CN109836478A - The preparation method and application of African swine fever virus P11.5 protein-specific polyclonal antibody - Google Patents
The preparation method and application of African swine fever virus P11.5 protein-specific polyclonal antibody Download PDFInfo
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Abstract
The invention discloses the preparation method and application of African swine fever virus P11.5 protein-specific polyclonal antibody, this method prepares specific polyclonal antibody using P11.5 polypeptide epitope immunization experiment animal such as mouse, rabbit etc..This method antigen purity is high, and the antibody specificity of preparation is strong, and affinity is high, and the amount of antigen used is few, easy to operate.The preparation method of the polyclonal antibody includes the selection of the specific antigen epitope of ASFV P11.5, the synthesis of polypeptide antigen, animal immune, the measurement of antibody and etc..Compared with the conventional method, having simplicity, quickly antigen purity is high, and the antibody specificity of preparation is strong, the few advantage of antigen usage amount.The polyclonal antibody can be used for African swine fever virus P11.5 Protein Detection, provide important technical method for the prevention and control of China's African swine fever.
Description
Technical field
The present invention relates to a kind of preparation method and applications of African swine fever virus P11.5 protein-specific polyclonal antibody.
Background technique
African swine fever is also known as East Africa swine fever or warthog disease, is a kind of acute heat type height for causing pig by African swine fever virus
Contagious disease.This disease only infects pig, and clinical symptoms are much like with swine fever with pathological change, is mainly shown as fever, skin
Cyanosis and lymph node, kidney, gastrointestinal tract mucous membrane bleeding etc..The death rate of this disease is very high, and during the epidemic up to 100%.Mesh
Preceding African swine fever has the tendency that continuing to expand in more than 30 national happening and prevelences, and the China on the 3rd of August in 2018 makes a definite diagnosis
The first African swine fever epidemic situation.The diagnosis of African swine fever connects frequently with such as hemadsorption test, direct immuno fluorescence test, animal
Kind test, indirect immunofluorescence assay, enzyme-linked immunosorbent assay, immunoelectrophoretic test, indirect enzyme-linked immunosorbent plaque assay etc.,
Relative specific reagent lacks in the world, brings many difficulties to control African swine fever.African swine fever virus is that one kind contains capsule
The double-stranded DNA of film, genome are linear dsdna, and length is between 170~190kb, 151 albumen of codified.The virus is
The unique member that African swine fever virus section African swine fever virus belongs to only has 1 serotype, but different regions ASFV separation strains at present
Genome has a certain difference.It can be at present 23 genotype by ASFV points according to P72 gene genetic evolutionary analysis.
Summary of the invention
In order to overcome drawbacks described above, the present invention provides a kind of African swine fever virus P11.5 protein-specific Anti-TNF-αs
The preparation method and application of body.P11.5 genetic comparison is stablized, very rich in infection cell, and the present invention utilizes P11.5 gene
The foundation such as expression, Peptide systhesis quickly detect African swine fever specific antibody and antigen, it is excellent to be provided for detection African swine fever
Matter reagent.
In order to achieve the above-mentioned object of the invention, a kind of the technical solution adopted by the present invention are as follows: African swine fever virus P11.5 albumen
Specific antigen polypeptide, the antigen polypeptide are P11.5-2 or P11.5-1；The sequence of P11.5-2 is
The sequence of PEERCTYKFNSYTKKMEL, P11.5-1 are DQEEKKALQNKETKNLGIP.
The antigen polypeptide is made by synthesis.
The present invention also provides a kind of African swine fever virus P11.5 protein immunogens, and the immunogene is directly by above-mentioned
African swine fever virus P11.5 protein-specific antigen polypeptide is constituted；Alternatively, the immunogene is by the African swine fever virus
P11.5 protein-specific antigen polypeptide and Freund's complete adjuvant mix；Alternatively, the immunogene is by the African swine fever disease
Malicious P11.5 protein-specific antigen polypeptide and Freund's incomplete adjuvant mix.
Invention additionally discloses a kind of preparation methods of African swine fever virus P11.5 protein-specific polyclonal antibody, including
Following steps:
A) African swine fever virus P11.5 protein-specific polypeptide epitope is selected:
P11.5-1—DQEEKKALQNKETKNLGIP；
Or P11.5-2-PEERCTYKFNSYTKKMEL；
B) African swine fever virus P11.5 protein-specific polypeptide epitope is synthesized；
C) African swine fever virus P11.5 protein-specific polypeptide epitope is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant first
Animal is immunized afterwards；Again by African swine fever virus P11.5 protein-specific polypeptide epitope direct immunization animal；
D) serum of separating immune animal contains anti-African swine fever virus P11.5 protein-specific Anti-TNF-α in serum
Body.
Preferably, the animal is mouse or rabbit.
The present invention also provides the anti-African swine fever virus P11.5 protein-specific Anti-TNF-αs that method above-mentioned prepares
Body and African swine fever virus P11.5 protein-specific polyclonal antibody in preparation detection African swine fever virus antigen and/or resist
Application in body kit.
Compared with the existing technology, beneficial effects of the present invention: the invention discloses a kind of quickly preparation African swine fever viruses
The method of P11.5 protein-specific polyclonal antibody, this method utilize P11.5 polypeptide epitope immunization experiment animal such as mouse, rabbit
Etc. preparing specific polyclonal antibody.This method antigen purity is high, and the antibody specificity of preparation is strong, and affinity is high, and what is used is anti-
Commercial weight is few, easy to operate.The preparation method of the polyclonal antibody includes the choosing of the specific antigen epitope of ASFV P11.5
It selects, the synthesis of polypeptide antigen, animal immune, the measurement of antibody and etc..Compared with the conventional method, there is easy to be quick, antigen
The antibody specificity of purity is high, preparation is strong, and antigen usage amount is few.The polyclonal antibody can be used for African swine fever virus P11.5
Protein Detection provides important technical method for the prevention and control of China's African swine fever.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.It should also be understood that after reading the content taught by the present invention, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within application the appended claims and limited
Range.
1. the screening of polypeptides epitope
According to medicine, disclosed African swine fever virus P11.5 protein amino acid sequence, screening design have hydrophily online
Good, antigenicity is strong, the specificity epitope with general character, such as sequence P11.5-1-DQEEKKALQNKETKNLGIP (SEQ ID
No.1), P11.5-2-PEERCTYKFNSYTKKMEL (SEQ ID No.2).
2. the synthesis of polypeptides epitope
Straight line polypeptide is synthesized, polypeptide is from C-terminal to N-terminal direction composition.
1. RINK resin 3g (degree of substitution 0.3mmol/g) is weighed in the reactor of 150ml, with the methylene chloride of 50ml
(DCM) it impregnates.
After 2.2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so weight
It is four times multiple, it is stand-by after resin is drained.
3. a certain amount of 20% piperidines (piperidines/DMF) is added into reactor, it is placed on decolorization swinging table and rocks 20min, with
This sloughs the Fmoc blocking group on resin.It is washed four times after having taken off protection with the DMF of 3 times of resin volumes, is then drained.
4. taking the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
5. it weighs that first amino acid of C-terminal is appropriate and 1- hydroxyl-benzene a pair of horses going side by side triazole (HOBT) is in right amount in the centrifuge tube of 50ml,
The DMF that 20ml is added is dissolved, and the N of 3ml is then added, N- diisopropylcarbodiimide (DIC) oscillation shakes up 1min, to molten
It is added in reactor after liquid clarification, then reactor is placed in 30 DEG C of shaking table and is reacted.
After 6.2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride: DIEA:DCM=1:1:2) half an hour, then with 3
The DMF of times resin volume is washed four times, is drained stand-by.
7. a certain amount of 20% piperidines (piperidines/DMF=1:4) is added into reactor, it is placed on decolorization swinging table and rocks
20min sloughs the Fmoc blocking group on resin with this.It is washed four times after having taken off protection with DMF, is then drained.
8. taking the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
9. weighing second amino acid next in right amount and HOBT being in right amount in the centrifuge tube of 50ml, the DMF general of 25ml is added
It is dissolved, and the DIC oscillation that 2.5ml is then added shakes up 1min, is added in reactor after solution clarification, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
After 10.1 hours, a small amount of resin is taken to detect, with ninhydrin method detection (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), if resin be it is colourless, illustrate fully reacting；If resin has color, illustrate condensation not exclusively, the reaction was continued.
11. after complete reaction, being washed resin four times with DMF, then drains, a certain amount of 20% is added into reactor
Piperidines (piperidines/DMF=1:4), be placed on decolorization swinging table and rock 20min, sloughs the Fmoc blocking group on resin with this.It is de-
It is washed four times after complete protection with DMF, then drains whether detection protection sloughs.
12. successively connecting subsequent amino acid according to step 9-11.
13. sloughing protection after connecting the last one amino acid, is washed four times with DMF, then taken out resin with methanol
It is dry.Then with 95 cutting liquids (trifluoroacetic acid: 1,2 dithioglycol: 3, isopropyl base silane: water=95:2:2:1) by polypeptide from resin
On cut down (every gram of resin adds 10ml cutting liquid), and with ice ether (cutting liquid: ether=1:9) centrifugal sedimentation four times.Most
It is isolated and purified, then is lyophilized with HPLC afterwards, obtain the polypeptide of certain purity.
Wherein HPLC purification condition:
Stationary phase: C18；
The configuration of mobile phase:
Pump A:V (TFA)/V (water)=1/1000
Pump B:V (TFA)/v (acetonitrile)=1/1000；
Flow velocity: 10mL/min；
Retention time: between 20-30min.
3. polypeptide is immune
A, polypeptide antigen (P11.5-1 or P11.5-2) is mixed with Freund's complete adjuvant, mouse or skin is immunized by abdominal channels
Interior and foot-pad immunization rabbit；Immunizing dose is every mouse 5ug, every 10ug of rabbit immunization dosage.B, by polypeptide antigen after 7 days
It is mixed with incomplete adjuvant, immunizing rabbit in mouse or intradermal and vola and lymph node is immunized by abdominal channels；Immunizing dose
For every mouse 10ug, every 50ug of rabbit immunization dosage.
C, polypeptide antigen is directly passed through into abdominal channels after 7 days, immunizing rabbit in mouse or intradermal and lymph node is immunized；Exempt from
Epidemic disease dosage is every mouse 20ug, every 100ug of rabbit immunization dosage.
D, it takes a blood sample after 7 days, separates serum, measure antibody response.Such as potency deficiency, c can be repeated and be immunized again once.
E, contain anti-African swine fever virus P11.5 protein-specific polyclonal antibody in the serum of separation.Use hemocyanin
The polypeptide of coupling carries out coated elisa plate, carries out ELISA detection, after serum 1:100 dilution, the reactivity of antibody, and OD450
Or OD490 is greater than 1.0 or more.
3. the application of polyclonal antibody
1) ELISA detects African swine fever virus P11.5 protein-specific polyclonal antibody
Using polypeptide antigen (P11.5-2) coated elisa plate of synthesis, other nonspecific activity bases are closed with confining liquid
Group, is added the anti-African swine fever virus P11.5 protein-specific polyclonal antibody of mouse to be checked after washing, 37 DEG C of incubations 1 are small
When after wash, then with enzyme label 37 DEG C of anti-mouse IgG whole immunoglobulin be incubated for 1 hour, developed the color with substrate, according to the strong of colour developing
Degree, determines whether there is African swine fever virus P11.5 protein-specific polyclonal antibody.Testing result such as table 1, table 2.
1 ELISA method of table detects African swine fever virus P11.5 protein-specific polyclonal antibody (p11.5-1)
2 ELISA method of table detects African swine fever virus P11.5 protein-specific polyclonal antibody (p11.5-2)
The antigenicity that result above can be seen that P11.5-2 polypeptide is better than P11.5-1.So selection P11.5-2 polypeptide is anti-
It is former.Wherein 1:100 refers to that mice serum is diluted by 1:100.Data in table are the readings of ELISA, and readings is higher, antibody titer
It is higher.
If detecting in Swine serum and whether containing African swine fever virus P11.5 protein-specific polyclonal antibody, can adopt
With same method and principle, technical staff is entirely capable of understanding and capable of operating, as long as serum is replaced with Swine serum, enzyme mark resists
Body replaces with anti-pig IgG and/or IgM enzymic-labelled antibody.
2) it can also be utilized with blocking ELISA method to detect African swine fever virus P11.5 protein-specific polyclonal antibody
Polypeptide antigen (P11.5-2) coated elisa plate of synthesis is closed other nonspecific activity groups with confining liquid, is added after washing
The anti-African swine fever virus P11.5 protein-specific polyclonal antibody of rabbit (or pig) to be checked, 37 DEG C be incubated for 30 minutes after wash,
Be added the anti-African swine fever virus P11.5 protein-specific polyclonal antibody of mouse, 37 DEG C be incubated for 30 minutes, after washing again with enzyme mark
37 DEG C of anti-mouse IgG whole immunoglobulin of note are incubated for 1 hour, are developed the color with substrate, according to the intensity of colour developing, are calculated OD value and are inhibited
Rate determines whether there is African swine fever virus P11.5 protein-specific polyclonal antibody, if OD value inhibiting rate reach 35% and with
On, it is judged to antibody positive.Such as the following table 3:
Table 3 blocks ELISA method to detect African swine fever virus P11.5 protein-specific polyclonal antibody
| Negative control | Blank control | Rabbit anteserum 1 | Rabbit anteserum 2 | Rabbit anteserum 3 | Rabbit anteserum 4 | |
| P11.5-2 | 1.150 | 0.06 | 0.312 | 0.223 | 0.212 | 1.10 |
| Inhibiting rate | 72.87% | 80.61% | 81.56% | 4.34% | ||
| As a result | It is positive | It is positive | It is positive | It is negative |
3) sandwich ELISA detects African swine fever virus P11.5 specific proteins
Utilize polyclonal antibody (the immune obtained rabbit anteserum of P11.5-2, IgG) after purification coating enzyme mark of anti-P11.5
Sample to be examined (the African swine fever virus P11.5 egg of expression is added by the unbonded non-specific group of confining liquid closing in plate
White or pig tissue samples), 37 DEG C are incubated for 1 hour, and the anti-P11.5 mouse IgG enzyme labelled antibody of enzyme label is added in washing
(P11.5-2 or the immune obtained mice serum of P11.5-1, IgG after purification), 37 DEG C are incubated for 1 hour, aobvious with substrate after washing
Color.According to the intensity of colour developing, determines whether contain African swine fever P11.5 proteantigen in sample, be shown in Table 4.
4 sandwich ELISA of table detects African swine fever virus P11.5 specific proteins antigen result
| Expression product | Blank control | Negative control | |
| OD650 value | 1.203 | 0.075 | 0.066 |
| As a result | It is positive | It is negative | It is negative |
4) other application of polyclonal antibody such as Immunofluorescence test etc.
First building expression African swine fever virus P11.5 albumen pcDNA-P11.5 carrier, by transfect vero cell, 48
After hour, it is incubated for above-mentioned polyclonal antibody (the immune obtained serum of P11.5-2), is washed after 30 minutes, and with
The anti-mouse IgG whole immunoglobulin of fluorescein FITC label is incubated for 30 minutes, and after washing plus 50% glycerol PBS buffer solution is sealed
Piece, in fluorescence microscopy microscopic observation, it can be observed that specific bright green fluorescence.
Example of the present invention is the description of the invention and cannot limit the present invention, with the comparable meaning of the present invention
Any change and adjustment in range, are all considered as within the scope of the invention.
Sequence table
<110>Yangzhou University
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<141> 2019-03-19
<160> 2
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1 5 10 15
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<400> 2
Pro Glu Glu Arg Cys Thr Tyr Lys Phe Asn Ser Tyr Thr Lys Lys Met
1 5 10 15
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Claims (7)
1. a kind of African swine fever virus P11.5 protein-specific antigen polypeptide, which is characterized in that the antigen polypeptide is
P11.5-2 or P11.5-1；The sequence of P11.5-2 is PEERCTYKFNSYTKKMEL, and the sequence of P11.5-1 is
DQEEKKALQNKETKNLGIP。
2. a kind of African swine fever virus P11.5 protein-specific antigen polypeptide according to claim 1, which is characterized in that
The antigen polypeptide is made by synthesis.
3. a kind of African swine fever virus P11.5 protein immunogen, which is characterized in that the immunogene is directly by claim 1
The African swine fever virus P11.5 protein-specific antigen polypeptide is constituted；Alternatively, the immunogene is by claim 1 institute
The African swine fever virus P11.5 protein-specific antigen polypeptide and Freund's complete adjuvant stated mix；Alternatively, the immunogene by
African swine fever virus P11.5 protein-specific antigen polypeptide and Freund's incomplete adjuvant described in claim 1 mix.
4. a kind of preparation method of African swine fever virus P11.5 protein-specific polyclonal antibody, includes the following steps:
A) African swine fever virus P11.5 protein-specific polypeptide epitope: P11.5-1-DQEEKKALQNKETKNLGIP is selected；
Or P11.5-2-PEERCTYKFNSYTKKMEL；
B) African swine fever virus P11.5 protein-specific polypeptide epitope is synthesized；
C) exempt from after mixing African swine fever virus P11.5 protein-specific polypeptide epitope with Freund's complete adjuvant or Freund's incomplete adjuvant first
Epidemic disease animal；Again by African swine fever virus P11.5 protein-specific polypeptide epitope direct immunization animal；
D) serum of separating immune animal contains anti-African swine fever virus P11.5 protein-specific polyclonal antibody in serum.
5. the preparation method according to claim 4, which is characterized in that the animal is mouse or rabbit.
6. the anti-African swine fever virus P11.5 protein-specific Anti-TNF-α that the method according to claim 11 prepares
Body.
7. African swine fever virus P11.5 protein-specific polyclonal antibody according to claim 6 is in preparation detection Africa
Application in CSFV antigen and/or antibody kit.
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Cited By (1)
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| CN114075264A (en) * | 2020-08-26 | 2022-02-22 | 中国农业科学院兰州兽医研究所 | Polypeptide for promoting swine organisms to generate African swine fever virus antigen specific immune response and application thereof |
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