CN104744572A - Avian and porcine hepatitis e virus shared antigen, monoclonal antibody and preparation method and application - Google Patents
Avian and porcine hepatitis e virus shared antigen, monoclonal antibody and preparation method and application Download PDFInfo
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Abstract
The invention relates to two monoclonal antibody hybridoma cell lines which secrete avian and porcine hepatitis e virus (HEV) shared epitope by virtue of a conventional hybridoma technique, wherein the lines are named 3E8 and 1B5 and the cell preservation numbers are respectively CCTCC No: C201395 and CCTCC No: C201396. The amino acid sequences of the shared epitope of the two lines identified by two monoclonal antibodies are identified by using a phage random 7 peptide library kit. The amino acid sequences are respectively V/IPHD (SEQ ID No: 1) and VKLYM (SEQ ID No: 2). Moreover, application of two mimic peptides of epitope in avian and porcine HEV serological diagnosis is analyzed. The two mimic peptides of the shared epitope of avian and porcine HEV capsid proteins disclosed by the invention can be used for rapid diagnosis of avian and porcine HEV and monitor of prevalence of disease. In addition, the two lines disclosed identify the monoclonal antibodies of the shared epitope and can be further used for rapidly diagnosing avian and porcine HEV.
Description
Technical field
The present invention relates to the aminoacid sequence that two fowl and pig hepatitis E virus have epitope, and the monoclonal antibody of above-mentioned total epitope can be identified, also relate to the simulating peptide of this epi-position and monoclonal antibody detect in vitro in application, belong to field of biology.
Background technology
Hepatitis E is the acute viral hepatitis caused by hepatitis E virus (Hepatitis E virus, HEV) infects, Major Epidemic in Asia, the developing country such as Africa and Latin America, also occasionally have Case report in developed country.In the acute viral hepatitis that China occurs, hepatitis E accounts for about 20%.Increasing research finds that hepatitis E is a kind of zoonosis, and therefore this disease is an important public health problem.
Studies have found that, HEV can infected pigs, chicken, sheep, the animal such as mouse and ox.Meng equals to be separated for 1997 and identifies first animal HEV strain isolated---pig HEV, and find that its homology with people HEV reaches about 97%, and also more and more evidence proves, pig HEV has infecting both domestic animals and human.Calendar year 2001, Haqshenas etc. isolate second animal strain isolated from the Fel Gallus domesticus suffering from hepatitis splenomegaly (Hepatitis-splenomegaly, HS) syndromes---fowl HEV.Although fowl HEV can infect the mankind also not have direct evidence to show at present, specific antibody the detecting in people and porcine blood serum of anti-fowl HEV, illustrate that fowl HEV has the possibility infecting the mankind or other domestic animal.
Fowl and pig HEV have dependency in heredity and antigenicity.Gene sequencing find, the full-length genome of fowl and pig HEV all containing 3 open reading frame (ORF), ORF1, ORF2 and ORF3.Wherein, the capsid protein of ORF2 encode viral is viral main structural protein, and containing viral main epitope.The capsid protein antigen that fowl HEV is being analyzed in relevant research finds, 268 amino acid regions of this PROTEIN C end can with the antiserum(antisera) generation cross reaction of people and pig HEV, illustrate that the total epitope of fowl and pig HEV is contained in this region.But about the accurate identification of total epitope, and the simulating peptide of this total epitope is not but still shown in there is relevant report in the serodiagnosis application of fowl and pig HEV.
Summary of the invention
An object of the present invention provide two strains to secrete monoclonal antibody hybridoma cell strain that anti-fowl and pig hepatitis E virus have epitope.
Two of object of the present invention is to provide two fowl and pig hepatitis E virus to have the aminoacid sequence of epitope.
Three of object of the present invention has been according to above-mentioned aminoacid sequence design and synthesis two polypeptide for fowl and pig hepatitis E antibody test, and sets up according to this polypeptide the serological diagnostic method that fowl and pig hepatitis E virus infect.
The technical problem that will solve required for the present invention, can be achieved through the following technical solutions:
As a first aspect of the present invention, the aminoacid sequence that fowl and pig HEV capsid protein have epitope is V/IPHD (SEQ ID No:1) and VKLYM (SEQ ID No:2).
As two aspects of the present invention, a kind of albumen, name is called monoclonal antibody 3E8 (monoclonal antibody 3E8), identify total epitope, the specific antibody of the sequence I/VPHD (SEQ ID No:1) of fowl and pig HEV ORF2 albumen, its hybridoma cell strain cyropreservation number is CCTCC No:C201395.
Further qualification finds, the aminoacid sequence of the simulating peptide of described epi-position is VPHD (SEQ IDNo:1).One or more amino acid wherein in V and D carry out lacking or replacing, and can affect the bonding force of polypeptide and monoclonal antibody, but not affect combination, and amino acid P and H is key amino acid, affect the combination of monoclonal antibody and polypeptide.
As a third aspect of the present invention, a kind of albumen, name is called monoclonal antibody 1B5 (monoclonal antibody 1B5), identify total epitope, the specific antibody of the sequence VKLYM (SEQ ID No:2) of fowl and pig HEV ORF2 albumen, the cyropreservation number of its hybridoma cell strain is CCTCC No:C201396.
Further, find that each aminoacid sequence all affects the combination of monoclonal antibody and polypeptide.
As a fourth aspect of the present invention, prepare two strains and secrete the monoclonal hybridoma system that anti-fowl and pig HEV capsid protein have epitope.
A preparation method for monoclonal antibody, is characterized in that, comprises the following steps:
It is characterized in that, comprise the following steps:
Utilize fowl hepatitis E virus Chinese pathogenic strain (Hepatitis E virus, HEV, CaHEV, Genbank number GU954430) ORF2 albumen be immunogen, immunity BABL/c mouse, adopt hybridoma cell technology to prepare two strains and secrete the hybridoma cell line that anti-fowl and pig HEV have epitope monoclonal antibody, called after 3E8 and 1B5 respectively.
Further, grand antibody 3E8 and 1B5 of monoclonal antibody of two total epitopes, heavy chain is IgG1, and light chain is κ chain.
As a fifth aspect of the present invention, polypeptide prepared by a kind of total epitope, is characterized in that, for the preparation of fowl and pig hepatitis E vaccine.Polypeptide prepared by a kind of above-mentioned total epitope and application, the aminoacid sequence improvement on synthesis of two epi-positions is utilized to be envelope antigen, indirect ELISA finds that it with the positive serum of fowl and pig HEV, strong antigen antibody reaction can occur, and does not react with negative serum.
As a sixth aspect of the present invention, described monoclonal antibody is for the preparation of the application test kit in fowl and pig hepatitis E virus diagnosis.
The detailed description of technical solution of the present invention:
Fowl hepatitis E virus Chinese pathogenic strain (the Hepatitis Evirus of the prokaryotic expression of the present invention's purifying, HEV, CaHEV, Genbank number GU954430) C of ORF2 holds 268 amino acid proteins to be immunogen, immunity BALB/c mouse, prepare anti-fowl hepatitis E virus Chinese pathogenic strain (Hepatitis E virus, HEV, CaHEV, Genbank number GU954430) monoclonal antibody of ORF2 albumen, successfully prepare two strain monoclonal antibody 3E8 and 1B5.And detected by indirect ELISA and Western blotting method and find, outside this two strains monoclonal antibody decapacitation specificity and fowl HEV ORF2 albumen test, can also with pig hepatitis E virus Shandong Province of China strain isolated (Hepatitis E virus, HEV, CHN-SD-sHEV, Genbank number KF176351) ORF2 albumen generation cross-immune reaction, show that the epi-position that 3E8 and 1B5 identifies is that fowl and pig HEV ORF2 albumen have epitope.
Utilize 3E8 and 1B5 two strain monoclonal antibody, wrap by ELISA Sptting plate, then utilize phage display random loops 7 peptide storehouse test kit (BioLabs
@inc) screen the mimic epitopes of above-mentioned two monoclonal antibody identifications respectively, find that an epitope motifs I/VPHD and monoclonal antibody 3E8 has binding activities, another epitope motifs VLYMSV and monoclonal antibody 1B5 has binding activities.
Beneficial effect of the present invention:
The simulating peptide of fowl of the present invention and pig hepatitis E virus capsid protein two total epitopes.
The total epitope that monoclonal antibody 3E8 identifies, its aminoacid sequence is made up of the sequence I/VPHD (SEQ ID No:1) of fowl and pig HEV ORF2 albumen.
The total epitope that monoclonal antibody 1B5 identifies, its aminoacid sequence is made up of the sequence VKLYM (SEQ ID No:2) of fowl and pig HEV ORF2 albumen.
This total epitope may be used for the quick diagnosis of fowl and pig viral hepatitis type E and the monitoring of disease popularity.For the preparation of fowl and pig hepatitis E vaccine, purity is greater than 98%.
The monoclonal antibody of the above-mentioned total epitope of two strain identification of the present invention, can quick diagnosis fowl and pig hepatitis E virus.
Accompanying drawing explanation
The enzyme of Fig. 1 goal gene recombinant expression plasmid cuts qualification.
The SDS-PAGE of Fig. 2 A target protein purifying analyzes (A).
The Western blot of Fig. 2 B target protein purifying identifies (B).
The antibody titer of monoclonal antibody 3E8 and 1B5 of Fig. 3 purifying.
The ELISA cross reaction result of Fig. 4 two strain monoclonal antibody 3E8 and 1B5 and fowl and pig HEV ORF2 albumen, Avian HEV is fowl Hepatitis E virus, and Swine HEV is pig Hepatitis E virus.
The cross reaction result of the Western blot of Fig. 5 A monoclonal antibody 3E8 and fowl and pig HEV ORF2 albumen.
The cross reaction result of the Western blot of Fig. 5 B monoclonal antibody 1B5 and fowl and pig HEV ORF2 albumen.
Fig. 6 A monoclonal antibody 3E8 screens the ELISA detection validation of positive bacteriophage.
Fig. 6 B monoclonal antibody 1B5 screens the ELISA detection validation of positive bacteriophage.
Fig. 7 simulates the polypeptide of total epitope and sudden change and disappearance and the ELISA reaction result of monoclonal antibody 3E8 and 1B5.
The monoclonal antibody 3E8 of hybridoma cell strain 3E8[fowl hepatitis E virus Chinese pathogenic strain (Hepatitis E virus, HEV, CaHEV, Genbank number GU954430) ORF2 albumen]
Depositary institution's title: China typical culture collection center (CCTCC)
Address: Wuhan City, Hubei Province Wuhan University
Preservation date: on July 17th, 2013
Deposit number: CCTCC NO:C201395.
The monoclonal antibody 1B5 of hybridoma cell strain 1B5[fowl hepatitis E virus Chinese pathogenic strain (Hepatitis E virus, HEV, CaHEV, Genbank number GU954430) ORF2 albumen]
Depositary institution's title: China typical culture collection center (CCTCC)
Address: Wuhan City, Hubei Province Wuhan University
Preservation date: on July 17th, 2013
Deposit number: CCTCC NO:C201396.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the condition that conditioned disjunction manufacturer as described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) provides is carried out.
The preparation of embodiment 1 mouse immune and monoclonal antibody
(1) expression, purifying fowl HEV ORF2 PROTEIN C end 268 amino acid
According to fowl hepatitis E virus Chinese pathogenic strain (Hepatitis E virus, HEV, CaHEV, Genbank number GU954430), design the primer ORF2-268-F:CAC with BamHI and XholI restriction enzyme site
gGATCCcAGTATATGTACGGC (SEQ ID No:3); ORF2-268-R:GAA
cTCGAGtTAGGGTGGTGAGGGGAAT (SEQ ID No:4), with CaHEV suspension for template, RT-PCR method obtains goal gene sequence, then goal gene and pET-28a (+) carrier are carried out double digestion with BamHI and XholI simultaneously, T4 ligase enzyme connects, and builds recombinant expression plasmid pET-ORF2-268.Recombinant plasmid is cut after qualification through enzyme and is obtained goal gene fragment 804bp and carrier segments 5300bp, after recombinant plasmid order-checking, CaHEV sequence alignment homology 100% on itself and GenBank, illustrates the prokaryotic expression carrier (Fig. 1) successfully constructing and comprise goal gene.
Be transformed into by positive recombinant expression plasmid in escherichia coli expression Host Strains Rosetta (DE3), picking positive monoclonal adds IPTG 1mM abduction delivering 6h in 37 DEG C of shaking table 225rpm.The centrifugal 10min of bacterium liquid 5000r/min, abandons supernatant, resuspended precipitation.Then ultrasonication thalline, working hour 1s, off time 3s, all times 15min, 225 times altogether.After ultrasonic, the centrifugal 10min of 12000r/min, collects inclusion body precipitation.Inclusion body is dissolved, incubated at room 1h with 5mL4M urea.Used the membrane filtration of 0.22 μm, then utilize Ni post to carry out the purifying of albumen.The albumen of purifying is analyzed through SDS-PAGE, has occurred a band at about 32kDa, and without other assorted bands (Fig. 2 A), illustrates and obtain purer target protein.Utilize fowl HEV antibody positive chicken serum as primary antibodie, Western blot qualification obtains the correction (Fig. 2 B) of target protein.It is 19.6mg/ml that the sample BCA determination of protein concentration test kit (Pierce BCA Protein Assay Kit) of purifying measures concentration, then frozen for subsequent use in-20 DEG C.
(2) immunity of mouse
A) immunogenic emulsification (freund's adjuvant):
Mixed according to 1:1 (V/V) with adjuvant by purifying protein, then shake emulsification on the oscillator, the immunogen that emulsification is good is dripped one and drops in the water of precooling, indiffusion in 30 minutes is that emulsification is complete.
B) immune programme for children
Select the BABL/c mouse in 6-8 week as immune animal.Tail vein blood sampling before each immunity, twice immunization interval is 2 weeks.First time Freund's complete adjuvant, albumen dosage be 75 μ g/ only, abdominal injection, total dose 200 μ l/ is only.Second time Freund's incomplete adjuvant, albumen dosage is still 75 μ g/.Third time immunity is carried out with reference to the identical dosage of second time and mode.Before merging, booster immunization once.
(3) cytogamy
The good BALB/c mouse of immunity, the aseptic splenocyte getting immune mouse merges by 5:1 with SP2/0 myeloma cell under 1mL50%PEG, then utilizes HAT substratum to carry out selection cultivation.
(4) screening of positive cell and subclone
With the fowl HEV ORF2-268 albumen of purifying for antigen (200ng/well) wrapper sheet, draw cells and supernatant with the cell clone of the indirect ELISA method screening positive, positive hole is shown to result, then continues to do subclone, simultaneously enlarged culturing frozen.Limiting dilution assay is adopted to carry out 2 subclones to selected hybridoma.Through twice subclone, the specific hybrid tumor cell strain obtaining the anti-target protein of the different female generation secretion of two strains after ELISA screening is 3E8 and 1B5, and its cyropreservation number is respectively: CCTCC No:C201395 and CCTCC No:C201396.
(5) preparation of odd contradictive hydroperitoneum and purifying
The hybridoma being cultured to vegetative period is blown down, then the cell suspension of mouse peritoneal inoculation 0.5mL.After inoculation, about 7-10 days, collects ascites.Ascites is put into the small beaker of 25mL, dropwise add isopyknic saturated ammonium sulphate, after slowly stirring 2h, 10000rpm is centrifugal, and the PBS of precipitation 0.01M pH7.2 is resuspended.Then monoclonal antibody is further purified with Protein G affinity column.Monoclonal antibody 3E8 and 1B5 of success preparation and purifying, itself and the specific ELISA of generation of target protein react, as the OD that monoclonal antibody and target protein ELISA react
450when=1.0, the antibody titers of monoclonal antibody 3E8 is 10
4~ 10
5, the antibody titers of monoclonal antibody 1B5 is 10
2~ 10
3(Fig. 3), illustrate that the monoclonal antibody of purifying obtains high antibody titer.
(6) monoclonal antibody hypotype qualification
Identify by the explanation of Sigma company mouse monoclonal antibody hypotype identification kit, be IgG1 through qualification 3E8 and 1B5 heavy chain subgroup, light chain is κ chain.
(7) cross reaction of monoclonal antibody and pig HEV ORF2 albumen
Prokaryotic expression pig HEV ORF2 (Swine Hepatitis E virus, CHN-SD-sHEV, Genbank number KF176351) PROTEIN C end 268 amino acid (SEQ ID NO.5), according to sequences Design two couples of primers F 15 '-CCGGGATCCCAGTTATTTTACTCC-3 ' (SEQID NO.6) and R15 '-ATGCTCGAGTCAATACTCCCGGGT-3 ' (SEQ IDNO.7), with reference to the expression strategy of fowl HEV ORF2 albumen, after great expression, purifying obtains albumen is sHEV-ORF2, be antigen by this albumen and fowl HEV ORF2-268 albumen, indirect ELISA and Western blot cross reaction is carried out with monoclonal antibody 3E8 and 1B5, result of indirect ELISA finds, monoclonal antibody 3E8 with 1B5 is the same with the result of fowl HEV ORF2 albumen with the reaction result of pig HEV ORF2 albumen, be the positive, and mice serum is negative before immunity, illustrate that two strain monoclonal antibodies with fowl and pig HEV ORF2 albumen, specific cross reaction (Fig. 4) can occur, and Western blot result confirms further, two strain monoclonal antibodies can with fowl and pig HEV ORF2 albumen generation cross-immune reaction (having occurred brown band the fowl of expecting and pig HEV ORF2 albumen size place) (Fig. 5 A and Fig. 5 B).Above-mentioned indirect ELISA and Western blot result jointly illustrate monoclonal antibody 3E8 and 1B5 can with fowl and pig HEV ORF2 albumen generation cross reaction, i.e. the epitope of this two strains monoclonal antibody identification is that fowl and pig HEV have epitope.
The qualification of embodiment 2 monoclonal antibody identification epitope aminoacid sequence
In order to identify the aminoacid sequence of the epitope that monoclonal antibody 3E8 and 1B5 identifies further, the present invention utilizes phage random ring seven peptide to open up test kit (NEW ENGLAND BioLab, the U.S.), then the mode that biological sieve is washed in a pan is adopted, sieve wash in a pan can with monoclonal antibody 3E8 and 1B5 can with the phage of its specific binding, by order-checking after concentrated for phage qualification, the nucleotide sequence of acquisition is shifted onto into aminoacid sequence, and compare, namely the amino acid that occurrence probability is the highest may be the amino sequence of monoclonal antibody identification epitope, then improvement on synthesis is passed through, the effect of accurate localize antigens epi-position different aminoacids sequence further.
(1) phage titre measures
Carry out titer determination to the phage that each takes turns sieve naughty, its method is summarized as follows: diluted by the incubated overnight liquid LB substratum 1:100 of intestinal bacteria ER2738.By respectively taking turns input, the phagocytosis body fluid of output gets 10 μ l and joins in the above-mentioned bacterial cultures of 200 μ l after diluting in proportion, short mix, the LB top-agar be ready in 50 DEG C of water baths is joined after incubated at room 5min, be spread evenly across containing on LB/IPTG/Xgal flat board after quick mixing, count after 37 DEG C of overnight incubation.
(2) biopanning
Utilize phage random ring seven peptide to open up test kit (NEW ENGLAND BioLab, the U.S.), by specification carries out the continuous elutriation of three-wheel.Be summarized as follows: be first buffered liquid (0.1mol/LNaHCO with bag
3, pH8.6) and dilute monoclonal antibody 3E8 and 1B5 bag respectively by 96 hole ELISA Sptting plates (100 μ g/ hole), 4 DEG C of overnight incubation; After add 300 μ l confining liquid (0.1M NaHCO
3, 5g/L bovine serum albumin, 0.2g/L NaN
3, pH8.6), 37 DEG C hatch 1h after, wash 10 μ l1 × 10 after plate
13the prophage 1:10 dilution of pfu/ml adds in reacting hole, washes plate after hatching 1h; Then add 100 μ l glycine-HCI damping fluid (0.2mol/L, pH2.2), incubated at room 10min, the phage of elution of bound, transfers to Eppendorf tube, adds in 150 μ l1M Tris-HCl (pH9.1) and elutriant.Get 2 μ l elutriants and measure titre, the phage of the enrichment specific combination that then increases.Carry out 2,3 again by above-mentioned steps and take turns elutriation, 2,3 take turns elutriation amplification eluate in 2 × 10
10the add-on of pfu is tested, and the concentration of Tween in washings is increased to 0.5%.Recording access amount and the elution amount of three-wheel elutriation pnagus medius by measuring phage titre, calculating productive rate=elution amount/access amount × 100%.Data presentation, along with elutriation number of times increases, productive rate improves gradually, and what prove specific binding phage arrives enrichment (table 1).
Table 1 phage random peptide library elutriation result
Note:
aphage quantity is added in biopanning experiment;
boften take turns wash-out in elutriation and obtain the phage quantity of specific binding;
cproductive rate represents the enrichment degree of often taking turns elutriation specific phage
(3) phage affinity capture ELISA
Each random picking 10 locus coeruleus the titer determination plate eluriating rear eluate are taken turns from two strain monoclonal antibodies the 3rd.Amplification also measures titre after purifying.Use NaHCO
3damping fluid (pH9.6) prepares 1 × 10
10the single cloned phage liquid of pfu/mL.Wrap by micro-reaction plate (100 μ l/ hole) with monoclonal antibody 3E8 and 1B5, after 4 DEG C of overnight incubation, plate washed by washings (PBST is containing the 0.01M pH7.2PBS of 0.5% tween 20), then confining liquid (containing 2.5% skim-milk PBST) closes 1h, PBST washes plate 3 times, add respectively and dilute 10 naughty strain phages of monoclonal antibody 3E8 and 1B5 sieve with coating buffer 1:100, after every hole adds 100 μ l incubated at room 1h, 5 times are washed again with PBST, add the HRP-mouse-anti M13 antibody (Pharmacia of 1:4000 dilution, USA) 100 μ l, after incubated at room 1h, PBST washes 5 times, nitrite ion 3, 3 ', 5, 5 '-tetramethyl benzidine two hydrochloric acid (TMB), 15min is hatched in 100 μ l/ holes, measure the absorbance of A490nm, light absorption value is greater than 0.5 and is judged to be the positive.The 10 strain phage clones that monoclonal antibody 3E8 and its sieve are washed in a pan all react (A490nm>0.8), illustrate that specific binding (Fig. 6 A and Fig. 6 B) all occurs with 3E8 10 strain phage clones.And 9 strains in monoclonal antibody 1B5 and 10 strain phage clones react (A490nm>0.8), illustrate that specific combination (Fig. 6 A and Fig. 6 B) occurs for 9 strain phage clones and 1B5.Specific combination can be there is in the polypeptide that the specific binding of phage clone and monoclonal antibody further illustrates phage display with monoclonal antibody.Finally determine that 10 strains with monoclonal antibody 3E8 and 9 strains and monoclonal antibody 1B5, the phage that combines can occur.
(4) preparation of phage single-chain DNA profiling and sequencing
After being checked order by the positive phage clones of above-mentioned acquisition, obtain the single-stranded DNA sequence of phage-displayed polypeptides, by DNAStar software, shift the aminoacid sequence obtaining DNA sequence encoding onto, namely obtain the aminoacid sequence of monoclonal antibody identification epitope.Specifically be implemented as follows: wash in a pan respectively to two strain monoclonal antibody sieves and identify that 10 strains of acquisition and 9 strain phage clone bacterium liquid add 200 μ l PEG/NaCl [20% (w/v) PEG-8000,2.5M NaCl], after mixing, 4 DEG C of standing 10min, the centrifugal 10min of 10000rpm, precipitation is dissolved in 100 μ l iodine damping fluids [10mM Tris-HCl (pH8.0), 1mM EDTA, 4M NaI] in 30 μ l ultrapure waters.Then send and checked order by Nanjing Jin Sirui biotechnology company, sequencing primer is 5 '-CCC TCA TAG TTA GCG TAA CG – 3 ' (SEQ ID No:8).The nucleotide sequence obtained after order-checking is derived corresponding exogenous amino acid sequences, and the aminoacid sequence of itself and known fowl and pig HEVORF2 albumen is carried out sequence analysis.Namely the aminoacid sequence that occurrence probability is the highest may be the aminoacid sequence of monoclonal antibody identification epitope.The aminoacid sequence obtaining monoclonal antibody 3E8 identification thus may be V/IPHD, and the aminoacid sequence that 1B5 identifies may be VKLYMS (table 2).
The exogenous amino acid sequences that table 2 positive bacteriophage is shown
Note: in dotted line is the amino acid that occurrence probability is the highest, it may be the aminoacid sequence of monoclonal antibody identification epitope
(5) monoclonal antibody 3E8 and 1B5 identifies the accurate location of the aminoacid sequence of epitope
According to the peptide sequence compare of analysis of the phage display that monoclonal antibody 3E8 combines, the polypeptide (suddenly change different aminoacid sequences) (table 3) that design and synthesis 6 sections may identify; Simultaneously according to the peptide sequence compare of analysis of the phage display of 1B5 combination, the polypeptide (table 3) that design and synthesis 2 sections of 1B5 may identify.Polypeptide send and is synthesized and coupling hemocyanin (KLH) by Nanjing Genscript Biotechnology Co., Ltd., Analysis and Identification purity >=98%.Then the polypeptide of above-mentioned synthesis is carried out indirect ELISA reaction with corresponding monoclonal antibody respectively, analyze the effect that in epitope, different aminoacids plays in it is combined with monoclonal antibody.The step that indirect ELISA reaction is implemented is as follows: wrap 8 polypeptide 100 μ l/ holes (2ng/ μ l) by elisa plate respectively, 4 DEG C of overnight incubation.PBST washes 3 times, adds 200 μ l confining liquids, incubated at room 1h, PBST washes 3 times again, adds 100 μ l (10ng/ μ l) monoclonal antibody 3E8 and 1B5, incubated at room 1h, after PBST washes 5 times, add the sheep anti-mouse igg (JackonImmunResearch, West Grove, PA) that 100 μ l (1:4000) HRP mark, after incubated at room 1h washing, add 100 μ lTMB, room temperature lucifuge leaves standstill 15min, adds 50 μ l3M H
2sO
4termination reaction, reads A450 value in spectrophotometer.Found that the polypeptide of P and H of sudden change can not react with 3E8, and the polypeptide of I and D that suddenly change still can react with 3E8, but bonding force weakens.Illustrate that the aminoacid sequence of the epitope that monoclonal antibody 3E8 identifies is I/VPHD, wherein its keying action of amino acid PH.And for 1B5, the polypeptide of delete amino acids MSV does not react with 1B5, illustrate that the aminoacid sequence of the epitope that monoclonal antibody 1B5 identifies is KLYMSV, aminoacid sequence can not shorten again.
Table 3 artificial sequence synthetic polypeptide
Embodiment 3 fowl and pig HEV have the application of epitope simulative peptide
The application of the amino sequence of total epitope is identified in order to monoclonal antibody 3E8 and 1B5 analyzing above-mentioned qualification, this patent is synthesized by Nanjing Genscript Biotechnology Co., Ltd. and the simulating peptide IPHD (3E8) of the total epitope of coupling hemocyanin (KLH) and KLYMSV (1B5), then by simulating peptide with 0.01M PBS (pH7.2) damping fluid bag by 96 hole elisa plates (100ng/ hole), plate is washed 5 times in (300 μ l/ hole) with the PBS washings (PBST) containing 0.5% tween 20 after 4 DEG C of overnight incubation, then confining liquid (PBST containing 2.5% skim-milk) closes 1h, after PBST washes 5 times, add 100 μ l fowl and pig HEV antibody positive and negative chicken and porcine blood serum (1:100), incubated at room 1h, PBST after washing 5 times respectively correspondence add 100 μ l (1:4000) HRP mark goat-anti chicken and pig IgG (Jackon ImmunResearch, West Grove, PA), after incubated at room 1h washing, add 100 μ l TMB, room temperature lucifuge leaves standstill 15min, add 50 μ l3M H
2sO
4termination reaction, reads A450 value in spectrophotometer.Found that two simulating peptide and fowl and pig HEV antibody positive chicken and porcine blood serum react, and do not react (table 4) with negative serum, illustrate that the simulating peptide IPHD (3E8) of the epitope that monoclonal antibody 3E8 and 1B5 of synthesis identifies and KLYMSV (1B5) can be good at being applied to the detection of fowl and pig HEV antibody and the serodiagnosis (Fig. 7) of fowl and pig HEV infection.
The indirect ELISA of the total mimetic peptide that the fowl that table 4 is different and pig HEV antibody positive and negative serum and 3E8 and 1B5 identify reacts
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.
Claims (7)
1. fowl and pig HEV capsid protein have the aminoacid sequence of epitope is V/IPHD (SEQ ID No:1) and VKLYM (SEQ ID No:2).
2. an albumen, name is called monoclonal antibody 3E8 (monoclonal antibody 3E8), be identify total epitope, the specific antibody of the sequence I/VPHD (SEQ ID No:1) of fowl and pig HEV ORF2 albumen, its hybridoma cell strain cyropreservation number is CCTCC No:C201395.
3. an albumen, name is called monoclonal antibody 1B5 (being called for short monoclonal antibody 1B5), identify total epitope, the specific antibody of the sequence VKLYM (SEQ ID No:2) of fowl and pig HEV ORF2 albumen, the cyropreservation number of its hybridoma cell strain is CCTCC No:C201396.
4. a preparation method for monoclonal antibody as claimed in claim 2 or claim 3, is characterized in that, comprises the following steps:
Utilize fowl hepatitis E virus Chinese pathogenic strain (Hepatitis E virus, HEV, CaHEV, Genbank number GU954430) ORF2 albumen be immunogen, immunity BABL/c mouse, adopt hybridoma cell technology to prepare two strains and secrete the hybridoma cell line that anti-fowl and pig hepatitis E virus have epitope monoclonal antibody, called after 3E8 and 1B5 respectively.
5. the preparation method of monoclonal antibody according to claim 4, is characterized in that, grand antibody 3E8 and 1B5 of described monoclonal antibody, heavy chain is IgG1, and light chain is κ chain.
6. the polypeptide having epitope and prepare as claimed in claim 1, is characterized in that, for the preparation of fowl and pig hepatitis E vaccine.
7. a monoclonal antibody as described in Claims 2 or 3, is characterized in that, for the preparation of fowl and pig hepatitis E virus test kit.
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| CN201410503871.5A CN104744572B (en) | 2013-09-27 | 2014-09-26 | Fowl and pig hepatitis E virus share antigen, monoclonal antibody and preparation method and application |
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| CN104744572B CN104744572B (en) | 2019-11-12 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109593122A (en) * | 2019-01-10 | 2019-04-09 | 西北农林科技大学 | Anti- pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application |
| CN109796531A (en) * | 2017-11-15 | 2019-05-24 | 中国农业科学院上海兽医研究所 | Pig Delta coronavirus N protein monoclonal antibody and its epitope and application |
| CN112285347A (en) * | 2020-09-30 | 2021-01-29 | 西北农林科技大学 | ELISA detection kit for pathogen antibody in porcine serum sample |
| CN114456263A (en) * | 2022-01-21 | 2022-05-10 | 西北农林科技大学 | Nano antibody specifically binding to hepatitis E virus capsid protein and application thereof |
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| DONG S 等: "Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies", 《J VIROL METHODS》 * |
| 王鑫杰 等: "应用噬菌体展示技术鉴定禽和猪戊型肝炎病毒衣壳蛋白一个共有抗原表", 《中国畜牧兽医学会兽医公共卫生学分会第三次学术研讨会论文集》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109796531A (en) * | 2017-11-15 | 2019-05-24 | 中国农业科学院上海兽医研究所 | Pig Delta coronavirus N protein monoclonal antibody and its epitope and application |
| CN109796531B (en) * | 2017-11-15 | 2022-03-11 | 中国农业科学院上海兽医研究所 | Monoclonal antibody of swine Delta coronavirus N protein, epitope and application thereof |
| CN109593122A (en) * | 2019-01-10 | 2019-04-09 | 西北农林科技大学 | Anti- pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application |
| CN112285347A (en) * | 2020-09-30 | 2021-01-29 | 西北农林科技大学 | ELISA detection kit for pathogen antibody in porcine serum sample |
| CN112285347B (en) * | 2020-09-30 | 2023-12-22 | 西北农林科技大学 | ELISA detection kit for pathogenic antibodies in pig serum sample |
| CN114456263A (en) * | 2022-01-21 | 2022-05-10 | 西北农林科技大学 | Nano antibody specifically binding to hepatitis E virus capsid protein and application thereof |
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| CN104744572B (en) | 2019-11-12 |
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