CN110456047A - A kind of infectious bursal disease virus antibody competition ELISA detection method and kit - Google Patents
A kind of infectious bursal disease virus antibody competition ELISA detection method and kit Download PDFInfo
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Abstract
The present invention relates to a kind of infectious bursal disease virus antibody competition ELISA detection method and kits, belong to field of biotechnology.IBDV VP2 gene optimization is designed, prokaryotic expression plasmid pET28+VP2 is constructed, converts Escherichia coli, the molecular weight of recombinant VP 2 albumen is about 50kDa.It uses the rVP2 albumen of purifying as screening antigen, screens IBDV VP2 monoclonal antibody.Use rVP2 albumen as envelope antigen, wherein one plant of VP2 monoclonal antibody of HRP- label is competition antibody, establishes IBDV antibody competition ELISA detection method (rVP2-cELISA).Compared with the IBDV antibody assay kit of existing commercialization, infectious bursal disease virus antibody competition ELISA detection method of the present invention, time-consuming shorter, specific more preferable, test sample range is wider, cost is less expensive and the advantages such as production is more convenient, has huge production application value.
Description
One, technical field
The present invention relates to a kind of infectious bursal disease virus antibody competition ELISA detection method and kits, belong to life
Object technical field.
Two, background technique
Gumboro disease (IBD) is caused by infectious bursal disease virus (IBDV) to encroach on the poult bursa of farbricius
For the infectious disease of main feature, which is to cause one of China and the serious Infectious Diseases of world's aviculture economic loss.It should
It is dead that disease not only causes infected animal, but also also results in immunity of organism inhibition, reduces the immune defense ability of body and to more
Kind vaccine immunization failure.Implementing vaccine inoculation to susceptible chicken group is the prevention disease most efficient method, but since various regions are flowed
The virulence of capable IBDV strain and antigenic difference, the every year economic loss as caused by IBD immuning failure or immunosupress
It is huge.In consideration of it, further analyzing the molecular epidemiology of current IBDV, developing economy, efficiently detection method is still current
Important topic urgently to be resolved in IBD prevention and control.
The main method of detection chicken IFN-γ V antibody has: neutralization test, fine jade expansion, indirect immunofluorescence (IFA) and indirect ELISA
(existing commercial kit envelope antigen is totivirus).Although neutralization test is the goldstandard of evaluation chicken group's antibody, but because of it
It is cumbersome, it takes a long time, and need to cultivate cell, is not suitable for the use of common farm.Although fine jade expands experiment, operation is not multiple
It is miscellaneous but not sensitive enough and take a long time, quick and sensitive purpose is not achieved.IFA operation is also comparatively laborious, needs to cultivate thin
Born of the same parents, and the fluorescence microscope for observing needs is expensive.And ELISA has special, easy, quick feature.Therefore, at present
The IBDV antibody level in chicken group is generally evaluated using indirect EILSA.But existing commercialization IBDV antibody ELISA detection examination
What agent box was all made of is indirect ELISA, and test sample needs two steps, time-consuming nearly 2 hours, and expensive.In view of this, originally grinding
Study carefully using competitive ELISA and detects IBDV antibody.This method only needs a step that can detect to blood serum sample, and time-consuming
30min is needed, compares indirect ELISA, more rapidly and efficiently.
Three, summary of the invention
Technical problem
Gumboro disease (IBD) is a kind of urgency of the chicken as caused by infectious bursal disease virus (IBDV) and turkey
Property, hot, highly contagious disease, are to cause one of China and the serious Infectious Diseases of world's aviculture economic loss.
Indirect EILSA is commonly used to evaluate the IBDV antibody level in chicken group.But the commercialization of existing import
IBDV antibody indirect ELISA detection kit is expensive, is unfavorable for promoting and applying；And domestic IBDV antibody indirect ELISA
Its envelope antigen of detection kit is the IBDV totivirus of purifying, and the culture of IBDV totivirus needs cell or chicken, culture
Higher cost, and IBDV purifying needs more expensive ultracentrifuge, takes a long time.And the IBDV antibody that this research is developed is competing
ELISA detection kit is striven, envelope antigen is used as using the recombinant VP 2 albumen (rVP2) of prokaryotic expression, the VP2 of HRP- label is mono-
Resist to compete antibody, the result of sample can be detected in 30min.It is detected than existing IBDV antibody indirect ELISA both at home and abroad
Kit has 4 big advantages: time-consuming shorter, specific more preferable, test sample range is wider, cost is less expensive and production is more convenient etc.
Advantage has huge production application value.
Technical solution
A kind of infectious bursal disease virus antibody competition ELISA detection method, which is characterized in that be with deposit number
The monoclonal antibody hybridoma cell strain 1H6 monoclonal of the infectivity resistant bursal disease poison VP2 albumen of CCTCC NO:C201966
Antibody is competition antibody.
Using rVP2 recombinant VP 2 albumen as envelope antigen, preparation method are as follows: IBDVVP2 gene optimization is designed, according to
The preferences optimization design VP2 gene of the listed IBDVVP2 sequence EU417824.1 of GenBank and e. coli codon,
The ATG of VP2 gene is removed, full genome synthesis, directed cloning enters in prokaryotic expression carrier pET-28a (+), constructs prokaryotic expression
Plasmid pET28+VP2 is converted Escherichia coli Rosetta (DE3)；After IPTG is induced, bacterium is collected by centrifugation, then according to
HisTrap HP specification purifies rVP2.
The detection method the specific steps are dilute the rVP2 recombinant protein of the purifying carbonate buffer solution of pH9.6
To 5 μ g/L, it is added to ELISA Plate, every 100 μ L of hole, 4 DEG C of coatings overnight, are closed, 37 with the PBST closing hydroful hole containing 10%FBS
DEG C, 2h, PBST are washed 3 times, and each 3min is patted dry；Diluted 50 96 parts of the hole μ L/ of clinical sample 1:500 is added, is added simultaneously
50 hole μ L/ of HRP-VP2 monoclonal antibody, HRP-VP2 monoclonal antibody 1:20000 dilution, 37 DEG C, 1h, PBST are washed 5 times, and each 3min is patted dry；
100 hole μ L/ of TMB color developing agent, magazine colour developing is added；2M H2SO4 is terminated, and surveys A450 value, determines result.Criterion: PI=
100- sample OD value/feminine gender mean OD value x100 is judged to the positive as PI > 50；As PI≤50, it is judged to feminine gender.
The infectious bursal disease virus antibody competition ELISA detection method can be applied directly.With the biography
Metachromia bursa of Fabricius virus antibody competition ELISA detection method can also construct kit application.
Beneficial effect
The infectious bursal disease virus antibody competition ELISA detection method that the present invention establishes, with existing commercialization
IBDV antibody assay kit is compared: time-consuming shorter, specific more preferable, test sample range is wider, cost is less expensive and production
The advantages such as more convenient have huge production application value.
The features and advantages of the invention are as follows:
The present invention is used as envelope antigen using the recombinant VP 2 albumen (rVP2) of prokaryotic expression, and the VP2 monoclonal antibody of HRP- label is
Antibody is competed, the result of sample can be detected in 30min.Than domestic and international existing IBDV antibody indirect ELISA detection reagent
Box has 4 big advantages；
1, specificity is more preferable: this kit uses the VP2 monoclonal antibody of HRP- label to compete antibody, and existing indirect
ELISA kit uses IBDV polyclonal antibody, it is well known that monoclonal antibody is more anti-with preferably specificity than more.
2, test sample range is wider: the VP2 monoclonal antibody that this kit uses HRP- to mark can not be examined to compete antibody
The limitation in sample source can detecte the sample of various animal origins or non-animal；And existing indirect ELISA reagent
Its ELIAS secondary antibody of box is the blood serum sample for chicken, and therefore, detection range can only be derived from the blood serum sample of chicken.
3, time-consuming shorter: this kit need to only compete measuring samples and HRP- the VP2 monoclonal antibody marked in test sample
Antibody is added simultaneously, only needs a step, detection time is in 30min or so；And existing indirect ELISA reagent kit, 2 steps are needed, are needed
It (to be taken: 1h or so) after serum to be checked is reacted with envelope antigen, then adding ELIAS secondary antibody reaction, (take: 1h is left
It is right), total time is in 2h or more.
4, cost is less expensive and production is more convenient: for from envelope antigen, the IBDV antibody competition of this research development
ELISA detection kit is used as envelope antigen using the recombinant VP 2 albumen (rVP2) of prokaryotic expression, only needs common shaking table just
Can be with the envelope antigen (rVP2) of our needs of mass production, and existing commercialization indirect ELISA reagent kit envelope antigen is
The culture of IBDV, IBDV need to use cell or chicken, high production cost, also inconvenience.In addition, the competition that this kit uses
Antibody is VP2 monoclonal antibody, only hybridoma need to be prepared ascites by mouse, 1 mouse can prepare 15ml
The ascites of left and right, and the VP2 monoclonal antibody that 1ml ascites is achieved with 4ml HRP- label also needs to mark HRP- in test sample
VP2 monoclonal antibody dilution 20000 or more, to sum up, 1 mouse preparation ascites can get HRP- label VP2 monoclonal antibody working solution it is total
Amount are as follows: 15ml ascites × 4mHRP- label VP2 monoclonal antibody × 20000=1.2 × 106Ml, the HRP- label that 1 kit needs
VP2 monoclonal antibody working solution be 100ml or so, so, the ascites of 1 mouse preparation can meet 10000 or more kits
Demand, cost is far below mostly anti-production cost.
Four, Detailed description of the invention
Fig. 1 10%SDS-PAGE analyzes expression of the VP2 overall length in Escherichia coli Transsetta (DE3)
The reactivity of Fig. 2 Westernblot analysis HRP-VP2 monoclonal antibody and rVP2
Biological deposits
Cell strain 1H6 is preserved in China typical culture collection center on May 9th, 2019, address: Wuhan, China is military
Chinese university, deposit number are CCTCC NO:C201966, classification naming: the monoclonal of infectivity resistant bursal disease poison VP2 albumen
Antibody hybridoma cell strain 1H6.
Five, specific embodiment
1 main experimental materials
The medium virulence attenuated vaccine strain of infectious bursal disease virus (IBDV) B87 is limited purchased from Nanjing day nation biotechnology
Company；E.coli Rosetta(DE)3Competent cell is purchased from Shanghai Wei Di Biotechnology Co., Ltd；Plasmid purification kit
Purchased from Omega company；DNA gel QIAquick Gel Extraction Kit is purchased from Quan Shijin Biotechnology Co., Ltd；Restriction enzyme is purchased from
TaKaRa company.
2 experimental methods
Expression of the 2.1 IBDV VP2 genes in Escherichia coli
2.1.1 the optimization of IBDV VP2 gene codon
Preferences according to the listed IBDVVP2 sequence (EU417824.1) of GenBank and e. coli codon are excellent
Change design VP2 gene, the ATG of VP2 gene is removed, make its merge pET28a (+) on His label, in Escherichia coli into
Row amalgamation and expression.
2.1.2 the building of IBDV VP2 DNA recombinant expression plasmid
By the VP2 gene of optimization design, full genome synthesis adds BamH I and I restriction enzyme site of Xho at its 5 ' end and 3 ' ends,
The VP2 gene of optimization design is synthesized by general biosystem (Anhui) Co., Ltd, and is cloned in prokaryotic expression carrier pET28a
In (+).It is identified with XhoI and MluI double digestion, and carries out sequencing analysis.The recombinant plasmid of acquisition is named as pET28-VP2 (4-
1350).It is analyzed with 1% agarose gel electrophoresis containing EB, it is seen that the DNA fragmentation of 2 length about 4.0Kb and 2.5Kb respectively, with
The theoretical value of pET28a (+) and VP2 genetic fragment is consistent.
2.1.3 expression of the IBDV VP2 gene in Escherichia coli
By recombinant plasmid pET28-VP2 (4-1350) Transformed E .coli Rosetta (DE)3Competent cell is coated on use
On 1.5% agar plate that LB is prepared, 37 DEG C of culture 14h choose single colonie and are inoculated with into LB, shaking culture 14h.Next day is with 1%
Amount is inoculated in LB culture medium (the 100 μ g/ml containing kalamycin) respectively, shaken cultivation, in A600When value is 0.8, it is separately added into dense eventually
Degree is the IPTG of 1.0mM, induces 4h respectively.The bacterial cultures of induction is taken, 5000g is centrifuged 5min, with 12%SDS-PAGE points
Analyse the expression of VP2 segment in thallus.Test synchronizes the E.coli Rosetta for setting up the conversion of pET28a (+) empty plasmid
(DE)3For control.Through 12%SDS-PAGE electrophoresis, there is the protein band of an entry at molecular weight about 50kDa, and pET28a (+)
E.coli Rosetta (the DE of conversion3) protein band then without 50kDa size.
2.1.4 Western blot analyzes the expression of IBDV VP2 albumen
The 3ml recombinant bacterium 5000g of inducing expression is centrifuged 10min, removes supernatant, 160 μ L deionized waters are added and are resuspended,
40 μ L 5 × SDS-Loading Buffer are added, carry out the SDS- of 12% separation gel and 5% concentration glue according to a conventional method
PAGE electrophoretic analysis, is transferred on nitrocellulose filter, and after transfer, nitrocellulose filter is sealed with 4 DEG C of 5% skimmed milk power
It closes overnight, then with primary antibody (His monoclonal antibody or chicken anti-ibd V hyper-immune serum), the secondary antibody (sheep anti-mouse igg antibody or HRP of HRP label
The rabbit-anti chicken igg antibody of label) it acts on therewith respectively, differential protein band is observed in the colour developing of DAB substrate.Test, which synchronizes, sets up sky
E.coli Rosetta (the DE of plasmid pET28a (+) conversion3) it is control.It develops the color through 12%SDS-PAGE electrophoresis, transfer and DAB
Afterwards, recombinant VP 2 albumen can have the protein band of an entry with His monoclonal antibody or chicken anti-ibd V hyper-immune serum at molecular weight about 50kDa,
And the E.coli Rosetta (DE of pET28a (+) conversion3) protein band then without 50kDa size.
2.1.5 the purifying of IBDV VP2 albumen
The culture for taking 200mL inducing expression, is collected by centrifugation bacterium, is resuspended with 30mL combination buffer, and 20 μ L are added
100mmol/L PMSF, ultrasonication is limpid to cell suspension under condition of ice bath, and 4 DEG C, it is broken that 10000g is centrifuged 30min removal cell
Piece takes 0.45 μm of aperture membrane filtration of supernatant, then purifies recombinant VP 2 protein fragments according to HisTrap HP specification.It is super
Contain a large amount of recombinant VP 2 albumen in sound precipitating, shows that recombinant VP 2 albumen mainly exists with inclusion bodies.
2.2 HRP mark IBDV VP2 gene monoclonal antibody
2.2.1 the preparation of IBDV VP2 gene monoclonal antibody
6~8 week old female BAl BIcs/c mouse is immunized with the IBDV of purifying, is immunized 1 time every 14d, after the 4th is immune, nothing
Bacterium takes immune BALB/c mice spleen cell, the SP2/0 cell fusion with logarithmic growth phase.Using rVP2 albumen as envelope antigen, use
Indirect elisa method surveys the culture supernatant of fused cell, has screened 8 plants of IBDVVP2 monoclonal antibodies.The positive hybridoma of selection is used
Limiting dilution assay is cloned.The positive hybridoma cell of cloning is expanded into culture, is injected intraperitoneally into 8~10 week old BALB/c
Female mice prepares ascites.
2.2.2 horseradish peroxidase (HRP) marks IBDV VP2 monoclonal antibody
Take 0.5ml ascites that the PBS dilution of equivalent is added, 3000r/min, 10min take supernatant with 0.45 μm of aperture filter membrane mistake
Then filter purifies VP2 monoclonal antibody according to HiTrap ProteinAHP specification.With horseradish peroxidase (HRP) by purifying
VP2 monoclonal antibody is marked with sodium periodate method[1]。
[1] Luo Jiali, Shen Ping Gui, Song Guangcheng, Chen Caiyun, Xue Hai raise the letter of horseradish peroxidase (HRP) labelled antibody
Easy method [J] Acta Biochimica et Biophysica Sinica, 1981 (01): 1-8.
2.2.3 western blot analyzes the reactivity of IBDV VP2 monoclonal antibody and rVP2
By pET28-VP2 (4-1350) Transformed E .coli Rosetta (DE)3With empty plasmid pET28a (+) conversion
E.coli Rosetta(DE3) inducing expression is carried out, SDS-PAGE electrophoretic analysis is transferred on nitrocellulose filter, 5% degreasing
Overnight, then using HRP-VP2 monoclonal antibody as primary antibody, differential protein band is observed in the colour developing of DAB substrate for 4 DEG C of milk powder closings.
After 10%SDS-PAGE electrophoresis, transfer and DAB colour developing, HRP-VP2 monoclonal antibody can be with the recombinant VP 2 egg of prokaryotic expression
The white protein band for having an entry at molecular weight about 50kDa, and the E.coli Rosetta (DE of pET28a (+) conversion3) then without
Protein band.The HRP-VP2 monoclonal antibody for being screened cell strain 1H6 can be with the recombinant VP 2 albumen of prokaryotic expression at molecular weight about 50kDa
There is the protein band of an entry.
The foundation of 2.3 IBDV antibody competition ELISA detection methods
The rVP2 recombinant protein of purifying is diluted to 5 μ g/L with the carbonate buffer solution of pH9.6, is added to ELISA Plate, often
100 μ L of hole, 4 DEG C of coatings overnight, are closed, 37 DEG C, 2h, PBST washing 3 times, often with the full hole confining liquid (PBST containing 10%FBS)
Secondary 3min, pats dry；Diluted 50 hole μ L/ (96 parts) of clinical sample 1:500 is added, while 50 hole μ L/ of HRP-VP2 monoclonal antibody is added
(HRP-VP2 monoclonal antibody 1:20000 dilution), 37 DEG C, 1h, PBST are washed 5 times, and each 3min is patted dry；100 μ of TMB color developing agent is added
The hole L/, magazine colour developing；2M H2SO4It terminates, surveys A450Value determines result.Criterion: PI=100- sample OD value/feminine gender is average
OD value x100 is judged to the positive as PI > 50；As PI≤50, it is judged to feminine gender.
The Preliminary Applications of 2.4 IBDV antibody competition ELISA detection methods
2.4.1 competitive ELISA
The rVP2 recombinant protein of purifying is diluted to 5 μ g/L with the carbonate buffer solution of pH9.6, is added to ELISA Plate, often
100 μ L of hole, 4 DEG C of coatings overnight, are closed, 37 DEG C, 2h, PBST washing 3 times, often with the full hole confining liquid (PBST containing 10%FBS)
Secondary 3min, pats dry；Diluted 50 hole μ L/ (96 parts) of clinical sample 1:500 is added, while 50 hole μ L/ of HRP-VP2 monoclonal antibody is added
(HRP-VP2 monoclonal antibody 1:20000 dilution), 37 DEG C, 1h, PBST are washed 5 times, and each 3min is patted dry；100 μ of TMB color developing agent is added
The hole L/, magazine colour developing；2M H2SO4It terminates, surveys A450Value determines result.IDEXX IBDV antibody indirect ELISA reagent is used simultaneously
Box detects clinical sample, as commercialization control group.
2.4.2 neutralization test
96 parts of clinical samples are carried out since 1: 2 to 2 multiple proportions serial dilutions respectively, are diluted to 1:64；100 μ L are respectively taken, point
It is not mixed with the cell adapted malicious dilution (viral level 200TCID50/0.1mL) of isometric IBDV B87, sets 37 DEG C of reactions
1h is inoculated in 96 well culture plates containing DF-1 single layer, 100 holes μ L/ (hole viral level 100TCID50/), every part of blood to be checked
4 repetitions are set clearly；Virus control, positive serum controls, negative serum control, cell controls are set up simultaneously.Set 37 DEG C, 5%CO2
Lesion is observed in culture daily, and record by the virucidin that Reed-Muench method calculates each blood serum sample as a result, imitated
On the contrary valence is then judged as positive according to national standard GB/T19167-2003, neutralization titer > 1:32, then be judged as feminine gender.
IBDV antibody competition ELISA, the IDEXX-ELISA (IBDV) and neutralization test established with recombinant VP 2 albumen is simultaneously
96 parts of clinical samples are detected, as a result: the I BDV antibody cELISA detection method of recombinant VP 2 albumen foundation, IDEXX-ELISA
(IBDV) compared with neutralization test, relative sensitivity is respectively 94.12% and 100%；Relative specificity is respectively 95.16% He
25.8%；Coincidence rate is respectively 94.79% and 52.08%.Concrete outcome is following (Tables 1 and 2).
1 rVP2-cELISA of table is compared with neutralization test
Relative sensitivity: 32/34 × 100%=94.12%；
Relative specificity: 59/62 × 100%=95.16%；
Coincidence rate: (32+59)/96 × 100%=94.79%
2 IDEXX-ELISA of table is compared with neutralization test
Relative sensitivity: 34/34 × 100%=100%；
Relative specificity: 16/62 × 100%=25.8%；
Coincidence rate: (34+16)/96 × 100%=52.08%
Claims (5)
1. a kind of infectious bursal disease virus antibody competition ELISA detection method, which is characterized in that be with deposit number
The monoclonal antibody hybridoma cell strain 1H6 monoclonal of the infectivity resistant bursal disease poison VP2 albumen of CCTCC NO:C201966
Antibody is competition antibody.
2. infectious bursal disease virus antibody competition ELISA detection method according to claim 1, it is characterised in that:
Using rVP2 recombinant VP 2 albumen as envelope antigen, preparation method are as follows: IBDVVP2 gene optimization is designed, according to
The preferences optimization design VP2 gene of the listed IBDVVP2 sequence EU417824.1 of GenBank and e. coli codon,
The ATG of VP2 gene is removed, full genome synthesis, directed cloning enters in prokaryotic expression carrier pET-28a (+), constructs prokaryotic expression
Plasmid pET28+VP2 is converted Escherichia coli Rosetta (DE3)；After IPTG is induced, bacterium is collected by centrifugation, then according to
HisTrap HP specification purifies rVP2.
3. infectious bursal disease virus antibody competition ELISA detection method according to claim 2, it is characterised in that:
The detection method the specific steps are, the rVP2 recombinant protein of purifying is diluted to 5 μ g/L with the carbonate buffer solution of pH9.6,
It is added to ELISA Plate, every 100 μ L of hole, 4 DEG C of coatings overnight, are closed, 37 DEG C, 2h with the PBST closing hydroful hole containing 10%FBS,
PBST is washed 3 times, and each 3min is patted dry；Diluted 50 96 parts of the hole μ L/ of clinical sample 1:500 is added, while HRP-VP2 is added
50 hole μ L/ of monoclonal antibody, HRP-VP2 monoclonal antibody 1:20000 dilution, 37 DEG C, 1h, PBST are washed 5 times, and each 3min is patted dry；TMB is added
100 hole μ L/ of color developing agent, magazine colour developing；2M H2SO4 is terminated, and surveys A450 value, determines result.Criterion: PI=100- sample
OD value/feminine gender mean OD value x100 is judged to the positive as PI > 50；As PI≤50, it is judged to feminine gender.
4. the application of infectious bursal disease virus antibody competition ELISA detection method described in one of claim 1-3.
5. the examination of infectious bursal disease virus antibody competition ELISA detection method building described in one of claim 1-3
Agent box.
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