CN101643720A - Antiviral serotype shared monoclonal antibody of foot-and-mouth disease and distinguished epitope thereof - Google Patents

Abstract

The invention discloses antiviral serotype shared monoclonal antibody of foot-and-mouth disease and a distinguished epitope thereof and the application, belonging to the prevention and control field of foot-and-mouth disease. The invention obtains two hybridoma cell lines which can secrete antiviral serotype shared monoclonal antibody of foot-and-mouth disease (microorganism maintenance numbers thereof are CGMCC No. 3103 and CGMCC No. 3104 respectively), and monoclonal antibodies 4B2 and 3D9 which are secreted by the hybridoma cell lines. ELISA and indirect immunofluorescence assay show that the two hybridoma cell lines both belong to FMDV shared monoclonal antibodies; wherein the 4B2 distinguishes one linear epitope of FMDV, and the 3D9 distinguishes one conformation epitope of FMDV. The4B2 and 3D9 are respectively used as a capture antibody and a detection antibody, a method for capturing ELISA by an antigen is adopted, specificity detection of FMDV can be carried out; the detectionmethod has the characteristics of high sensibility, strong specificity and good repeatability.

Description

Foot-and-mouth disease virus resistant serotype is shared monoclonal antibody and identified epitope thereof

Technical field

The present invention relates to monoclonal antibody, relate in particular to the serotype sharing monoclonal antibody of two strain foot-and-mouth disease virus resistants (FMDV) and a strain monoclonal antibody institute identified epitope wherein, the application in foot and mouth disease diagnosis or control of said monoclonal antibody of the present invention and identified epitope thereof belongs to the anti-system field of foot and mouth disease.

Background technology

(Foot and mouth disease virus FMDV) belongs to the member of Picornaviridae Hostis to foot and mouth disease virus, and its genome is the sub-thread positive chain RNA, the about 8.5kb of total length.Foot and mouth disease is one of serious harm important transmissible disease artiodactylous, and this disease can cause that cub death, milk yield descend, meat reduces, meat descends, the production performance of animal reduces, and causes enormous economic loss.In addition, owing to the restriction of trade with forbid that the loss that causes is bigger, the direct economic loss that the whole world causes every year thus can reach tens billion of dollars.Asia1 type FMD (Asia1/JS/CHA/05, GenBank accession number: EF149009),, be known as Asia1 type Jiangsu pedigree have been broken out since 2005 owing at first separate in China Jiangsu Province on China Jiangsu, Shandong, Gansu, Beijing, Hebei and other places.Once popular, propagation trend is swift and violent in China cows for this pedigree foot and mouth disease virus, and the loss that causes is very serious.The beginning of this century, O type foot and mouth disease virus Pan Asia pedigree was at China (O/Tibet/CHA/99, GenBank accession number: AJ539138) and surrounding countries even Europe (Mason PW et al.J Gen Virol.2003,84 (Pt 6): 1583-93) all have popularly, bring about great losses for many countries.Therefore, actively develop the research of Asial type and O type foot and mouth disease (FMD), the prevention and control of China FMD are had great importance.

The antigenic structure of foot and mouth disease virus is very complicated, and its epitope of the foot and mouth disease virus of different serotypes, genotype and strain isolated and antigen site all are not quite similar, and have antigenic variation widely.Utilize sequential analysis of monoclonal antibody escape mutants and cross neutralization test, identified the antigen site of some different serotypes FMDV, relevant research is mainly from O, A, C and Asial type FMDV.The neutrality monoclonal antibody plays a leading role in protection susceptible animal opposing foot and mouth disease virus course of infection, and some conservative relatively immunodominant epitopes can induce body to produce the shared monoclonal antibody of serotype, has important value for the FMDV diagnosis of infection.In recent years, for the existing report of the antigenicity research of conservative relatively VP2 albumen N-stub area, in the monoclonal antibody at FMDV VP2 N-end of Yang report, 9B4,15F7 and F1412SA epi-position are the trypsinase responsive type, behind the trypsin treatment epitope peptide, epi-position disappears to the activity of monoclonal antibody, shows that its epi-position belongs to conformation type, and comprises Lys ²(K ₂)-Lys ³(K ₃) or Arg ¹³(R ₁₃) residue; Monoclonal antibody 4B2 situation is similar among the monoclonal antibody 13A6 of Freiberg report and the present invention, the identification polypeptide section is identical and all be non-trypsinase responsive type monoclonal antibody, show that both epitope regions are same or similar, therefore two monoclonal antibody weight chain variabl area sequences are compared, the result proves that the two has close part but is not identical monoclonal antibody; Wang in 2007 etc. have carried out the immunogenicity analysis to VP1 and the last main antigen section of VP2 of Asia1 type strain YNBS/58, found that, D7 (133-163aa of VP1) and two sections of B1 (1-33aa of VP2), can both induce cavy lymphadenosis, B1 does not have the neutralization activity but D7 has the neutralization activity.When with D7 and the common immune guinea pig of B1, can induce the neutralizing antibody reaction of high level, illustrate that B1 (VP2N-end) has promoter action to the immunogenicity of D7 (VP1 133-163aa).There is immunocompetence site in 1-33aa zone at VP2, although do not belong to the neutrality site, the site in this section in main and the immune response of avtive spot have and strengthen and promoter action.For the location of epi-position and qualitative, these researchs abroad are with the screening of monoclonal antibody pressure or with synthetic pepscan, can only determine amino acid or peptide section that epi-position is correlated with, can not accurately determine the position and the character of epi-position.Though China has the article of Asia1 type and O type FMDV monoclonal antibody to deliver, yet do not see the report of sharing the epi-position monoclonal antibody as yet; About FMDV shares the accurate location of epi-position and qualitative, still there is not research report both at home and abroad.

Summary of the invention

One of the object of the invention provides two strains can secrete the monoclonal antibody 4B2 of anti-FMDV serotype dependent/non-dependent and the hybridoma cell line of 3D9 respectively;

Two of the object of the invention provides the total epi-position aminoacid sequence of all serotype FMDV that discerned by said monoclonal antibody 4B2;

Three of the object of the invention is that said monoclonal antibody or sharing epi-position are used for diagnosis or prevention foot and mouth disease.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

One strain can be secreted the hybridoma cell line 4B2 of the non-serotype dependency of anti-FMDV monoclonal antibody, and its microbial preservation number is: CGMCC No.3103; Classification name: monoclonal antibody hybridoma cell; The preservation time is: on May 18th, 2009; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.

The secreted monoclonal antibody of above-mentioned hybridoma cell line 4B2.

The present invention has further determined the non-serotype dependency of the FMDV that is discerned by this monoclonal antibody linear epitope, and the amino-acid residue of this linear epitope motif is the proteic Glu of FMDV-VP2 ⁶-Thr ⁷-Thr ⁸-X-Leu ¹⁰-Glu ¹¹(SEQ ID NO:1), wherein, X is selected from any one amino-acid residue, and key amino-acid residue is Glu ⁶, Thr ⁷, Thr ⁸, Leu ¹⁰And Glu ¹¹, decisive amino acid is Glu ¹¹This epi-position optimum activity unit is a dodecapeptide: ₂KKTEETTLLEDR ₁₃(SEQ IDNO:2), wherein, ₂KKTE ₅With ₁₂DR ₁₃For epi-position primary activity unit Glu ⁶-Thr ⁷-Thr ⁸-X-Leu ¹⁰-Glu ¹¹Has synergism.

One strain can be secreted the hybridoma cell line 3D9 of the non-serotype dependency of anti-FMDV monoclonal antibody, and its microbial preservation number is: CGMCC No.3104; Classification name: monoclonal antibody hybridoma cell; The preservation time is: on May 18th, 2009; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.

Non-serotype dependency monoclonal antibody by the secreted anti-FMDV of this hybridoma cell line 3D9.

3D9 is a capture antibody with hybridoma cell line 3D9 excretory monoclonal antibody, as detecting antibody, adopts antigen capture ELISA method with biotin labeled hybridoma cell line 4B2 excretory monoclonal antibody, can be applicable to detect FMDV.

The detailed description of overall technical architecture of the present invention:

The present invention prepares the monoclonal antibody of anti-Asia1 type Jiangsu pedigree FMDVAsia1/YS/CHA/05 and O type FMDV Pan Asia pedigree O/YS/CHA/05 strain with the totivirus immunity BALB/c mouse of purifying, obtains monoclonal antibody 4B2 and 3D9 that two strains have strong reactive behavior respectively.ELISA and indirect immunofluorescence detect and show that this two strains monoclonal antibody all belongs to FMDV sharing monoclonal antibody; Indirect immunofluorescence and Western blot detect and show, linear epitope of 4B2 identification FMDV, conformation type epi-position of 3D9 identification FMDV.

The present invention further utilizes phage display to screen the mimic epitopes of above-mentioned monoclonal antibody 4B2 identification in 12 peptide storehouses at random, finds that an epi-position motif ETTXLE (X represent 20 seed amino acid residues any one) has the activity of combining with monoclonal antibody 4B2.

In view of there is the height homology in 6～11 amino acids sequences of the VP2 albumen N-end of all 7 serotype FMDV that announce among monoclonal antibody 4B2 mimic epitopes motif ETTXLE and the GenBank, with the relevant small peptide of a series of N-stub area of intestinal bacteria amalgamation and expression epi-position, and carry out Western blot and analyze, determine that ETTLLE six peptides are the primary activity unit of monoclonal antibody 4B2 epi-position.And then the optimum activity unit that determines this epi-position is ₂KKTEETTLLEDR ₁₃6 amino-acid residues in primary activity unit of monoclonal antibody 4B2 identification epi-position are carried out the amino acid replacement analysis one by one determine that wherein 5 amino acid all are important for the epi-position activity, thereby the key amino-acid residue of determining this epi-position is Glu ⁶, Thr ⁷, Thr ⁸, Leu ¹⁰And Glu ¹¹, amino-acid residue Glu wherein ¹¹Play a decisive role for this epi-position activity; ₂KKTE ₅With ₁₂DR ₁₃Unit ETTLLE all has synergism for the epi-position primary activity.

In view of above-mentioned two monoclonal antibodies have non-serotype dependency, and has very high specificity aspect the external FMDV diagnosis, the present invention so with monoclonal antibody 3D9 be capture antibody, biotin labeled monoclonal antibody 4B2 as detecting antibody, set up the antigen capture ELISA method that detects FMDV.The best bag of determining to catch monoclonal antibody 3D9 is 2.5 μ g/mL by concentration, and the best working concentration that detects monoclonal antibody 4B2 is 0.9 μ g/ml.Sensitivity test shows that this method is 1.8 * 10 to the cytotoxic limit of identification of Asial FMDV ⁴TCID ₅₀, be 7.9 * 10 to the cytotoxic limit of identification of O type FMDV ³TCID ₅₀The accordance test shows that the recall rate of this method pair cell poison is 100%, and histotoxic recall rate is 73.3%.Viruses such as bovine tuberculosis, pleuropneumonia, Niu Liure, Akabane Disease, ox infective rhinitis are detected, and all negative, no cross reaction takes place.The FMDV antigen capture ELISA method that this research is set up has the characteristics of susceptibility height, high specificity and good reproducibility, can be used for the specific detection of FMDV.

Two strain monoclonal antibodies among the present invention and epi-position thereof are all significant for the diagnostic reagent exploitation of FMDV, and research also has the potential using value for epiposition vaccine.

Description of drawings

The cell conditioned medium of Fig. 1 monoclonal antibody 4A8,4B2 and 1F6 detects at O type and the antigenic IFA of Asia1 type; A-E:O type FMDV infected B HK21 cell antigen; F-J:Asia1 type FMDV infected B HK21 cell antigen; A-C, F-H: be respectively 4B2,1F6 and 4A8; D, I: negative serum contrast; E, J: positive serum contrast.

The specificity IFA of Fig. 2 monoclonal antibody 3D9,4B2 identifies.

Monoclonal antibody 3D9, the 4B2 of Fig. 3 SDS-PAGE purification Identification; M: molecular weight of albumen mark, 1: purified monoclonal antibody 3D9,2: purified monoclonal antibody 4B2.

Fig. 4 ELISA detects 24 phage clones with monoclonal antibody 4B2 specific combination; The affine phage of ELISA method screening monoclonal antibody 4B2 specificity; Add in the microwell plate with monoclonal antibody 4B2 bag quilt with each phage clone of identical plaque number, and establish the monoclonal antibody 1F6 of isoantibody hypotype, the confining liquid that contains 1%BSA and original peptide storehouse (Phage Library, PL) in contrast.

The ELISA of seven positive phage clones of Fig. 5 detects; The affine phage of ELISA method screening monoclonal antibody 4B2 specificity; Each phage clone with identical plaque number adds in the microwell plate of using monoclonal antibody 4B2 bag quilt, and establishes the monoclonal antibody 1F6 of isoantibody hypotype, the confining liquid that contains 1%BSA and original peptide storehouse in contrast.

The aminoacid sequence comparison of 7 positive phage clones 12 peptides of Fig. 6.

All VP2 aminoacid sequence comparison results among Fig. 7 12 peptides and the GenBank.

Fig. 8 carrier pGEX-6p-1 cuts evaluation through BamHI and XhoI enzyme; 1:Marker; 2: carrier pGEX-6p-1.

The PCR that 5 small peptides of Fig. 9 insert carrier pGEX-6p-1 identifies; M:Marker; 1:Y15-1; 2:Y15-2; 3: six peptides; The 4:5 peptide; 5: tetrapeptide; The 6:H2O contrast.

The PCR that 9 small peptides of Figure 10 insert carrier pGEX-6p-1 identifies; M:Marker; 1-9:L5 ( ₁ DKKTEETTLLE ₁₁), L4 ( ₂ KKTEETTLLE ₁₁), L3 ( ₃ KTEETTLLE ₁₁), L2 ( ₄ TEETTLLE ₁₁), L1 ( ₅ EETTLLE ₁₁), R1 ( ₆ETTLLE D ₁₂), R2 ( ₆ETTLLE DR ₁₃), R3 ( ₆ETTLLE DRI ₁₄) and R4 ( ₆ETTLLE DRIL ₁₅).

The PCR that 7 small peptides of Figure 11 insert carrier pGEX-6p-1 identifies; M:Marker; 1-7:Y12 ( ₂KKTE ETTLLEDR ₁₃), S-1 ( ₂KKTE ATTLLEDR ₁₃), S-2 ( ₂KKTE EATLLEDR ₁₃), S-3 ( ₂KKTE ETALLEDR ₁₃), S-4 ( ₂KKTE ETTALEDR ₁₃), S-5 ( ₂KKTE ETTLAEDR ₁₃) and S-6 ( ₂KKTE ETTLLADR ₁₃).

Figure 12 SDS-PAGE (A) and Western blot (B) analyze, and determine that epi-position primary activity unit is ₆ETTLLE ₁₁A, B:1. albumen Marker; 2: induce preceding PGEX-6P-1; 3: induce back PGEX-6P-1; 4-8: the Y15-1 of expression, Y15-2, P6, P5 and P4.

Figure 13 designs and merges proteic SDS-PAGE of epi-position (A) and Western blot (B) analysis, and the result has determined that the active amino-acid residue of obvious enhancing epi-position is ₂KK ₃And R ₁₃Marked albumen Marker; L5 ( ₁ DKKTEETTLLE ₁₁), L4 ( ₂ KKTEETTLLE ₁₁), L3 ( ₃ KTEETTLLE ₁₁), L2 ( ₄ TEETTLLE ₁₁), L1 ( ₅ EETTLLE ₁₁), R1 ( ₆ETTLLE D ₁₂), R2 ( ₆ETTLLE DR ₁₃), R3 ( ₆ETTLLE DRI ₁₄) and R4 ( ₆ETTLLE DRIL ₁₅).

Figure 14 designs the fusion proteic SDS-PAGE of epi-position (left side) and Wes tern blot analysis (right side) identifies that epi-position optimum activity unit is ₂KKTE ETTLLEDR ₁₃1: albumen Marker; 2:Y15-1; 3:Y12; 4:L4; 5:R2; 5:Y15-2.

Figure 15 designs and merges proteic SDS-PAGE of epi-position (A) and Western blot (B) analysis.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.

One, material and method

1. virus, cell, bacterial strain and laboratory animal

Hoof-and-mouth disease strain Asia1/YS/CHA/05 belongs to Asia1 type FMDV Jiangsu pedigree, this pedigree strain since 2005 in ground eruption and prevalence (Asia1/JS/CHA/05, GenBank accession number: EF149009) such as China Jiangsu, Shandong, Gansu, Beijing, Hebei; The O/YS/CHA/05 strain belongs to O type Pan Asia pedigree, this pedigree strain beginning of this century last century Mo is in China (O/Tibet/CHA-/99, GenBank accession number: AJ539138) and surrounding countries even Europe (Mason PW et al.J Gen Virol.2003,84 (Pt 6): 1583-93) all have popularly, bring about great losses for many countries.Breast hamster nephrocyte BHK-21, SP2/0 myeloma cell preserve for this laboratory; Plasmid pGEX-6p-1 and recipient bacterium BL21 are preserved by this laboratory; The female BALB/c mouse of cleaning level, SPF level BALB/c suckling mouse are purchased the Experimental Animal Center in Harbin Veterinary Medicine Inst., China Academy of Agriculture.

2. main agents

The sheep anti-mouse igg of fusogen PEG/DMSO (Mw, 1450), HAT salt (50x), HT salt (50x), HRP or FITC mark, available from Sigma company; France white-oil adjuvant MONTANIDE ISA 206VG is available from SEPPIC company; Monoclonal antibody subgroup identification test kit is available from Southern Biotech company; L-glutaminate (glutamine), glycine are available from Amresco company; Dimethyl sulfoxide (DMSO) (DMSO), O-Phenylene Diamine (OPD), PEG6000 are available from Solarbio company; Ph.D.-12 ^TMPeptide storehouse test kit is available from New England Biolabs company; The anti-M13 phage antibody of HRP mark is available from GE Healthcare company; Restriction enzyme BamHI, XhoI are available from TaKaRa company; The T4DNA ligase enzyme is available from New England Biolabs company; High sugared DMEM dry powder is available from GIBCO company; Import top grade foetal calf serum (PAA) is available from Spain Na lgene company; 96 porocyte culture plates are available from Canadian JET Biochemical company.

3. mouse immunity and MONOCLONAL ANTIBODIES SPECIFIC FOR

(1) the cell adapted poison of O type FMDV is inoculated in the BHK-21 cell that covers with individual layer, treats that 75% above cell occurs receiving poison after the pathology.With the cell culture fluid multigelation three times of results, preliminary lysing cell, releasing virus.Viral liquid after the freeze thawing added TritonX-100 and adding formaldehyde cracking in 4: 10000 with 1: 1000 and more than the deactivation 48h, the viral liquid 1mL that gets deactivation goes up BHK-21 cell blind passage three generations, detects whether deactivation is thorough.Nutrient solution behind freezing-thawing and cracking is made 9 000rpm (Beckman supercentrifuge, JA-10 rotor), 4 ℃ of centrifugal 90min, reclaim viral liquid supernatant, discard cell precipitation.Volume by viral liquid supernatant adds the PEG6000 of 80g/L and the NaCl of 40g/L, and stirring at room 2h is to dissolving fully, 4 ℃ of precipitations of spending the night.Abandon supernatant with supernatant 9 000rpm, 4 ℃ of centrifugal 90min next day, reclaims precipitation.Under low temperature, will precipitate resuspended gently with the NET damping fluid.With resuspended supernatant 29000rpm, 4 ℃ of centrifugal 2h, reclaim precipitation, following in low temperature with the resuspended gently precipitation of an amount of NET damping fluid, fully packing behind the mixing ,-70 ℃ are frozen standby.Purified antigen is carried out 10 times, 100 times, 1000 times dilutions with the NET damping fluid, with the proteinic concentration of spectrophotometer measurement.

(2) the O type FMDV antigen 200 μ g/200 μ L with purifying add equal-volume France white-oil adjuvant 206VG, and are fully emulsified after back subcutaneous injection female BALB/c mouse in 6 age in week; Carry out the 2nd immunity after 2 weeks; After 2 weeks, carry out the 3rd immunity at interval with the equivalent antigen that does not add adjuvant.For stimulating mouse to produce the strong immunization reaction fast, the immunization ways that 3d adopts tail vein and spleen injection to combine before merging carries out booster immunization, injection qdx antigen.

(3) cytogamy

A) preparation of feeder layer cells: cytogamy is carried out the preparation of feeder layer cells the day before yesterday, and test tools mades such as eye scissors, ophthalmology tweezers, plate are with preceding high temperature dry sterilization, and the HAT complete culture solution is done steriling test.Get 2 BALB/c mouse and extract the eyeball bloodletting, separate negative serum according to a conventional method.Draw the neck dislocation to put to death mouse, it is soaked in 75% alcohol, move into Bechtop behind the 10min.Mouse web portion upwards is fixed on the mouse frame, mentions the positive middle part of abdomen skin with tweezers, eye scissors is laterally cut an osculum, is sure not to break peritonaeum, tears skin up and down with scissors and tweezers, fully exposes peritonaeum.Mention peritonaeum gently with tweezers, the 8-10ml HAT complete culture solution of drawing in the 10ml syringe is injected the abdominal cavity, be sure not to puncture internal organs and enteron aisle, syringe is not extracted, aspirate 5-10 time back and forth at intraperitoneal,, aspirate again about 30 seconds with tweezers massage both sides belly then, repeat above operation 2-3 time.With syringe resorption intraperitoneal liquid.Note avoiding mesentery and fatty tissue, in order to avoid stop up syringe needle.The abdominal cavity cell that will take out from 2 mouse adds in the 55ml HAT nutrient solution, dispels the mixing cell, is sub-packed in 6 96 well culture plates, 100 μ l/ holes.Place 37 ℃, 5%CO ₂Cultivate in the incubator.

B) myeloma cell's preparation: 36-48h before merging with myeloma cell's enlarged culturing, makes cell be in logarithmic phase.Merge the same day, cell is blown down from the bottle wall, be collected in the 50ml centrifuge tube with 15ml DMEM basic culture solution.The centrifugal 10min of 1000rpm.Put in the 20mlDMEM basic culture solution mixing with cell precipitation is resuspended.The myeloma cell's suspension that takes a morsel, platform is expected blue dyeing counting, and is standby.

C) preparation of immune spleen cell: extract the eyeball of mouse blood sampling before merging, the preparation positive serum.Draw the neck dislocation to cause death mouse, be soaked in 75% alcohol, be put in super clean bench behind the 10min.The aseptic abdominal cavity of opening is separated reticular tissue and is taken out spleen, and spleen is put into the plate that the sterilization nylon wire is housed and fills the 15mlDMEM basic culture solution, grinds spleen with sterilization glass syringe inner core, and splenocyte is all entered in the plate by mesh.Splenocyte solution is changed in the 50ml centrifuge tube, add the DMEM basic culture solution approximately to 30ml, mixing.The centrifugal 8min of 1000rpm abandons supernatant.Cell precipitation is suspended from the 10mlDMEM basic culture solution mixing.Obtained cell suspension, platform are expected blue dyeing counting, and be standby.

D) splenocyte and myeloma cell are merged: get the 65mlHAT nutrient solution, and 15ml basis DMEM nutrient solution and 1ml 50%PEG preheating in 37 ℃ of water-baths, other fills the 200ml beaker of 37 ℃ of water fully.Add 1 part of myeloma cell's number by 5 parts of spleens cell numbers and get corresponding cell suspension amount, add in the 50ml glass centrifuge tube, add the DMEM basic culture solution to 30ml, mixing.The centrifugal 10min of 1000rpm abandons supernatant, falls to do as far as possible.Touch the centrifuge tube bottom with palm, make sedimentation cell loose evenly in the pasty state.Centrifuge tube is put into the 200ml beaker that fills 37 ℃ of water, evenly rotate centrifuge tube on the other hand, another hand is drawn 37 ℃ of 50%PEG solution 1ml with the 1ml pasteur pipet, adds in the 1min, leaves standstill 2min.Add DMEM basic culture solution termination reaction soon after slow earlier subsequently, 10min is left standstill in 37 ℃ of water-baths.The centrifugal 10min of 1000rpm abandons supernatant, adds 65ml HAT substratum, dispels cell lightly, and every hole 0.1ml is inoculated in and cultivates 6 96 well culture plates that feeder cell are arranged, puts 37 ℃, 5% CO ₂Cultivate in the incubator.The later half amount of 5d is changed liquid, changes liquid behind the 8d entirely, treats that the clone gets supernatant when growing to hole floorage 1/4-1/3 and detects and change the HT nutrient solution.

4. antibody test ELISA

With the virus antigen bag of purifying by in microwell plate, add 100 μ l Hybridoma Cell Culture supernatants, 37 ℃ of incubation 1h after scouring, the sheep anti mouse two that adds the HRP mark of dilution in 1: 5000 resists, the washing back adds substrate OPD lucifuge colour developing 10min, measures light absorption value in wavelength 492nm.

5. indirect immunofluorescence

Microwell plate is cultivated the BHK-21 cell to individual layer, inoculation foot and mouth disease virus 4 * 10 ³TCID ₅₀/ well, occur fixing with cold dehydrated alcohol before the CPE, add 50 μ l Hybridoma Cell Culture supernatants, the PBS washing is three times behind 37 ℃ of incubation 40min, the fluorescein-labelled sheep anti-mouse igg antibody that adds dilution in 1: 200,37 ℃ of incubation 40min, the washing back is observed under inverted fluorescence microscope, with+number number record fluorescence intensity.

6. micro-cell neutralization test

(1) mensuration of viral malicious valency: virus inoculation in monolayer cell, is added behind 37 ℃ of absorption 1h and keeps liquid, put incubator and cultivate; Day by day observe, treat that cytopathy (CPE) reaches more than 75%, results viral suspension freeze thawing 3 times with the centrifugal 10min of 3000r/min, is got supernatant liquor, and quantitatively being distributed into the 1ml bottle, to put-70 ℃ of preservations standby, and the virus of selecting for use must be that pair cell has more stable virulence.Take from one bottle in the virus that-70 ℃ of refrigerators preserve, virus is done 10 times of dilutions of going forward one by one on 96 well culture plates be 10 ^-1, 10 ^-2, 10 ^-11..., every hole viral suspension amount is 50 μ l, and each extent of dilution is done 8 holes, and every hole adds 100 cell suspensions, and last column of every block of plate is established the contrast of 8 porocytes, and the concentration of preparation cell suspension is degree so that cell covers with individual layer in 24h.Culture plate is put 5%CO ₂37 ℃ of cultivations of incubator, from 48-72h observation of cell pathology day by day, the record result.

Press Reed and Muench Liang Shi method and calculate TCID ₅₀

Table 1

TCID ₅₀Calculate (dosage of inoculation 50 μ l)

Ig TCID ₅₀=be higher than the logarithm of the logarithm-distance proportion * dilution factor of 50% viral dilution degree

The logarithm that is higher than 50% viral dilution degree is-6, and distance proportion is 0.26, and the logarithm of dilution factor is-1.

IgTCID ₅₀＝-6+0.26×(-1)

＝-6.3

TCID then ₅₀=10 ^-6.3, 50 promptly viral works 10 ^-6.3Dilution, 50 μ l are inoculated in every hole, can make half histocyte pipe generation pathology.

(2) neutralization test: in the animal ascites, containing in the multiple proteins composition antagonist has booster action with virus, as complement, immunoglobulin (Ig) and immuno conglutinin etc.For getting rid of these heat labile nonspecific reaction factors, the serum or the ascites that are used for neutralization test must be through heating 56 ℃ of 30min inactivation treatment.Get the odd contradictive hydroperitoneum of inactivation treatment, on 96 holes trace Tissue Culture Plate, make a series of doubling dilutions with the DMEM that does not contain serum, its extent of dilution is respectively 1: 4,1: 8,1: 16,1: 32,1: 64 of former ascites or the like, every hole content is 50 μ l, and each extent of dilution is done 4 holes.Get the viral liquid that-70 ℃ of refrigerators are preserved, original malicious valency is diluted to 200 TCID ₅₀, (mix with equivalent ascites, its malicious valency is 100TCID to 50 μ l ₅₀).As virus titer is 10 ^-6.3, 50 μ l.So virus should be done 2 * 10 ^-4.3Dilution.Every hole adds 50 μ l viral dilution liquid, seals lid, places 37 ℃ of incubators and 1h.When the preparation cell suspension, its concentration is to cover with individual layer degree of being in 24h: take out in serum and the virus He behind the 1h, every hole adds 100 μ l cell suspensions.Put 5%CO ₂37 ℃ of incubators are cultivated, and cultivate 48h certainly and begin observed and recorded day by day, and 120h declares eventually.Be warranty test result's accuracy, each test all must be provided with following contrast.The positive and negative (sp2/0) ascites and ascites to be checked are carried out parallel test, and positive ascites cytopathy do not occur to correlating, and negative ascites cytopathy occurs to correlating.All set up virus control on each each piece plate of test, earlier virus is made 0.1,1,10,100,1000 TCID ₅₀Dilution, each extent of dilution is done 4 holes, and every hole adds 50 μ l.Every then hole 100 μ l cell suspensions.0.1TCID TCID ₅₀Should not cause cytopathy and 100TCID ₅₀Must cause cytopathy, otherwise should test untenable.For checking that the pair cell of tested ascites own has or not any toxic action, it is necessary setting up tested ascites toxicity contrast.The ascites to be checked (the minimum extent of dilution that is equivalent to tested ascites in the neutralization test) that promptly in histocyte, adds the low power dilution.Normal cell contrasts promptly the not cell suspension hole of virus inoculation and ascites to be checked.Normal cell causes testing error to correlate form and the characteristic of life that keeps good in whole neutralization test always for avoiding culture plate itself, should all set up this contrast on every block of plate.When virus recurrence test, the positive, feminine gender, normal cell when ascites toxicity contrasts all establishments, just can judge that tested ascites hole 100%CPE occurs and is judged to feminine gender to taking a picture, and it is positive that the preserver appears in 50% above cell; The result of fixed virus dilution ascites neutralization test calculates, and is to calculate to protect 50% cell hole not produce cytopathic ascites extent of dilution, and this extent of dilution is the NAT of this part serum.With Reed and Muench Liang Shi method (or Karber method) calculation result.

Table 2

Fixed virus-dilute serum method NAT calculates

With Reed and Muench Liang Shi method (or Karber method) calculation result

80％-50％

Distance proportion=---=0.5

80％-20％

IgPD ₅₀=be higher than the logarithm of the logarithm-distance proportion * dilution factor of 50% serum dilution

IgPD ₅₀＝-1.5-0.5×(-0.3)＝-1.35

PD then ₅₀=10 ^-1.36, 50 μ l

Because of 10 ^-1.35=1/22, promptly 1: 22 serum can protect 50% cell not produce pathology, and be exactly the NAT of this part serum at 1: 22.

7. biological the elutriation

Ascites is slightly carried through sad-ammonium sulfate method earlier, used NAb then ^TMProtein G SpinPurification Kit (PIERCE) carries out affinitive layer purification, is used for biological the elutriation after SDS-PAGE identifies purity, with the linear at random dodecapeptide of M13 phage display storehouse monoclonal antibody is carried out epitope mapping.The biological reference reagent box specification sheets of eluriating carries out: the 1st takes turns and eluriates in microwell plate bag by 10 μ g monoclonal antibodies, and the 2nd, 3 take turns and wrap respectively by 5 μ g, 1 μ g, and 4 ℃ are spent the night; After the 0.05%TBST washing,, add 1.5 * 10 with the 10g/LBSA sealing ¹¹Pfu/100 μ l shows the M13 phage of dodecapeptide, room temperature jog 1h, TBST elutes the bonded phage with elution buffer after washing 10 times in room temperature jog 10min, add in the damping fluid and the phage of wash-out, inoculation intestinal bacteria ER2738 increases.Titre is reclaimed and measured to phage after the amplification, get 1.5 * 10 ¹¹The pfu phage enters next round and eluriates.The 4th takes turns elutriation adopts Protein G prize law to carry out the liquid phase combination, and the monoclonal antibody usage quantity is 300ng.The single phage clone of picking increases, and ELISA identifies its activity with the phage affinity capture, and the single stranded DNA to positive bacteriophage carries out sequential analysis at last.

8. phage affinity capture ELISA

Bag is by 100ng monoclonal antibody (every hole 100 μ l, 0.1M sodium bicarbonate pH8.6) in 96 hole polyethylene enzyme plates, and 4 ℃ are spent the night, and set up repeating hole, sets up the monoclonal antibody that derives from same mouse preparation of the different epi-positions of identification simultaneously, and the 1%BSA confining liquid is contrast.Behind the room temperature sealing 2h, with 0.1%TBST dilution phage clone to 10 ¹⁰Pfu/100 μ l, add every hole, room temperature jog reaction 1h, after washing six times with 0.1%TBST, the mouse-anti M13 phage antibody that adds the HRP mark of dilution in 1: 5000, use substrate A BTS[2,2 '-azinobis (3-ethylbenzthiazolinesulfonicacid)] develop the color and afterwards survey absorbancy in 405nm, with the positive findings criterion of P/N＞2.1 as sample to be checked.

9. the preparation of phage single-chain dna profiling and order-checking

The 3rd, 4 take turns elutriation after, the random choose phage clone is inoculated into the ER2738 mid-log phase culture that contains 1ml 1: 100 dilution respectively, cultivates 4.5h in 37 ℃ of concussions.The centrifugal 10min of 12 000rpm respectively gets 500 μ l supernatants in new pipe, and every pipe adds 200 μ l PEG/NaCl, behind the mixing, leave standstill 10min, the centrifugal 10min of 12 000rpm, precipitation is dissolved in the 100 μ l sodium iodide damping fluids, adds 250 μ l dehydrated alcohols again, and room temperature leaves standstill 10min, the centrifugal 10min of 12 000rpm, abandon supernatant, precipitation is used 70% washing with alcohol, after the seasoning, with the dissolving of 30 μ l pure water, promptly can be used as sequencing template.Order-checking is finished by Shanghai biotechnology company limited, and sequencing primer is-96gIII that sequence is: 5 '-CCC TCA TAG TTA GCG TAA CG-3 '.

10.SDS-PAGE and Western blot analyzes

Operations such as SDS-PAGE, transfer printing, sealing, undertaken by the laboratory ordinary method, ascites monoclonal antibody (1: 1000) with the confining liquid dilution reacts 1h for 37 ℃ again, Tris-C1 washing lotion washing 3 times, each 10min is then with 1: 5000 times of sheep anti-mouse igg room temperature reaction 1h that dilutes the HRP mark, wash 3 times, each 10min places DAB substrate colour developing liquid to develop the color the observed and recorded result at last.

The small peptide 11. intestinal bacteria amalgamation and expression epi-position is correlated with

Two complementary oligonucleotide chains of synthetic coding epi-position and brachymemma epi-position DNA are shown in table 3,4,5.Insert the fragment upstream and introduce BamHI viscosity extending end (small letter part), terminator codon and XhoI viscosity extending end (underscore part) are introduced in the downstream, and carry out codon optimized according to the e. coli codon preference to this epitope gene.

The complementary oligonucleotide chain of relevant small peptide and primers designed coding DNA is identified in table 3. epi-position primary activity unit

Table 4. epi-position synergy amino acid is identified the complementary oligonucleotide chain of relevant small peptide coding DNA

The key amino acid of table 5. epi-position is identified the complementary oligonucleotide chain of relevant small peptide and primers designed coding DNA

Two complementary strands are directly annealed forms the double-stranded DNA of band sticky end, inserts between the BamHI and XhoI of pGEX-6p-1, transforms the BL21 competent cell, cuts the evaluation recombinant plasmid with the PstI enzyme, name respectively (showing 3-5).Single positive bacterium colony incubated overnight of picking and sequence verification, dilution in 1: 100 is inoculated in the fresh liquid LB substratum then, and 37 ℃ of concussions are cultured to OD _600nm=0.6, adding inductor IPTG is 1mmol/L to final concentration, and 37 ℃ are continued to cultivate 5h, collect thalline and resuspended with an amount of PBS, standby.

12.SDS-PAGE electrophoresis and Western blot detect

SDS-PAGE electrophoresis (15%Tris-glycine) divides isolated fusion protein, be transferred to (90V on the nitrocellulose filter, 45min), spend the night in 4 ℃ of sealings with 5% skimming milk-PBS confining liquid, with monoclonal antibody 8E8 in 37 ℃ of incubation 1h, with the sheep anti-mouse igg of HRP mark in room temperature effect 1h, DAB (6mg/10mL) colour developing.

13. Purification of Monoclonal Antibodies and biotin labeling

Monoclonal antibody 3D9,4B2 earlier through sad-ammonium sulfate method preliminary purification, are carried out affinitive layer purification with the NAbTM Spin Purification Kits of PIERCE company then.

13.1 sad-ammonium sulfate method preliminary purification key step is as follows:

(1) gets the pretreated ascites of 3ml and add 6ml 0.06mol/L PH4.8 acetate buffer solution, stir; Add 99 μ l sad (it is sad dropwise to add under the ice bath) in the ascites and stirred 30 minutes, 4 ℃ left standstill more than 2 hours; Take out 11000rpm centrifugal 30 minutes, and abandoned precipitation; Supernatant is transferred PH to 7.4 (only need add about 1 μ l and get final product) with 2mol/LNaOH;

(2) add saturated ammonium sulphate to 50% saturation ratio down at 4 ℃, ice bath stirring action 30 minutes, 4 ℃ left standstill more than 2 hours; 11000 left the heart 30 minutes, abandoned supernatant; Precipitate among the PBS of the PH7.4 that is dissolved in 5ml 0.1M;

(3) in suspension just stir the limit and add an amount of saturated ammonium sulphate solution to precipitation, make that concentration is 30%--40% in the ammoniumsulphate soln, ice bath stirring action 30 minutes, 4 degree are static more than 2 hours; 11000 left the heart 30 minutes, got precipitation, precipitation were incorporated among the PBS of PH7.4 of 2ml 0.1M;

(4) will precipitate suspension and pack in the dialysis band, 4 ℃ of PBS dialysis with the PH7.4 of 0.1M were changed liquid once in 2 hours, dialysed 2 days.

13.2 the affinitive layer purification operation steps is as follows:

(1) vortex mixing Protein G filler is drawn 200 μ l Protein G and is added adsorption column;

(2) add 300 μ l binding buffer liquid, mixings gently;

The centrifugal 1min of (3) 5000 * g takes out adsorption column, discards the liquid in the collection tube;

(4) again adsorption column is put into collection tube, add 400 μ l binding buffer liquid, the centrifugal 1min of 5000 * g behind the mixing;

(5) take out adsorption column, discard the liquid in the collection tube, adsorption column is reentered into collection tube;

(6) add 200 μ l sad-the monoclonal antibody sample of ammonium sulfate method preliminary purification is in adsorption column, room temperature effect 30min shakes every now and then and guarantees that gel filler is in suspended state all the time;

The centrifugal 1min of (7) 5000 * g is in adsorption column immigration-individual new collection tube;

(8) in adsorption column, add 400 μ l binding buffer liquid, mixing suspension gel filler, the centrifugal 1min. of 5000 * g discards the liquid in the collection tube. adsorption column moved in the collection tube again. repeat twice;

(9) adsorption column is moved into a new collection tube, add 400 μ l elution buffers, mixing gel filler 5min;

The centrifugal 1min of (10) 5000 * g changes adsorption column over to new collection tube then, collects the albumen elutriant;

(11) ultraviolet spectrophotometer is measured the elutriant protein content, packing in a small amount, and-20 ℃ are frozen standby.

13.3 biotin labeling monoclonal antibody 4B2:

Use EZ-

The Sulfo-NHS-LC-Biotinylation test kit carries out biotinylation to monoclonal antibody 4B2, carries out biotin labeling according to the method on the product description, and concrete steps are as follows:

(1) preparation of Sulfo-NHS-LC-vitamin H.From-20 ℃ of refrigerators, take out the Sulfo-NHS-LC-vitamin H, its temperature and room temp are consistent.Get 2.2mg Sulfo-NHS-LC-vitamin H, be dissolved in the deionized water of 400 μ l, be formulated as the 10mMSulfo-NHS-LC-vitamin H, matching while using.

(2) preparation of MAb purifying thing.Take out the monoclonal antibody of affinitive layer purification, it is mixed with the protein liquid of 2mg/ml with PBS.

(3) biotinylation of MAb purifying thing.According to MAbs purifying thing mole number: the Sulfo-NHS-LC-vitamin H is that the ratio of 1: 20 (the Sulfo-NHS-LC-vitamin H is fully excessive) is mixed, the 10mM Sulfo-NHS-LC-vitamin H of 27 μ l is added in the 1ml 2mg/mlMAbs purifying thing, places in the ice and hatch 2h.

(4) dialysis is desalted.Adopt test kit with Zeba vitamin H small molecules and the salt that post will not react that desalt dispose.

(5) the biotinylated MAb after the collection dialysis.

Two, test-results

(1) evaluation of 4B2 and 3D9 MONOCLONAL ANTIBODIES SPECIFIC FOR and 4B2 identification epi-position

1. at the screening and the evaluation of the non-serotype dependency of FMDV linear epitope monoclonal antibody

The Hybridoma Cell Culture supernatant of immune mouse spleen cell and SP2/0 cytogamy detects and IFA verifies with indirect ELISA, and positive criterion is, with the hybridoma culture supernatant to the antigenic OD of FMDV ₄₉₂Absorbance value and normal BALB/c mouse serum, SP2/0 cell conditioned medium are to FMDV antigen OD ₄₉₂The ratio of value is all greater than 2.1, and the hybridoma supernatant is negative to the immunofluorescence result of normal BHK-21 cell, is judged to positive hybridoma cell.Through screening and three limiting dilution assay clones, obtain 2 strain positive hybridoma cells, respectively called after 4B2 and 3D9.Identify the immunoglobulin subclass of 2 strain of hybridoma secretory antibodies with the hybridoma supernatant, the result shows that 3D9 heavy chain type is IgG2b, and 4B2 heavy chain type is IgG1, and 2 strain monoclonal antibody light chains are κ type (table 6).Show that with the neutralization test of O/YS/CHA/05 strain and Asia1/YS/CHA/05 virus strain monoclonal antibody 3D9 and 4B2 fail to reach 1: 32 thinning ratio, being judged to be does not have the neutralization activity.2 strain monoclonal antibodies have been carried out totivirus Western blot analyzed, 3D9 does not see the specific reaction band, and 4B2 has the significant reaction band.Therefore think that the 2 strain monoclonal antibody 3D9 that obtained discern the conformation type epi-position and 4B2 identification linear epitope.

The anti-Asia1 type of table 6 foot and mouth disease virus monoclonal antibody biological characteristics is identified

2.4B2 immunofluorescence detects

When the serotype specificity of monoclonal antibody is analyzed, carry out O type and the antigenic IFA detection of Asia1 type (Fig. 1) with 4B2 and other 2 strain O type FMDV monoclonal antibodies.The result proves that 4B2 can discern all detected objects, can determine that this monoclonal antibody is that non-serotype dependency FMDV shares the epi-position monoclonal antibody.

3.3D9, the antigenic indirect immunofluorescence of 4B2 and many serotype FMDV detects

Represent the BHK-21 of virus strain infection cell with Asia1 type, O type and each genotype of A type FMDV respectively, set up the contrast of BHK-21 cell simultaneously, through the fixing laggard immunofluorescence experiment that in the ranks connects of dehydrated alcohol, identify the reactivity of monoclonal antibody 3D9,4B2 and different serotypes and genotype FMDV, result such as Fig. 2 show that on behalf of strain, monoclonal antibody 3D9,4B2 and whole 6 FMDV all respond.Thereby, determine that monoclonal antibody 3D9,4B2 are each genotype sharing monoclonal antibody of FMDV.

4. monoclonal antibody 3D9,4B2 purifying

Monoclonal antibody 3D9,4B2 identify purity through SDS-PAGE behind sad-ammonium sulfate and affinitive layer purification, the result greatly about the visible Ig heavy chain of 55kDa and 25kDa place and two specific bands of light chain, conforms to expection respectively as shown in Figure 3; The protein content of measuring both through ultraviolet spectrophotometer is respectively 2.5mg/mL, 2.6mg/mL.

(2) the accurate location and the evaluation of 4B2 identification epi-position

1. the mimic epitopes motif of monoclonal antibody 4B2 identification is ETTXLE

For identifying the linear epitope of monoclonal antibody 4B2 identification, this experiment utilize a displaying at random the phage library of linear dodecapeptide the monoclonal antibody 4B2 of purifying carried out 4 take turns biological eluriate (table 7), taking turns 36 phage clones of common picking the 3rd, 4 increases, ELISA identifies its reactive behavior with the phage affinity capture, set up the monoclonal antibody 1F6 of isoantibody hypotype of same mouse preparation and confining liquid BSA in contrast, to get rid of the interference of antibody constant region and BSA, final 24 positive phage clones at antibody variable region (Fig. 4) that obtain all have higher avidity.Single stranded DNA to these positive phage clones carries out sequential analysis, obtains 7 12 different peptide sequences, called after P9, P12, P14, P11, P22, P16 and P8.Again these 7 different bacteriophages positive colonies are further detected with ELI SA, and build figure (Fig. 5) according to the ordering of 3 empirical average value sizes.7 12 different peptide ammino acid sequences are carried out sequence alignment, from the result, can derive 7 phage clones and all contain a characteristic sequence ETTXLE (x represent 20 seed amino acid residues any one) (Fig. 6), with the motif of its tentative linear epitope of discerning for monoclonal antibody 4B2.With all 7 serotype FMDV P1 Argine Monohydrochloride sequence alignments among motif ETTXLE and the GenBank, find itself and VP2N-end the 6-11 amino acids ( ₆ETTLLE ₁₁) demonstrate high similarity (Fig. 8), and this zone is conservative at all serotype FMDV camber.Therefore proposing 4B2 may be the shared monoclonal antibody of all serotype FMDV.

When motif was derived, the inventor noticed, P9, P12, P14 and P11 for TTXLE determine play a key effect, especially the aminoacid sequence TTXLE among the P14 has more cogency.P22, P16 and P8 are for E ₆Really tailor-made with also very clear and definite.In amino acid character and function aspects, S and T, D and E are because amino acid character and functional similarity can mutual alternative; And E and G also have close place amino acid whose aspect some, for example structure and solvability etc.Therefore, though the reactive behavior of P8 reduces, still can be discerned by 4B2.

Table 7 utilizes phage display peptide library that 4B2 is carried out 4 and takes turns biological elutriation result

1.4B2 epi-position is accurately located

For describing the 4B2 epi-position in detail, need at first to verify whether 4B2 can special reaction take place with ETTLLT.Strategy of the present invention is to comprise with intestinal bacteria amalgamation and expression VP2N-end ₆ETTLLE ₁₁Two 15aa overlapping peptide Y15-1 ( ₁DKKTE ETTLLEDRIL ₁₅) and Y15-2 ( ₆ ETTLLEDRILTTRNG ₂₀), six peptides ( ₆ETTLLE ₁₁) and the pentapeptide of N-end brachymemma ( ₇TTLLE ₁₁) and tetrapeptide ( ₈TLLE ₁₁).Analyze by Western blot, determine six peptides ( ₆ETTLLE ₁₁) whether be the active elementary cell of this epi-position.Secondly, increase amino-acid residue respectively one by one in the elementary cell left and right sides with 9 oligopeptides of amalgamation and expression, R1 ( ₆ETTLLE D ₁₂), R2 ( ₆ETTLLE DR ₁₃), R3 ( ₆ETTLLE DRI ₁₄), R4 ( ₆ETTLLE DRIL ₁₅), L1 ( ₅ EETTLLE ₁₁), L2 ( ₄ TEETTLLE ₁₁), L3 ( ₃ KTEETTLLE ₁₁), L4 ( ₂ KKTEETTLLE ₁₁) and L5 ( ₁ DKKTEETTLLE ₁₁), all can obviously strengthen this elementary cell activity to verify bright any two amino acid (left side) and (right side), be considered as strengthening the active critical amino acid of this epi-position.The result proves ₂K ₃K and R ₁₃For obviously strengthening the active critical amino acid of this elementary cell.On this basis, design express Y12 ( ₂KKTE ETTLLEDR ₁₃), to analyze through carrying out Western blot together with Y15-1, L4, R2 and Y15-2 with previous each peptide, comparatively validate determines that Y12 is this epi-position optimum activity unit.Then be with reference to peptide with Y12, will be wherein the primary activity unit ( ₂ETTLLE ₁₁) amino-acid residue use L-Ala (A) to replace one by one, Western blot analyzes demonstrations, with definite this epi-position activity is had the amino acid of the property contributed, promptly key amino acid.

2.1 enzyme is cut and product connects qualification result

Carrier pGEX-6p-1 cuts qualification result through BamHI and XhoI enzyme, shows that carrier is accurately cut, and size is about 4900bp, as shown in Figure 8.Designed 5,9 insert fragments with 7 small peptides is connected after enzyme is cut qualification result with carrier pGEX-6p-1, designs the about 1000bp of clip size that comprises restriction enzyme site, shown in Fig. 9,10 and 11.The result proves that the external source fragment is connected into correctly, and the amalgamation and expression and the Western blot that can be used for next step analyze.

2.24B2 epi-position primary activity unit be six peptides ( ₆ETTLLE ₁₁)

Amalgamation and expression oligopeptides Y15-1 ( ₁DKKTE ETTLLEDRIL ₁₅), Y15-2 ( ₆ ETTLLEDRILTTRNG ₂₀), six peptide P6 ( ₆ETTLLE ₁₁), pentapeptide P5 ( ₇TTLLE ₁₁) and tetrapeptide P4 ( ₈TLLE ₁₁), carry out Western blot with 4B2 and analyze.Establish the empty carrier contrast simultaneously.The result shows that Y15-1 and 4B2 are strong reaction; Y15-2 takes second place; Six peptides are the most weak; Pentapeptide, tetrapeptide and vehicle Control all do not have band.Can determine thus six peptides ( ₆ETTLLE ₁₁) be the active least unit of epi-position (Figure 12, B).For guaranteeing result's verity, Super super quick colouring reagents box of Ecl Plus and FUJIFILM luminescence imaging analyser LAS-3000 are adopted in this test, have carried out the high sensitivity analysis for the reaction band.

2.34B2 the active best unit of epi-position is 12 peptides ₂KKTEETTLLEDR ₁₃

According to the experimental design of front, amalgamation and expression R1 ( ₆ETTLLE D ₁₂), R2 ( ₆ETTLLE DR ₁₃), R3 ( ₆ETTLLE DRI ₁₄), R4 ( ₆ETTLLE DRIL ₁₅), L1 ( ₅ EETTLLE ₁₁), L2 ( ₄ TEETTLLE ₁₁), L3 ( ₃ KTEETTLLE ₁₁), L4 ( ₂ KKTEETTLLE ₁₁) and L5 ( ₁ DKKTEETTLLE ₁₁), with the expression product (quantitatively) of L5-R4 respectively with the 4B2100ug/lane of purifying carry out SDS-PAGE (Fig. 4-13, A) and Western blotv (Figure 13 B) analyzes.The result shows, from L4 and R2, react band along with ₂K ₃K and R ₁₃Adding respectively and obviously strengthen explanation ₂K ₃K and R ₁₃Important participation this epi-position immune response is all had tangible strengthening effect.Thereby can judge ₂K ₃K and R ₁₃For for the active basic critical amino acid of unit activity enhanced of epi-position.Therefore, determined the active minimum extent of enhancing elementary cell ETTLLE basically.

According to the front result, again amalgamation and expression Y12 ( ₂KKTEETTLLEDR ₁₃), (Figure 14, A) (Figure 14 B) analyzes with Western blot to carry out SDS-PAGE together with Y15-1, L4, R2 and Y15-2.Found that the band of Y15-1 and Y12 obviously is better than other L4, R2 and Y15-2, this result has further verified the strong activity of Y12.Since to select the shorter and the strongest active expression of peptides of aminoacid sequence, therefore, comparatively speaking, selection Y12 ( ₂KKTEETTLLEDR ₁₃) comparatively reasonable as epi-position optimum activity unit.

3.4B2 key amino acid Glu in the epi-position ⁶(E ₆), Thr ⁷(T ₇), Thr ⁸(T ₈), Leu ¹⁰(L ₁₀) and Glu ¹¹(E ₁₁)

With Y12 is with reference to peptide, to primary activity unit in the epi-position ₆ETTLLE ₁₁Amino acid use L-Ala (A) to replace one by one, called after S-1 (KKTE ATTLLEDR), S-2 (KKTE EATLLEDR), S-3 (KKTE ETALLEDR), S-4 (KKTE ETTALEDR), S-5 (KKTE ETTLAEDR) and S-6 (KKTE ETTLLADR).Each metathetical small peptide of prokaryotic expression also checks the activity of each substituted peptide to subdue situation with Western blot.Oligopeptides of expressing and Y12 together carry out SDS-PAGE and Westernblot and analyze, and the result shows that S-2, S-3 and S-5 significant reaction weaken, and S-6 is reactionless, explanation ₆ETTLLE ₁₁Middle T ₇, T ₈, L ₁₀And E ₁₁This epi-position all there is contribution; E wherein ₁₁This epi-position activity is played a decisive role,, cause this epi-position not discerned (Figure 15) by 4B2 once replacement.In conjunction with 2.4 results, determine E at last ₆, T ₇, T ₈, L ₁₀And E ₁₁Be the key amino acid of this epi-position.

VP2 albumen is carried out the homology modeling show, the VP2 albumen N-end at 4B2 epi-position place just is near the albumen bottom the threefold axis, and has covered the Ca on the virus particle just ²⁺The site.Though this position has disguise, but still can be discerned by specific antibody.Therefore, monoclonal antibody 4B2 can overcome the serotype restriction of FMD diagnosis, is applied to the exploitation of universal diagnostic reagent.

The effect test of test example 1 monoclonal antibody of the present invention

1. Sheet is anti-and detect determining of monoclonal antibody working concentration

Adopt among the embodiment 1 13.1 method Sheets anti-and detect the monoclonal antibody working concentration, the result shows: to detect monoclonal antibody 4B2 be dilution in 1: 100 when (0.9 μ g/ml) when catching monoclonal antibody 3D9 dilution (2.5 μ g/mL) in 1: 1000, biotin labeling, OD _450nmValue is near 1.0 and P/N ratio maximum.The result is as shown in table 8.

Table 8 is caught the best bag of monoclonal antibody by concentration and the best working concentration square formation of detection monoclonal antibody titration results

2. sensitivity test

With Asia1FMDV cell toxicant (TCID ₅₀=10 ^6.75) and O type FMDV cell toxicant (TCID ₅₀=10 ^6.5) carry out gradient dilution, carry out ELISA and detect, the result shows that this method is 1.8 * 10 to the cytotoxic limit of identification of Asia1FMDV ⁴TCID ₅₀, be 7.9 * 10 to the cytotoxic limit of identification of O type FMDV ³TCID ₅₀As shown in table 9.

Table 9 susceptibility measuring result

1: 50 times of positive bovine serum of the anti-Asia1 type FMDV of dilution, the positive guinea pig serum of resisting O-type FMDV and corresponding negative serum are mixed 37 ℃ of water-bath 2h with Asia1 type and O type FMDV equal-volume respectively.By good enzyme plate, behind 37 ℃ of sealing 1h, the sample that adds above-mentioned blocking-up sample successively and do not block detects monoclonal antibody with monoclonal antibody 3D9 bag, the Streptavidin of HRP mark, and substrate colour developing liquid is measured the OD value.According to document: blocking-up rate %=[(negative serum blocking-up OD value-positive serum blocking-up OD value)/(negative serum is blocked OD value-blank OD value)] * 100%; If the blocking-up rate greater than 50%, then is judged to be the positive.Shown in the table 10.

The test of table 10 specific inhibition

Simultaneously, bovine tuberculosis, pleuropneumonia, Niu Liure, Akabane Disease, ox infective rhinitis antigens such as (ox pass nose) are detected, set up the standard FMDV positive and negative control with the antigen capture ELISA method of setting up.The result shows that test sample is negative entirely, and no cross reaction takes place.

3. antigen capture ELISA method detects FMDV cell toxicant and tissue poison

3.1 antigen capture ELISA method detects each genotype FMDV cell toxicant

Obtain 13 parts of cell toxicants after representing the BHK-21 of virus strain infection cell with Asia1 type, O type and each genotype of A type FMDV respectively, detect with antigen capture ELISA method, the result shows that 13 parts of cell toxicants are all positive, and recall rate is respectively 100%.Sample is followed successively by in proper order: cell contrast, Asia1 standard cell lines poison, O type standard cell lines poison, Asia1-1, Asia1-2, Asia1-3, Asia1-4, Asia1-5, Asia1-6, Pan Asia-1, Pan Asia-2, Pan Asia-3, South East Asia, A type, sinotype-1, sinotype-2.The result is as shown in table 11.

Table 11 antigen capture ELISA method detects each genotype FMDV cytotoxic reaction result

3.2 antigen capture ELISA method detects each genotype FMDV tissue poison

13 parts of FMDV that this laboratory is preserved organize pathological material of disease, detect with antigen capture ELISA method, and the result shows that 13 parts of cell toxicants are all positive, and recall rate is respectively 73.3%.Sample is followed successively by in proper order: negative control, Asia1 fat corrected milk(FCM) sodoku, O type fat corrected milk(FCM) sodoku, Asia1-1, Asia1-2, Asia1-3, Asia1-4, Asia1-5, Asia1-6, Pan Asia-1, Pan Asia-2, Pan Asia-3, South East Asia, sinotype-1, sinotype-2, sinotype-3.

Table 12 antigen capture ELISA method detects each genotype FMDV and organizes malicious reaction result

Sequence table-determine original text 20090731.txt

Sequence table

＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture

＜120〉foot-and-mouth disease virus resistant serotype is shared monoclonal antibody and identified epitope thereof

<130>KLPI05676

<160>2

<170>PatentIn?version?3.1

<210>1

<211>6

<212>PRT

<213>Foot?and?mouth?disease?virus

<400>1

G1u?Thr?Thr?X?Leu?Glu

1 5

<210>2

<211>12

<212>PRT

<213>Foot?and?mouth?disease?virus

<400>2

Lys?Lys?Thr?Glu?Glu?Thr?Thr?Leu?Leu?GLu?Asp?Arg

1 5 10

Claims (10)

1, a strain can be secreted the hybridoma cell line 4B2 of the non-serotype dependency monoclonal antibody of foot-and-mouth disease virus resistant, and its microbial preservation number is: CGMCC No.3103.

2, by the described hybridoma cell line excretory of claim 1 monoclonal antibody.

3, the motif of the linear epitope of the foot and mouth disease virus VP2 albumen N-end discerned of the described monoclonal antibody of claim 2, it is characterized in that: the motif of this linear epitope is made up of the aminoacid sequence on the VP2 albumen N-end of foot and mouth disease virus, its aminoacid sequence is shown in the SEQ ID NO:1, wherein, X is any one amino-acid residue.

4, according to the motif of the described linear epitope of claim 3, it is characterized in that: its key amino-acid residue is Glu ⁶, Thr ⁷, Thr ⁸, Leu ¹⁰And Glu ¹¹Wherein, decisive amino-acid residue is Glu ¹¹

5, according to the motif of claim 3 or 4 described linear epitopes, it is characterized in that: this epitope sequences is that seven serotype FMDV share, and optimum activity unit is a dodecapeptide, and its aminoacid sequence is shown in the SEQ ID NO:2.

6, a strain can be secreted the hybridoma cell line 3D9 of the non-serotype dependency of anti-FMDV monoclonal antibody, and its microbial preservation number is: CGMCC No.3104.

7, by the secreted monoclonal antibody of the described hybridoma cell line of claim 6.

8, claim 1 or 6 described hybridomas tie up to preparation diagnosis, prevention or the reagent of treatment foot and mouth disease or the purposes in the medicine.

9, claim 2 or 7 described monoclonal antibodies are in preparation diagnosis, prevention or the reagent of treatment foot and mouth disease or the purposes in the medicine.

10, the purposes of the motif of claim 3 or 4 described linear epitopes in reagent, vaccine or the medicine of preparation diagnosis, prevention or treatment foot and mouth disease.

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