CN106380515A - Hybridoma cell strain for stable secretion of anti-PEDV monoclonal antibody and secreted antibody and application thereof - Google Patents
Hybridoma cell strain for stable secretion of anti-PEDV monoclonal antibody and secreted antibody and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Abstract
The invention discloses a hybridoma cell strain for stable secretion of anti-PEDV monoclonal antibody and the secreted antibody and application thereof. The hybridoma cell strain capable of stably secreting the anti-PEDV monoclonal antibody is named as 1B9, and the microbial preservation number is CGMCC No.12695. Researches prove that recognition epitope of the monoclonal antibody secreted by the hybridoma cell strain is conformational epitope. It shows through virus neutralization tests that the monoclonal antibody can effectively neutralize the strain of PEDV G2 subtype but has no neutralization activity to a strain of PEDV G1 subtype. In view of biological characteristics of the monoclonal antibody, the monoclonal antibody can be used for identification of the strains of PEDV G1 and G2 subtypes, and on this basis, a series of diagnostic methods are established. In addition, the monoclonal antibody also can be used for treating diseases caused by porcine epidemic diarrhea virus. The invention provides a novel technological means for diagnosis and treatment of PEDV G1 subtype.
Description
Technical field
The present invention relates to the antibody of a strain of hybridoma strain and its secretion and application, resist particularly to one plant of stably excreting
The antibody of the hybridoma cell strain of PEDV monoclonal antibody and its secretion and application, the invention belongs to biological technical field.
Background technology
Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) are to cause in pig industry
One of piglet diarrhea, the important pathogen body become thin, be dehydrated, bring heavy economic lossess to pig industry.Research shows, PEDV has two
Individual gene hypotype, i.e. G1 and G2 hypotype.2010 before, and, based on G1 hypotype, 2010 afterwards for PEDV epidemic isolates, and PEDV flows
Row strain is based on G2 hypotype.Gene sequencing shows, the Main Differences of PEDV G1 and G2 hypotype are on S gene.G1 hypotype
Strain S gene is made up of 4152nt, and the S gene of G2 is constituted for 4161nt, increased 9 bases on mrna length.Gene
Inside there is also larger difference.In PEDV genome, maximum variation is located at S gene, and the mutation in S gene is concentrated mainly on N
End.
NPL-PEDV/2013/P10 compared with NPL-PEDV/2013, ORF1a/b albumen, nucleoprotein, NS3B, memebrane protein with
And envelope protein is completely the same on amino acid levels.And contain six nucleotide polymorphisms in spike protein (S), lead to ammonia
Base acid site change：F375L, T486P, D856E, A1081V, A1099S and Y1253D.2 aminoacid are had to become in S1 region
Different, there are 4 amino acid variations (Lawrence et al., 2014) in S2 region.Coronavirus S protein can induce neutrality
The generation of antibody, S genovariation inevitably result in S protein induction produce neutrality antibody neutralization virus in terms of on presence poor
Different.
Inventor is anti-with the virion of Porcine epidemic diarrhea virus (PEDV) the epidemic isolates LNCT2 of purification
Former, immune BALB/c mouse, the hybridoma cell strain of the anti-PEDV of 1 plant of stably excreting of acquisition, it is named as 1B9.IFA detection finds
1B9 monoclonal antibody can be reacted with PEDV-LNCT2, and Western-blot detection then finds 1B9 monoclonal antibody with PEDV-LNCT2 not
Reaction, illustrates that the epi-position that 1B9 monoclonal antibody is identified is comformational epitope.Virus neutralization tests shows, 1B9 monoclonal antibody can effectively neutralize
The strain of this three plants of PEDV-G2 hypotypes of PEDV-LNCT2, PEDV-LNSY and PEDV-Hjms, but to PEDV G1 hypotype strain
CV777 does not have neutralization activity, and PEDV-LNCT2, PEDV-LNSY and PEDV-Hjms and CV777 on S gene, difference is relatively
Greatly, in conjunction with IFA experimental result and Genetic Variation Analysis result, show that the antigen of 1B9 monoclonal antibody identification is S protein.
1B9 monoclonal antibody effectively can neutralize virus so as to lose the ability of infection cell, its epitope identifying and combining
Require study, by studying the epitopic regions of its combination, it is possible to obtain the molecule machine of this MAbs blocking PEDV infection cell
System.
In view of 1B9 monoclonal antibody can neutralize PEDV G2 (LNCT2, LNSY and Hjms) hypotype strain it is impossible to neutralize PEDV G1
(CV777) characteristic of hypotype strain, therefore this monoclonal antibody can be used to prepare diagnostic kit differentiation PEDV G1 and G2 hypotype poison
Strain；Can be used to after this monoclonal antibody is labeled develop ELISA, Ag-capture ELISA, PEDV colloidal gold colloidal gold detection test paper strip and competition
ELISA, possesses wide using value and researching value.
Content of the invention
The technical problem to be solved is to provide one kind can effectively neutralize Porcine epidemic diarrhea virus
The monoclonal antibody of (Porcine Epidemic Diarrhea Virus, PEDV) and the hybridization secreting this monoclonal antibody
Tumor cell strain.
In order to achieve the above object, present invention employs following technological means：
Inventor is anti-with the virion of Porcine epidemic diarrhea virus (PEDV) the epidemic isolates LNCT2 of purification
Former, immune BALB/c mouse, the hybridoma cell strain of the anti-PEDV monoclonal antibody of 1 plant of stably excreting of acquisition, it is named as 1B9.Should
Hybridoma Cell Culture supernatant ascites antibody potency is respectively 1：1000 and 1:100000.1B9 monoclonal antibody subclass is IgG2b, gently
Chain is κ type.IFA (indirect immunofluorescene assay) detection finds, 1B9 monoclonal antibody can be reacted with PEDV-LNCT2, but
Western blot result shows, 1B9 monoclonal antibody is not reacted with PEDV particle, S protein, and no specific reaction band is above-mentioned
Two kinds of results show that the epitope that this monoclonal antibody is identified is comformational epitope.Virus neutralization experiment shows, this monoclonal antibody can be effective
Neutralize the G2 gene hypotype strain such as PEDV-LNCT2, PEDV-LNSY and PEDV-Hjms so as to lose the ability of infection cell, but
CV777 strain (G1 gene hypotype) can not be neutralized.In view of the biological characteristicses of this monoclonal antibody, 1B9 monoclonal antibody can be used to differentiate G1 and
G2 hypotype strain, and set up a series of blocking-up ELISA, capture ELISA, indirect ELISA, competitive ELISA and colloid gold test paper
The diagnostic methods such as bar.
In the present invention, the described one plant hybridoma cell strain being capable of stably excreting anti-PEDV monoclonal antibody, name
For 1B9, Classification And Nomenclature is mice Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus cell strain, is deposited in Chinese microorganism strain and protects
Hide administration committee's common micro-organisms center, address is in the microorganism of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Institute, its microbial preservation number is：CGMCC No.12695, the preservation time is on July 21st, 2016.
Brief description
Fig. 1 is electron microscopic observation PEDV virion；
Fig. 2 detects the reaction of 1B9 monoclonal antibody and PEDV-LNCT2 for IFA；
1 is the Vero-E6 cell effect result of 1B9 monoclonal antibody and inoculation LNCT2 strain；2 is 1B9 monoclonal antibody and healthy Vero-
E6 cell effect result；
Fig. 3 verifies the response situation of 1B9 monoclonal antibody and PEDV protein component for Western-blot；
1：PEDV-LNCT2S albumen；2：PEDV-LNCT2；3：PEDV-CV777；4：Vero-E6 cell；
Fig. 4 identifies the composition of 1B9 monoclonal antibody for Western-blot；
Fig. 5 is 1B9 monoclonal antibody and virus neutralization measuring.
Specific embodiment
To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
Apparent.But these embodiments are only exemplary, any restriction is not constituted to the scope of the present invention.People in the art
Member should be understood that can be to enter to the details of technical solution of the present invention and form under without departing from the spirit and scope of the present invention
Row modification or replacement, but these modifications and replacement each fall within protection scope of the present invention.
The preparation of the anti-PEDV monoclonal antibody of embodiment 1 and identification
1 materials and methods
1.1 cells, experimental animal, antibody and reagent
SP2/0 and Vero-E6 cell line is by Harbin Veterinary Medicine Inst., China Academy of Agriculture's swine alimentary canal infectious disease wound
New team preserves；RPMI1640 culture fluid (Gibco)；HAT and HT culture medium, PEG1450, Freund's complete adjuvant, Freund be not complete
Full adjuvant is purchased from Sigma company；BALB/c mouse is by this institute Experimental Animal Center on behalf of purchase.
The culture of 1.2PEDV and purification
1.2.1PEDV-LNCT2 the culture of strain
After Vero-E6 cell covers with formation monolayer, discard nutritional solution, with PBS (phosphate buffered solution, pH7.2) washing
Three times, inoculate PEDV-LNCT2 strain.Viral inoculum dose is inoculated according to 5% concentration, and virus inoculation adds pancreatin, pancreatin simultaneously
Final concentration of 10 μ g/ml.Observation of cell pathological changes, when pathological changes in 80%～90% cell, by frozen for virus to -20 ice
Case, receives poison, standby.
1.2.2PEDV-LNCT2 purification
PEDV-LNCT2 virus liquid removes after cell debriss through 5000g centrifugation 5min, with supercentrifuge 100000g centrifugation
2h, abandoning supernatant, collect precipitation.Precipitation uses sucrose density gradient centrifugal purification, altogether 5 sucrose concentrations of setting, 20%,
30%th, 40%, 50% and 60%.Supercentrifuge SW32 rotor 100000g centrifugation 2h after, collect 50%～60% sucrose layer it
Between sample, sample carries out desaccharide process through SW32 rotor 100000g centrifugation 2h, and abandoning supernatant collects precipitation.Precipitation is
The virion of purification.
1.3 the preparation of anti-PEDV monoclonal antibody
1.3.1BALB/c the immunity of mice
The PEDV-LNCT2 virion of purification is inoculated 6 week old BALB/c mouse with equivalent Freund adjuvant mixing pneumoretroperitoneum
5, every antigen inoculation dosage is 100 μ g.The antigen that same dose incomplete Freund's adjuvant is processed is injected after 2 weeks.Cell melts
3d before conjunction, is not added with antigen (every 200 μ g) the lumbar injection booster immunization 1 time of adjuvant, you can carry out cell fusion with equivalent.
1.3.2 the foundation of monoclonal antibody screening technique
Set up indirect IFA method according to a conventional method.After Vero-E6 cell forms monolayer, inoculate PEDV-LNCT2, see after 24h
Examine cytopathy, when pathological changes in 50% cell, fix cell with 4% paraformaldehyde, 4 DEG C or -20 DEG C save backup.Little
Mus negative serum, positive serum or hybridoma later to be checked as sample, with DyLightTMThe goat of 488 labellings resists
Mouse IgG is anti-as two, observes, record has the cell hole of positive cell after reaction 45min under inverted fluorescence microscope.
1.3.3 cell fusion and the foundation of positive hybridoma cell strain
Immune mouse spleen cell and SP2/0 cell are according to 5：Add PEG after 1 ratio mixing to merge, 37 DEG C in 96 well culture plates,
Cultivate in CO2 incubator.When the culture fluid of hybridoma starts to turn yellow, or hybridoma covers with to the 1/10 of bottom hole area
When, Aspirate supernatant, screens positive hybridoma cell through IFA method.With limiting dilution assay 3 sub-clones continuous to positive hole, extremely
Till all monoclonal holes are the positive.Build strain by after the final monoclonal amplification culture obtaining, frozen.
1.3.4 the mensure of the preparation of odd contradictive hydroperitoneum and potency
Take 8 week old BALB/c mouse lumbar injections sterilizing paraffin oil (every 0.5ml), will be good for growth conditions miscellaneous after 7d
Hand over oncocyte, with every about 5 × 105Individual lumbar injection BALB/c mouse.7d about mouse web portion start substantially to expand, repeatedly take out
Take ascites, cell component is removed with 3000r/min centrifugation 10min and other precipitate, collect supernatant.Add 0.02% after purification
Sodium azide, subpackage, -70 DEG C of preservations.Ascites is incremented by dilution with PBS, measures the potency of monoclonal antibody with IFA.
1.3.5 the Western-blot analysis of monoclonal antibody
It is transferred to NC by half-dried for the PEDV-LNCT2 virion protein matter of purification under Non SDS-PAGE method
Film, after NC film is closed through 2% skimmed milk, with 1:10000 odd contradictive hydroperitoneum effect 2h, wash 3 times, add 1: 10000 dilution
Mountain sheep anti-mouse igg (H+L) the effect 1h of HRP labelling, washs 3 times, scanning imagery.
1.3.6 hypotype identification monoclonal antibody hypotype identification
Monoclonal antibody subclass is identified using Mouse MonoAb-ID Kit (HRP), obtains in above test by kit specification
To monoclonal antibody carry out hypotype identification.
1.3.7 positive hybridoma cell strain secretes the mensure of monoclonal antibody stability
By the positive hybridoma cell recovery of frozen 3 months, 24 porocyte plates cultures, whether detection supernatant is positive to sentence
Whether the positive cell of fixed recovery has the ability of secretory antibody.Will be thin for hybridoma that the positive rate through 3 sub-clones reaches 100%
Born of the same parents carry out continuous passage culture in vitro 3 months, detect 1 cell supernatant every 2 weeks.
1.3.8 monoclonal antibody neutralization activity measures
Odd contradictive hydroperitoneum after 0.22 μm of membrane filtration, with 2 times of doubling dilutions of RPMI1640 culture fluid；PEDV-LNCT2、
PEDV-CV777, PEDV-LNSY and PEDV-Hjms tetra- strain virus are diluted to every 100 μ L virus liquids and contain 200 TCID50, virus liquid
Mix with antibody equal-volume, 37 DEG C of reaction 1h, add pancreatin, the final concentration of 10 μ g/ml of pancreatin, inoculating cell, be placed in cell
Incubator 5 days, observation of cell pathological changes situation.
2 results
The purification of 2.1PEDV-LNCT2 virion
Identified sucrose density gradient virion after purification is concentrated mainly between the 50%-60% layer of sucrose, sample
Product see Fig. 1 through electron microscopic observation result it is seen that complete PEDV-LNCT2 virion, and recording its concentration with extinction photometer method is
0.424mg/ml.
The preparation of 2.2 anti-PEDV monoclonal antibodies
2.2.1 the foundation of monoclonal antibody hybridoma cell strain
With the PEDV-LNCT2 virion of purification for immunogen immune BALB/c Mus, the hybridoma of fusion is through IFA
After screening positive clone, positive hole is carried out with 3 sub-clones, the hybridoma of the anti-PEDV monoclonal antibody of 1 plant of stably excreting of acquisition is thin
Born of the same parents' strain, is named as 1B9, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its microbial preservation
Number it is：CGMCC No.12695.
2.2.2 the IFA analysis of monoclonal antibody
IFA detection finds, 1B9 Mab supernatant can be reacted with the Vero-E6 cell of inoculation PEDV-LNCT2 strain,
Do not react with healthy Vero-E6, result is as shown in Figure 2.
2.2.3 the Western-blot analysis of monoclonal antibody
Using this plant of monoclonal antibody of Western-blot verification experimental verification and PEDV response situation, result is as shown in figure 3, this result table
Bright, 1B9 monoclonal antibody and PEDV-LNCT2, CV777 and healthy Vero-E6 cell all do not react, with LNCT2S albumen also not
There is specific reaction thus it is speculated that the epitope of 1B9 identification is space conformation epi-position.
2.2.4 odd contradictive hydroperitoneum collection and the identification of monoclonal antibody hypotype
Extract its ascites after mouse web portion expands, 3000r/min centrifugation 10min takes supernatant.Using in the detection of IFA method
Cleer and peaceful ascites antibody potency.Result shows：1B9 monoclonal antibody cell supernatant antibody titer is higher than 1：1000, ascites antibody potency is high
In 1：100000.The monoclonal antibody of this test preparation is carried out hypotype identification, result shows that 1B9 monoclonal antibody is IgG2b hypotype, gently
Chain is κ chain.It is Western-blot with goat anti-mouse IgG (H+L) antibody of HRP labelling to find, this monoclonal antibody is by heavy chain and light
Chain two parts form, and result is as shown in Figure 4.
2.2.5 fusion cell line secretes the Stability Determination of monoclonal antibody
Freeze-stored cell strain recovery is cultivated in 24 well culture plates, indirect ELISA detects cell supernatant, result OD405
Value is still very high.By passage 3 months, detected 1 supernatant every 2 weeks, result shows OD405Value stabilization.
2.2.6 monoclonal antibody neutralization titer measures
Result is as shown in figure 5, this result shows that PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms can be by 1B9 monoclonal antibodies
With, 1B9 monoclonal antibody 1：160、1：80 and 1：PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms can be neutralized completely during 160 dilution
Three strains, but 1B9 monoclonal antibody can not neutralize PEDV-CV777 strain.
Claims (5)
1. one plant can stably excreting porcine epidemic diarrhea resisting virus (Porcine Epidemic Diarrhea Virus,
PEDV) hybridoma cell strain of monoclonal antibody, is named as 1B9, is deposited in China Committee for Culture Collection of Microorganisms general
Logical microorganism center, its microbial preservation number is：CGMCC No.12695, the preservation time is on July 21st, 2016.
2., by the monoclonal antibody of the porcine epidemic diarrhea resisting virus of the hybridoma cell strain secretion described in claim 1, it is special
Levy and be that the epitope that described monoclonal antibody is identified is comformational epitope.
3. purposes in preparing medicine for the monoclonal antibody of the porcine epidemic diarrhea resisting virus described in claim 2, described medicine
Thing is used for the disease that treatment is caused by Porcine epidemic diarrhea virus.
4. the monoclonal antibody of the porcine epidemic diarrhea resisting virus described in claim 2 is in preparation detection Porcine epidemic diarrhea virus
Purposes in G2 hypotype reagent.
5. the monoclonal antibody of the porcine epidemic diarrhea resisting virus described in claim 2 is in preparation difference Porcine epidemic diarrhea virus
Purposes in G2 hypotype and Porcine epidemic diarrhea virus G1 hypotype reagent.
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| CN108586609A (en) * | 2018-05-24 | 2018-09-28 | 青岛博隆基因工程有限公司 | A kind of monoclonal antibody of porcine epidemic diarrhea resisting virus and application |
| CN110144330A (en) * | 2019-05-29 | 2019-08-20 | 暨南大学 | Hybridoma, monoclonal antibody and the application of anti-PEDV monoclonal antibody can be secreted |
| CN110187097A (en) * | 2019-05-28 | 2019-08-30 | 中国农业科学院哈尔滨兽医研究所 | Porcine epidemic diarrhea virus fluorogenic quantitative detection test strips and its preparation method and application |
| CN110564696A (en) * | 2019-09-06 | 2019-12-13 | 中国农业大学 | Monoclonal antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity |
| CN112680421A (en) * | 2021-02-01 | 2021-04-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof |
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| CN108586609A (en) * | 2018-05-24 | 2018-09-28 | 青岛博隆基因工程有限公司 | A kind of monoclonal antibody of porcine epidemic diarrhea resisting virus and application |
| CN108586609B (en) * | 2018-05-24 | 2021-10-08 | 青岛博隆基因工程有限公司 | Monoclonal antibody for resisting porcine epidemic diarrhea virus and application |
| CN110187097A (en) * | 2019-05-28 | 2019-08-30 | 中国农业科学院哈尔滨兽医研究所 | Porcine epidemic diarrhea virus fluorogenic quantitative detection test strips and its preparation method and application |
| CN110187097B (en) * | 2019-05-28 | 2021-09-14 | 中国农业科学院哈尔滨兽医研究所 | Fluorescent quantitative detection test strip for porcine epidemic diarrhea virus and preparation method and application thereof |
| CN110144330A (en) * | 2019-05-29 | 2019-08-20 | 暨南大学 | Hybridoma, monoclonal antibody and the application of anti-PEDV monoclonal antibody can be secreted |
| CN110564696A (en) * | 2019-09-06 | 2019-12-13 | 中国农业大学 | Monoclonal antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity |
| CN112680421A (en) * | 2021-02-01 | 2021-04-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof |
| CN112680421B (en) * | 2021-02-01 | 2022-09-13 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof |
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