CN105440132A - Monoclonal antibody against porcine epidemic diarrhea virus N protein and application thereof - Google Patents
Monoclonal antibody against porcine epidemic diarrhea virus N protein and application thereof Download PDFInfo
- Publication number
- CN105440132A CN105440132A CN201410384478.9A CN201410384478A CN105440132A CN 105440132 A CN105440132 A CN 105440132A CN 201410384478 A CN201410384478 A CN 201410384478A CN 105440132 A CN105440132 A CN 105440132A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- protein
- epidemic diarrhea
- porcine epidemic
- diarrhea virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention discloses a monoclonal antibody against porcine epidemic diarrhea virus N protein. The present invention also discloses a porcine epidemic diarrhea virus infection diagnosis kit comprising the monoclonal antibody. The present invention also discloses a preparation method and application of the monoclonal antibody. The monoclonal antibody against the porcine epidemic diarrhea virus N protein has good reactivity with porcine epidemic diarrhea virus, is suitable for rapid and accurate diagnosis of the PEDV (porcine epidemic diarrhea virus), meanwhile provides an important technical support for surveillance and prevention of the PEDV, and has good application prospects.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to a kind of monoclonal antibody and application thereof of porcine epidemic diarrhea resisting viral N proteins.
Background technology
Porcine epizootic diarrhea (porcineepidemicdiarrhea, PED) be by the Porcine epidemic diarrhea virus (porcineepidemicdiarrheavirus of coronavirus genus, PEDV) one caused is acute, high degree in contact pig infectious intestinal disease, this disease with the vomiting of pig, diarrhoea, dehydration and to sucking piglets height lethality rate for principal character.1978, this disease Belgium with Britain by reported first.Subsequently, also this disease is found successively in countries such as Hungary, Italy, China, Japan, Thailand, the U.S. and Korea S.Since the end of the year 2010, PEDV varient will cause huge financial loss to Chinese pig industry.2013, PEDV variant viral was also broken out in the U.S..In recent years, PEDV is the important pathogen body causing the death of China and U.S.A piglet.PEDV full-length genome is about 28kb, comprise 7 open reading frame can to encode 4 main structural protein: spike protein (spike, S), envelope protein (envelope, E), membrane glycoprotein (membrane, M), nucleocapsid protein (nucleocapsid, N) and 3 kinds of Nonstructural Protein: replicative enzyme 1a and 1b, ORF3.The large percentage that wherein N protein is shared in the structural protein of PEDV, there is stronger antigenicity, host T cell and B cell immune response can not only be induced, and it is early stage at virus infection, anti-N protein antibody (Sun Dongbo, Feng Li, the Shi Hongyan of higher level can be produced in body, Deng, 2006).N protein provides an architecture basics for helical virion, is a basic phosphoprotein be associated with genome.
After pig infects PEDV, if be only difficult to make correct diagnosis according to clinical symptom, pathological change and epidemiology, particularly not easily distinguish with transmissible gastroenteritis (TGE), laboratory technique must be relied on just to make correct diagnosis.The method that current diagnosis PEDV infects has indirect hemagglutination test (IHA), immuno-electron microscope (IEM), enzyme linked immunosorbent assay (ELISA), immunofluorescence (IF), RT-PCR etc., wherein immunofluorescence and enzyme linked immunosorbent assay comparatively common (Deng Zuli grain husk, 2013; Zheng Fengmei, Zhao Jun, Huo Jinyao, etc., 2013).But these existing diagnostic techniquess are comparatively consuming time, effort, cannot widespread use clinically.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of monoclonal antibody of porcine epidemic diarrhea resisting viral N proteins, and this monoclonal antibody can be used for diagnosing Porcine epidemic diarrhea virus (PEDV) rapidly and accurately.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of monoclonal antibody of porcine epidemic diarrhea resisting viral N proteins is provided.
Preferably, described Porcine epidemic diarrhea virus N protein has aminoacid sequence shown in SEQIDNO.1.
Preferred, described antibody is the monoclonal antibody of being secreted by hybridoma cell strain CCTCCNO.C2014136.
In another aspect of this invention, additionally provide a kind of recombinant vectors, it comprises the nucleotide sequence of aminoacid sequence shown in coding SEQIDNO.1.
The empty carrier of described recombinant vectors comprises prokaryotic expression carrier or carrier for expression of eukaryon.
In the present invention, the preferred pColdIDNA prokaryotic expression carrier of prokaryotic expression carrier.
In another aspect of this invention, additionally provide a kind of host cell, it comprises above-mentioned recombinant vectors.
In another aspect of this invention, additionally provide a kind of recombinant protein, this recombinant protein is obtained through expression by above-mentioned recombinant vectors.
In another aspect of this invention, additionally provide a kind of preparation method of said monoclonal antibody, comprise the following steps:
Build the recombinant expression vector containing Porcine epidemic diarrhea virus N protein gene;
Described recombinant expression vector is transformed into host cell and carries out abduction delivering, obtain the Porcine epidemic diarrhea virus recombinant N protein of solubility;
By immune mouse after described recombinant N protein purifying, get the Mouse spleen cells after immunity, merge with SP2/0 oncocyte, screening obtains the monoclonal hybridoma of secreting specificity antibody;
Described hybridoma is injected into mouse peritoneal, after mouse web portion swells, gathers ascites, obtain the monoclonal antibody of porcine epidemic diarrhea resisting viral N proteins.
In another aspect of this invention, additionally provide a kind of test kit diagnosing Porcine epidemic diarrhea virus to infect, comprise said monoclonal antibody.
In another aspect of this invention, the application of said monoclonal antibody in the product of preparation diagnosis Porcine epidemic diarrhea virus infection is additionally provided.
In another aspect of this invention, the application of said monoclonal antibody in the product of preparation prevention Porcine epidemic diarrhea virus infection is additionally provided.
The monoclonal antibody of porcine epidemic diarrhea resisting viral N proteins of the present invention, with Porcine epidemic diarrhea virus, there is good reactivity, be applicable to diagnose PEDV rapidly and accurately, simultaneously for monitoring, prevention PEDV provide important technical support, there is good application prospect.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is that the PEDVN gene PCR of the embodiment of the present invention 1 increases (1A) and after structure prokaryotic expression carrier, PCR (1B) and enzyme cut qualification (1C) result figure;
Fig. 2 is the N protein expression product SDS-PAGE (2A) of the embodiment of the present invention 1 purifying and westernblot (2B) the result figure of recombinant N protein;
Fig. 3 is that the embodiment of the present invention 3 porcine epidemic diarrhea resisting viral N proteins MAb mediated ELISA is tired qualification result figure;
Fig. 4 is the embodiment of the present invention 4 Porcine epidemic diarrhea virus N protein monoclonal antibody specificity qualification figure;
Fig. 5 is the embodiment of the present invention 5 porcine epidemic diarrhea resisting viral N proteins monoclonal antibody Westernblot analytical results figure;
Fig. 6 is the embodiment of the present invention 5 porcine epidemic diarrhea resisting viral N proteins monoclonal antibody IFA analytical results figure.
Monoclonal antibody hybridoma cell line of the present invention, be preserved in China typical culture collection center on August 5th, 2014 and (be called for short CCTCC, address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center), preserving number is CCTCCNO.C2014136, and its Classification And Nomenclature is hybridoma cell strain 2B8.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions, usual condition routinely, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, the chief editors such as J.G. Sai Deman, Ma Xuejun, Su Yuelong translates. Beijing: Science Press, 2004) described in method carry out.
In order to diagnose PEDV rapidly and accurately, the present invention have developed can have the monoclonal antibody of sound response with Porcine epidemic diarrhea virus N protein.Specific monoclonal antibody is obtained especially by following process:
1, according to Porcine epidemic diarrhea virus JS-2013 strain nucleotide sequence information, the primer of design pair for amplification N full length gene.Extract virus genome RNA, reverse transcription synthesis cDNA, with primer N-F, N-R amplification total length N gene, be cloned on Pcold-I expression vector, cut through enzyme, check order and identify correctly, this plasmid is proceeded in competent cell BL21 (DE3), utilize IPTG abduction delivering.With Ni-NTA post, purifying is carried out to recombinant protein, obtain solubility recombinant N protein, as the immunogen of the follow-up monoclonal antibody of preparation.
2, using recombinant N protein after purifying as immunogen, immunity 6 week age female BABL/c mouse.First immunisation uses complete Freund's adjuvant, and two exempt to select incomplete Freund's adjuvant, and recombinant N protein mixes with freund's adjuvant 1:1 ratio, and first immunisation protein content doubles.Third and fourth direct immunization recombinant N protein.Merge front 3 ~ 4d, the pure antigen booster immunization of abdominal injection.Get the Mouse spleen cells after immunity, merge with the ratio of SP2/0 oncocyte in 5:1, screening positive hybridoma cell, adopt limiting dilution assay to choose the monoclonal hybridoma of secreting specificity antibody, by 10 through 4 subclones
6individual cell/only immunity BABL/c mouse in 8 week age, takes mouse ascites after 7 days, obtains monoclonal antibody.
The recombinant expressed Porcine epidemic diarrhea virus N protein of embodiment 1
According to Porcine epidemic diarrhea virus JS-2013 strain nucleotide sequence information, the primer of design pair for amplification N full length gene (1326bp, N full length gene sequence is as shown in SEQIDNO.2), is synthesized by Shanghai Sheng Gong biotechnology limited-liability company.Upstream primer sequence is: N-F:5 '-GCTCGGTACCCTCGAGATGGCTTCTGTCAGTTTTCAGGATC-3 ' (SEQIDNO.3); Downstream primer sequence is N-R:5 '-ATTCGGATCCCTCGAGTTAATTTCCTGTGTCGAAGATCTCG-3 ' (SEQIDNO.4), and restriction enzyme site is XhoI.Extract virus genome RNA, reverse transcription synthesis cDNA, with primer N-F, N-R amplification total length N gene, PCR primer 1% agarose gel electrophoresis Testing and appraisal reclaims, go out by pcr amplification the N gene that size is about 1300bp, conform to expected results (Figure 1A).Carrier pColdIDNA XhoI enzyme cuts rear and each 100ng of PCR primer, DE3 competent cell is transformed after connecting 15min with 5XIn-FusionHDEnzymePremix50 DEG C, be spread evenly across on the LB agar plate containing ammonia benzyl resistance, cultivate 14h ~ 16h picking colony for 37 DEG C and shake bacterium, whether PCR and enzyme cut qualification recombinant expression plasmid correct, PCR qualification result as shown in Figure 1B, amplify the N gene that size is about 1300bp, enzyme cuts qualification result as shown in Figure 1 C, obtains the N gene endonuclease bamhi of about 1300bp.PCR is identified correct plasmid is served Hai Shenggong biotechnology limited-liability company and carried out order-checking qualification.Sequencing result shows vector construction success.Through the positive expression vector plasmid called after pColdI-N that order-checking qualification is correct.
Prokaryotic expression, purification of Recombinant Porcine epidemic diarrhea virus N protein program are as follows:
To be accredited as positive pColdI-N/DE3 microbionation 2 × YT substratum, inoculative proportion 1:100, shake after bacterium to bacterium liquid OD value reaches 0.6 through 37 DEG C of vibrations, adding IPTG to final concentration is 0.5mM, and 15 DEG C are continued to induce 12h.Collected by centrifugation bacterium liquid after cleaning 2 times with PBS, after broken bacterium damping fluid is resuspended, ultrasonicly to clarify to bacterium liquid, centrifugal segregation ultrasound precipitation, purifying supernatant, according to the N protein that His-Binding-Resin protein purification test kit specification sheets purifying is expressed, and Bradford quantification of protein test kit is used to carry out quantitative analysis to target protein.The target protein of purifying is transferred on pvdf membrane after SDS-PAGE electrophoresis, reacts with the positive porcine blood serum of PEDV, then add sheep anti-mouse igg and the ECL colour developing of horseradish peroxidase-labeled.SDA-PAGE and Weaternblot result shows, and the recombinant protein size of Prokaryotic expression, purification is about 52kDa, and conform to (Fig. 2) with expection albumen size, the aminoacid sequence of Porcine epidemic diarrhea virus N protein is as shown in SEQIDNO.1.In Fig. 2, Fig. 2 A is the SDS-PAGE electrophoresis result figure that pColdIDNA prokaryotic expression carrier expresses Porcine epidemic diarrhea virus recombinant N protein, and wherein M is protein molecular weight standard; 1 is 0.5mMIPTG abduction delivering bacterium liquid ultrasound precipitation; 2 is the ultrasonic supernatant of 0.5mMIPTG abduction delivering bacterium liquid; 3 is purification of Recombinant N protein; Fig. 2 B is that mouse-anti His tag monoclonal antibody Westernblot identifies recombinant protein result figure, and wherein M is protein molecular weight standard; 1 is pColdIDNA empty carrier induced product; 2 is the recombinant N protein of purifying.
The preparation of embodiment 2 porcine epidemic diarrhea resisting viral N proteins mouse resource monoclonal antibody
If the Recombinant Swine epidemic diarrhea virus N protein of purification in embodiment 1 is as immunogen, immune mouse, for the preparation of monoclonal antibody.Select BABL/c female mice in 6 week age, the N protein of Immune expression purifying.First immunisation using the N protein of purifying as immunogen, after mixing with Freund's complete adjuvant 1:1 ratio, by the antigen amount of 50ug/ only (200ul/ is only) through abdominal injection.After two weeks, then through abdominal injection, by the same doses of antigen of Freund's incomplete adjuvant process, repeated immunity once every two weeks.Before each immunity, eye socket is taken a blood sample, and ELISA identifies antibody titer.Third time immunity starts, and does not add adjuvant.Merge front 3 ~ 4d, the pure antigen booster immunization of abdominal injection.Get mouse spleen and SP2/0 cytogamy, carry out monoclonal antibody preparation.After merging two weeks, indirect ELISA method screening positive hybridoma cell, concrete grammar is as follows:
1, bag quilt: purification of Recombinant Porcine epidemic diarrhea virus N protein and pColdIDNA expression vector His label protein wrap by ELISA enzyme plate respectively, and every hole package amount is 200ng/100 μ L, 4 DEG C of bags are spent the night.
2, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ tween-20.Each 5 minutes.
3, close: 5% skimming milk 37 DEG C hatches 2 hours, 200 μ L/ holes.
4, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ Tween-20.Each 5 minutes.
5, add cell culture supernatant: cells and supernatant adds the enzyme plate of bag reorganized N protein and His label protein respectively, every hole culture supernatant 50 μ L, skimming milk 50 μ L, mix, 37 DEG C of incubator effects 1 hour.
6, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ Tween-20.Each 5 minutes.
7, add two to resist: after doubly diluting HRP sheep anti-mouse igg with 5% skimming milk by 1:10000, every hole 100 μ L adds enzyme plate, hatches 1 hour for 37 DEG C.
8, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ Tween-20.Each 5 minutes.
9, develop the color: every hole adds 50 μ LTMB nitrite ions, lucifuge incubated at room 10 minutes.
10, stop: every hole adds 50 μ L2MH
2sO
4.
11, OD value detects: microplate reader measures OD
450value.
After Recombinant Swine epidemic diarrhea virus N protein and the dual ELISA of pColdIDNA carrier tag albumen are screened, choose the clone of carrier tag albumen test feminine gender, recombinant N protein reacting positive, carry out 4 subclones after enlarged culturing, after each subclone, all carry out dual ELISA Screening and Identification positive colony.After 4 subclones, obtain the hybridoma cell strain of 3 strain stably excreting monoclonal antibodies.A last preferred wherein strain of hybridoma strain 2B8 is preserved in China typical culture collection center on August 5th, 2014 and (is called for short CCTCC, address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center), preserving number is CCTCCNO.C2014136, and its Classification And Nomenclature is hybridoma cell strain 2B8.The positive hybridoma cell of screening, through enlarged culturing, is prepared for odd contradictive hydroperitoneum.
Prepared by odd contradictive hydroperitoneum, concrete steps are as follows: choose BABL/c female mice in 8 week age, abdominal injection pristane, and 200 μ L/ only.Immunity pristane is after one week, and positive hybridoma cell counts, and basic DMEM substratum is resuspended, and 10
6/ Mice Inoculated abdominal cavity.Observe mouse web portion change after one week, if occur, belly swells, and namely carries out ascites collection, gathers the centrifugal 10min of ascites 4,000rpm/min, supernatant packing-80 DEG C preservation.
Embodiment 3 porcine epidemic diarrhea resisting viral N proteins monoclonal antibody ELISA tires qualification
As the ascites of preparation in embodiment 2, as primary antibodie, the ELISA that indirect ELISA method detects ascites tires, and concrete grammar is as follows:
1, bag quilt: purification of Recombinant Porcine epidemic diarrhea virus N protein and pColdIDNA expression vector His label protein wrap by ELISA enzyme plate respectively, and every hole package amount is 200ng/100 μ L, 4 DEG C of bags are spent the night.
2, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ tween-20.Each 5 minutes.
3, close: 5% skimming milk 37 DEG C hatches 2 hours, 200 μ L/ holes.
4, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ Tween-20.Each 5 minutes.
5, primary antibodie is added: after adopting 5% skimming milk the ascites gathered to be diluted according to 1:10000, add the enzyme plate of the reorganized Porcine epidemic diarrhea virus N protein of bag and His label protein respectively, every hole 100 μ L, 37 DEG C act on 1 hour.
6, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ Tween-20.Each 5 minutes.
7, add two to resist: after doubly diluting by 1:10000 with 5% skimming milk, every hole 100 μ L adds enzyme plate, hatches 1 hour for 37 DEG C.
8, wash: wash 3 times, 200 μ L/ holes with the PBST containing 5 ‰ Tween-20.Each 5 minutes.
9, develop the color: every hole adds 50 μ LTMB nitrite ions, lucifuge incubated at room 10 minutes.
10, stop: every hole adds 50 μ L2MH
2sO
4.
11, OD value detects: microplate reader measures OD
450value.
As shown in Figure 3, titer of ascites OD450 result is shown as 1.491 to result.In Fig. 3, MAb is porcine epidemic diarrhea resisting viral N proteins monoclonal antibody; Positive serum be purification of Recombinant N protein immunity 4 times after BABL/c mice serum; Negative serum is not immune BABL/c mice serum; His label protein is the label protein obtained after pColdIDNA empty carrier induction purifying.
Embodiment 4 porcine epidemic diarrhea resisting viral N proteins monoclonal antibody specificity is identified
As the ascites of preparation in embodiment 2, as primary antibodie, detect the atopy of porcine epidemic diarrhea resisting viral N proteins monoclonal antibody.Concrete operations are as follows:
Collect cell sample: from China Agriculture Academe Shanghai Veterinary Institute's swine disease laboratory, obtain Pestivirus suis (CSFV), porcine reproductive and respiratory syndrome virus (PRRS), PRV (Pseudorabies virus) (PRV), the cell sample that encephalitis b virus (JEV) infects, the Vero cell sample infected with Porcine epidemic diarrhea virus JS-2013.According to 4:1 volume ratio mixing 5 × sample buffer, after boiling 10min ,-20 DEG C save backup.
Westernblotting:10 μ L equivalent total protein loading carries out SDS-PAGE electrophoresis, and after electrophoresis, adopt the half-dried transferring film instrument of Bole, voltage 15V, transfer printing 50min, be transferred to albumen on nitrocellulose membrane (NC film).Subsequent experimental concrete operations are carried out as follows after transfer printing:
1, close: close NC film 1 hour by 5% skimming milk room temperature.
2, wash: wash NC film 3 times with the TBST containing 1 ‰ Tween-20, each 10 minutes.
3, primary antibodie is added: the ascites of preparation in embodiment 2, by the dilution proportion primary antibodie of the TBST buffered soln containing 5%BSA according to 1:500-1000, room temperature effect 1 hour or 4 DEG C of overnight incubation.
4, wash: wash NC film 3 times with the TBST containing 1 ‰ Tween-20, each 10 minutes.
5, add two to resist: company of Zhong Shan Golden Bridge HRP marks goat anti-mouse IgG antibody, adds after doubly diluting with TBST by 1:6000, room temperature effect 1h.
6, wash: wash NC film 3 times with the TBST containing 1 ‰ Tween-20, each 10 minutes.
7, develop: adopt Thermo company ECL luminescence reagent box to carry out exposure imaging.
As shown in Figure 4, the specificity of porcine epidemic diarrhea resisting viral N proteins monoclonal antibody prepared by embodiment 2 is good for result.In Fig. 4, PEDV is Porcine epidemic diarrhea virus; PRRS is porcine reproductive and respiratory syndrome virus; JEV is encephalitis b virus; CSFV is Pestivirus suis; PRV is PRV (Pseudorabies virus).
Embodiment 5 indirect immunofluorescence and Westernblotting detect the specificity of porcine epidemic diarrhea resisting viral N proteins monoclonal antibody respectively
As the ascites of preparation in embodiment 2, as primary antibodie, use indirect immunofluorescence (IFA) and Westernblotting to detect the specific reaction of ascites respectively, the concrete steps of IFA are as follows:
1, be paved with vero cell in cell infection PEDV:6 porocyte culture plate, infect vero cell, after 12h, with ice formaldehyde in-20 DEG C of fixing 10min with PEDV;
2, wash: wash 4 times with PBS, 700 μ L/ holes, each 5 minutes;
3, close: 37 DEG C, the PBS buffered soln containing 5%BSA hatches 1 hour;
4, wash: wash 4 times with PBS, 700 μ L/ holes, each 5 minutes;
5, add primary antibodie: the ascites of preparation in embodiment 2, by the dilution proportion primary antibodie of PBS buffered soln according to 1:1000-4000, SP2/0 cell culture supernatant does negative control, hatches 1 hour for 37 DEG C;
6, wash: wash 4 times with PBS, 700 μ L/ holes, each 5 minutes;
7, add two to resist: FITC sheep anti-mouse igg available from Sigma, adds six orifice plates after doubly diluting with PBS by 1:1000, and lucifuge 37 DEG C hatches 1 hour;
8, wash: wash 4 times with PBS, 700 μ L/ holes, each 5 minutes;
9, fluorescence microscope.
Westernblotting experimental procedure is with embodiment 4.
As shown in Figure 5, in Fig. 5,1 is that SP2/0 cell culture supernatant carries out Westernblot result to the vero cell infecting PEDV to Westernblot result; 2 is that porcine epidemic diarrhea resisting viral N proteins monoclonal antibody MAb2B8 carries out Westernblot result to the vero cell infecting PEDV.
As shown in Figure 6, in Fig. 6,5A is that MAb2B8 carries out indirect IF staining result to the vero cell infecting PEDV to IFA result, and cell cytosol and after birth are the reaction of glittering green fluorescence; 5B is that SP2/0 cell culture supernatant carries out indirect IF staining result to the vero cell infecting PEDV, and cell is there are no bright green fluorescent dye.
From above experimental result, porcine epidemic diarrhea resisting viral N proteins monoclonal antibody specificity of the present invention is fine, is applicable to Rapid&Early diagnosis PEDV.
The above embodiment only have expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. the monoclonal antibody of a porcine epidemic diarrhea resisting viral N proteins.
2. monoclonal antibody according to claim 1, is characterized in that, described Porcine epidemic diarrhea virus N protein has aminoacid sequence shown in SEQIDNO.1.
3. monoclonal antibody according to claim 1 and 2, is characterized in that, this antibody is the monoclonal antibody of being secreted by hybridoma cell strain CCTCCNO.C2014136.
4. a recombinant vectors, is characterized in that, comprises the nucleotide sequence of aminoacid sequence shown in coding SEQIDNO.1.
5. a recombinant protein, is characterized in that, this recombinant protein is obtained through expression by recombinant vectors described in claim 4.
6. the preparation method of monoclonal antibody described in claim 1, is characterized in that, comprises the following steps:
Build the recombinant expression vector containing Porcine epidemic diarrhea virus N protein gene;
Described recombinant expression vector is transformed into host cell and carries out abduction delivering, obtain the Porcine epidemic diarrhea virus recombinant N protein of solubility;
By immune mouse after described recombinant N protein purifying, get the Mouse spleen cells after immunity, merge with SP2/0 oncocyte, screening obtains the monoclonal hybridoma of secreting specificity antibody;
Described hybridoma is injected into mouse peritoneal, after mouse web portion swells, gathers ascites, obtain the monoclonal antibody of porcine epidemic diarrhea resisting viral N proteins.
7. the test kit diagnosing Porcine epidemic diarrhea virus to infect, is characterized in that, comprises monoclonal antibody according to claim 1.
8. the application of monoclonal antibody described in claim 1 in the product of preparation diagnosis Porcine epidemic diarrhea virus infection.
9. the application of monoclonal antibody described in claim 1 in the product of preparation prevention Porcine epidemic diarrhea virus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410384478.9A CN105440132B (en) | 2014-08-07 | 2014-08-07 | The monoclonal antibody and its application of porcine epidemic diarrhea resisting viral N proteins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410384478.9A CN105440132B (en) | 2014-08-07 | 2014-08-07 | The monoclonal antibody and its application of porcine epidemic diarrhea resisting viral N proteins |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105440132A true CN105440132A (en) | 2016-03-30 |
| CN105440132B CN105440132B (en) | 2019-03-29 |
Family
ID=55550816
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410384478.9A Expired - Fee Related CN105440132B (en) | 2014-08-07 | 2014-08-07 | The monoclonal antibody and its application of porcine epidemic diarrhea resisting viral N proteins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105440132B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434568A (en) * | 2016-09-18 | 2017-02-22 | 广东温氏大华农生物科技有限公司 | Hybridoma cell line for resisting porcine epidemic diarrhea virus S proteins and application of hybridoma cell line |
| CN107099506A (en) * | 2017-04-24 | 2017-08-29 | 江苏省农业科学院 | Porcine epidemic diarrhea virus double-antibody sandwich elisa immue quantitative detection reagent box and its application |
| CN109061159A (en) * | 2018-05-24 | 2018-12-21 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PEDV immune detection chromatograph test strip and its preparation method and application |
| CN109678953A (en) * | 2018-12-24 | 2019-04-26 | 西北农林科技大学 | A kind of construction method and purposes of the VHH phage display library of anti-PEDV N protein |
| CN109734802A (en) * | 2019-03-18 | 2019-05-10 | 扬州大学 | A kind of preparation method recombinating Porcine epidemic diarrhea virus N, S protein monoclonal antibody |
| 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 |
| CN112898417A (en) * | 2020-12-17 | 2021-06-04 | 河南九天生物科技有限公司 | Preparation and process of efficient monoclonal antibody for resisting porcine epidemic diarrhea |
| CN113583113A (en) * | 2020-12-31 | 2021-11-02 | 安徽九川生物科技有限公司 | Single-chain antibody for resisting porcine epidemic diarrhea virus and preparation method thereof |
| CN117683127A (en) * | 2023-12-05 | 2024-03-12 | 华中农业大学 | ELISA detection kit for capturing porcine epidemic diarrhea virus antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969450A (en) * | 2014-05-26 | 2014-08-06 | 江苏省农业科学院 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody |
-
2014
- 2014-08-07 CN CN201410384478.9A patent/CN105440132B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969450A (en) * | 2014-05-26 | 2014-08-06 | 江苏省农业科学院 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody |
Non-Patent Citations (6)
| Title |
|---|
| GAO,Y.,等: "nucleocapsid protein，GenBank：AFC98507.1", 《GENBANK》 * |
| 吴丹: "猪流行性腹泻病毒N蛋白单抗的制备及其模拟表位鉴定", 《中国优秀硕士学位论文数据库》 * |
| 时晓丽，等: "猪流行性腹泻病毒N蛋白单克隆抗体的制备与鉴定", 《畜牧与兽医》 * |
| 童光志,冯力，等: "猪流行性腹泻病毒N蛋白单克隆抗体的制备与鉴定", 《中国预防兽医学报》 * |
| 童光志，单同领: "病毒宏基因组学分析腹泻仔猪肠道病毒群落及变异PEDV的分离和鉴定", 《中国畜牧兽医学会家畜传染病学分会第八届全国会员代表大会暨第十五次学术研讨会论文集》 * |
| 裴敦国，等: "抗猪流行性腹泻病毒N蛋白单克隆抗体制备及部分特性鉴定", 《东北农业大学学报》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434568A (en) * | 2016-09-18 | 2017-02-22 | 广东温氏大华农生物科技有限公司 | Hybridoma cell line for resisting porcine epidemic diarrhea virus S proteins and application of hybridoma cell line |
| CN107099506A (en) * | 2017-04-24 | 2017-08-29 | 江苏省农业科学院 | Porcine epidemic diarrhea virus double-antibody sandwich elisa immue quantitative detection reagent box and its application |
| CN107099506B (en) * | 2017-04-24 | 2020-04-03 | 江苏省农业科学院 | Double-antibody sandwich ELISA quantitative detection kit for porcine epidemic diarrhea virus and application thereof |
| 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 |
| CN109061159A (en) * | 2018-05-24 | 2018-12-21 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PEDV immune detection chromatograph test strip and its preparation method and application |
| CN109678953A (en) * | 2018-12-24 | 2019-04-26 | 西北农林科技大学 | A kind of construction method and purposes of the VHH phage display library of anti-PEDV N protein |
| CN109734802A (en) * | 2019-03-18 | 2019-05-10 | 扬州大学 | A kind of preparation method recombinating Porcine epidemic diarrhea virus N, S protein monoclonal antibody |
| CN112898417A (en) * | 2020-12-17 | 2021-06-04 | 河南九天生物科技有限公司 | Preparation and process of efficient monoclonal antibody for resisting porcine epidemic diarrhea |
| CN113583113A (en) * | 2020-12-31 | 2021-11-02 | 安徽九川生物科技有限公司 | Single-chain antibody for resisting porcine epidemic diarrhea virus and preparation method thereof |
| CN113583113B (en) * | 2020-12-31 | 2023-02-03 | 安徽九川生物科技有限公司 | Single-chain antibody for resisting porcine epidemic diarrhea virus and preparation method thereof |
| CN117683127A (en) * | 2023-12-05 | 2024-03-12 | 华中农业大学 | ELISA detection kit for capturing porcine epidemic diarrhea virus antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105440132B (en) | 2019-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105440132A (en) | Monoclonal antibody against porcine epidemic diarrhea virus N protein and application thereof | |
| CN103760365B (en) | A kind of BAS-ELISA kit detecting CSFV Erns and E2 antigen | |
| CN108107217B (en) | Classical swine fever virus truncated E2 protein and application thereof | |
| Ma et al. | Two-way antigenic cross-reactivity between porcine epidemic diarrhea virus and porcine deltacoronavirus | |
| Langedijk et al. | Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein Erns for serologic diagnosis of pestivirus infections in swine | |
| CN102023217A (en) | Double-antibody biotin-Avidin ELISA (enzyme-linked immuno sorbent assay) detection kit for cattle viral diarrhea virus and application method thereof | |
| CN109187968B (en) | Bivalent gold-labeled test paper for detecting classical swine fever virus and porcine pseudorabies virus and preparation method thereof | |
| CN104862285A (en) | Porcine epidemic diarrhea virus antibody capture based ELISA detection method and application | |
| CN105527442A (en) | CSFV antibody detection system and preparation method thereof | |
| CN102236017A (en) | Indirect ELISA kit for detecting African swine fever virus antibody and application thereof | |
| Ji et al. | An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells | |
| CN109187993A (en) | A kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit and its application | |
| CN103570830A (en) | Preparation method of anti-canine parvovirus protein VP2 specific IgY | |
| CN103499693A (en) | Competitive Alpha LISA (linked immuno sorbent assay) detection kit for classical swine fever virus (CSFV) antibody and detection method thereof | |
| CN103616509B (en) | Detect E III-indirect ELISA antibody assay kit and the application of Latex agglutination test | |
| Lei et al. | Specific recombinant proteins of porcine epidemic diarrhea virus are immunogenic, revealing their potential use as diagnostic markers | |
| Fu et al. | Development of a dot immunoblot method for differentiation of animals infected with foot-and-mouth disease virus from vaccinated animals using non-structural proteins expressed prokaryotically | |
| CN102533663B (en) | Foot-and-mouth disease hybridoma cell line, monoclonal antibody, detection reagent and test kit | |
| CN104744572A (en) | Avian and porcine hepatitis e virus shared antigen, monoclonal antibody and preparation method and application | |
| CN101799471A (en) | Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus NS3 | |
| CN103983782A (en) | ELISA kit for detecting hog cholera virus Erns IgM antibody | |
| CN103255111B (en) | Green fluorescent protein marked recombinant swine fever virus, its rescue method and application | |
| Chen et al. | The identification of a B-cell epitope in bovine viral diarrhea virus (BVDV) core protein based on a mimotope obtained from a phage-displayed peptide library | |
| CN107557344A (en) | One plant of expression CSFV E 2 protein monoclonal antibody hybridoma cell line CSFV 3H3G6 and antibody and kit | |
| Fregeneda-Grandes et al. | Detection of rainbow trout antibodies against viral haemorrhagic septicaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190329 Termination date: 20200807 |