPV infections (e.g., types 16 and 18) cause most cervical, penile, perineal, vaginal, anal and oropharyngeal cancers and precancerous lesions; whereas non-oncogenic, low risk types of HPV infection (e.g. types 6 and 11) can lead to genital warts and recurrent respiratory papillomatosis.
Cervical cancer is the only tumor of established etiology at present, and is the result of persistent infection with Human Papillomavirus (HPV). During carcinogenesis, HPV DNA is integrated into the genome of human cells, and HPV E6 and E7 proteins are continuously expressed in transformed cells. The HPV E6 protein is mediated by E6-AP (E6 associated protein), binds to and degrades oncostatin p 53; the HPV E7 protein is combined with the cancer suppressor protein pRb, so that the pRb releases a nuclear transcription factor E2F, the cell hyperproliferation is caused, the cell cycle regulation is out of control, the cell immortalization is caused, and the generation of cervical cancer is promoted.
The research on the structure of the HPV E7 protein solution shows that the highly conserved N end of the E7 protein is unstructured and may be one reason of low immunogenicity, and the intracellular localization of the high-risk HPV E7 protein changes with different states of cell fusion, i.e. the distribution of the HPV E7 protein in cells is in a dynamic state, and the HPV E7 protein can be in cytoplasm and nucleus.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the monoclonal antibody capable of specifically recognizing the HPV18E7 protein, has no cross reaction with other proteins, obviously improves the specificity, accuracy and reliability of HPV18E7 protein detection, truly reflects the expression level of the HPV18E7 protein in a cell nucleus, and can be applied to the potential of preparing medicines for preventing and treating cancers such as cervical cancer, penile cancer, perineum cancer, vaginal cancer, anal cancer, oropharyngeal cancer and the like.
In the first aspect of the invention, the heavy chain variable region of the monoclonal antibody comprises HCDRs with amino acid sequences shown as SEQ ID NO. 1-3, and the light chain variable region of the monoclonal antibody comprises L CDRs with amino acid sequences shown as SEQ ID NO. 4-6;
further, the monoclonal antibody is a human, murine, rabbit or chimeric antibody;
further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 7 is shown in the specification; the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 8 is shown in the specification;
further, the monoclonal antibody is an antibody specific to HPV; preferably, the monoclonal antibody is an antibody specific against HPV 18; more preferably, the monoclonal antibody is an antibody specific against the HPV18E7 protein, and the protein is localized in the nucleus.
In a second aspect of the invention, there is provided a recombinant protein comprising a monoclonal antibody according to any one of the first aspect of the invention, and optionally a tag sequence to facilitate expression and/or purification;
further, the tag sequence comprises a6 × His tag;
further, the recombinant protein is a specific anti-HPV recombinant protein; preferably, the recombinant protein is a recombinant protein specific against HPV 18; more preferably, the recombinant protein is a recombinant protein specifically resisting HPV18E7 protein, and the HPV18E7 protein is positioned in cell nucleus.
In a third aspect of the invention, there is provided a nucleic acid molecule capable of encoding the variable region of the heavy and/or light chain of a monoclonal antibody according to any one of the first aspects of the invention or a recombinant protein according to the second aspect of the invention;
further, the nucleic acid molecule encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 9; and/or the nucleic acid molecule encoding the amino acid sequence of the variable region of the monoclonal antibody light chain has the amino acid sequence of SEQ ID NO: 10, or a nucleotide sequence shown in the figure.
In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to any one of the third aspects of the invention;
further, the vector is selected from prokaryotic or eukaryotic expression vectors;
still further, the carrier is preferably selected from: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.
In the fifth aspect of the present invention, a protein or polypeptide is provided, wherein the protein or polypeptide is obtained by expressing the vector of the fourth aspect of the present invention through an expression system;
further, the expression system is a bacterial, yeast, filamentous fungus, eukaryotic mammalian cell, insect cell, plant cell or cell-free expression system.
In a sixth aspect of the invention, there is provided a host cell capable of expressing the monoclonal antibody, recombinant protein, vector or protein or polypeptide described above.
In a seventh aspect of the invention, there is provided a conjugate comprising a monoclonal antibody according to any one of the first aspect of the invention, and an additional biologically active substance;
further, the monoclonal antibody is coupled with other bioactive substances directly or through a connecting fragment;
further, the coupling moiety comprises: a detectable label, drug, toxin, cellular molecule, radionuclide or enzyme;
still further, the coupling moiety comprises a fluorescent or luminescent label, a radiolabel, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., I L-2, etc.), an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPH L)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, and the like.
In an eighth aspect of the present invention, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the monoclonal antibody, the recombinant protein, the nucleic acid molecule, the vector, the protein or the polypeptide, the host cell or the conjugate, and optionally a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances;
further, the pharmaceutical composition is in an injection form;
further, the application of the pharmaceutical composition in preparing a medicament for preventing or treating tumors;
further, the tumor is cervical cancer, penile cancer, perineal cancer, vaginal cancer, anal cancer, or oropharyngeal cancer.
In a ninth aspect, the present invention provides a monoclonal antibody according to any one of the first aspect of the present invention, a recombinant protein according to the second aspect of the present invention, a protein or polypeptide according to the fifth aspect of the present invention, or a conjugate according to the seventh aspect of the present invention, for use in the preparation of a medicament for preventing or treating a tumor;
further, the tumor is cervical cancer, penile cancer, perineal cancer, vaginal cancer, anal cancer, or oropharyngeal cancer.
In a tenth aspect of the invention, there is provided a test device, preferably selected from a pharmaceutical agent, reagent, test plate or kit, comprising a monoclonal antibody according to any one of the first aspect of the invention, a recombinant protein according to the fourth aspect of the invention, a protein or polypeptide according to the fifth aspect of the invention or a conjugate according to the seventh aspect of the invention;
further, the use of the medicament in the preparation of a medicament for preventing or treating a tumor expressing HPV18E7 protein; specifically, the tumor is cervical cancer, penile cancer, perineal cancer, vaginal cancer, anal cancer or oropharyngeal cancer; more specifically, the reagent comprises a chip and immune microparticles coated with antibodies;
further, the reagent, the detection plate or the kit is used for detecting HPV18E7 protein in a sample; and/or detecting the endogenous HPV18E7 protein in a tumor cell; and/or detecting a tumor cell expressing HPV18E7 protein; and/or identifying the type of HPV;
further, the detection plate comprises a substrate (support plate) and a test strip containing the monoclonal antibody according to any one of the first aspect of the present invention or the conjugate according to the seventh aspect of the present invention; specifically, the test strip further comprises an antigenic spot region; more specifically, the test strip is formed by sequentially overlapping sample filtering paper, a chromatographic material, a nitrocellulose membrane and absorbent paper;
further, the kit comprises a first container containing the monoclonal antibody according to any one of the first aspect of the present invention; and/or a second container comprising a secondary antibody directed against a monoclonal antibody according to any one of the first aspect of the invention; and/or a third container comprising a cell lysis reagent; or a detection plate according to the invention;
further, the antibody in the first container is detectably labeled;
further, the antibody in the second container is detectably labeled.
In an eleventh aspect of the present invention, there is provided a method for detecting HPV E7 protein in a sample, the method comprising:
(1) contacting the sample with an antibody according to the first aspect of the invention;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of a complex is indicative of the presence of HPV E7 protein in the sample, preferably by the method of E L ISA;
further, the HPV E7 protein comprises HPV18E7 protein;
further, in (1), the sample is contacted with two antibodies against HPV E7 protein, at least one of which is an antibody according to any one of the first aspect of the present invention, and in (2), the detection is performed by the E L ISA method;
further, the "antigen-antibody complex" is a "first antibody-antigen-second antibody" ternary complex; wherein the first antibody is an antibody according to any one of the first aspect of the invention and the binding epitope of the second antibody is different from the binding epitope of the first antibody;
further, in (1), after the sample is contacted with the antibody according to the first aspect of the present invention, a third antibody against the first antibody is further added to the reaction system, and the formation of an "antigen-first antibody-third antibody" complex is detected in (2);
further, the first antibody, the second antibody or the third antibody carries a detectable label thereon;
further, the detectable label is a biotin label, a colloidal gold label, a horseradish peroxidase label, a radionuclide label or a fluorescein label;
further, the sample comprises: human or animal tissue samples, tumor resection samples, exfoliated cell samples.
Compared with the prior art, the invention has the beneficial effects that: the monoclonal antibody provided by the invention has the advantages of high specificity, strong affinity, stable expression and good repeatability, can specifically recognize HPV E7 protein, particularly HPV18E7 protein, and has the potential of being used for preparing medicines for preventing and treating cancers such as cervical cancer, penile cancer, perineum cancer, vaginal cancer, anal cancer, oropharyngeal cancer and the like.
Drawings
FIG. 1 is a SDS-PAGE result of the purification of the monoclonal antibody against HPV18E7 protein of strain 8 in example 1.
FIG. 2 shows the titer results of the ISA test of the monoclonal antibody E L of the 8 strain anti-HPV 18E7 protein in example 1.
FIG. 3 shows the result of immunohistochemical staining detection of cervical cancer tissue sections by the 8 strain anti-HPV 18E7 protein monoclonal antibody in example 2.
FIG. 4 shows the results of E L ISA detection of E7 protein of 14 HPV subtypes by monoclonal antibody AT-18-34 in example 4.
Fig. 5 shows the distribution of the sites on 40 different cervical tissue chips in example 5.
FIG. 6 shows the result of immunostaining of cervical tissue chips with monoclonal antibody AT-18-34 of example 5.
FIG. 7 shows the results of immunohistochemical staining of cervical lesion tissues with the monoclonal antibody AT-18-34 of example 5.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. It will be understood by those skilled in the art that, based on the disclosure of the present application in combination with common general knowledge in the art, the nucleotide and amino acid sequences disclosed in the present application can be synthesized by other methods commonly used in the art, for example, by chemical synthesis to obtain the sequences disclosed in the present application. Furthermore, the skilled person can construct the novel nucleotide sequences obtained in the present application into any suitable vector, host cell. The following is merely an exemplary illustration of the scope of the invention as claimed, and various changes and modifications of the invention of the present application may be made by those skilled in the art based on the disclosure, which also fall within the scope of the invention as claimed.
The present invention will be further described below by way of specific examples. The various chemicals used in the examples of the present invention were obtained by conventional commercial routes unless otherwise specified.
The invention provides a monoclonal antibody for resisting HPV E7 protein, in particular HPV18E7 protein, and a preparation method and application thereof. The monoclonal antibody can be combined with recombinant HPV18E7 protein, can be used for detection of formalin-fixed paraffin-embedded clinical tissue sample sections, and can be positioned in cell nuclei. The invention provides a method for detecting and/or detecting immobilized HPV18E7 protein, a kit containing the monoclonal antibody and a detection plate.
The invention provides a monoclonal antibody, wherein a heavy chain variable region of the monoclonal antibody comprises HCDR with an amino acid sequence shown in SEQ ID NO. 1-3, and a light chain variable region of the monoclonal antibody comprises L CDR with an amino acid sequence shown in SEQ ID NO. 4-6.
Further, the invention provides a monoclonal antibody AT-18-034 specifically binding to HPV18E7 protein, wherein the variable regions of the heavy chain and the light chain of the monoclonal antibody consist of the amino acid sequences.
The preparation method of the monoclonal antibody comprises the following steps of immunizing a Balb/c mouse by adopting purified GST-HPV 18E7 recombinant protein (the amino acid sequence of the GST-HPV 18E7 recombinant protein is shown as SEQ ID NO: 11), fusing mouse spleen cells with SP2/0 cells, using His-HPV18E7 recombinant protein (the amino acid sequence of the His-HPV18E7 recombinant protein is shown as SEQ ID NO: 12) as a screening antigen, screening positive hybridoma cells by using an E L ISA method, and finally screening to obtain 9 monoclonal strains, wherein the monoclonal antibodies are further detected by an E L ISA method and subjected to immunohistochemical tests to finally obtain a monoclonal antibody AT-18-034 capable of specifically identifying the nuclear HPV18E7 protein, and the antibody subtype is Ig2a or Kappa.
The invention also provides application of the monoclonal antibody AT-18-034 in preparation of an immunodetection tool for detecting HPV18E7 protein.
Specifically, the immunodetection tool is a kit, a chip or test paper.
In a specific embodiment, the invention provides an immunohistochemical detection kit, which comprises the monoclonal antibody AT-18-034 and can detect the expression condition of HPV18E7 protein in tissue cells.
The invention also provides application of the monoclonal antibody in preparing a kit for detecting tumors, wherein the tumors specifically refer to tumors closely related to HPV E7 protein expression, and include but are not limited to cervical cancer, penile cancer, perineum cancer, vaginal cancer, anal cancer, oropharyngeal cancer, precancerous lesion and other related tumors.
Compared with the prior art, the invention provides a preparation method and a sequence of a monoclonal antibody AT-18-034, and also provides application of the monoclonal antibody in preparation of an immunodetection tool for detecting HPV E7 protein, an immunohistochemical kit containing the monoclonal antibody AT-18-034, and application of the monoclonal antibodies in preparation of a kit for detecting tumors. The monoclonal antibody can be combined with HPV18E7 protein in a cell nucleus, and has no cross with other proteins in the cell, so that the specificity of HPV18E7 protein detection is obviously improved, and the monoclonal antibody can be applied to detection of the expression level of HPV18E7 protein in related tumor tissue cells of cervical cancer, penile cancer, perineum cancer, vagina, anus and oropharyngeal cancer, precancerous lesion and the like.
The invention provides a monoclonal antibody for detecting HPV18E7 protein and application thereof, and raw materials and reagents used in a diagnostic tool containing the monoclonal antibody can be purchased from the market.
The present invention is further illustrated below with reference to specific examples, which are intended to illustrate the invention only and not to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the following examples, are generally performed according to conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1: preparation of monoclonal antibody against HPV18E7 protein
(1) Animal immunization
Six female BA L B/C mice of 4-6 weeks are taken, the immunogen is GST-HPV 18E7 protein, complete Freund adjuvant emulsification is adopted, subcutaneous or intraperitoneal injection is adopted for immunization, the immunization dose is 50 mu g/mouse, after three times of immunization, tail blood is taken, His-HPV18E7 recombinant protein (5 mu g/ml) is taken as detection antigen, E L ISA method gradient dilution is carried out to determine the serum titer, whether the immunization is strengthened or not is determined according to the result, and the mice with the highest antibody titer are selected for cell fusion.
(2) Cell fusion and culture
Spleen cells of immunized mice were collected by a conventional method and fused with myeloma cells SP2/0 at a ratio of: SP2/0 ═ 5:1, and the fused cells were distributed in 96-well plates and incubated at 37 ℃ under 5% CO2Selectively culturing in constant-temperature incubator, changing HAT culture medium for 3 times after fusion, and detecting E L ISA when hybridoma cells grow over a microscope.
(3) Screening and culture of monoclonal antibodies
When the detection is carried out, indirect E L ISA detection is carried out by adopting an enzyme label plate coated by His-HPV18E7, E L ISA screening results are positive holes with OD values larger than 1, recheck is carried out, after the positive holes are continuously detected for positive twice, the positive holes are amplified, cryopreservation and limited dilution are carried out in time, a 96-hole cell culture plate is taken, 150 mu l of HAT culture solution is added into each hole, the positive holes needing to be subcloned are lightly blown and suspended, 100 mu l of cell suspension is sucked and added into the 96-hole cell plate, dilution is carried out in a multiple ratio from the first hole, holes with about 100 cells in the holes are counted, the holes are added into a sample adding groove containing 6ml of HAT culture solution, the cells are added into a feeder layer-paved cell plate according to 100 mu l/hole, the samples are added, 3ml of HAT culture medium is added into the feeder layer-paved cell plate, 100 mu l/hole is added into the feeder layer-paved, the cells are added into the feeder layer-paved with 5ml of HAT culture medium, 100 mu l/hole, six rows of AT 18-18 cell plates, AT 18-18 cell suspension protein is added, AT 18-AT-18-AT-18 cell culture plate is stably cultured, AT-18-AT-18-cell-protein stable-AT-protein is obtained, AT-18-protein is added, and the AT-18-AT.
(4) Preparation of ascites by monoclonal antibodies
Culturing hybridoma cells in large amount, pre-sensitizing 8-10 week-old BA L B/C female mice with liquid paraffin, and injecting monoclonal cell suspension into abdominal cavity with cell dosage of 1x106Observing after one week, after the abdomen of the mouse obviously expands to a certain degree, extracting ascites by using a 9-gauge needle, removing fibrin from the ascites fluid, purifying by Protein A/G affinity column chromatography, collecting Protein peak effluent, dialyzing by using Phosphate Buffer Solution (PBS), determining the concentration of antibody Protein by using an ultraviolet spectrophotometer OD260 and 280, detecting the purity by SDS-PAGE, detecting the antibody titer by indirect E L ISA, subpackaging and freezing the antibody AT-20 ℃, wherein the detection result of the SDS-PAGE is shown in figure 1, and the result shows that 8 monoclonal antibodies can be simply and effectively purified by affinity chromatography, the detection result of E L ISA is shown in figure 2, and from figure 2, when His-18E 7 recombinant Protein is taken as an antigen, the titer of AT-18-04 is the highest, the titer of the HPV-18-25 is the lowest, wherein the AT-18-04 reaches the peak value when the concentration is 62.5/ml, and the rest antibodies except the AT-18-25 reach the peak value AT 250 ng/ml.
Example 2: screening of specific anti-nuclear HPV18E7 protein monoclonal antibody
Respectively using monoclonal antibodies AT-18-04, AT-18-05,the cervical cancer tissues are subjected to immunohistochemical staining by AT-18-25, AT-18-26, AT-18-27, AT-18-28, AT-18-29 and AT-18-34. The method comprises the following steps: soaking pathological paraffin sections in dimethylbenzene twice for 15 minutes each time; then soaking in 100% ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes respectively, and finally washing twice with deionized water; placing the tissue slices into a boiled 0.01M sodium citrate buffer solution (pH6.0), and carrying out high temperature and high pressure for 5 minutes; cooling at room temperature for 30min, washing with TBST for three times, 5min each time; addition of 3% H for inactivation of endogenous peroxidase2O2Treating at room temperature for 10 min; TBST washing for 3 times, each time for 5 min; adding TBST containing 10% calf serum, and sealing at room temperature for 20 min; discarding the confining liquid, adding the 8 strains of antibodies with the concentration of 20ug/ml respectively, and incubating at room temperature for 1 h; after full washing, adding Anti-Mouse/Rabbit IgG-HRP (Dako K5007) dropwise, and incubating for 30min at room temperature; DAB (Dako) color development for 2-5min (observation under microscope) after full washing, and flushing for 5min with running water; counterstaining with hematoxylin for 1min, washing with tap water, and returning to blue; dehydrating step by step, sequentially soaking in 75% ethanol, 95% ethanol, and anhydrous ethanol for 5 min; finally, soaking the mixture twice in dimethylbenzene for 5min each time; then sealing the neutral gum into a piece; and finally, observing and photographing through a microscope.
The results of immunohistochemical staining of cervical cancer sections with the 8 monoclonal antibodies are shown in FIG. 3, wherein the 8 monoclonal antibodies showed large differences in performance on the same cervical cancer tissue, 3 monoclonal antibodies failed to stain cervical cancer tissues, AT-18-25, AT-18-27 and AT-18-29, 1 monoclonal antibody AT-18-26 stained the sections but non-diseased tissue, 3 monoclonal antibodies stained cervical tissue cytoplasm from strong to weak, AT-18-28> AT-18-05> AT-18-04, only AT-18-34 stained nuclei, AT-18-25, AT-18-27, AT-18-29-26 and AT-18-26 were detected in E L, which bound to recombinant His-HPV18E7 protein but not to HPV18E 4 protein inside the tissue cells, AT-18-28, AT-18-05 and AT-18-04, which bound to the recombinant HPV18E7 protein, and AT-9-18-9 protein 6859 were detected in E L, which the HPV18E protein was found to bind to the HPV18E 9 protein, AT-18 protein, which was found in the HPV-9 monoclonal antibodies and the HPV-9 monoclonal antibodies were found to bind to the HPV-9, which only the HPV-9, and the HPV-9 monoclonal antibodies were found in the HPV-18-35, which bind to the HPV-9, which binds to the HPV-9 monoclonal antibodies in the HPV-9 monoclonal antibodies.
Example 3: monoclonal antibody AT-18-34 gene cloning and sequencing
Total RNA of hybridoma producing the monoclonal antibody AT-18-34 was extracted with reference to the Invitrogen "TRIZO L Reagent" operating manual, reverse transcription was performed with reference to the Fermantas "RevertAid First Strand cDNA Synthesis Kit" instructions, and 5 'and 3' end primers were designed and synthesized, the resulting cDNA was used as a template to amplify the monoclonal antibody gene under the reaction conditions of 94 ℃, 1min, 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min (30 cycles), 72 ℃, 5min, recovery of the PCR product band, ligation to pMD-19T vector, transformation of TOP10 competent cells, plating on a L B plate with IPTG, X-gal for white spot screening culture (37 ℃, overnight), selection of white single colonies to 2ml L B medium (Amp +), extraction of DNA after 37 ℃ culture, sequencing of the monoclonal antibody, sequencing of the heavy chain region of the monoclonal antibody, SEQ ID-18-34, and further analysis of the heavy chain variable region of the monoclonal antibody, SEQ ID-18, SEQ ID-34, SEQ ID-18-34, and the heavy chain variable region of the monoclonal antibody, SEQ ID region of the heavy chain variable region of the monoclonal antibody, SEQ ID region, SEQ ID of SEQ ID NO: SEQ ID NO 18-34, SEQ ID region, SEQ ID NO: SEQ ID NO 18-34, SEQ ID region of the monoclonal antibody, SEQ ID region, SEQ ID NO, SEQ ID region, SEQ ID.
Example 4 ISA method of E L detection of specificity of monoclonal antibody AT-18-34 recognition of subtype HPV
The antigen respectively selects high-risk His-HPV 16E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 13), His-HPV18E7 recombinant protein, His-HPV 31E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 14), His-HPV 33E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 15), His-HPV 35E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 16), His-HPV 39E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 17), His-HPV 45E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 17)The sequence is shown as SEQ ID NO: 18) and His-HPV 52E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 19), His-HPV 56E 7 recombinant protein (amino acid sequence shown in SEQ ID NO: 20), His-HPV 58E 7 recombinant protein (amino acid sequence shown in SEQ ID NO: 21), His-HPV 59E 7 recombinant protein (amino acid sequence shown in SEQ ID NO: 22), His-HPV68 recombinant protein (amino acid sequence shown in SEQ ID NO: 23) and low-risk His-HPV 6E 7 recombinant protein (amino acid sequence is shown as SEQ ID NO: 24), His-HPV 11E 7 recombinant protein (amino acid sequence shown in SEQ ID NO: 25), 1. mu.g/ml plate, 4 ℃ overnight; PBST is washed and dried; adding 5% skimmed milk powder, sealing, and acting at 37 deg.C for 2 hr; PBST is washed and dried; adding 1 mu g/ml AT-18-34 monoclonal antibody, and reacting for 1h AT 37 ℃; PBST is washed and dried, and then goat anti-mouse-HRP secondary antibody (Sigma A2554) (1: 10000) is added for reaction at 37 ℃ for 1 h; TMB color development after PBST Wash patting to dryness 2M H2SO4 is terminated; OD450 nm. The results are shown in FIG. 4, in which 12 kinds of them are high-risk subtypes, except that HPV6 and HPV11 are low-risk subtypes. The AT-18-34 monoclonal antibody only binds with high-risk His-HPV18E7 recombinant protein, slightly binds with His-HPV 39E 7 recombinant protein, and does not bind with HPV E7 protein of other subtypes. The experimental result shows that the monoclonal antibody AT-18-34 can be specifically combined with the HPV18E7 protein without cross reaction with other proteins.
Further, the monoclonal antibody AT-18-34 purified in the section (4) of example 1 was diluted with PBS 1: 10000 according to the specification of the subtype identification kit from Sigma, and the experimental results show that: the AT-18-34 monoclonal antibody is IgG2a, kappa.
Example 5: application of monoclonal antibody AT-18-34 in immunohistochemistry of cervical tissue
(1) Application of immunohistochemistry in cervical chip tissue
The cervical tissue chip has 40 different cervical cancer tissues, the distribution of each point on the chip is shown in figure 5, and the specific information of each point is shown in table 1. The chip was subjected to immunohistochemical detection by the same procedure as in example 2, using monoclonal antibody AT-18-34 as the primary antibody AT a concentration of 20. mu.g/ml. The tissue chip had 43 samples, of which a6 and a7, A8 and B1, and E8 and F1 were different sites of the same case tissue, and thus 40 different case samples were obtained. As shown in FIG. 6, in the test specimen, there was no brown stain at four points of C6, C7, C8 and D1, and there was a brown nuclear stain in the remaining chips. That is, only four samples, 20, 21, 22, and 23, were not detected as positive, and the rest were all positive. The experimental result shows that the positive detection rate of the monoclonal antibody AT-18-34 in the cervical chip tissue is 90 percent (36/40).
TABLE 1 cervical tissue chip information
Site of the body Organizing information Site of the body Organizing information Site of the body Organizing information Site of the body Organizing information Site of the body Organizing information Site of the body Organizing information
A1 Squamous carcinoma of cervix B1 Cervical cancer C1 Cervical cancer D1 Cervical cancer E1 Cervical cancer F1 Adenocarcinoma
A2 Squamous carcinoma of cervix B2 Cervical cancer C2 Cervical cancer D2 Cervical cancer E2 Cervical cancer F2 Cervical cancer
A3 Cervical cancer B3 Cervical cancer C3 Cervical cancer D3 Cervical cancer E3 Squamous carcinoma of cervix F3 Cervical cancer
A4 Squamous carcinoma of cervix B4 Cervical cancer C4 Squamous carcinoma of cervix D4 Squamous carcinoma of cervix E4 Cervical cancer
A5 Cervical cancer B5 Cervical cancer C5 Cervical cancer D5 Squamous carcinoma of cervix E5 Squamous carcinoma of cervix
A6 Cervical cancer B6 Cervical cancer C6 Cervical cancer D6 Squamous carcinoma of cervix E6 Squamous carcinoma of cervix
A7 Cervical cancer B7 Cervical cancer C7 Cervical cancer D7 Squamous carcinoma of cervix E7 Cervical cancer
A8 Cervical cancer B8 Cervical cancer C8 Cervical cancer D8 Adenocarcinoma E8 Cervical cancer
(2) Application of immunohistochemistry in cervical lesion tissue
Further explores the application of the monoclonal antibody AT-18-34 in cervical lesion tissues. Cervical Intraepithelial Neoplasia (CIN) is a precancerous lesion of cervical cancer, including low cervical grade (CIN grade 1), moderate cervical grade (CIN grade 2), high grade dysplasia, and carcinoma in situ (CIN grade 3). Paraffin sections of cervical inflammatory tissues (negative control) and cervical lesion tissues (positive control) are selected, and monoclonal antibody AT-18-34 is adopted to carry out immunohistochemical staining detection, and the specific operation refers to example 2. The results are shown in FIG. 7: fig. 7A is a cervical inflammation sample, no brown nuclear staining detected on the sample; FIG. 7B shows the staining result of CIN1-2 grade sample, when heterotypic cells begin to appear in the basal lamina and monoclonal antibody AT-18-34 is used for detection, brown nuclear staining begins to appear in the basal lamina, and the brown nuclear staining part occupies about 1/3 of the whole epithelial layer; FIG. 7C shows the result of staining CIN2-3 grade sample, with brown nuclear stained cells increased to about 1/2 as diseased cells increased; FIG. 7D shows staining results of CIN3 grade sample, wherein brown stained nuclear cells cover the epithelial layer above 2/3; fig. 7E shows cervical cancer samples with infiltrations as positive controls, where the infiltrates were all seen to be stained with brown nuclei. Therefore, as cervical lesions become more severe, HPV E7 protein expression increases, staining of the monoclonal antibody AT-18-34 increases in each grade group, and the staining localizes in the nucleus.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Attapulgin biomedical (Suzhou) Co., Ltd
<120> monoclonal antibody for detecting HPV18E7 protein, and preparation and application thereof
<130>20181119
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Gly Ile Asp Leu Ser Arg Tyr Ala
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Ile Gly Ser Ser Gly Ser Thr
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Ala Arg Gly Ala Ile Pro Asp Leu
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Gln Ser Val Tyr Asp Asn Asn Trp
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Gly Ala Ser
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Ala Gly Gly Tyr Ser Gly Asn Ile Tyr Gly
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Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly Ser
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Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Arg Tyr Ala
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Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
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Ile Ile Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
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Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr
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Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Ala
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Ile Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Lys
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Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu
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Ile Tyr Gly
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cagtcgttgg aggagtccgg gggtcgcctg gtaacgcctg gaggatccct gacactcacc 60
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cctatgctga cccagactgc atcgtccgtg tctgcggctg tgggaggcac agtcaccatc 60
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ccagggcagc gtcccaagcg cctgatctat ggtgcatcca atctggcatc tggggtccca 180
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Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
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Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
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Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
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Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
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Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
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Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
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Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
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Gly Ser His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu
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Glu Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu
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Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln
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His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys
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Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser
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Phe Val Cys Pro Trp Cys Ala Ser Gln Gln
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His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu Pro
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Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys
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Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala Asp
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Asp Leu Arg Ala Phe Gln Gln Leu PheLeu Asn Thr Leu Ser Phe Val
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Cys Pro Trp Cys Ala Ser Gln Gln Lys Leu Ala Ala Ala Leu Glu His
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His His His His His
115
<210>13
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Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln
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Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp
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Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr
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Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu
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Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln
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Lys Pro
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Met Arg Gly Glu Thr Pro Thr Leu Gln Asp Tyr Val Leu Asp Leu Gln
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Asp Glu Glu Asp Val Ile Asp Ser Pro Ala Gly Gln Ala Lys Pro Asp
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Glu Leu Leu Met Gly Ser Phe Gly Ile Val Cys Pro Asn Cys Ser Thr
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Arg Leu
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Met Arg Gly His Lys Pro Thr Leu Lys Glu Tyr Val Leu Asp Leu Tyr
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Asp Glu Asp Glu Gly Leu Asp Arg Pro Asp Gly Gln Ala Gln Pro Ala
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Thr Ala Asp Tyr Tyr Ile Val Thr Cys Cys His Thr Cys Asn Thr Thr
50 55 60
Val Arg Leu Cys Val Asn Ser Thr Ala Ser Asp Leu Arg Thr Ile Gln
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Gln Leu Leu Met Gly Thr Val Asn Ile Val Cys Pro Thr Cys Ala Gln
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Leu
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Met His Gly Glu Ile Thr Thr Leu Gln Asp Tyr Val Leu Asp Leu Glu
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Thr Leu Arg Leu Cys Val Gln Ser Thr His Ile Asp Ile Arg Lys Leu
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Glu Asp Leu Leu Met Gly Thr Phe Gly Ile Val Cys Pro Gly Cys Ser
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Gln Arg Ala
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Met Arg Gly Pro Lys Pro Thr Leu Gln Glu Ile Val Leu Asp Leu Cys
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Gln His Gln Leu Leu Ala Arg Arg Asp Glu Pro Gln Arg His Thr Ile
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Ala Ser Arg Asp Thr Leu Arg Gln Leu Gln Gln Leu Phe MetAsp Ser
85 90 95
Leu Gly Phe Val Cys Pro Trp Cys Ala Thr Ala Asn Gln
100 105
<210>18
<211>106
<212>PRT
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Met His Gly Pro Arg Ala Thr Leu Gln Glu Ile Val Leu His Leu Glu
1 5 10 15
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20 25 30
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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
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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
<210>19
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<212>PRT
<213>Artificial sequence
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Met Arg Gly Asp Lys Ala Thr Ile Lys Asp Tyr Ile Leu Asp Leu Gln
1 5 10 15
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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>20
<211>105
<212>PRT
<213>Artificial sequence
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Met His Gly Lys Val Pro Thr Leu Gln Asp Val Val Leu Glu Leu Thr
1 5 10 15
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20 25 30
Glu Asp Glu Asp Glu Asp Glu Val Asp His Leu Gln Glu Arg Pro Gln
35 40 45
Gln Ala Arg Gln Ala Lys Gln His Thr Cys Tyr Leu Ile His Val Pro
50 55 60
Cys Cys Glu Cys Lys Phe Val Val Gln Leu Asp Ile Gln Ser Thr Lys
65 70 75 80
Glu Asp Leu Arg Val Val Gln Gln Leu Leu Met Gly Ala Leu Thr Val
85 90 95
Thr Cys Pro Leu Cys Ala Ser Ser Asn
100 105
<210>21
<211>88
<212>PRT
<213>Artificial sequence
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Tyr Ile Leu Asp Leu His Pro Glu Pro Thr Asp Leu Phe Cys Tyr Glu
1 5 10 15
Gln Leu Cys Asp Ser Ser Asp Glu Asp Glu Ile Gly Leu Asp Gly Pro
20 25 30
Asp Gly Gln Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile Val Thr Cys
35 4045
Cys Tyr Thr Cys Gly Thr Thr Val Arg Leu Cys Ile Asn Ser Thr Thr
50 55 60
Thr Asp Val Arg Thr Leu Gln Gln Leu Leu Met Gly Thr Cys Thr Ile
65 70 75 80
Val Cys Pro Ser Cys Ala Gln Gln
85
<210>22
<211>107
<212>PRT
<213>Artificial sequence
<400>22
Met His Gly Pro Lys Ala Thr Leu Cys Asp Ile Val Leu Asp Leu Glu
1 5 10 15
Pro Gln Asn Tyr Glu Glu Val Asp Leu Val Cys Tyr Glu Gln Leu Pro
20 25 30
Asp Ser Asp Ser Glu Asn Glu Lys Asp Glu Pro Asp Gly Val Asn His
35 40 45
Pro Leu Leu Leu Ala Arg Arg Ala Glu Pro Gln Arg His Asn Ile Val
50 55 60
Cys Val Cys Cys Lys Cys Asn Asn Gln Leu Gln Leu Val Val Glu Thr
65 70 75 80
Ser Gln Asp Gly Leu Arg Ala Leu Gln Gln Leu Phe Met Asp Thr Leu
8590 95
Ser Phe Val Cys Pro Leu Cys Ala Ala Asn Gln
100 105
<210>23
<211>110
<212>PRT
<213>Artificial sequence
<400>23
Met His Gly Pro Lys Pro Thr Val Gln Glu Ile Val Leu Glu Leu Cys
1 5 10 15
Pro Cys Asn Glu Ile Glu Pro Val Asp Leu Val Cys His Glu Gln Leu
20 25 30
Gly Asp Ser Asp Asp Glu Ile Asp Glu Pro Asp His Ala Val Asn His
35 40 45
His Gln His Gln Leu Leu Ala Arg Arg Asp Glu Gln Gln Arg His Thr
50 55 60
Ile Gln Cys Thr Cys Cys Lys Cys Asn Asn Leu Leu Gln Leu Val Val
65 70 75 80
Glu Ala Ser Arg Glu Asn Leu Arg Lys Leu Gln Leu Leu Phe Met Asp
85 90 95
Ser Leu Asn Phe Val Cys Pro Trp Cys Ala Thr Glu Thr Gln
100 105 110
<210>24
<211>98
<212>PRT
<213>Artificial sequence
<400>24
Met His Gly Arg His Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln
1 5 10 15
Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser
20 25 30
Ser Glu Asp Glu Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu
35 40 45
Lys Gln His Phe Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn
50 55 60
Val Arg Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln
65 70 75 80
Gln Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro
85 90 95
Lys Thr
<210>25
<211>98
<212>PRT
<213>Artificial sequence
<400>25
Met His Gly Arg Leu Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln
1 5 10 15
Pro Pro Asp Pro Val Gly LeuHis Cys Tyr Glu Gln Leu Glu Asp Ser
20 25 30
Ser Glu Asp Glu Val Asp Lys Val Asp Lys Gln Asp Ser Gln Pro Leu
35 40 45
Thr Gln His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Asp Ser Asn
50 55 60
Val Arg Leu Val Val Glu Cys Thr Asp Gly Asp Ile Arg Gln Leu Gln
65 70 75 80
Asp Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro
85 90 95
Lys Pro

Claims (12)

1. A monoclonal antibody for detecting HPV18E7 protein is characterized in that a heavy chain variable region of the monoclonal antibody comprises HCDR1-HCDR3 with amino acid sequences shown as SEQ ID NO 1-3 respectively, and a light chain variable region of the monoclonal antibody comprises L CDR 1-L CDR3 with amino acid sequences shown as SEQ ID NO 4-6 respectively.
2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO: 7 is shown in the specification; the amino acid sequence of the variable region of the light chain of the monoclonal antibody is SEQ ID NO: shown in fig. 8.
3. A recombinant protein comprising the monoclonal antibody of claim 1 or 2 and a tag sequence to facilitate expression and/or purification.
4. A nucleic acid molecule capable of encoding the monoclonal antibody of claim 1 or 2 or the recombinant protein of claim 3.
5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule encoding the amino acid sequence of the heavy chain variable region of said monoclonal antibody is as set forth in SEQ ID NO: 9; and/or nucleic acid molecules encoding the amino acid sequence of the variable region of the monoclonal antibody light chain are shown in SEQ ID NO: 10, or a nucleotide sequence shown in the figure.
6. A vector comprising the nucleic acid molecule of claim 4 or 5.
7. A protein or polypeptide expressed from the vector of claim 6 by an expression system; the expression system is a bacterial, bacteriophage, yeast, filamentous fungus, mammalian cell, insect cell, plant cell or cell-free expression system.
8. A host cell capable of expressing the monoclonal antibody of claim 1 or 2, the recombinant protein of claim 3, the vector of claim 6, or the protein or polypeptide of claim 7.
9. A conjugate comprising the monoclonal antibody of claim 1 or 2, and an additional biologically active substance; the monoclonal antibody is coupled with other bioactive substances directly or through a connecting fragment.
10. A pharmaceutical composition comprising the monoclonal antibody of claim 1 or 2, the recombinant protein of claim 3, the nucleic acid molecule of claim 4 or 5, the vector of claim 6, the protein or polypeptide of claim 7, the host cell of claim 8 or the conjugate of claim 9, and a pharmaceutically acceptable carrier or excipient, and other biologically active substances.
11. Use of the monoclonal antibody of claim 1 or 2, the recombinant protein of claim 3, the protein or polypeptide of claim 7, or the conjugate of claim 9 for the preparation of a kit for detecting a tumor closely associated with HPV E7 protein expression, wherein the tumor is cervical cancer, penile cancer, perineal cancer, vaginal cancer, anal cancer, or oropharyngeal cancer.
12. A test device comprising the monoclonal antibody of claim 1 or 2, the recombinant protein of claim 3, the protein or polypeptide of claim 7, or the conjugate of claim 9.
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