CN103130890A - Human papillomavirus (HPV) 16E7 monoclonal antibody and relevant hybridoma cell strain and application thereof - Google Patents
Human papillomavirus (HPV) 16E7 monoclonal antibody and relevant hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention provides a human papillomavirus (HPV) 16E7 monoclonal antibody. The HPV 16E7 monoclonal antibody combines protein specificity shown in SEQ ID NO:2, and the monoclonal antibody is obtained by animal immunity, wherein a fusion protein formed by the protein shown in the SEQ ID NO:2 and a known label and the fusion protein is used as antigen fro the animal immunity. The monoclonal antibody is obtained by two hybridoma cell lines with preservation numbers of CGMCC519 or CGMCC5200. The invention further provides application technology of relevant bybridoma cell strains and the antibody for detecting a biomarker, especially for detecting a HPV carcinogenic protein which is specifically expressive to tumor cells. The HPV 16E7 monoclonal antibody is capable of specifically detecting HPV16E7, can be used as a reagent of high sensitivity and high specificity, and is used for establishing of cancer detection method such as developing body external kit products. The antibody is capable of specifically combining cervical cancer cells transformed by HPV virus, can be used for researching of treatment for cancers, and developing target treating medicines. The HPV 16E7 monoclonal antibody is high in detection agent specificity, flexible in reaction, low in cost, and suitable for large-scaled popularization.
Description
Technical field
The present invention relates to genetically engineered and immunological technique field, more specifically, relate to the monoclonal antibody technique field, refer to especially a kind of HPV16E7 monoclonal antibody, relevant hybridoma cell strain and application.
Background technology
Cervical cancer is the common cancer of female reproductive system, occupies women's malignant tumour second.Large quantity research discovery, human papillomavirus HPV (human papilloma virus) is the arch-criminal who causes cervical cancer, can also cause multiple other tumour, comprises reproductive tract, mammary gland, digestive tube, reaches respiratory cancer.The HPV propagation in population of China in recent years is growing on and on, and the research work of the cancerous precaution of the Forbidden City neck, treatment is extremely important.
1949, at first Sttauss observed human papillomavirus (HPV) particle under Electronic Speculum in wart body leach liquor.Zur Hansen in 1976 propose HPV may be spread through sex intercourse carcinogenic factor after, HPV infects the heat subject that becomes the tumour virus etiological study with the research of Cervical Cancer.HPV be a kind of there is species specificity have a liking for epithelium virus, belong to the small DNA virus of double-stranded closed loop, comprise approximately 8000 base pairs.Comprising 8 early stage open reading frames (E1-E8), 2 late period single open reading frame and 1 non-coding Chang Kong district.In open reading frame, E6 and E7 gene pairs Growth of Cells stimulate the most important in early days, and E6, the E7 albumen of E6, E7 coding cause the cervical epithelial cells immortalization.And late period reading frame L1 and encode the respectively main and less important capsid protein of HPV of L2 gene, be assembled into the capsid of HPV.
The E6 of high-risk hypotype HPV, the cause-effect relationship that E7 albumen causes normal epithelium cell to change into cancer cell is proved by science.Therefore, the hybridoma of a kind of HPV16E7 monoclonal antibody and secretion HPV16E7 monoclonal antibody need to be provided, this hybridoma energy stably excreting HPV16E7 monoclonal antibody, be the further function of research HPV16E7, for the research of cancer therapy and cancer detection, lays the foundation.
Summary of the invention
Main purpose of the present invention is exactly the problems and shortcomings for above existence, a kind of HPV16E7 monoclonal antibody, related manufacturing processes, hybridoma cell strain and application are provided, this HPV16E7 monoclonal anti physical efficiency specific detection HPV16E7, can be used for the infection of the high-risk oncovirus hypotype of HPV16, and the specific detection of cervical cancer early diagnosis.Specificity is high, is quick on the draw, and cost is low, is suitable for large-scale promotion application.
In order to solve above-mentioned purpose, in a first aspect of the present invention, provide a kind of HPV16E7 monoclonal antibody, be characterized in, described HPV16E7 monoclonal antibody is the monoclonal antibody to the albumen energy specific binding shown in SEQ ID NO:2.
" specificity " described in the present invention refers to the recognition capability of monoclonal antibody to corresponding antigens or approximate antigenic substance.Specificity is high, just strong to the recognition capability of antigen.Therefore, above-mentioned HPV16E7 monoclonal antibody can specific recognition and in conjunction with the albumen shown in SEQ ID NO:2.Albumen shown in SEQ ID NO:2 can directly synthesize, and also can prepare by gene engineering method.In a specific embodiment of the present invention, the albumen shown in SEQ ID NO:2 can be to be produced by the 1st to 291 nucleotide codings of SEQ ID NO:1.
Preparing monoclonal antibody of the present invention antigen used is to obtain by engineered method, utilizes and express the polynucleotide sequence SEQ ID NO:1 that contains the antigen of the present invention of encoding in prokaryotic organism.Those skilled in the art knows the carrier that is applicable to express antigen of the present invention usually.Can select suitable carrier according to selected suitable promotor and goal gene sequence to be expressed.Can use any suitable host cell expression antigen of the present invention.Suitably host's example comprises: prokaryotic cell prokaryocyte is as intestinal bacteria, genus bacillus, streptomycete etc.The method of transduction, conversion or transfection is known in the art, includes, but not limited to virus infection, calcium chloride infection protocol, liposome transfection method, electroporation or microprojectile bombardment methods etc.In order to activate promotor, to select transformant or the required gene that increases, can in the conventional nutritional medium of suitably modifying, cultivate by the host cell of transduction, transfection or conversion.The culture condition such as the temperature of using in cultivation, pH value are all generally to be determined by the host cell of selected expression specified protein, and these conditions are all well known to those skilled in the art.For a large amount of recombinant proteins that obtain, can adopt inducible promoter, and utilize the expression of inductor induced gene.In fact, " inductor " can be any material that can induce genetic expression in the host, can be chemical substance or environmental stimulus.
Preferably, described monoclonal antibody is that the fusion rotein that adopts the albumen shown in SEQ ID NO:2 and known label to form obtains as antigen-immunized animal.
More preferably, described known label is, but is not limited to histidine-tagged (His label), glutathione S-transferase (GST label), Trx label, S-tag label, HA label, HSV label, Myc label or VSV-G label.In a specific embodiment of the present invention, described known label is the GST label, with the albumen shown in SEQ ID NO:2, forms the GST-HPV16E7 fusion rotein.
More preferably, described monoclonal antibody adopts described fusion rotein to obtain as the antigen immune mouse.
Further, described monoclonal antibody has specificity to HPV16E7, and HPV16L2 is not had to specificity.
Preferably, the hybridoma cell strain that described monoclonal antibody is CGMCC5199 or CGMCC5200 by preserving number produces.
In a second aspect of the present invention, two kinds of hybridoma cell strains are provided, be characterized in, described hybridoma cell strain is for generation of above-mentioned HPV16E7 monoclonal antibody, and the preserving number of described hybridoma cell strain is CGMCC5199 or CGMCC5200.
Above-mentioned hybridoma cell strain has been deposited in one of international depositary institution, and " (China General Microbiological Culture Collection Center; CGMCC; address: great Tun road, Chaoyang District, BeiJing, China city; Institute of Microorganism, Academia Sinica; postcode: 100101); preservation date: on September 8th, 2011, Classification And Nomenclature is mouse hybridoma cell (Mouse hybridoma) to China Committee for Culture Collection of Microorganisms's common micro-organisms " center ".
As antigen, prepared by the fusion rotein that the preparation method of above-mentioned HPV16E7 monoclonal antibody adopts the albumen shown in SEQ ID NO:2 and known label to form.
Preferably, described known label is, but is not limited to histidine-tagged (His label), glutathione S-transferase (GST label), Trx label, S-tag label, HA label, HSV label, Myc label or VSV-G label.In a specific embodiment of the present invention, described known label is the GST label, with the albumen shown in SEQ ID NO:2, forms the GST-HPV16E7 fusion rotein.
Preferably, described fusion protein immunization mouse, the splenocyte and the myeloma cell that get mouse are merged, filter out and can secrete the hybridoma that the albumen shown in SEQ ID NO:2 is had to the monoclonal antibody of specific reaction, in the animal ascites from the culture supernatant of described hybridoma or from injecting described hybridoma, obtain described monoclonal antibody.
More preferably, the preserving number of described hybridoma cell strain is CGMCC5199 or CGMCC5200.
In of the present invention one preferred specific embodiment, using the GST-HPV16E7 fusion rotein as the antigen immune mouse, get the splenocyte of mouse and the myeloma cell of syngeneic animal and merged.Screening can produce the hybridoma of purpose antibody, carries out the cloning cultivation, and sets up the hybrid cell strain.Aforesaid method is only exemplary, and other Mammalss of immunity that for example can use the same method, using its splenocyte as immunocyte.Can also select suitable myeloma cell for merging, the myeloma cell who for example is used for from rat, mouse or hamster.Immunocyte and myeloma cell's fusion can be carried out according to ordinary method.
The hybridoma that screening produces purpose antibody carries out mono-clonal.The hybridoma cell strain of the generation monoclonal antibody of the present invention obtained can go down to posterity and cultivate or preserve for a long time in liquid nitrogen in ordinary culture medium.While from hybridoma, collecting monoclonal antibody of the present invention, can from hybridoma vitro culture supernatant liquor, obtain antibody, or hybridoma is injected to suitable Mammals and obtains antibody from animal ascites.A kind of front method is suitable for obtaining highly purified antibody, and a kind of rear method is suitable for obtaining in a large number antibody.The antibody obtained by aforesaid method, can purify by ordinary method, such as saltouing, the method such as gel-filtration, affinity chromatography.
In a third aspect of the present invention, provide the above-mentioned application of HPV16E7 monoclonal antibody in the diagnostic tool of cervical cancer or other human cancer due to preparation detection and/or auxiliary diagnosis human papilloma virus infection.
In a fourth aspect of the present invention, a kind of immunoassay is provided, be characterized in, described immunoassay adopts above-mentioned HPV16E7 monoclonal antibody.
Preferably, described immunoassay is ELISA immunoassay, immunochormatography, immunocytochemical stain assay method or immunohistochemical staining assay method.
In a fifth aspect of the present invention, a kind of test kit is provided, be characterized in, described test kit comprises above-mentioned HPV16E7 monoclonal antibody.
For a person skilled in the art, according to content of the present invention, utilize said monoclonal antibody, prepare corresponding testing product, such as Kit and/or Rapid detection test strip etc., be apparent.Therefore, be also the present invention's content required for protection.
Beneficial effect of the present invention is:
1, HPV16E7 monoclonal antibody of the present invention is that the albumen shown in SEQ ID NO:2 is had to specific monoclonal antibody, can be specifically in conjunction with the albumen shown in SEQ ID NO:2, thereby can be used for the cervical cancer cell that HPV infects, its cell expressing HPV16E7 albumen, the HPV16E7 monoclonal antibody can provide specific detection, and specificity is high.
2, HPV16E7 monoclonal antibody of the present invention has specificity to HPV16E7, HPV16L2 is not had to specificity, thereby can carry out the specific detection that due to the HPV mankind's normal cell due to infecting changes into intraepithelial neoplasia cells (CIN) or cancer cells, make up the deficiency that there is no at present the HPV method for detecting specificity, utilize the HPV16E7 monoclonal antibody to carry out detection specificity high, be quick on the draw.
3, immunoassay of the present invention and test kit can be used for the species specificity detection of HPV positive tumor cell, make up the deficiency that there is no at present the cervical cancer method for detecting specificity.The biomarker of HPV16E7 monoclonal antibody in can the specific binding tumour cell, HPV16E7 or HPV18E7 albumen.Provide specificity high, be quick on the draw, cost hangs down diagnostic techniques, is suitable for high throughput testing and large-scale promotion application.
The accompanying drawing explanation
Fig. 1 is the ELISA detected result of serum titer after 6 immunity of fusion mouse.
Fig. 2 is the SDS-PAGE qualification result of the HPV16E7 monoclonal antibody after purifying, wherein, and the numbering of the monoclonal antibody that E1-E4 is the different batches wash-out.
Fig. 3 and Fig. 4 be the HPV16E7 monoclonal antibody that produces of different hybridomas respectively with HPV16E7L2 and HPV18E7 in conjunction with detected result.
Fig. 5, Fig. 6 and Fig. 7 are the result that HPV16E7 monoclonal antibody 8C10 antibody and 8E5 antibody carry out the immunocytochemical stain experiment to HPV positive cervical cancer cell SiHa respectively, the wherein negative control mice IgG of Fig. 5.
Fig. 8 and Fig. 9 are that HPV16E7 monoclonal antibody 1A5 cuts into slices and carries out the result of immunohistochemical experiment, the wherein negative contrast of Fig. 8 Cervix Squamous Cell carninomatosis reason.
Embodiment
The inventor, through extensive and deep research, obtains a kind of HPV16E7 monoclonal antibody, and this HPV16E7 monoclonal antibody has excellent specific binding effect to HPV16E7.Completed on this basis the present invention.
In order more clearly to understand technology contents of the present invention, especially exemplified by following examples, be described with reference to the accompanying drawings.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.In embodiment, various chemical reagent commonly used used, be commercially available prod.
1, experiment material and method
1.1 reagent and medicine
Fu Shi fully, Freunds incomplete adjuvant (Freund ' s Adjuvant complete or incomplete); PEG (Polyethyleenglycol4000*PEG*Macrogolum 4,000 lot#BCBCO873), purchased from Fluka company; Horseradish peroxidase-labeled sheep anti-mouse igg (Goat anti-mouse IgG-HRP lot#15-035-164), purchased from Jackson company; DMEM (Dulbecco ' s Modified Eagle Medium, lot#SH3002.01B), purchased from HyClone company; Foetal calf serum, purchased from Hyclone company; TMB (Tetramethylbenzidine), purchased from Sigma company; Mouse monoclonal antibody subclass parting kit (Mouse Monoclonal Antiboay Isotyping Reagents, 058K4836); IHC test kit (Dako, EnVision+System-HRP Labelled Polymer Anti-mouse, K4000); Other related reagent is the pure or analytical pure of top grade.
1.2 instrument and consumptive material
Superpurgative working table (Boxun is purchased from Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); CO
2constant incubator (German Thermo); Ultralow Temperature Freezer (Forma-86C Germany Thermo); YDS-50B-125 type liquid nitrogen biological container (Cengdu Jinfeng Liquid Nitrogen Container Co. Ltd.); Simple microscope (XDS-1A); Electronic balance (plum Teller-Tuo benefit instrument Shanghai company limited); 85-1 constant temperature blender with magnetic force (Community of Jin Tan County city Jie Ruier Electrical Appliances Co., Ltd); Table-type high-speed refrigerated centrifuge (German eppendorf); HHS type electric-heated thermostatic water bath (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); MULTISKAN MK3 microplate reader (Thermo company); Ultrapure water preparation device (U.S. MILLPORE company); GZX-9240MBE digital display air dry oven (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); WellWASK4 MK2 washes plate machine (purchased from Thermo company); 96 holes, 24 porocyte culture plates (NUNC company); 96 hole enzyme plates (NUNC company).Single track, multichannel pipettor (German Eppendorf company); Tissue Culture Flask, improvement ox Boydii blood cell counting plate etc.
1.3 cell strain, tissue sample and animal
SP2/0 myeloma cell, clone SiHa:SP2/0 is given by teacher Shou Chengchao of Beijing Tumour Hospital laboratory, and clone SiHa is purchased from Shanghai Inst. of Life Science, CAS cell resource center.
Tissue slice: tumor biopsy is bought in Xi'an Ai Lina bio tech ltd.
4～6 week age female BALB/c mouse: buy the Experimental Animal Center in Yangzhou University.
1.4 main solution system
Coated damping fluid (carbonate solution): get 0.2mol/L Na
2cO
38mL, with 0.2mol/L NaHCO
317mL mixes, then adds 75mL distilled water, adjust pH to 9.6.
Dilution buffer liquid (PBS, without calcium and magnesium):
Lavation buffer solution (PBS-Tween 20,0.5 ‰): Tween-200.5mL, join in 1L dilution buffer liquid (PBS), and stirring and evenly mixing, between adjust pH to 7.0～7.2.
20 * substrate is used liquid A: get TMB 21mg, fully be dissolved in 5mL ethanol.
100 * substrate is used liquid B: get urea hydrogen peroxide 16.5mg, be dissolved in the 1mL distilled water.
Stop buffer (2mol/L H
2sO
4): distilled water 178.3mL drips the vitriol oil (98%) 21.7mL.
Incomplete DMEM substratum: be purchased from Hyclone company.
Complete DMEM substratum: be purchased from Hyclone company.
The HAT substratum: complete DMEM substratum 99mL, add 500 * HAT stock solution 1mL, under aseptic condition, prepare.
1.5 the preparation of monoclonal antibody
1.5.1 antigen preparation:
Prepare prokaryotic expression carrier and express GST-HPV16E7L2 (seeing the aminoacid sequence shown in SEQ ID NO:3) as immunizing antigen, using HPV16E7L2-His (seeing the aminoacid sequence shown in SEQ ID NO:4) as detectable antigens.The detection protokaryon protein sequence that all the other relate to is: GST-HPV16E7 (seeing the aminoacid sequence shown in SEQ ID NO:5), GST-HPV16L2 (seeing the aminoacid sequence shown in SEQ ID NO:6), GST-HPV18E7 (seeing the aminoacid sequence shown in SEQ ID NO:7).
1.5.2 mouse immune program
Take GST-HPV16E7L2 as immunogen, and immune programme for children is in Table 1.
Table 1 mouse immune program
Annotate: three exempt from after blood sampling in 7 days survey and tire
1.5.3 cytogamy and cultivation
1.5.3.1 the preparation of feeder layer cells (merge and get Turnover of Mouse Peritoneal Macrophages the day before yesterday)
Get above ICR mouse in 8 week age, de-cervical vertebra is lethal, 75% alcohol-pickled 5min; Add the 10ml substratum in the 15ml centrifuge tube; Tear mouse Dermal exposure peritonaeum with autoclaving scissors, tweezers, with the 5ml sterilizing syringe, draw RPMI-DMEM 5ml; Tweezers are mentioned peritonaeum, and substratum is injected to abdominal cavity, massage gently belly 1min, extract substratum; The cell of extraction is spread in 96 orifice plates.
1.5.3.2 cytogamy
Myeloma cell SP2/0 and splenocyte are merged, merged ratio splenocyte: SP2/0=5: 1, fused cell is sub-packed in 96 well culture plates, is placed in 37 ℃, 5%CO
2in constant incubator, selectivity is cultivated.Entirely change liquid 3 times with the HAT substratum after merging; When hybridoma covers with a field of microscope, carry out the ELISA detection.
1.5.3.3 the screening of positive hybridoma
During detection, the screening of employing indirect ELISA: antigen is selected the HPV16E7L2-His fusion rotein, the 2ug/ml wrapper sheet, 4 ℃ are spent the night, after the PBST washing pats dry, add 5% skim-milk sealing, 37 ℃ of effects 2h or 4 ℃ spend the night, after the PBST washing pats dry, add the hybridoma supernatant and the positive (P), negative (N) and blank (blank directly adds ELIAS secondary antibody) are set, 37 ℃ of reaction 1h, add the anti-37 ℃ of reaction 30～45min of sheep anti mouse-HRP bis-again after the PBST washing pats dry, the PBST washing pats dry rear TMB colour developing and 2M H
2sO
4stop.
The ELISA the selection result with the OD value be greater than 0.6 for going out positive hole, and rechecked.After twice continuous detecting is positive, this positive porocyte is increased, in time frozen and subclone.
1.5.3.4 the subclone in positive hole, enlarged culturing and frozen
For 2 screenings positive hole all, adopt limiting dilution assay to carry out subclone, get 96 porocyte culture plates, add 150ul HAT nutrient solution in every hole; The positive hole that needs subclone is blown and beaten to suspension gently, draw the 100ul cell suspension and be added in 96 porocyte plates, since the first hole doubling dilution.Kong Zhongyue is chosen in cell counting to be had the hole of 100 cells and joins in the loading slot that contains 6ml HAT nutrient solution, by the 100ul/ hole, is added in the cell plate of paving feeder layer, adds first three columns; Add HAT substratum 3ml, the 100ul/ hole is added in the cell plate of paving feeder layer, three row in adding again; Add HAT substratum 5ml, the 100ul/ hole is added in the cell plate of paving feeder layer again, six row after adding.When the positive rate that calculates every block of plate reaches 100%, can obtain stable cell line.Transfer in Tissue Culture Plate, enlarged culturing is also frozen in a large number.
1.5.3.5 a large amount of productions of monoclonal antibody (ascites preparation)
A large amount of hybridomas (8C10) of cultivating, with 1 * 10
6cells/ amount abdominal injection is only used the BALB/C female mice in 8-10 age in week of whiteruss sensitization in advance, gather ascites after about 10 days, detect the ascites positive and tire by ELISA with HPV16E7L2-His is coated, by mouse ascites 2ml, utilizing 10 times of Binding buffer dilutions.Prepare Protein G, 2ml beads is added in post, utilize 200ml Binding buffer to carry out balance simultaneously.After balance, lightly sample is added in post, flow rate control is at per minute 0.5ml/min.After sample is crossed post, carry out Wash Step, utilize 10ml Binding buffer and 10ml Washing buffer to be cleaned, the Washing buffer after last 1ml cleans carries out OD260, and 280 determination of protein concentration, so that the proof cleaning performance.Before carrying out wash-out, first add 100ul Neutralizing Buffer in the EP pipe, carry out wash-out, add Elution buffer, each 1ml, collect altogether 6ml, afterwards, then uses the excessive wash-out of 10ml Elution buffer.After recycling 20ml Binding buffer (plus NaN3 0.05%) carries out balance, be stored in Binding buffer-NaN3.
1.5.4 the Function Identification of monoclonal antibody
1.5.4.1 the specificity of monoclonal antibody and cross reaction are identified
Detect monoclonal antibody to HPV16E7L2-His, HPV18E7-His, GST-HPV16E7, GST-HPV16L2, the reaction of several albumen of GST-HPV18E7.Adopt the ELISA method to be identified: antigen is selected HPV16E7L2-His, GST-HPV16E7, GST-HPV16L2, GST-HPV18E7 and HPV18E7-His fusion rotein, the equal 2ug/ml wrapper sheet of antigen, 4 ℃ are spent the night, after the PBST washing pats dry, add 5% skim-milk sealing, 37 ℃ of effects 2h or 4 ℃ spend the night, after the PBST washing pats dry, add the positive colony supernatant and negative SP2/0 (N) is set and blank (blank directly adds ELIAS secondary antibody), 37 ℃ of reaction 1h, after patting dry, the PBST washing adds again the anti-37 ℃ of reaction 30～45min of sheep anti mouse-HRP bis-, the PBST washing pats dry rear TMB colour developing and 2M H
2sO
4stop.
1.5.4.2 the hypotype of monoclonal antibody is identified
Adopt the hypotype identification kit of Sigma company, article No. is: 058K4836.Testing sequence is pressed the shop instruction operation.
1.5.4.3 monoclonal antibody specificity is identified ELISA:
Use GST-HPV16L2, GST-HPV18E7 and GST-HPV16E7 identify, 4ug/ml spends the night coated, and 5% 37 ℃ of skim-milks sealing 2 hours, carry out specificity identification to 13 strain HPV16 monoclonal antibodies, 37 ℃ act on 1 hour, PBST machine washing 4 times, the anti-37 ℃ of effects of dilution sheep anti mouse two in 1: 8,000 45 minutes, TMB colour developing after PBST machine washing 4 times, stop reading.
1.5.4.4 monoclonal antibody specificity is identified Western blot:
1.5.4.4.1 the evaluation of monoclonal antibody
With the albumen 1ug/ of HPV16-E7L2 and HPV18-E7 prokaryotic expression band loading, 100V voltage, transferring film 45min, 5% skim-milk room temperature sealing 2 hours, add 4 ℃ of positive hybridoma supernatants to spend the night, and adds sheep anti mouse-HRP, room temperature reaction 1h, DAB colour developing after washing 4 times.
1.5.4.4.2 the evaluation of monoclonal antibody
With the albumen 1ug/ of HPV16-L2 and HPV16-E7 prokaryotic expression band loading, 100V voltage, transferring film 45min, 5% skim-milk room temperature sealing 2 hours, add 4 ℃ of monoclonal antibodies to spend the night, and adds sheep anti mouse-HRP, room temperature reaction 1h, DAB colour developing after washing 4 times.
1.5.4.5 monoclonal antibody detects in conjunction with tumour cell: immunocytochemical stain (ICC)
Select cervical cancer cell strain Siha cell to carry out the immunocytochemical stain test to monoclonal antibody.Concrete experimental technique is as follows: 1. the Siha cell divides in 96 orifice plates, adherent growth 2 days; 2. 4% paraformaldehyde is processed respectively 10min with 0.1%Triton, 3%H2O2 after fixing; 3. 37 ℃ of sealings of 5% bovine serum added antibody (5 μ g/ml) after 2 hours, and 37 ℃ are reacted 2 hours; 4. PBS adds sheep anti mouse two dilutions in anti-1: 2000 after washing 3 times, and 37 ℃ are reacted 1 hour; 5. after PBS washes 3 times, the DAB colour developing, hatch 10min, take pictures for 37 ℃.
1.5.4.6 monoclonal antibody is in conjunction with the detection of tumor biopsy: immunohistochemical methods (IHC)
Select the pathological section of kinds cancer to be detected monoclonal antibody.Section is put into dimethylbenzene 15 minutes, then puts into successively dehydrated alcohol, 95%, 90%, 80%, 70% alcohol 5 minutes.Section is put into to antigen retrieval liquid, more than 90 ℃ 15 minutes.To be cooled to room temperature, use 1%Trion to process 10 minutes, using 3% hydrogen peroxide to process 10 minutes.Room temperature sealing 2 hours.Then add antibody (5 μ g/ml), 4 ℃ are spent the night.Wash gently 3 times, each 3 minutes, add EnVision+System-HRP Labelled Polymer Anti-mouse, room temperature 30min.Wash gently 5 times, each 3 minutes, add DAB room temperature 2-5 minute, washing.Haematoxylin redyeing 2 minutes, washing.Dehydration is put into 70%, 80%, 90%, 95% each 2 minutes, dehydrated alcohol 4 minutes successively, is putting into dimethylbenzene 5 minutes, mounting, microscopic examination.
2 results
2.1 merging the mouse positive serum tires
Shown in Figure 1, after 6 immunity, the ELISA of serum titer detects and reaches 1: 25, more than 000.
2.2 screening after merging
Merged altogether 8 blocks of 96-porocyte plates, fusion rate is in 65% left and right, and positive rate is 9.78% (with positive hole, OD >=0.6), and wherein OD >=1.0 has 12 strains, and second day carries out the ELSIA reinspection, picks out the positive holes of 17 strains and carries out subclone.
2.3 screen after subclone
The subclone cell plate change liquid entirely through twice, the ELISA detected result: 17 strains have 5 strains to turn out cloudy in positive hole, and all the other 12 strains are still positive, and positive rate is 70.6%.
2.4 the cross reactivity of monoclonal antibody anti-HPV16E7 to HPV-18E7 albumen
Detect the emiocytosis liquid of monoclonal antibody strain by the ELISA method.Two kinds of albumen of HPV16E7L2-His and HPV18E7-His are all used the 2ug/ml wrapper sheet.Found that: the emiocytosis liquid of monoclonal antibody strain 4G1,6F2,3G5,3A5,1A5,4C8,4G5 is to HPV18 type albumen (E7) no cross reaction.The emiocytosis liquid of monoclonal antibody strain 8E5,8C10,8G9 has stronger cross reaction to HPV18E7-His albumen.The emiocytosis liquid of monoclonal antibody strain 8B12,1F6 has weak cross reaction.Result is summed up in Table 2.Wherein monoclonal antibody strain 1A5 (cell strain ATTO-11-1) is Eclectics's oncocyte that preserving number is CGMCC5199, monoclonal antibody strain 8C10 (cell strain ATTO-11-2) is Eclectics's oncocyte that preserving number is CGMCC5200, be deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preservation date: on September 8th, 2011.
Table 2ELISA detects monoclonal antibody to the HPV18E7 cross reaction
2.5 clone's Subtype is identified
Ask for an interview table 3, the result that the antibody that 12 strain clones are secreted carries out Subtype is: 1F6,4G5,8B12,8G9 antibody are the IgG1 type, and 3A5,3G5,8C10,8E5 antibody are the IgG2b type, and 1A5,6F2,4G1 antibody are the IgG2a type, and 4C8 antibody is the IgG3 type.
The hypotype of table 3 monoclonal antibody is identified
| Antibody | Hypotype |
| 8E5 | IgG2b |
| 8C10 | IgG2b |
| 8G9 | IgG1 |
| 8B12 | IgG1 |
| 1F6 | IgG1 |
| 1A5 | IgG2a |
| 3A5 | IgG2b |
| 3G5 | IgG2b |
| 4C8 | IgG3 |
| 4G1 | IgG2a |
| 4G5 | IgG1 |
| 6F2 | IgG2a |
2.6 ELISA identifies
Identify the result obtained through ELISA, see the following form: 4G1 and 4G5 antibody are for HPV16-L2, and wherein 4G5 and E7 antibody have cross reaction; 1A5,3A5,3G5,6F2,8C10 and 8G9 antibody are all for HPV16-E7.
2.7 SDS-PAGE identifies
Monoclonal antibody after purifying (8C10 antibody), through the SDS-PAGE qualification result, obtains purity at the monoclonal antibody 8C10 antibody more than 95%, sees Fig. 2.
2.8 Western blot identifies
Collect 12 strain clone supernatants and do Western blot detection, wherein the SP2/0 cell conditioned medium is done negative control, GST antibody is done positive control, refer to Fig. 3 and 4, result is: 12 strain monoclonal antibodies all have combination with HPV16E7L2, wherein with the HPV16E7L2 protein-specific, be combined and with HPV18E7 albumen no cross reaction 7 strain 1A5,3A5,3G5,4G1,4G5,6F2,8G9 arranged, 4G1 and HPV16L2 have combination, all the other antibody all belong to HPV16E7.
2.9 immunocytochemical stain (ICC) is identified
Utilize mIgG (negative control), monoclonal antibody 8C10,8E5 carry out the immunocytochemical stain evaluation to the cervical cancer SiHa cell of cultivating.Result shows that cervical cancer tumer line dyeing negative control mIgG that monoclonal antibody 8C10 and 8E5 can make HPV16 infect specifically fails to make cell dyeing, and in the cell of stained positive, HPV16E7 is arranged in tenuigenin and nucleus, sees Fig. 5, Fig. 6 and Fig. 7.
3.0 tumor biopsy immunohistochemical staining test (IHC)
Utilize HPV16E7 monoclonal antibody 1A5 to carry out the immunohistochemical methods detection to the kinds cancer pathological section.Result shows that monoclonal antibody 1A5 has positive staining (presenting brown colouring in Fig. 9) to Cervix Squamous Cell cancer, endometrioid adenocarcinoma, ovary mucinous adenocarcinoma, breast ductal cancer patient's paraffin section sample in cytoplasm.By HC2 bis-generations hybrid capture technology, confirmed that these samples are by the HPV virus infection before this.As shown in Figure 8, not use sample that this monoclonal antibody hatches as negative control.And the sample that uses monoclonal antibody 1A5 to hatch presents the positive staining result, progressive one has confirmed that the HPV16E7 of this monoclonal antibody and sample is specific binding.The place of arrow indication is specific stain, sees Fig. 9.
In sum, HPV16E7 monoclonal anti physical efficiency specific detection HPV16E7 of the present invention, can be used for HPV and cause cancerous tumor cell, comprises the specific detection of precancerous lesion or cervical cancer cell.
In this specification sheets, the present invention is described with reference to its specific embodiment.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Claims (10)
1. a HPV16E7 monoclonal antibody, is characterized in that, described HPV16E7 monoclonal antibody is the monoclonal antibody to the albumen energy specific binding shown in SEQ ID NO:2.
2. HPV16E7 monoclonal antibody according to claim 1, is characterized in that, described monoclonal antibody is that the fusion rotein that adopts the albumen shown in SEQ ID NO:2 and known label to form obtains as antigen-immunized animal.
3. HPV16E7 monoclonal antibody according to claim 2, is characterized in that, described monoclonal antibody adopts described fusion rotein to obtain as the antigen immune mouse.
4. HPV16E7 monoclonal antibody according to claim 3, is characterized in that, described monoclonal antibody has specificity to HPV16E7, and HPV16L2 is not had to specificity.
5. HPV16E7 monoclonal antibody according to claim 1, is characterized in that, the hybridoma cell strain that described monoclonal antibody is CGMCC5199 or CGMCC5200 by preserving number produces.
6. a hybridoma cell strain, is characterized in that, described hybridoma cell strain is for generation of HPV16E7 monoclonal antibody according to claim 1, and the preserving number of described hybridoma cell strain is CGMCC5199 or CGMCC5200.
HPV16E7 monoclonal antibody according to claim 1 preparation detect and/or the diagnostic tool of the human cancer disease that the auxiliary diagnosis human papilloma virus infection causes in application.
8. an immunoassay, is characterized in that, described immunoassay adopts HPV16E7 monoclonal antibody according to claim 1.
9. immunoassay according to claim 8, is characterized in that, described immunoassay is ELISA immunoassay, immunochormatography, immunocytochemical stain assay method or immunohistochemical staining assay method.
10. a test kit, is characterized in that, described test kit comprises HPV16E7 monoclonal antibody according to claim 1.
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| CN110196332A (en) * | 2019-05-22 | 2019-09-03 | 艾托金生物医药（苏州）有限公司 | A kind of method and its application detecting more hypotype HPV E7 albumen |
| CN112362874A (en) * | 2020-10-30 | 2021-02-12 | 武汉呵尔医疗科技发展有限公司 | Cervical cancer screening kit |
| CN112430583A (en) * | 2020-10-30 | 2021-03-02 | 武汉呵尔医疗科技发展有限公司 | Monoclonal antibody against HPV E7, cell strain and application thereof |
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| CN115112885A (en) * | 2022-06-18 | 2022-09-27 | 广州臻卓生物技术有限公司 | HPV detection kit and preparation method and application thereof |
| CN115112885B (en) * | 2022-06-18 | 2023-07-28 | 杭州爱光健康科技有限公司 | HPV detection kit and preparation method and application thereof |
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