CN109593122A - Anti- pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application - Google Patents

Anti- pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application Download PDF

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CN109593122A
CN109593122A CN201910023151.1A CN201910023151A CN109593122A CN 109593122 A CN109593122 A CN 109593122A CN 201910023151 A CN201910023151 A CN 201910023151A CN 109593122 A CN109593122 A CN 109593122A
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monoclonal antibody
virus
pig
albumen
hepatitis
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赵钦
孙亚妮
周恩民
陈宜阳
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Northwest A&F University
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Abstract

The present invention discloses a kind of anti-pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application, relate generally to field of biotechnology, the monoclonal antibody is mainly secreted by hybridoma cell strain 1E4, pig hepatitis E virus ORF2 PROTEIN C 267, end amino acid using Prokaryotic expression, purification is envelope antigen, monoclonal antibody mAb-1E4 for the affinitive layer purification in the region is blocking antibody, the monoclonal antibody specific marked by HRP, the monoclonal antibody can be directly detected by the substrate TMB or DAB of HRP, and it is applied in the drug of preparation detection pig hepatitis E virus.

Description

Anti- pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application
Technical field
The present invention relates to field of biotechnology, anti-more particularly to a kind of anti-pig hepatitis E virus ORF2 protein monoclonal Body and its preparation and application.
Background technique
Hepatitis E virus (Hepatitis E virus, HEV) belongs to Hepatitis E virus section Hepatitis E virus category, is By transmission without coating single strand plus RNA virus.Although Hepatitis E is self-limited disease, but its case fatality rate is higher than A type and hepatitis B are even up to 20%-30% in pregnant woman.
Mammal HEV is broadly divided into 4 genotype, but an only serotype.Wherein 3 type of gene and 4 type HEV are people Illness poison altogether is raiseeed, pig and wild deer are its main natural reservoirs.Pig and the open country of HEV infection are eaten by mistake about people at present The report of deer food and Infection outbreak Hepatitis E case.Other than pig, other known animal sources host further include birds, Rat, rabbit, mongoose, goat, potential animal reservoir include dog, cat, monkey etc..
Pig HEV is unstable degradable, it is difficult to it is separated from carrying in malicious sample, and so far still without effective cell culture Model.Therefore, the inspection of antiviral antibody in the detection of nucleic acids of viral RNA and serum is mainly passed through to the diagnosis of the virus infection It surveys.Currently, the antibody report of identification pig HEV capsid protein is less.Only several plants of monoclonal antibodies identify comformational epitope, mainly Applied to the detection of virus infection host cell restrovirus, and it is found to have certain neutralizing effect.But above-mentioned monoclonal antibody is to answer Employment HEV capsid protein is prepared as immunogene, still needs in-depth study to the affinity of pig HEV capsid protein.
Indirect ELISA is mostly used for the detection of HEV antibody in Swine serum at present, it is required that the envelope antigen of high-purity and pole It is also easy to produce non-specific result.
Summary of the invention
The object of the present invention is to provide a kind of monoclonal antibody preparation for detecting pig hepatitis E virus ORF2 albumen and answer With method, Anti-HEV antibody in Swine serum is detected using blocking ELISA, to improve the spy detected to Anti-HEV antibody in Swine serum Anisotropic and sensitivity.
To achieve the above object, the present invention provides following schemes:
The present invention provides a kind of pig hepatitis E virus Shandong Province of China separation strains (Hepatitis E virus, HEV, CHN- SD-sHEV, GenBank number KF176351) capsid protein, the albumen separates by pig hepatitis E virus Shandong Province of China 267, the ORF2 PROTEIN C end Amino acid profile of strain, amino acid sequence (QLFYSRPVVSANGEPTVKLYTSVENAQQDKGI AIPHDIDLGESRVVIQDYDNQ HEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSGTVTF VNVATGAQGVSRSLDWSKVTLDGR PLTTIQQYSKTFYVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAA GHRVCISTYTTNLGSGPVSIAAVGVLA PHTALAVLEDTVDYPARAHTFDDFCPECRALGLQGCAFQSTVAELQRL KMKVGKTREY.) identical as SEQ ID NO.1.
The present invention also provides the hybridoma cell strain of one plant of anti-pig hepatitis E virus ORF2 protein monoclonal antibody of secretion, The hybridoma cell strain is named as 1E4, microbial preservation number are as follows: CCTCC NO:C2018250；Depositary institution are as follows: Chinese allusion quotation Type culture collection, depositary institution, Hubei Wuhan, address, Wuhan University.
As another aspect of the present invention, the present invention also provides a kind of monoclonal antibody of hybridoma cell strain secretion, Identify the 625th to 628 amino acid on pig HEV ORF2 protein sequence (625EPTV628) it is named as 1E4.
Preferably, the label that horseradish peroxidase is carried out to the monoclonal antibody, is named as HRP-1E4.
As another aspect of the present invention, the present invention also provides a kind of albumen for being used to prepare hybridoma cell strain 1E4, It is characterized in that, the albumen is the recombinant protein of prokaryotic expression, sHEV-ORF2-C is named as, as envelope antigen, amino acid Sequence is identical as SEQ ID NO.1.
The present invention also provides a kind of methods for preparing albumen described in claim 5, pass through albumen described in prokaryotic expression.
The present invention also provides application of the monoclonal antibody in the drug of preparation detection pig hepatitis E virus.
The present invention has following technical effect that
The present invention provides a kind of anti-pig HEV ORF2 protein monoclonal antibodies, after which is marked with HRP, can utilize it It establishes and blocks ELISA method, the method for foundation is remarkably improved specificity and sensitivity to HEV antibody test in Swine serum.
Detailed description of the invention
Fig. 1 is the purifying figure that SDS-PAGE analyzes destination protein sORF2-C purifying and monoclonal antibody 1E4；Wherein swimming lane M: albumen Maker；Band 1-2: sORF2-C albumen and monoclonal antibody 1E4 after purification.
Fig. 2 is the potency figure that indirect ELISA detects 1E4 after HRP label；
Fig. 3 is the result for blocking ELISA to detect 56 parts of positive serum samples with indirect ELISA established using HRP-1E4 Compare figure；56 parts of blood serum samples are received after 5 first tap poison CHN-SD-sHEV in 0,7,14,21,28,35,42,49 and 56dpi The Swine serum collected.Solid line and dotted line respectively represent the cut-off value for blocking ELISA and indirect ELISA.
Fig. 4 is the cut-off value for blocking ELISA and the cross reaction figure using blocking ELISA and different virus serum.
Specific embodiment
It is clearly and completely described below in conjunction with the technical solution in the embodiment of the present invention, it is clear that described reality Applying example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is general Logical technical staff every other embodiment obtained without making creative work belongs to what the present invention protected Range.
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, With reference to embodiment The present invention is described in further detail.
The protokaryon table of 1 267 amino acid (sORF2-C) in destination protein pig hepatitis E virus ORF2 PROTEIN C end of embodiment It reaches
(1) expression and purification of restructuring destination protein
Using pig HEV ORF2 overall length plasmid as template, according to 267, ORF2 PROTEIN C end amino acid design primer amplified fragments Afterwards, it after by III double digestion of restriction enzyme BamH I and Hind, connects after also with above-mentioned enzyme double digestion It on pET-21b carrier, then converts to 5 α of clone bacterium Trans, plasmid is extracted after culture and is saved.
The sORF2-C positive recombinant plasmid of above-mentioned preservation is converted into Bacillus coli expression host strain BL21 (DE3), is extracted single Colony inoculation is in the LB culture medium of the 5mL resistance of benzyl containing ammonia, at 37 DEG C, overnight shaking culture under the conditions of 225rpm.Next day presses 1: 20 ratio is inoculated into fresh LB culture medium, and 37 DEG C, 225rpm shaken cultivation to OD600nmIsopropyl is added to 0.6 or so in value Base-β-D thiogalactoside (IPTG) continues to cultivate, collects large intestine after being added IPTG 6 hours to final concentration of 1mmol/L Bacillus bacterium solution, 5000rpm are centrifuged 10min, discard supernatant.
(2) purifying of restructuring destination protein
After the precipitating ultrasound cracking after centrifugation, inclusion body is dissolved, is carried out according to Ni-NTA (Invitrogen) specification The purifying of destination protein, SDS-PAGE analyzes the purification effect of albumen, as a result as shown in Figure 1, having obtained as the result is shown purer Destination protein, wherein swimming lane M: albumen Maker；Band 1-2: sORF2-C albumen and monoclonal antibody 1E4 after purification, each The applied sample amount of band is about 7.5 μ g, and monoclonal antibody heavy and light chain relative molecular mass respectively may be about 50ku and 25ku, SORF2-C albumen is about 40ku.
The preparation of the monoclonal antibody of the anti-pig hepatitis E virus sORF2-C albumen of embodiment 2 and HRP label
(1) emulsification of albumen:
The target protein of purifying is mixed according to 1:1 (V/V) and emulsified with incomplete Freund's adjuvant with complete.
(2) immune programme:
Select 6-8 weeks BABL/c mouse as immune animal, immune preceding tail vein is taken a blood sample every time, twice immunization interval It is 2 weeks, uses Freund's complete adjuvant for the first time, albumen dosage is 75 μ g/, and intraperitoneal injection, 200 μ L/ of accumulated dose is only.Second of use Incomplete Freund's adjuvant, albumen dosage are still 75 μ g/.Third time is carried out with reference to second of identical dosage and mode to be immunized, Booster immunization is primary before merging.
(3) cell fusion:
Disconnected neck puts to death the BALB/c mouse be immunized, the sterile splenocyte for taking immune mouse and SP2/0 myeloma cell by 5:1 is merged at 1mL50%PEG, then carries out selection culture using HAT culture medium.
(4) screening of positive cell and subclone:
Using the target protein of purification as antigen (200ng/well) wrapper sheet, cells and supernatant is drawn with indirect ELISA method Positive cell clone is screened, positive hole, continues to be subcloned as the result is shown, while expanding and cultivating and freeze.Using limited dilute Interpretation of the law carries out 2 subclones to selected hybridoma, and screening one plant of positive hybridoma cell strain is 1E4, the hybridization Tumor cell strain is preserved in China typical culture collection center, preservation address is Hubei Wuhan, military 03xx days in December, 2018 Chinese university, deposit number are CCTCC NO:C2018250.
(5) preparation and purifying of monoclonal antibody ascites:
The hybridoma for collecting culture to growth period is washed with 0.01M PBS (containing K+, PH 7.2) and is counted afterwards twice then Every mouse peritoneal injectionThe cell suspension of a cell (volume 0.5mL).7-10 days after inoculation, observe small Slowly, abdomen obviously swells for mouse activity, and intraperitoneal, collection ascites is inserted into syringe needle.Ascites is put into the small burning of 25mL In cup, isometric saturated ammonium sulfate is added dropwise, after slowly stirring 2h, 10000rpm centrifuged deposit, precipitating is with 0.01M's PBS (pH7.2) is resuspended, and monoclonal antibody then is further purified with Protein G affinity column, sees Fig. 1.
(6) monoclonal antibody of HRP label after purification:
1) material prepares
A) horseradish peroxidase (HRP): article No. Cat.P8375, type VI, Sigma.
b)0.1M NaIO4: weigh 0.214g NaIO4, it is dissolved in 10mL tetra- and steams in water.
C) it 1mM sodium-acetate buffer (pH4.4): weighs 0.1361g crystallization sodium acetate and is dissolved in the steaming water of 1L tetra-, use glacial acetic acid Adjust pH to 4.4.
D) 0.2M carbonate buffer solution (pH9.5): by 0.32g Na2CO3,0.586g NaHCO3It is dissolved in 50mL tetra- and steams water In, adjusting pH is 9.5.
E) 0.02M carbonate buffer solution (pH9.5): 0.02M Na is prepared respectively2CO3With 0.02M NaHCO3In 200mL's In reagent bottle, it is slowly added into the Na of 0.02M2CO3, adjust pH to 9.5.
f)NaBH4(4mg/mL)
g)0.01M PBS(pH7.2)
H) IgG of purifying IgG: is concentrated into 10mg/mL. using Millipore concentrator
2) HRP labeled monoclonal antibody
A) 5mg HRP is weighed in teat glass, 1.25mL tetra- is added and steams water, and after mixing, Fresh is added 0.25mL0.1M NaIO4At room temperature, it is protected from light, is slowly stirred 20min.
B) 4 DEG C of dialysed overnights, the outer liquid of bag filter is 1mM sodium-acetate buffer (pH4.4).
C) 25 μ L 0.2M carbonate buffer solutions (pH9.5) are added, the IgG that 100 μ L concentration are 10mg/mL, room is then added Temperature is protected from light, and slowly stirs 2h.
D) NaBH of 100 μ L Fresh is added4(4mg/mL), 4 DEG C of standing 2h after mixing.
E) 4 DEG C of dialysed overnights, the outer liquid of bag filter is 0.01M PBS (pH7.2).
F) HRP-IgG is taken out from bag filter, isometric glycerol is added, and the BSA that concentration is 1mg/mL is then added, point Dress is stored in -20 DEG C.
3) titration of HRP-1E4
HRP-1E4 gradient dilution (10-1To 10-4) after, it is reacted by indirect ELISA with the sORF2-C albumen of purifying.When OD450When nm value is 0.3, the titre of HRP-1E4 is 10-2, as a result as shown in Figure 2.HRP-1H5 is as irrelevant monoclonal antibody pair According to (HRP-1H5 be with fowl HEV ORF2 protein immunization mouse after, by cell fusion, subclone screening and monoclonal antibody screening with The monoclonal antibody obtained after purification).
The foundation of 3 pig hepatitis E virus specific antibody of embodiment blocking ELISA diagnostic method
(1) optimization of ELISA method optimum reaction condition is blocked
1) solution is prepared
A) it is coated with buffer liquid: 0.01M PBS (500mL, pH=7.2 ± 0.1): NaCl 4.25g, NaH2PO4·2H2O 0.178g, Na2HPO4·12H2O 1.386g, dissolution are settled to 500mL, and pH value determination can be reserved for one week in 7.1-7.3, room temperature；
B) cleaning solution (PBST): 5mL Tween-20 is added in every 1L 0.01M PBS (pH=7.2 ± 0.1), mixes well；
C) confining liquid: 2.5g skimmed milk power is dissolved in 100mL PBST, and short-term preservation is in 4 DEG C, and long-term preservation is in -20 ℃；
D) substrate TMB (developing solution): A liquid (1L): weighing 66.5063g potassium citrate (potassium citrate), uses 800mL tetra- steams water dissolution and adjusts pH value to 4.0 with concentrated hydrochloric acid, and 3-4 μ L H is added2O2, it is settled to 1L, 4 DEG C of preservations.B liquid (30mL): weighing 0.2956g tetramethyl benzidine (TMB), and 0.0633g tetrabutyl ammonium borohydride (TBABH) is dissolved in 30mL diformazan Yl acetamide (DMA), 4 DEG C are kept in dark place.A liquid and B liquid are mixed with 39:1 ratio when use, it is ready-to-use；
E) terminate liquid: the dense H of 3M2SO4。
2) envelope antigen is determined with antibody optium concentration is blocked
It is carried out by chessboard square matrix method, the recombinant protein sORF2-C of pig HEV is diluted to final concentration with coating buffer respectively 2 μ g/mL, 4 μ g/mL, 8 μ g/mL, 4 DEG C of coatings overnight, then use PBST (the 0.1M PBS containing 0.05%Tween-20) board-washing, The PBST (confining liquid) for containing 2.5% skimmed milk power is added, 200 holes μ L/ are incubated at room temperature 1h, and it is added after washing and presses 1 with PBST: 100, the diluted HRP-1E4 monoclonal antibody of 1:1000,1:10000 carries out indirect ELISA, is incubated at room temperature 1h, after washing plate, TMB (100 hole μ L/) is added color development at room temperature 15 minutes, 3M H is then added2SO4Reaction is terminated, ELISA Plate surveys OD450Nm value.As a result As shown in table 1 below, the results show that it is that best effort is dense that envelope antigen, which is diluted to 8 μ g/mL, HRP-1E4 monoclonal antibodies to be diluted to 1:1000, It spends, at this time OD450The value of nm is about 1.0.
1 antigen coat concentration of table and the just suboptimization of monoclonal antibody dilution
3) optimum dilution degree of Swine serum to be checked determines
ELISA reaction is carried out according to the antigen of above-mentioned optimization and the best effort concentration of HRP-1E4 monoclonal antibody, confining liquid is added After washing, it is first separately added into the diluted pig HEV antibody positive of 1:10,1:20 and 1:40 and negative Swine serum, 1h is incubated at room temperature, washes After plate, the HRP-1E4 monoclonal antibody of best effort concentration is added, 100 μ L developing solutions are then added according to the method described above and 50 μ L are terminated Then liquid is read.As a result as shown in table 2 below, the results show that according to the blocking rate of negative and positive Swine serum, 1:20 is dilute Releasing Swine serum is best dilution ratio.
The selection of the Swine serum dilution ratio to be checked of table 2
4) ELISA optimum reaction condition is blocked
According to above-mentioned steps 2) and 3) determine optium concentration come determine block ELISA optimum reaction condition, specific steps Are as follows:
A) final concentration of 8 μ g/mL recombinant protein sORF2-C is added in every hole in 96 hole elisa Plates, and 100 μ L, 4 DEG C were coated with Night, PBST board-washing；
B) PBST (confining liquid) for containing 2.5% skimmed milk power is added in every hole, 200 holes μ L/ are incubated at room temperature 1h, and PBST is washed Plate；
C) the 100 diluted Swine serums to be checked of μ L 1:20, diplopore reinspection is added in every hole, while setting up standard positive and shining and mark Quasi- negative control is incubated at room temperature 1h, PBST board-washing；
D) the 100 μ L diluted HRP-1E4 of 1:1000 are added in every hole, are incubated at room temperature 1h, PBST board-washing；
E) 100 μ L TMB developing solutions are added in every hole, are incubated at room temperature 15min, and 50 μ L 3M H are added in every hole2SO4Terminate reaction；
F) automatic microplate reader reads OD450Nm calculates blocking rate (PI (%)=[1- (the OD450nm/ feminine gender of positive serum The OD450nm of serum)] × 100%.).
(2) determination of critical value
230 parts of Swine serums being negative with commercial reagents box (ten thousand safe biologies, Beijing) detection and 56 parts of clinics are taken to collect Pig HEV Positive Sera sample (indirect ELISA detection is negative), utilizes above-mentioned steps 4) what is determined blocks the reaction of ELISA Condition is detected, and calculates blocking rate, and the average blocking rate (X) for calculating blood serum sample is 6.4%, and standard deviation (S) is 3.5%.According to formula: critical value=6.4%+3 (or 2) × 3.5%, the critical value that yin and yang attribute criterion is calculated are 16.9%.It is the positive when sample blocking rate >=16.9%, is feminine gender as blocking rate < 13.4%.When 13.4% < sample hinders Disconnected rate < 16.9% is judged as suspicious, need to repeat to detect, if being still below 16.9%, be judged to feminine gender.
(3) repetitive test of ELISA is blocked
The OD of 56 parts of clinical serums by commercial ELISA Kit tests positive450Nm is divided into 6 groups from high to low (0.8) 0.3-0.4,0.4-0.5,0.5-0.6,0.6-0.7,0.7-0.8, are greater than, a sample is extracted in every group to measure resistance The repeatability of disconnected ELISA.Interior repetitive test is carried out between criticizing and criticized with 3 elisa plates of same batch and different batches, is calculated The coefficient of variation.As a result 3 be see the table below, the results show that the coefficient of variation of 6 parts of serum between criticizing and in batch interior experiment is respectively less than 10%, Prove that the blocking ELISA detection method has good repeatability.
The repetition test result of the blocking of table 3 ELISA
(4) the cross reaction experiment of ELISA reaction is blocked
According to above-mentioned steps 4) determine blocking ELISA reaction condition, to existing known 40 parts of PRRS virus (PRRSV), the positive serum of 17 parts of swine fever virus (CSFV), 37 parts of pig circular ring virus (PCV) is detected, while with 56 portions of pigs HEV positive serum is as positive control.As a result as shown in figure 4, the results show that except pig HEV antibody positive Swine serum is positive anti- Should be outer, and be that feminine gender is answered, it was demonstrated that the blocking ELISA method can specific detection pig HEV antibody, not with other several swine diseases Positive serum react.
(5) the sensibility and specificity test of ELISA is blocked
2542 parts of clinical Swine serum samples are used to measure what established blocking ELSIA method and Wang et al. were established The consistency of indirect ELISA method, wherein the inconsistent serum of result continues on for measurement blocking ELSIA and Western Immuno prints The consistency of mark (western blot) and commercial reagents box (ten thousand safe biologies, Beijing), calculation formula are as follows: consistency (%)= (B+D)/(A+C) × 100%.As a result 4 be see the table below, the results show that block ELISA and indirect ELISA, western blot and The consistency of commercial reagents box is respectively that 93.23%, 92% and 95%, Kappa value is all larger than 0.45%.
4 comparison of coherence of table
Note: consistency (%)=(B+D)/(A+C) × 100；Kappa value, which be used to measure, blocks ELISA and other methods Consistency, value, which is greater than 0.45, is considered variant significant.(+/- respectively indicate with specific ELISA method testing result is sun Property+or negative-, A, B, C and D are the expressions of the correspondence computational item for consistency formula.)
Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention Various changes and improvements, should all fall into claims of the present invention determine protection scope in.
Sequence table
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>anti-pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 267
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr
1 5 10 15
Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile
20 25 30
Ala Ile Pro His Asp Ile Asp Leu Gly Glu Ser Arg Val Val Ile Gln
35 40 45
Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala
50 55 60
Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu
65 70 75 80
Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr
85 90 95
Asn Pro Met Tyr Val Ser Gly Thr Val Thr Phe Val Asn Val Ala Thr
100 105 110
Gly Ala Gln Gly Val Ser Arg Ser Leu Asp Trp Ser Lys Val Thr Leu
115 120 125
Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr
130 135 140
Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr
145 150 155 160
Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile
165 170 175
Leu Ile Glu Asn Ala Ala Gly His Arg Val Cys Ile Ser Thr Tyr Thr
180 185 190
Thr Asn Leu Gly Ser Gly Pro Val Ser Ile Ala Ala Val Gly Val Leu
195 200 205
Ala Pro His Thr Ala Leu Ala Val Leu Glu Asp Thr Val Asp Tyr Pro
210 215 220
Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Ala Leu
225 230 235 240
Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val Ala Glu Leu Gln Arg
245 250 255
Leu Lys Met Lys Val Gly Lys Thr Arg Glu Tyr
260 265

Claims (7)

1. a kind of pig hepatitis E virus capsid protein, which is characterized in that the albumen is by pig hepatitis E virus Shandong Province of China 267, the ORF2 PROTEIN C end Amino acid profile of separation strains, amino acid sequence are SEQ ID NO.1:
2. a kind of hybridoma cell strain for secreting anti-pig hepatitis E virus ORF2 protein monoclonal antibody, which is characterized in that institute It states hybridoma cell strain and is named as 1E4, microbial preservation number are as follows: CCTCC NO:C2018250.
3. the monoclonal antibody secreted by hybridoma cell strain as claimed in claim 2, is named as mAb-1E4.
4. monoclonal antibody according to claim 3, it is characterised in that: carry out horseradish peroxidating to the monoclonal antibody The label of object enzyme, is named as HRP-1E4.
5. a kind of albumen for being used to prepare hybridoma cell strain 1E4 as claimed in claim 2, which is characterized in that the albumen is The recombinant protein of prokaryotic expression, is named as sHEV-ORF2-C, as envelope antigen, amino acid sequence and SEQ ID NO.1 phase Together.
6. a kind of method for preparing albumen described in claim 5, which is characterized in that pass through albumen described in prokaryotic expression.
7. application of the monoclonal antibody according to claim 3 in the drug of preparation detection pig hepatitis E virus.
CN201910023151.1A 2019-01-10 2019-01-10 Anti- pig hepatitis E virus ORF2 protein monoclonal antibody and its preparation and application Pending CN109593122A (en)

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CN116554316A (en) * 2023-05-29 2023-08-08 西北农林科技大学 Nano antibody specifically binding with hepatitis E virus capsid protein and application thereof

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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
CN116554316A (en) * 2023-05-29 2023-08-08 西北农林科技大学 Nano antibody specifically binding with hepatitis E virus capsid protein and application thereof

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