CN105585631A - Human-papilloma-virus 16-type monoclonal antibody and application thereof - Google Patents
Human-papilloma-virus 16-type monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides a human-papilloma-virus 16-type (HPV16) specific monoclonal antibody. The specific monoclonal antibody is free of obvious cross reaction with other-type HPV, and has the advantages of being high in specificity and sensitivity when used for detection, the content of HPV16-type vaccine with biological activity in vaccine seeds can be accurately detected, and the specific monoclonal antibody can be widely applied in the clinical detection process and the vaccine producing process.
Description
Technical field
The present invention relates to Molecular Virology and field of immunology, particularly, the present invention relates to HPV 16The monoclonal antibody of hybridoma cell line and generation thereof and their sequence of encoding, and they are diagnosed, prevent and treatApplication.
Background technology
HPV (HPV) is nonencapsulated double-stranded small DNA virus, mainly invades and people's epithelial tissue, and then luresIt is good to send out various, neoplasm pathology. High-risk HPV infects relevant to the generation of Several Kinds of Malignancy, and the HPV of low risk infectsCause anus and reproduction wart. The Epidemic Scope that HPV infects is wide, the malignant tumour of the relevant lethal causing and multiple spreading through sex intercourseDisease has serious harmfulness to human health, and exploitation prevention safely and effectively or therapeutic vaccine are significant.
HPV virion diameter is 55～60nm, and nucleocapsid is 20 body symmetries, by five of 72 Major capsid protein L1sAggressiveness and less important capsid protein L2 composition. Studies confirm that in a large number, HPVL1 albumen is the main target protein of HPV vaccine. MultipleThe HPVL1 albumen of expressing in expression system can be formed on morphosis and natural viral Particle Phase seemingly without L2 albumen is auxiliaryViruslike particle (Virus-L1keParticle, VLP). Restructuring HPVL1-VLP vaccine is successfully listing for preventionThe disease such as cervical carcinoma, condyloma acuminatum that HPV infects and causes thus, and fully proved that L1-VLP has and wild homologous virusIdentical antigenicity and immunogenicity, its structure is highly similar to natural HPV, has retained overwhelming majority's neutralization table of natural viralPosition, can induce the neutralizing antibody of high titre.
State Food and Drug Administration (CFDA) is at " vaccine preclinical study technological guidance principle for prevention " middle fingerGo out: " product in links and the step of production technology all should be set up corresponding Standard of Monitoring, so that the entering of subsequent techniqueOK, ensure the quality of product, the stability of technique ", require " at least to comprise at semi-finished product and the quality control in finished product stage simultaneouslyDiscrimination test "
In HPV vaccine R&D process, need to carry out the mensuration of four aspects, type identification experiment, antigenic content detect andVitro efficacy is measured. Its method all can adopt DAS-ELISA. Therefore,, in vaccine research, monoclonal antibody isThe important tool of vaccine antigen Quality Control, especially has the active antibody of specificity and neutralization and has and can not replace in vaccine research and developmentThe effect in generation.
The Human-papilloma Vaccine of having gone on the market at present abroad mainly contains three kinds, respectively: Initial Public Offering in 2006 by16, the tetravalent vaccine (Gardasil) that 18,6 and 11 type human mammilla tumor virus L 1 recombinant proteins form, Initial Public Offering in 2007The bivalent vaccine (Cervarix) being formed by 16 and 18 type papillomavirus L1 recombinant proteins, and Initial Public Offering in December, 2014The nine valency vaccine (Gardasil that formed by 6,11,16,18,31,33,45,52 and 58 type human mammilla tumor virus L 1 recombinant proteins9). The invention provides at most and can, for the specificity of HPV16 within the scope of nine valencys and the active monoclonal antibody of neutralization, utilize itIn the ELISA detection kit prepared of two strains, can be specifically for differentiating fast and quantitative HPV16L1 albumen, canBe widely used in quality inspection, the sound development to women and public defending in Clinical detection and current vaccine manufacturer production vaccine processBiological and ecological methods to prevent plant disease, pests, and erosion control will have great importance.
Summary of the invention
First object of the present invention is to provide the monoclonal antibody that can identify HPV16 and the hybridization that produces this antibodyOncocyte system.
Second object of the present invention is to provide a kind of double-antibody sandwich elisa kit for detection of HPV16.
The 3rd object of the present invention is to provide the preparation method of monoclonal antibody.
The 4th object of the present invention is the preparation method of the antigen detection kit that HPV is provided.
Experiment purpose of the present invention is to be achieved through the following technical solutions:
Monoclonal antibody provided by the invention, it is using HPV16L1 pentamer albumen as immunogene, prepares. ParticularlySay that HPV16L1 pentamer albumen is as immunogen immune mouse, adopt hybridoma technology to obtain through Fusion of Cells screeningEnergy continues, the hybridoma cell strain of the anti-HPV16L1 of stably excreting, obtains monoclonal antibody by each cell line secretion.
Identify in a preferred embodiment the monoclonal antibody of HPV 16, by hybridoma cell strain4G12 produces, and its CGMCC preserving number is 11298.
Disclosed by the inventionly produced by hybridoma cell strain 4G12, its CGMCC preserving number is 11298 monoclonal antibodies, bagContaining at least 1 antibody heavy chain variable region and at least 1 antibody chain variable region, and wherein said antibody chain variable region hasCDR sequence C DRL1, CDRL2 are or/and CDRL3, wherein:
CDRL1 comprises SEQIDNO:1;
CDRL2 comprises SEQIDNO:2;
CDRL3 comprises SEQIDNO:3.
By hybridoma cell strain, 4G12 produces, and its CGMCC preserving number is that 11298 monoclonal antibody variable region of heavy chain have choosingFrom CDRH1, CDRH2 or/and CDRH3, wherein:
CDRH1 comprises SEQIDNO:4;
CDRH2 comprises SEQIDNO:5;
CDRH3 comprises SEQIDNO:6.
The monoclonal antibody of L 1 Protein of Human Papillomavirus Type 16 or antigen binding fragment in a preferred embodimentSection, comprises at least 1 antibody chain variable region and comprises that SEQIDNO:7 and at least 1 antibody heavy chain variable region comprise SEQIDNO：8。
The invention provides in a preferred embodiment a kind of nucleic acid of separation, this nucleic acid coding antibody of the present inventionAt least one variable region of light chain SEQIDNO:7 and variable region of heavy chain SEQIDNO:8.
The invention provides in a preferred embodiment a kind of expression vector of nucleic acid, this carrier transfection host cellShi Suoshu nucleic acid is effectively connected with the control sequence of host cell identification.
The invention provides in a preferred embodiment a kind of host cell of expression vector.
The invention provides in a preferred embodiment the monoclonal antibody of another kind of HPV 16, itsBe characterised in that this monoclonal antibody is produced by hybridoma cell strain 5A6, its CGMCC preserving number is 11299.
By hybridoma cell strain, 5A6 produces, and the monoclonal antibody that its CGMCC preserving number is 11299 comprises at least 1 and resistsBody weight chain variable region and at least 1 antibody chain variable region, and wherein said antibody chain variable region has CDR sequenceCDRL1, CDRL2 are or/and CDRL3, wherein:
CDRL1 comprises SEQIDNO:9;
CDRL2 comprises SEQIDNO:10;
CDRL3 comprises SEQIDNO:11.
Disclosed by the inventionly produced by hybridoma cell strain 5A6, the monoclonal antibody that its CGMCC preserving number is 11299Variable region of heavy chain has the CDRH1 of being selected from, CDRH2 or/and CDRH3, wherein:
CDRH1 comprises SEQIDNO:12;
CDRH2 comprises SEQIDNO:13;
CDRH3 comprises SEQIDNO:14.
The invention provides in a preferred embodiment a kind of monoclonal antibody of identifying HPV 16Or Fab, it is characterized in that wherein comprising at least 1 antibody chain variable region and comprise SEQIDNO:15 and at least 1Individual antibody heavy chain variable region comprises SEQIDNO:16.
The invention provides in a preferred embodiment a kind of nucleic acid of separation, its code book invention antibody at leastVariable region of light chain SEQIDNO:15 and variable region of heavy chain SEQIDNO:16.
The invention provides in a preferred embodiment a kind of expression vector of nucleic acid, its carrier transfection host cellShi Suoshu nucleic acid is effectively connected with the control sequence of host cell identification.
The invention provides in a preferred embodiment a kind of host cell that comprises expression vector.
The invention provides on the other hand the kit of a kind of HPV16L1 of detection, comprise monoclonal antibody disclosed by the inventionOr Fab.
The kit of detection HPV16L1 disclosed by the invention is the double-antibody sandwich for detection of HPV16L1 antigenELISA kit, also comprises detectable mark: radio isotope, fluorescent material, luminescent substance, coloring matter and/orEnzyme.
The invention provides on the other hand a kind of combination that detects a kind of specific detection L 1 Protein of Human Papillomavirus Type 16Thing, said composition comprises monoclonal antibody of the present invention or Fab.
The monoclonal antibody of HPV 16 can be prepared as follows:
1) pentamer of L 1 Protein of Human Papillomavirus Type 16 utilizing is as immunogene, immune Balb/c mouse after purifying, andBlood sampling, detects serum titer with indirect ELISA method, selects the high Balb/c mouse of serum titer to carry out booster immunization, and fromIn this Mice Body, prepare immune spleen cell;
2) prepare myeloma cell's (SP2/0) suspension and inject Balb/c mouse, after mouse grows solid tumor, preparing marrowOncocyte, merges the immune spleen cell described in myeloma cell and step 1), prepares hybridoma delicate, detects screeningTire high hybridoma cell strain and clone expand cultivate;
3) by step 2) the described hybridoma of building after strain expands and cultivates, and collects supernatant pneumoretroperitoneum injection mouse, collectMouse ascites, detects through specificity identification and neutralization activity the monoclonal antibody that obtains HPV 16 after purifying.
The preparation method of HPV double-antibody sandwich elisa antigen detection kit:
Neutralize active monoclonal antibody specific 5A6 as catching using preserving number as 11299 possessing of hybridoma secretionObtain antibody coated elisa plate, neutralize active specific monoclonal taking preserving number as 11298 possessing of hybridoma secretionAntibody 4G12 as detecting antibody, does with recombinant human papilloma virus-16 type VLP albumen after horseradish peroxidase-labeledFor the contrast of standard items preparation calibration curve. This kit also comprises concentrated cleaning solution, sample diluting liquid dry powder, enzyme labelled antibody dilutionLiquid dry powder, substrate solution A, substrate solution B and stop buffer etc. Wherein said concentrated cleaning solution, substrate solution A, substrate solution B, stop bufferComponent and proportioning as follows:
Concentrated cleaning solution: Nacl8.18g, Na2HP0412H2O3.58g, KCL0.2g, KH2PO40.25g, adds distilled waterTo 1000mL;
Substrate solution A:3,3 ', 5 ', 5-tetramethyl biphenyl diamines 200mg, absolute ethyl alcohol 100mL, adds distilled water to 1000mL;
Substrate solution B:Na2HPO414.6g, citric acid 9.3g, the hydrogen peroxide urea 6.4mL of 0.75% concentration, adds distilled waterTo 1000mL, regulate pH to 5.0 ~ 5.4;
Stop buffer: 1mol/L sulfuric acid solution.
The invention provides on the other hand a kind of kit in preparation prevention or detect that HPV 16 L1 infectsApplication in detection composition.
The invention provides on the other hand a kind of composition in preparation prevention or detect that HPV 16 L1 infectsApplication in detection composition.
The monoclonal antibody that the present invention obtains has good specificity, and experiment shows all do not have with other eight types of HPVHave cross reaction, at the vaccine product having gone on the market at present, nine valencys are products of maximum types, and indirect ELISA shows that these are anti-Body has higher tiring simultaneously, and the monoclonal antibody that therefore the present invention obtains can be used for divalence, trivalent or tetravalence, up-to-date at presentNine valency vaccines of listing and comprise the vaccine of HPV16 type or composition in about the specific detection of HPV16 albumen.
The present invention adopts double antibodies sandwich method, utilizes two monoclonal antibodies to carry out specific detection and quantitative to HPV16, canThe pentamer that detects HPV16, also can detect HPV16VLP, thereby, the invention provides a kind of for HPV16 specific detection and fixedThe kit of amount, its detectability: 0.03ug/ml, the range of linearity: 10-0.1ug/ml, for detection of having high specific, Gao MinThe advantage of perception, can accurately detect the level in sample with bioactive HPV16, in Clinical detection and vaccine producerThe quality inspection of producing in vaccine process will be used widely.
Brief description of the drawings
Accompanying drawing 1:SDS-PAGE analyzing and testing result, detection display, the purity of purified each hypotype monoclonal antibody is equalReach more than 95%.
Accompanying drawing 2:ELISA double antibody sandwich method detects HPV16L1VLP calibration curve, and ordinate is OD450. abscissaFor the logarithm value of HPV16L1VLP concentration.
Specific implementation method
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention. Do not deviating from thisIn the situation of bright spirit and essence, the amendment that the inventive method, step or condition are done or replacement, all belong to model of the present inventionEnclose.
If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.In embodiment, the reagent in unreceipted source is the conventional reagent in this area or commercially available reagent.
The foundation of embodiment 1, hybridoma cell line
1, animal immune
1) antigen preparation: utilize escherichia expression system, the pentamer albumen of the L1 albumen of preparation HPV16 type, transmission electron microscopeObserve (100,000 times), result demonstration, the pentamer that in the visual field, visible diameter is 10nm left and right, is diluted to pentamer albumen10μg/ml。
2) fundamental immunity: antigen is mixed and fully emulsified to branch hypodermic injection, every with Freund's complete adjuvant equal-volumeBalb/c mouse per injection amount is 10 μ g.
3) booster immunization: booster immunization adopts the emulsion of antigen and freund 's incomplete adjuvant. Carrying out before Fusion of Cells 3My god, the normal saline solution through lumbar injection containing 15ug antigen.
2, the preparation of hybridoma
Splenocyte and the SP2/0 cell of collecting according to a conventional method mouse melt with the PEG4000 of 500g/L in the ratio of 10:1Close. Select to cultivate with HAT nutrient solution, merge latter 10～15 days, get supernatant and adopt indirect elisa method screening hybridoma cell strain.Adopt limiting dilution assay to carry out subclone to gained positive clone strain. The operating procedure of indirect elisa method is as follows: use 200ng/ holeHPV16VLP wrapper sheet, as positive control, just cleer and peaceful on the culture medium without clonal growth with immune serum 1:2000Normal mice serum is as negative control, and every hole adds 1:2000HRP-goat anti-mouse IgG 100 μ l, finally measures 450nmODValue. All OD450 values are greater than the more than 2 times person of negative control, can the positive clone of preliminary judgement.
3, the foundation of hybridoma cell line
The positive colony that step 2 is obtained continues cloning, above-mentioned hybridoma is tied up to the DMEM that contains 10% hycloneIn culture medium, proceed to cultivate, go down to posterity, cultivate after 10 generations, hybridoma cell line still can well-grown, stable going down to posterity,Obtain the hybridoma cell line of stably excreting monoclonal antibody.
The preparation of the monoclonal antibody of embodiment 2 anti-HPV16
Select the BALB/c mouse of growing up, intraperitoneal inoculation norphytane, every mouse 0.5ml. In 7-10 days pneumoretroperitoneum the 16th generations of inoculation, are assortedHand over oncocyte, every mouse 1 × 106-2×106Individual. Behind 5 days, interval, treat that belly obviously expands, while touch with hand, skin has tightlyOpen sense, available No. 16 syringe needles gather ascites.
By centrifugal ascites (13000r/min30 minute), remove cell component and other sediment, collect supernatant. WithProteinG～SepharoseCL-4B carries out purifying, the PBS buffer solution that upper prop liquid is 20mM, and column chromatography eluent is:PH2.7, the glycine buffer of 20mM, obtains the monoclonal antibody of anti-HPV16. SDS-PAGE detection display, purified listThe purity of clonal antibody all reaches more than 95%, specifically sees accompanying drawing 1.
Embodiment 3: the hypotype qualification of antibody
Adopt indirect elisa method, use antibody that the above-mentioned hybridoma of the Identification of the antibodies of the various IgG hypotypes of anti-mouse producesIgG hypotype.
Result shows, 17 strain clones, and 6C7 clone is IgG2b, all the other are IgG1, the results are shown in Table 1.
Table 1ELISA qualification antibody subtype (OD450 value *)
*: all numerical value is the mean value in multiple hole
Embodiment 4:ELISA detects antibody and HPV16VLP reactivity
Verify by indirect ELISA the detection HPV16 that can antibody purification easily and fast, and further by makingWith the antibody of variable concentrations, can tentatively judge the affinity of antibody.
HPV16VLP is coated in to 96 orifice plates with 200ng/ hole, then with 1ug/ml, 0.2ug/ml and 0.04ug/mlAntibody working concentration add in each hole, detect the combination of each monoclonal antibody purification and HPV16VLP by ELISAIntensity. Result shows, 17 strain antibodies all can be combined with HPV16VLP in the concentration using in this experiment, and signal strength signal intensity is all largeIn 1, the results are shown in Table 2.
Table 2ELISA detects antibody and HPV16VLP bond strength
*: all numerical value is the mean value in multiple hole
Embodiment 5: the specificity identification that detects antibody
HPV-16L1VLP, HPV-18L1VLP and HPV-58L1VLP, by alkaline denaturation and thermal denaturation processing, make it secondaryOr tertiary structure destruction, primary structure is preserved. React with monoclonal antibody again, adopt indirect ELISA to examine itSurvey. By this experiment, not only can identify that antibody is to HPV-16L1VLP, HPV-18L1VLP and HPV-58L1VLP tri-Plant the identification situation of VLP, can also identify whether this antibody is conformation type identification antibody. If react with monoclonal antibody after protein denaturationOD450 value obviously reduces, and proves that this monoclonal antibody is conformation type identification antibody.
Experimental procedure: VLP albuminate processing: 0.2M sodium carbonate, 0.01MDTT, pH10.6 incubated at room 30 minutesAfter boil 5 minutes. Coated: VLP albumen is diluted to 2 μ g/ml. Add to 96 hole ELISA Plates with 100 μ l/ holes, 4 DEG C were coated withNight. Sealing: coated good ELISA Plate is dried. Add in plate with 300 μ l/ hole confining liquids (2%BSA), it is little that room temperature is placed 1-2Time. Sample Dilution: antibody sample is diluted to respectively to 0.3 μ g/ml with sample diluting liquid, mixes, 100 μ l/ holes add to ELISA PlateIn, room temperature is placed 1h. Adding two resists: after sealing, ELISA Plate dries, and HRP mark sheep anti mouse two is anti-with 1:4000 concentration, 100 μ l/Hole adds in ELISA Plate, and room temperature is placed 1h. Colour developing: every hole 300 μ l wash plate 5 times, dries. With toilet paper wiping bottom. With 100μ l/ hole adds nitrite ion, room temperature lucifuge colour developing 10 minutes. Stop: add stop buffer, cessation reaction with 100 μ l/ holes. Reading: willELISA Plate is put into ELIASA, and OD450 carries out reading and data analysis.
The specific detection result of antibody is as shown in table 3. Result shows, except clone 2C2 has and intersects with HPV58VLPReaction, other is the special clone of HPV16VLP. These clones identify the not numerical value of sex change HPV16VLP and are all greater than 1, forObviously positive findings, and the numerical value of identification sex change HPV16VLP is all less than 0.1, negative result. Show that these monoclonal antibodies are structureResemble type identification monoclonal antibody.
The specific ELISA of table 3 antibody detects (OD450. numerical value *)
* all numerical value is the mean value L in multiple hole: not albuminate D: degenerative treatments albumen
Embodiment 6: the active detection of neutralization of antibody
By in pseudovirus-cell and model, detect the active ability of neutralization of each strain antibody.
First each hypotype antibody is diluted to 200ug/ml with PBS, then antibody is carried out to 4 multiple proportions gradient dilutions, get 50ulThe HPV16 pseudovirus of each concentration antibody and 50ul suitable concn in 96 orifice plates 4 DEG C hatch one hour, with pseudovirus and PBSMixed liquor for contrast. Then each mixed liquor is added respectively in 96 orifice plates that are covered with in advance 293FT cell at cell culture incubatorMiddle cultivation 72 hours. Collecting cell afterwards, uses Flow cytometry fluorescence, calculates Control of Fluorescence rate, Control of Fluorescence rate=(1-experimental group/control group) × 100%. Using Control of Fluorescence rate be greater than 50% and 90% respectively as this monoclonal antibody to HPV16 vacationVirus in and titre. Table 4 result shows, the 50% inhibiting rate titre of 5D12 is 25ug/ml, and the titre of 90% inhibiting rate is greater than100ug/ml, the 50% inhibiting rate titre of 5D8 and 1H5 is 0.1ug/ml, the titre of 90% inhibiting rate is 0.39ug/ml. And itsHe clones, and the titre of 50% inhibiting rate titre and 90% inhibiting rate is respectively 0.01ug/ml and 0.02ug/ml. These result tablesBright, these monoclonal antibodies of the present invention all have neutralization active.
The inhibiting rate titre of the each hypotype antibody of table 4
Embodiment 7: in antibody and the external ELISA competitive assay of serum
Utilize competitive ELISA experiment, detect the anti-HPV16 neutralizing antibody state of the antibody of preparation and the World Health Organization (WHO)Border standard items (1stWHOReferenceReagent2007, Anti-humanpapillomavirustype16Serum, NIBSCcode05/134) whether competitive. The standard items of WHO are the polyvalent antibodies in people source, purchased from state of BritainFamily's biological products assay institute (NationalInstituteforBiologicalStandardsandControl,NIBSC). By this experiment, can analyze the epi-position of each strain monoclonal antibody and the correlation of international standard substance epi-position.
First, HPV16VLP is diluted to 100ng/ml, 100ul/ hole adds in 96 orifice plates, 4 DEG C of overnight incubation. By 96After orifice plate washing with 2%BSA confining liquid room temperature sealing 2 hours, then by the international standard substance of 100ul0.5IU/ml andThe HRP labelled antibody of 100ul0.3ug/ml adds in 96 orifice plates, the contrast using 100ulPBS as international standard substance. Room temperatureHatch after 1.5 hours and wash plate, cessation reaction, colour developing, with ELIASA OD450 reading. Calculating inhibiting rate=(1-experimental group OD./Control group OD.) × 100%.
In antibody and the external ELISA competitive assay of serum result as shown in table 5, international standard substance is to clonal antibody 1E5,1H4,2A1,2C2,4G12,4H7,5A6,5E2,6C7,7C10 all has significantly strong inhibition (inhibiting rate > 40%), international standardProduct have weak inhibition (inhibiting rate < 20%) to clonal antibody 1H5 and 2D11, and international standard substance is to clonal antibody 3D5,5D8,5F11There is no inhibitory action with 7B9. Above result shows, except clone 3D5,5D8, the epi-position of 5F11 and 7B9 and international standard substanceThere is no correlation, other clonal antibody all has certain correlation.
Embodiment 8: the affinity qualification of antibody 4G12 and antibody 5A6
Use BIACORE3000(GE) biosensor analysis antigen-antibody in conjunction with and the dynamics of dissociating, calculated Dan KeThe binding constant K of grand antibody 4G12 and 5A6 and HPV16VLPa, dissociation constant KdWith affinity KD, with reflect this antibody withThe power of the combination degree of HPV16VLP.
HPV16VLP is diluted to 40 μ g/ml with acetic acid-sodium-acetate buffer of pH5.5, according to the explanation of manufacturerBook (BIACORE3000 biology sensor, GE company of the U.S.), by HPV16VLP coupling coremaking sheet, arranges coupling level and is4000RU。
Dilute respectively antibody to 0.3125 with PBS buffer solution, 0.625,1.25,2.5,5,10 and 20nmol/L. DetectTime, first antibody sample introduction 60 seconds, then in conjunction with 60 seconds, dissociates 500 seconds, finally uses acetic acid-sodium acetate buffer of the pH5.0 of 10mMLiquid regeneration chip. Carry out the dynamic analysis of antigen-antibody combination according to the description of manufacturer, and data Biacore3000Evaluation software is analyzed. The affinity testing result of antibody 4G12,5A6 and HPV16VLP is as shown in table 6.Result shows that 4G12,5A6 antibody are specific, is only combined with HPV16VLP, is not all combined with other 8 kinds of HPVVLP.
The affinity qualification of table 6 antibody 4G12 and antibody 5A6
aN/A:NotApplicable, represents not combination.
Embodiment 9: the variable region sequences of clone 4G12 and clone 5A6 is measured
The 4G12 of acquisition and 5A6 monoclonal cell are extracted respectively to mRNA, and reverse transcription is cDNA, uses variable region universal primer to enterRow High fidelity PCR amplification, is inserted into PCR product fragment in T carrier, to carry out determined dna sequence, and by the sequence translation obtainingBecome the amino acid sequence of protein. After the sequence of acquisition is compared, do not show identical sequence, obtained sequence is describedFor special sequence.
Sequence is shown in sequence table.
Utilize the sequence of above-mentioned qualification, by known antibody engineering technology, can prepare range gene engineered antibody, exampleAs chimeric antibody, humanized antibody, single-chain antibody, double antibody etc., and retain the biological characteristics of its monoclonal antibody being derived fromProperty.
The assembling of embodiment 10:HPV16 detection kit
To possess the active monoclonal antibody specific 5A6 of neutralization as capture antibody coated elisa plate, active to possess neutralizationMonoclonal antibody specific 4G12 after horseradish peroxidase-labeled as detect antibody, with recombined human papillomatosisPoison-16 type VLP albumen are as the contrast of standard items preparation calibration curve.
Coated antibody 5A6 is diluted to 10 μ g/mL with the carbonate buffer solution of pH9.60.05mol/L, at ELISA PlateEvery hole adds 100 μ L, coated spending the night at 4 DEG C, and the coating buffer that inclines, with PBST washing 2 times, pats dry, and then in every hole, adds 200 μ L3% bSA (BSA), puts into 37 DEG C of insulating boxs and seals after 2 hours, with PBS washing 1 time, adds 10% sugarcaneSugar aqueous solution, room temperature protection 1 hour, fills after drying aluminium foil bag and vacuumizes rear 4 DEG C of preservations after patting dry.
With the antibody of horseradish peroxidase-labeled 4G12, obtain 4G12-HRP and preserve. In ELISA Plate, add respectively VLPSample 100 μ L/ holes, hatch 1.5 hours for 37 DEG C, add 4G12-HRP(0.5ug/ml after washing plate again) 100 μ L/, hatch 1 for 37 DEG CHour, after patting dry, washing add developer to develop the color, and 37 DEG C of incubation 10min, add stop buffer 50 μ L/ holes, use ELIASA450nm wavelength carries out reading.
This kit also comprises concentrated cleaning solution, sample diluting liquid dry powder, enzyme labelled antibody dilution dry powder, substrate solution A, the endThing liquid B and stop buffer etc. Wherein said concentrated cleaning solution, substrate solution A, substrate solution B, component and the proportioning of stop buffer are as follows:
Concentrated cleaning solution: Nacl8.18g, Na2HP0412H2O3.58g, KCL0.2g, KH2PO40.25g, adds distilled waterTo 1000mL;
Substrate solution A:3,3 ', 5 ', 5-tetramethyl biphenyl diamines 200mg, absolute ethyl alcohol 100mL, adds distilled water to 1000mL;
Substrate solution B:Na2HPO414.6g, citric acid 9.3g, the hydrogen peroxide urea 6.4mL of 0.75% concentration, adds distilled waterTo 1000mL, regulate pH to 5.0 ~ 5.4;
Stop buffer: 1mol/L sulfuric acid solution.
The linearity of embodiment 11:HPV16 detection kit detects with repeatability
Application ELISA double antibody sandwich method, clone 4G12 and clone 5A6 antibody do pairing experiment, determine taking clone 5A6 as bagBy antibody, as detecting antibody, determine ELISA detection method with HRP marker clone 4G12, kit detects the range of linearityAs table 7. Fig. 2 is shown in by accompanying drawing, and the range of linearity is 0.1ug/ml-10ug/ml.
The table 7 kit range of linearity detects
Antigen is diluted according to 10ug/ml, 3ug/ml, 1ug/ml, 0.3ug/ml, 0.1ug/ml successively, according to above-mentionedELASA experimental implementation step criticize between and batch in repeated experiment. Every duplicate samples criticize interior and batch between test all duplicate detection 10Inferior, and calculate standard deviation and the coefficient of variation. Mean standard deviation is 0.262, and average coefficient of variation 5.545%, illustrates the present inventionThe double antibodies sandwich ELASA antigen detection method of setting up has good repeatability.
Embodiment 12:HPV16 detection kit specific test
Carry out HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 by the method for above-mentioned foundationThe sample detection of VLP, the processing of VLP albuminate: 0.2M sodium carbonate, 0.01MDTT, pH10.6 incubated at room is after 30 minutesBoil 5 minutes. 10ug/mlVLP100ul is detected in the 96 every holes of orifice plate. The results are shown in Table 8, result shows, this kit detects notSex change HPV16VLP signal is good, the HPV16VLP of nonrecognition sex change, and with other type HPVVLP without intersecting.
Above result shows, kit can have bioactive HPV16VLP for special detection, therefore, and canTo be widely used in the research and development of HPV16 vaccine.
Table 8ELISA method detects HPV16VLP Evaluation on specificity result (OD450) *
*: all numerical value is the mean value in multiple hole
The mensuration program of embodiment 13:HPV16 detection kit
1, preparation sample
1) washing lotion preparation: take out concentrated washing lotion, add distilled water, be settled to 1L and fully mix for subsequent use;
2) sample diluting liquid preparation: to 50ml, fully mix for subsequent use with the rare dry powder of gained washing lotion diluted sample in 1;
3) enzyme labelled antibody diluent preparing: dilute the rare dry powder of enzyme to 50ml by gained washing lotion in 1, fully mix for subsequent use;
4) with the rare dilution standard product of sample that prepare, dilution gradient is set. Concentration be respectively 30ug/ml, 10ug/ml, 3ug/ml,1ug/ml, 0.3ug/ml, 0.1ug/ml, 0.03ug/ml, for subsequent use.
5) with the enzyme dilution enzyme labelled antibody preparing, get 1300 times of dilutions of appropriate enzyme labelled antibody, for subsequent use.
2, determination step
1) application of sample: the standard items that prepare and testing sample are added in coated plate, and 100ul/well arranges multiple hole and the moon simultaneouslyProperty control wells. (negative control is empty is sample diluting liquid) covers shrouding film and puts into 37 DEG C of incubation 45min.
2) add enzyme labelled antibody: ELISA Plate is taken out from 37 DEG C of incubators, discard liquid in hole, add washing lotion 300ul/well,Wash plate 5 times. Discard in hole and on toilet paper, pat dry after liquid for the last time. Add the enzyme labelled antibody solution 100ul/ preparingWell, covers cover plate film and puts into 37 DEG C of incubation 45min.
3) colour developing: ELISA Plate is taken out from 37 DEG C of incubators, discard liquid in hole, add washing lotion 300ul/well, wash plate 10Inferior. Discard in hole and on toilet paper, pat dry after liquid for the last time. Get volume required chromogenic substrate A liquid and chromogenic substrate B liquidRatio according to 1:1 mixes, and 100ul/well adds in ELISA Plate, room temperature lucifuge colour developing 5 minutes.
4) cessation reaction: get volume required stop buffer and add in ELISA Plate, 50ul/well.
5) value of reading: ELISA Plate is put into ELIASA, and OD450nm carries out reading.
6) result is judged: Cutoff value=2.1 × NC average, is judged to be the positive higher than the sample detection result of this numerical value.
The application of embodiment 14:HPV16 detection kit
Get standard items HPV16VLP albumen and carry out gradient dilution, adopt double-antibody sandwich elisa antigen prepared by the present invention to detectKit detects sample HPV16, HPV18 after diluting, the L110 μ g/ml pentamer antigen of HPV58, so as to evaluatingThe Preliminary Applications effect of this kit. Result demonstration, the ELISA method that the present invention sets up can detect HPV16 pentamerAlbumen, and there is specificity.
Table 9ELISA method detects HPV16 pentamer Evaluation on specificity result (OD450)
SEQUENCELISTING
<110>Beijing Health Guard Biotechnology Co., Ltd.
<120>HPV 16 monoclonal antibody and application thereof
<130>2015
<160>16
<170>PatentInversion3.3
<210>1
<211>10
<212>PRT
<213>artificial sequence
<400>1
SerAlaSerSerSerValSerTyrMetHis
1510
<210>2
<211>7
<212>PRT
<213>artificial sequence
<400>2
SerThrSerAsnLeuAlaSer
15
<210>3
<211>8
<212>PRT
<213>artificial sequence
<400>3
GlnArgSerSerTyrProTrpThr
15
<210>4
<211>9
<212>PRT
<213>artificial sequence
<400>4
PheAsnIleLysAspThrTyrMetHis
15
<210>5
<211>17
<212>PRT
<213>artificial sequence
<400>5
ArgIleAspProAlaAsnGlyAsnProGlnTyrAspProLysPheGln
151015
Gly
<210>6
<211>9
<212>PRT
<213>artificial sequence
<400>6
GluValGlyGlyTyrTyrPheAspTyr
15
<210>7
<211>105
<212>PRT
<213>artificial sequence
<400>7
AspIleGlnLeuThrArgSerProAlaIleMetSerAlaSerProGly
151015
GluLysValThrIleThrCysSerAlaSerSerSerValSerTyrMet
202530
HisTrpPheGlnGlnLysProGlyThrSerProLysLeuTrpIleTyr
354045
SerThrSerAsnLeuAlaSerGlyValProAlaArgPheSerGlySer
505560
GlySerGlyThrSerTyrSerLeuThrIleSerArgMetGluAlaGlu
65707580
AspAlaAlaThrTyrTyrCysGlnGlnArgSerSerTyrProTrpThr
859095
PheGlyGlyGlyThrLysLeuGluIle
100105
<210>8
<211>117
<212>PRT
<213>artificial sequence
<400>8
ValLysLeuGlnGlnSerGlyAlaGluLeuValLysProGlyAlaSer
151015
ValLysLeuSerCysThrAlaSerGlyPheAsnIleLysAspThrTyr
202530
MetHisTrpValLysGlnArgProGluGlnGlyLeuGluTrpIleGly
354045
ArgIleAspProAlaAsnGlyAsnProGlnTyrAspProLysPheGln
505560
GlyLysAlaThrIleThrAlaAspThrSerSerAsnThrAlaCysLeu
65707580
GlnLeuSerSerLeuThrSerGluAspThrAlaValPheTyrCysAla
859095
ArgGluValGlyGlyTyrTyrPheAspTyrTrpGlyGlnGlyThrThr
100105110
ValThrValSerSer
115
<210>9
<211>11
<212>PRT
<213>artificial sequence
<400>9
GlnAlaThrGlnAspIleValLysAsnLeuAsn
1510
<210>10
<211>7
<212>PRT
<213>artificial sequence
<400>10
TyrAlaThrGluLeuAlaGlu
15
<210>11
<211>8
<212>PRT
<213>artificial sequence
<400>11
GlnPheTyrGluPheProLeuThr
15
<210>12
<211>9
<212>PRT
<213>artificial sequence
<400>12
PheAsnIleLysAspThrTyrMetHis
15
<210>13
<211>17
<212>PRT
<213>artificial sequence
<400>13
ArgIleAspProAlaAsnGlyAsnThrGluTyrAspProAsnPheGln
151015
Gly
<210>14
<211>9
<212>PRT
<213>artificial sequence
<400>14
GluValGlyGlyTyrTyrPheAspTyr
15
<210>15
<211>106
<212>PRT
<213>artificial sequence
<400>15
AspIleGlnLeuThrGlnSerProSerSerMetSerAlaSerLeuGly
151015
AspArgIleThrIleThrCysGlnAlaThrGlnAspIleValLysAsn
202530
LeuAsnTrpTyrGlnGlnLysProGlyLysProProSerPheLeuIle
354045
TyrTyrAlaThrGluLeuAlaGluGlyValProSerArgPheSerGly
505560
SerGlySerGlySerAspTyrSerLeuThrIleSerAsnLeuGluSer
65707580
GluAspPheAlaAspTyrTyrCysLeuGlnPheTyrGluPheProLeu
859095
ThrPheGlyAlaGlyThrLysLeuGluIle
100105
<210>16
<211>117
<212>PRT
<213>artificial sequence
<400>16
ValLysLeuGlnGlnSerGlyAlaGluLeuValLysProGlyAlaSer
151015
ValLysLeuSerCysThrAlaSerGlyPheAsnIleLysAspThrTyr
202530
MetHisTrpValLysGlnArgProGluGlnGlyLeuGluTrpIleGly
354045
ArgIleAspProAlaAsnGlyAsnThrGluTyrAspProAsnPheGln
505560
GlyLysAlaThrIleThrSerAspThrSerSerAsnThrAlaTyrLeu
65707580
HisLeuSerSerLeuThrSerGluAspThrAlaValTyrTyrCysAla
859095
ArgGluValGlyGlyTyrTyrPheAspTyrTrpGlyGlnGlyThrThr
100105110
ValThrValSerSer
115
Claims (19)
1. identify a monoclonal antibody for HPV 16, it is characterized in that this monoclonal antibody is by hybridomaStrain 4G12 produces, and its CGMCC preserving number is 11298.
2. monoclonal antibody as claimed in claim 1, is characterized in that described antibody comprises at least 1 antibody heavy chain variable regionAt least 1 antibody chain variable region, and wherein said antibody chain variable region there is CDR sequence C DRL1, CDRL2 or/andCDRL3, wherein:
CDRL1 comprises SEQIDNO:1;
CDRL2 comprises SEQIDNO:2;
CDRL3 comprises SEQIDNO:3.
3. monoclonal antibody as claimed in claim 2, is characterized in that wherein said antibody heavy chain variable region has to be selected fromCDRH1, CDRH2 are or/and CDRH3, wherein:
CDRH1 comprises SEQIDNO:4;
CDRH2 comprises SEQIDNO:5;
CDRH3 comprises SEQIDNO:6.
4. identify monoclonal antibody or the Fab of HPV 16, it is characterized in that wherein comprising toFew 1 antibody chain variable region comprises that SEQIDNO:7 and at least 1 antibody heavy chain variable region comprise SEQIDNO:8.
5. a nucleic acid for separation, is characterized in that at least one variable region of light chain SEQ of described nucleic acid coding antibody of the present inventionIDNO:7 and variable region of heavy chain SEQIDNO:8.
6. comprise an expression vector for nucleic acid described in claim 5, while it is characterized in that carrier transfection host cell described in coreAcid is effectively connected with the control sequence of host cell identification.
7. one kind comprises the host cell of claim 6 expression vector.
8. identify a monoclonal antibody for HPV 16, it is characterized in that this monoclonal antibody is by hybridomaStrain 5A6 produces, and its CGMCC preserving number is 11299.
9. monoclonal antibody as claimed in claim 8, is characterized in that described antibody comprises at least 1 antibody heavy chain variable regionAt least 1 antibody chain variable region, and wherein said antibody chain variable region there is CDR sequence C DRL1, CDRL2 or/andCDRL3, wherein:
CDRL1 comprises SEQIDNO:9;
CDRL2 comprises SEQIDNO:10;
CDRL3 comprises SEQIDNO:11.
10. monoclonal antibody as claimed in claim 9, is characterized in that wherein said antibody heavy chain variable region has to be selected fromCDRH1, CDRH2 are or/and CDRH3, wherein:
CDRH1 comprises SEQIDNO:12;
CDRH2 comprises SEQIDNO:13;
CDRH3 comprises SEQIDNO:14.
Identify monoclonal antibody or the Fab of HPV 16, it is characterized in that wherein comprising for 11. 1 kindsAt least 1 antibody chain variable region comprises that SEQIDNO:15 and at least 1 antibody heavy chain variable region comprise SEQIDNO:16.
The nucleic acid of 12. 1 kinds of separation, is characterized in that at least one variable region of light chain SEQ of described nucleic acid coding antibody of the present inventionIDNO:15 and variable region of heavy chain SEQIDNO:16.
13. 1 kinds comprise the expression vector of nucleic acid described in claim 12, while it is characterized in that carrier transfection host cell described inNucleic acid is effectively connected with the control sequence of host cell identification.
14. 1 kinds comprise the host cell of claim 13 expression vector.
15. 1 kinds are detected the kit of HPV16L1, and it comprises arbitrary claim 1-4 and/or arbitrary claim 8-11 instituteThe monoclonal antibody of stating or Fab.
The kit of 16. detection HPV16L1 as claimed in claim 15, also comprises detectable mark: radioactivity coordinationElement, fluorescent material, luminescent substance, coloring matter and/or enzyme.
The composition of 17. 1 kinds of specific detection L 1 Protein of Human Papillomavirus Type 16s, it is characterized in that said composition comprise appointMonoclonal antibody or Fab described in one claim 1-4 and/or arbitrary claim 8-11.
18. as described in claim 15 or 16 kit in preparation prevention or detect the detection that HPV 16 L1 infectsApplication in composition.
The 19. detection combinations that composition prevents or detect HPV 16 L1 to infect in preparation as claimed in claim 17Application in thing.
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| CN112125972A (en) * | 2019-06-25 | 2020-12-25 | 神州细胞工程有限公司 | anti-HPV 16L 1protein monoclonal antibody and detection method using same |
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| US10844119B2 (en) * | 2016-10-11 | 2020-11-24 | Agenus Inc. | Anti-LAG-3 antibodies and methods of use thereof |
| CN109021097A (en) * | 2017-06-08 | 2018-12-18 | 艾托金生物医药（苏州）有限公司 | A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45 |
| CN108276491B (en) * | 2018-02-05 | 2021-01-12 | 南京薇熙生物医药科技有限公司 | Monoclonal antibody capable of specifically recognizing HPV18L1 protein and application thereof |
| CN111662378B (en) * | 2019-12-30 | 2022-05-31 | 中生方政生物技术股份有限公司 | Monoclonal neutralizing antibody of HPV18L1 and application thereof |
| CN111205364B (en) * | 2020-02-26 | 2022-05-17 | 中生方政生物技术股份有限公司 | Monoclonal neutralizing antibody against HPV31L1 and application thereof |
| CN113512109B (en) * | 2021-03-09 | 2022-03-25 | 北京康乐卫士生物技术股份有限公司 | Monoclonal neutralizing antibody against human papillomavirus 31 and application thereof |
| CN116199772B (en) * | 2022-11-16 | 2023-10-17 | 怡道生物科技(苏州)有限公司 | HPV31 type capsid protein L1 monoclonal antibody, preparation method and application |
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| CN105669859A (en) | 2016-06-15 |
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