CN103554234A - Competitive ELISA method based on foot-and-mouth disease A type VP1 protein and its monoclonal antibody - Google Patents

Competitive ELISA method based on foot-and-mouth disease A type VP1 protein and its monoclonal antibody Download PDF

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CN103554234A
CN103554234A CN201310399981.7A CN201310399981A CN103554234A CN 103554234 A CN103554234 A CN 103554234A CN 201310399981 A CN201310399981 A CN 201310399981A CN 103554234 A CN103554234 A CN 103554234A
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foot
mouth disease
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熊毅
蒋家霞
冯淑萍
颜健华
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GUANGXI ZHUANG AUTONOMOUS REGION CENTER FOR ANIMAL DISEASE CONTROL AND PREVENTION
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Abstract

The invention relates to a competitive ELISA method based on a foot-and-mouth disease A type VP1 protein and its monoclonal antibody, also relates to a preparation method of the foot-and-mouth disease A type VP1 protein, and a preparation method of the monoclonal antibody of the foot-and-mouth disease A type VP1 protein, and belongs to the technical field of animal immunological detection. In the invention, a primer pair C1 and C2 and a primer pair E1 and E2 are amplified to obtain a gene sequence of the foot-and-mouth disease A type VP1 protein, the foot-and-mouth disease A type VP1 protein is obtained by constructing an expression plasmid, introducing the expression plasmid into a prokaryotic expression host and carrying out inducible purification, the foot-and-mouth disease A type VP1 protein monoclonal antibody is obtained by treating the foot-and-mouth disease A type VP1 protein as an antigen through a hybridomas technology, and the competitive ELISA method used for detecting a foot-and-mouth disease A type antibody is established based on the foot-and-mouth disease A type VP1 protein and its monoclonal antibody. The detection method has a strong specificity and a good stability, and can be used for detecting a foot-and-mouth disease A type serum antibody. By comparing a result obtained through the detection method with a liquid phase blocking ELISA kit, the coincidence rate is 95.8%.

Description

Competitive ELISA method based on foot and mouth disease A type VP1 albumen and monoclonal antibody thereof
Technical field
The invention belongs to animal immunology detection technique field, particularly relate to a kind of foot and mouth disease A type VP1 albumen and preparation method thereof, and a kind of foot and mouth disease A type VP1 albumen based on preparing and the competitive ELISA method of monoclonal antibody thereof.
Background technology
Foot and mouth disease is the highly infective being caused by foot and mouth disease virus (FMDV) and the animal epidemic with Important Economic meaning, infected cattle, pig, sheep, goat and other artiodactyl.After zoogenetic infection foot and mouth disease, the positions such as mouth, hoof, nipple there will be bubble to form erosion, and the animal of morbidity is to be generally optimum process.Although foot and mouth disease mortality ratio is low, because its sickness rate height almost reaches 100%, and velocity of propagation is very fast, has restricted the international trade that participates in animal and animal product between the development of livestock industry and country, and OIE is classified as category-A animal epidemic.
Foot and mouth disease exists seven serotypes (O, A, C, SAT1, SAT2, SAT3 and Asia1) and more than 80 hypotype, in each serotype, there are antigenicity and epidemiological features separately, do not have cross immunity, immune One serotype vaccine can not be resisted another serotype; In each serotype, also having its different genotype, also there is difference to a certain degree in antigenicity.This species diversity, mainly due to high mutation rate and the regroup of virus, has brought difficulty also to diagnosis, control and the elimination of foot and mouth disease disease.O type is most popular serotype in global range, spreads all over middle country in Southeast Asia; Asia1 starts to be confined to Asia, has progressively intruded in recent years middle east; The tool diversity of A type antigen, other serotype is more easily recombinated relatively, mainly in Asia, Europe, Africa and Latin America distributes; South Africa 1,2,3 types (SAT1-3) are mainly popular in Africa.
, since the foot and mouth disease of O, A, these three kinds of serotypes of Asia-I occurs reported first in 1958, there is thereafter generation in China once in a while.A type foot and mouth disease has been imported Xinjiang region in the 1964 Nian Cong Soviet Union and has again been caused popular, then popular on a large scale less than occurring.Until within 2009, break out A type foot and mouth disease in Wuhan, Hubei, current popular strain confirms the strain similar (Thailand, Malaysia) separated with 2008-2009 Nian country in Southeast Asia according to foot and mouth disease VP1 gene genetic evolutionary analysis, belongs to A/ASIA/Sea-97 pedigree; The same year in Chinese Shanghai, Shandong, Jiangsu, Guangxi, Guizhou and six, Xinjiang province also reported breaking out of A type foot and mouth disease, within 2010, in the Korea S without the nonimmune area of foot and mouth disease, occurred too.Nick J. Knowles [20]deng what report country in Southeast Asia A type foot and mouth disease in recent years, popularly after O and Asia1 type, again caused breaking out of East Asia Region, this transnational propagation of foot and mouth disease, make the foot and mouth disease epidemic situation of China more complicated, epidemic preventing working is more rigorous, to the research of A type foot and mouth disease, should more go deep into.The south ，Yu country in Southeast Asia that Guangxi is located in China's Mainland is adjacent, and the liberalization of Association of South-east Asian Nations trade area, is faced with the threat of invasion, and this needs us to carry out foot and mouth disease monitoring and prevention and control.
ELISA is the abbreviation of enzyme linked immuno-sorbent assay, be that enzyme connects immunosorption experiment, be the important method that current foot-and-mouth disease antibody detects, the method is divided into LPB-ELISA (LPB-ELISA), and solid phase competitive ELISA (SPC-ELISA).Wherein SPC-ELISA, with its hypersensitivity and high correlation, becomes the foot-and-mouth disease antibody detection method of main flow just gradually.But the weakness of SPC-ELISA is operation more complicated, even experienced operator also must need just can complete for 4-5 hour.The current research situation for foot and mouth disease aspect, as patent CN201110001730.X, this invention is combined in foot-and-mouth disease antibody on elisa plate by coating technique, then pass through Ag-Ab association reaction by foot-and-mouth disease antigen mortise elisa plate hole, through stabilizer treatment with after being dried, carry out vacuum packaging, finally set low warm prolonged preservation, when needs detect foot-and-mouth disease antibody, this elisa plate can be directly used in to experiment, but the elisa plate prolonged preservation of this invention, to produce certain error for this detection method, and can not be by O, A, the foot and mouth disease of these three kinds of serotypes of Asia-I distinguishes, be unfavorable for promoting the use of.
Summary of the invention
The object of the invention is detection technique for existing foot and mouth disease A type serum exist testing cost high, detect the deficiencies such as troublesome poeration, resultant error are large, a kind of competitive ELISA method based on foot and mouth disease A type VP1 albumen and monoclonal antibody thereof is provided, invention relates to the preparation of A type fmd protein VP1 monoclonal antibody, and applies to the detection of cause of disease, antibody and the development of vaccine etc.; The method of having set up monoclonal antibody competitive ELISA detects A type antibody, forms A type foot and mouth disease immunologic surveillance technology platform.
The technical scheme that the present invention realizes is: a kind of foot and mouth disease A type VP1 albumen, comprise foot and mouth disease A type VP1 albumen 1 and foot and mouth disease A type VP1 albumen 2 that two kinds of different phraseologies obtain, foot and mouth disease A type VP1 albumen 1 and foot and mouth disease A type VP1 albumen 2 aminoacid sequences are shown in SEQ ID NO:1.
A kind of method of preparing foot and mouth disease A type VP1 albumen, the foot and mouth disease virus FMDV genome of take is template, utilize a pair of clone's primer and a pair of expression primer to carry out pcr amplification and obtain FMDV-A-VP1 gene, by FMDV-A-VP1 gene respectively with pET-32a, pGEX-6p-1 plasmid connects structure and obtains expression plasmid PET-32a-VP1, pGEX-6p-1-VP1, again expression plasmid PET-32a-VP1 and pGEX-6p-1-VP1 are proceeded to respectively to abduction delivering in prokaryotic expression host e. coli BL21 and obtain fusion rotein 1 and fusion rotein 2 respectively, fusion rotein 1 and the fusion rotein 2 respectively purified aminoacid sequence that obtains are the foot and mouth disease A type VP1 albumen 1 shown in SEQ ID NO:1 and foot and mouth disease A type VP1 albumen 2.
As the present invention, prepare the further restriction of the method for foot and mouth disease A type VP1 albumen, described a pair of clone's primer comprises primer C1:5 '-TACCAAATTACACGGGAA-3 ', primer C2:5 '-GACATGTCCTCCTGCATCTG-3 '.
As the present invention, prepare the further restriction of the method for foot and mouth disease A type VP1 albumen, described a pair of expression primer comprises primer E1:5 '-CCGGAATTCACCACCACTGTTGAGAACTACG-3 ', primer E2:5 '-CCGCTCGAGTCATTACAGGAGTTGCTTTGCAGGTGC-3 '.
As the present invention, prepare the further restriction of the method for foot and mouth disease A type VP1 albumen, in described pcr amplification process, annealing temperature is 54.5 ~ 57.5 ℃; Described fusion protein purification is process His and GST column chromatography purification.
It is abundant for the present invention is disclosed, the structure recombinant gene expression vector relating in of the present invention, abduction delivering recombination, chromatogram purification obtain foot and mouth disease A type VP1 protein process step, according to the conventional existing method in laboratory, can realize, the concrete steps of wherein said pcr amplification are as follows:
Steps A: the foot and mouth disease A C-type virus C gene DNA of take is template, utilize first couple of clone primer C1, C2 amplification to obtain the sequence (886bp) that comprises FMDV-A-VP1 gene, then by second couple of expression primer E1, E2 this sequence that increases, obtain FMDV-A-VP1 gene (639bp).
Step B: the FMDV-A-VP1 gene (639bp) obtaining with steps A amplification carries out purifying, connects pMD18-T carrier, builds pMD18-T-VP1 cloning vector.
Step C: it is template that the step C of take builds pMD18-T-VP1, utilize expression primer E1, E2 to carry out pcr amplification, purifying reclaims PCR product, with EcoR I and Xhol I restriction endonuclease, this PCR purified product and expression vector pET-32a, pGEX-6p-1 is carried out respectively to double digestion.
The implementation procedure of above-described pcr amplification relies on existing round pcr, by pcr amplification repeatedly, and genetically engineered double digestion technology, realizes the prokaryotic expression of albumen.
A method of preparing the above foot and mouth disease A type VP1 albumen monoclonal antibody, this preparation method comprises the following steps:
Step is animal immune a): the BALB/c female mice in 5 ~ 7 week age of foot and mouth disease A type VP1 albumen 1 immunity of utilizing that expression plasmid PET-32a-VP1 induction obtains, after 3 immunity, filter out the tire mouse of > 1:12800 of immunoassay.
Step b) cytogamy: the splenocyte of getting the immune mouse that step a) obtains, carry out cytogamy with myeloma cell SP2/0, utilize expression plasmid pGEX-6p-1-VP1 to induce the foot and mouth disease A type VP1 albumen 2 obtaining as envelope antigen, the cell after merging is carried out to indirect ELISA screening and obtain hybridoma.The identical sequence albumen that described foot and mouth disease A type VP1 albumen 1, foot and mouth disease A type VP1 albumen 2 obtain for different phraseologies, both Argine Monohydrochloride sequences are shown in SEQ ID NO:1.
Step c) hybridoma a large amount of clones of monoclonal antibody: by step b) obtaining is expelled in mouse peritoneal, and the mouse after raising injection 1 ~ 3 week, collects the mouse ascites that belly expands, and ascites is carried out to purifying and obtain foot and mouth disease A type VP1 albumen monoclonal antibody.
A kind of competitive ELISA method based on foot and mouth disease A type VP1 albumen and monoclonal antibody thereof, the method comprise diluted sample, antigen coated, sealing, application of sample and add monoclonal antibody, add enzymic-labelled antibody, colour developing and termination, measured value, data processing, this competitive ELISA detection method step comprises:
Step 1) diluted sample: the dilution of serum sample is original volume 2 ~ 4 times, obtain test serum sample.
Step 2) antigen coated: envelope antigen used is the foot and mouth disease A type VP1 albumen 2 that expression plasmid pGEX-6p-1-VP1 induction obtains, and the coated concentration of envelope antigen is 0.5 ~ 0.7ug/mL.As the further restriction that the present invention is based on the competitive ELISA method of foot and mouth disease A type VP1 albumen and monoclonal antibody thereof, described foot and mouth disease A type VP1 albumen 2 its aminoacid sequences are shown in SEQ ID NO:1.
Step 3) sealing: by confining liquid sealing step 2) coated antigen, be 40 ~ 60min off-period, the enzyme plate after being sealed.
Step 4) application of sample with add monoclonal antibody: by step 1) the test serum sample that obtains joins step 3) in enzyme plate after the sealing that obtains, serum antibody is combined with envelope antigen, adding extent of dilution is the foot and mouth disease A type VP1 albumen monoclonal antibody of 1:300 ~ 1:500 again, make foot and mouth disease A type VP1 albumen monoclonal antibody in conjunction with not with the residue envelope antigen of serum antibody response, obtain sample, the reacted enzyme plate of monoclonal antibody.
Step 5) adds enzymic-labelled antibody: after the sample obtaining in step 4), monoclonal antibody reaction, in enzyme plate, add enzyme labelled antibody, the weaker concn of enzymic-labelled antibody is 1:4500 ~ 6000, enzymic-labelled antibody is combined with foot and mouth disease A type VP1 albumen monoclonal antibody, and be not combined with serum antibody, obtain adding the enzyme plate after enzyme labelling monoclonal antibody.
Step 6) colour developing with stop: adding of obtaining in step 5) adds freshly prepared substrate solution in the enzyme plate after enzymic-labelled antibody, the rear room temperature lucifuge of vibration mixing is hatched 10 ~ 20min and is developed the color, every hole adds stop buffer again, blending incubation 5 ~ 10min termination reaction, obtains the solution that can measure.
Step 7) measured value: the solution of measuring determination step 6) obtaining is at OD 450nmthe light absorption value A at place 450nm;
Step 8) data processing: the light absorption value A that utilizes step 7) to measure 450nmcalculate testing sample inhibiting rate, described testing sample inhibiting rate PI%=(1-A 450sample/A 450negative control) * 100.
As the further restriction that the present invention is based on the competitive ELISA method of foot and mouth disease A type VP1 albumen and monoclonal antibody thereof, described confining liquid be in 1%BSA solution, 1% gelatin solution, 5% foetal calf serum solution, 5% skim-milk solution any.
As the further restriction that the present invention is based on the competitive ELISA method of foot and mouth disease A type VP1 albumen and monoclonal antibody thereof, the antibody of described enzymic-labelled antibody is goat anti-mouse IgG-HRP, and the antibody in enzymic-labelled antibody is for take two antibody goat anti-mouse IgG-HRP of foot and mouth disease A type VP1 albumen monoclonal antibody isolated anti-foot and mouth disease A type VP1 albumen monoclonal antibody after antigen is expelled to goat; Described A 450negative control is the light absorption value A out of the determination of serum with not infecting FMDV virus 450.
Substantive distinguishing features of the present invention and marked improvement are:
(1) to utilize RT-PCR and albumen pronucleus expression technology successfully to prepare purity higher and have good specificity and immunogenic foot and mouth disease A type VP1 albumen 1 and foot and mouth disease A type VP1 albumen 2 in the present invention.The foot and mouth disease A type VP1 albumen 1 of acquisition of the present invention and foot and mouth disease A type VP1 albumen 2 can be used as the preparation of its monoclonal antibody, due to the foot and mouth disease A type VP1 albumen that adopts two kinds of different phraseologies to obtain, make monoclonal antibody preparation process efficiency higher, the probability that obtains monoclonal antibody is larger.
(2) adopt hybridoma technology, the foot and mouth disease A type VP1 albumen 1 of take is immunogen, foot and mouth disease A type VP1 albumen 2 has been set up indirect ELISA screening method for envelope antigen, screen, prepared the hybridoma cell strain of anti-foot and mouth disease A type VP1 albumen, hybridoma cell strain monoclonal antibody all can be blocked by foot and mouth disease A type positive serum, the qualification result of monoclonal antibody biological nature shows to select this monoclonal antibody as the detection antibody of competitive ELISA, and the monoclonal antibody that has solved the specific recognition of competitive ELISA detection method obtains a difficult problem.
(3) take foot and mouth disease A type VP1 albumen 1 is envelope antigen, the monoclonal antibody of foot and mouth disease A type VP1 albumen is competition antibody, the monoclonal antibody competitive ELISA method that detects foot and mouth disease A type serum antibody has been optimized in foundation, obtains that a kind of high specificity, stability are high, the antibody detection method of foot and mouth disease A type quickly and easily.
(4) determined competitive ELISA negative and positive judgement inhibiting rate threshold value, (PI) >=30% is positive for inhibiting rate, and < 30% is negative for inhibiting rate (PI).The present invention is based on foot and mouth disease A type VP1 albumen and antibody thereof and distinguish the competitive ELISA method that foot and mouth disease A type infects, compare with LPB-ELISA detection kit, coincidence rate is 95.8%, the advantages such as this explanation detection method high specificity of the present invention, good stability, can be used for the detection of foot and mouth disease A type serum antibody, this provides technology platform for the monitoring of foot and mouth disease A type immune antibody level detection, epidemic situation and epidemiology survey.
Accompanying drawing explanation
The amplification of the sequence (886bp) of Fig. 1 .FMDV-A-VP1 gene, in figure, left arrow indication is FMDV-A-VP1 gene (886bp).
The amplification of Fig. 2 .FMDV-A-VP1 gene (639bp), in figure, left arrow indication is FMDV-A-VP1 gene (639bp).
Fig. 3. the SDS-PAG electrophorogram of foot and mouth disease A type VP1 albumen 1, in figure, right arrow indication is foot and mouth disease A type VP1 albumen 1 band.
Fig. 4. the SDS-PAG electrophorogram of foot and mouth disease A type VP1 albumen 2, in figure, right arrow indication is foot and mouth disease A type VP1 albumen 2 bands.
Embodiment
Below in conjunction with Figure of description and embodiment, a kind of competitive ELISA method and application thereof based on foot and mouth disease A type VP1 albumen monoclonal antibody of the present invention described, these descriptions are not that content of the present invention is further limited, in following examples, all reagent used all can be bought by commercial means, and the operation steps in following examples is all chamber routine operation method realizations by experiment if no special instructions.
the acquisition of embodiment 1 foot and mouth disease A type VP1 albumen
(1) design of primers
The foot and mouth disease virus A type gene order of having delivered according to GenBank, designs the sequence that a pair of clone's primer (C1, C2) amplification comprises FMDV-A-VP1 gene, and size is 886bp; A pair of for the specific expressed primer of complete VP1 gene (639bp) (E1, E2) according to prokaryotic expression carrier and goal gene sequences Design, and insert EcoR I and two restriction enzyme sites of Xhol I.Primer sequence send Dalian Bao Bio-Engineering Company synthetic.
The primer nucleotide sequence of table 1 amplification FMDV-A-VP1 gene
Figure 25036DEST_PATH_IMAGE001
(2) gene clone
The foot and mouth disease A C-type virus C gene DNA of take is template, utilize first couple of clone primer C1, C2 amplification to obtain the sequence (886bp) that comprises FMDV-A-VP1 gene, then by second couple of expression primer E1, E2 this sequence that increases, obtain FMDV-A-VP1 gene (639bp).As shown in Figure 1, FMDV-A-VP1 gene (639bp) clone result as shown in Figure 2 for the sequence of FMDV-A-VP1 gene (886bp) clone result.Reaction system and reaction process in gene clone process are as shown in table 2.
Table 2. polymerase chain reaction (PCR) system:
(3) expression vector establishment, abduction delivering, purifying
FMDV-A-VP1 gene is connected with pET-32a, pGEX-6p-1 plasmid respectively to build and obtains expression plasmid PET-32a-VP1, pGEX-6p-1-VP1, expression plasmid PET-32a-VP1 and pGEX-6p-1-VP1 are proceeded to respectively to abduction delivering in prokaryotic expression host e. coli BL21 again and obtain fusion rotein 1 and fusion rotein 2 respectively, fusion rotein 1 and fusion rotein 2 are respectively through obtaining foot and mouth disease A type VP1 albumen 1 and foot and mouth disease A type VP1 albumen 2 through His and GST column chromatography purification.The electrophorogram of fusion rotein is as Fig. 3, Fig. 4.The identical sequence albumen that foot and mouth disease A type VP1 albumen 1, foot and mouth disease A type VP1 albumen 2 obtain for different phraseologies, both protein sequences are shown in SEQ ID NO:1.
the acquisition of embodiment 2 foot and mouth disease A type VP1 albumen monoclonal antibodies
Step is animal immune a): the BALB/c female mice in 7 week age of foot and mouth disease A type VP1 albumen 1 immunity of utilizing that expression plasmid PET-32a-VP1 induction obtains, after 3 immunity, filters out each immunoassay and tire and be greater than the mouse of 1:12800.
Step b) cytogamy: the splenocyte of getting the immune mouse that step a) obtains, carry out cytogamy with myeloma cell SP2/0, utilize expression plasmid pGEX-6p-1-VP1 to induce the foot and mouth disease A type VP1 albumen 2 obtaining as envelope antigen, the cell after merging is carried out to indirect ELISA screening and obtain hybridoma.
Step c) hybridoma a large amount of clones of monoclonal antibody: by step b) obtaining is expelled in mouse peritoneal, and the mouse after raising injection, after 1 week, is collected the mouse ascites that belly expands, and ascites is carried out to purifying and obtain foot and mouth disease A type VP1 albumen monoclonal antibody.
The identical sequence albumen that foot and mouth disease A type VP1 albumen 1, foot and mouth disease A type VP1 albumen 2 obtain for different phraseologies, both protein sequences are shown in SEQ ID NO:1.
the competitive ELISA detection method of embodiment 3 Schweineseuche A type serum
Get the porcine blood serum of 6 doubtful infection foot and mouth disease A types, this serum is carried out to yin and yang attribute judgement, the method comprise diluted sample, antigen coated, sealing, application of sample and add monoclonal antibody, add enzymic-labelled antibody, colour developing and termination, measured value, data processing, in this competitive ELISA detection method:
Step 1) diluted sample: the dilution of serum sample is original volume 4 times, obtain test serum sample.
Step 2) antigen coated: envelope antigen used is the foot and mouth disease A type VP1 albumen 2 that expression plasmid pGEX-6p-1-VP1 induction obtains, and the coated concentration of envelope antigen is 0.625ug/mL.
Step 3) sealing: by 1%BSA solution confining liquid sealing step 2) coated antigen, be 40min off-period, the enzyme plate after being sealed.
Step 4) application of sample with add monoclonal antibody: by step 1) the test serum sample that obtains joins step 3) in enzyme plate after the sealing that obtains, serum antibody is combined with envelope antigen, adding extent of dilution is the foot and mouth disease A type VP1 albumen monoclonal antibody of 1:400 again, make foot and mouth disease A type VP1 albumen monoclonal antibody in conjunction with not with the residue envelope antigen of serum antibody response, obtain sample, the reacted enzyme plate of monoclonal antibody.
Step 5) adds enzymic-labelled antibody: after the sample obtaining in step 4), monoclonal antibody reaction, in enzyme plate, add enzyme labelled antibody, the weaker concn of enzymic-labelled antibody is 1:4500, enzymic-labelled antibody is combined with foot and mouth disease A type VP1 albumen monoclonal antibody, and be not combined with serum antibody, obtain adding the enzyme plate after enzymic-labelled antibody.The antibody of enzymic-labelled antibody used is goat anti-mouse IgG-HRP.
Step 6) colour developing with stop: adding of obtaining in step 5) adds freshly prepared substrate solution in the enzyme plate after enzymic-labelled antibody, the rear room temperature lucifuge of vibration mixing is hatched 20min and is developed the color, every hole adds stop buffer again, and blending incubation 5min termination reaction, obtains the solution that can measure.
Step 7) measured value: the solution of measuring determination step 6) obtaining is at OD 450nmthe light absorption value A at place 450nm;
Step 8) data processing: the light absorption value A that utilizes step 7) to measure 450nmcalculate testing sample inhibiting rate, described testing sample inhibiting rate PI%=(1-A 450sample/A 450negative control) * 100.Detected result is as shown in table 3, and accuracy rate and LPB-ELISA detection method are compared and calculated.
the competitive ELISA detection method of 4 Ns of foot and mouth disease A type serum of embodiment
Get the bovine serum of 6 doubtful infection foot and mouth disease A types, this serum is carried out to negative and positive judgement, the method comprise diluted sample, antigen coated, sealing, application of sample and add monoclonal antibody, add enzymic-labelled antibody, colour developing and termination, measured value, data processing, in this competitive ELISA detection method:
Step 1) diluted sample: the dilution of serum sample is original volume 2 times, obtain test serum sample.
Step 2) antigen coated: envelope antigen used is the foot and mouth disease A type VP1 albumen 2 that expression plasmid pGEX-6p-1-VP1 induction obtains, and the coated concentration of envelope antigen is 0.7ug/mL.
Step 3) sealing: by 1% gelatin solution confining liquid sealing step 2) coated antigen, be 60min off-period, the enzyme plate after being sealed.
Step 4) application of sample with add monoclonal antibody: by step 1) the test serum sample that obtains joins step 3) in enzyme plate after the sealing that obtains, serum antibody is combined with envelope antigen, adding extent of dilution is the foot and mouth disease A type VP1 albumen monoclonal antibody of 1:300 again, make foot and mouth disease A type VP1 albumen monoclonal antibody in conjunction with not with the residue envelope antigen of serum antibody response, obtain sample, the reacted enzyme plate of monoclonal antibody.
Step 5) adds enzymic-labelled antibody: after the sample obtaining in step 4), monoclonal antibody reaction, in enzyme plate, add enzyme labelled antibody, the weaker concn of enzymic-labelled antibody is 1:6000, enzymic-labelled antibody is combined with foot and mouth disease A type VP1 albumen monoclonal antibody, and be not combined with serum antibody, obtain adding the enzyme plate after enzyme labelling monoclonal antibody.The antibody of enzymic-labelled antibody used is goat anti-mouse IgG-HRP.
Step 6) colour developing with stop: adding of obtaining in step 5) adds freshly prepared substrate solution in the enzyme plate after enzymic-labelled antibody, the rear room temperature lucifuge of vibration mixing is hatched 10min and is developed the color, every hole adds stop buffer again, and blending incubation 10min termination reaction, obtains the solution that can measure.
Step 7) measured value: the solution of measuring determination step 6) obtaining is at OD 450nmthe light absorption value A at place 450nm;
Step 8) data processing: the light absorption value A that utilizes step 7) to measure 450nmcalculate testing sample inhibiting rate, described testing sample inhibiting rate PI%=(1-A 450sample/A 450negative control) * 100.Detected result is as shown in table 3, and accuracy rate and LPB-ELISA detection method are compared and calculated.
the competitive ELISA detection method of embodiment 5 goat foot and mouth disease A type serum
Get the lowlenthal serum of 6 doubtful infection foot and mouth disease A types, this serum is carried out to negative and positive judgement, the method comprise diluted sample, antigen coated, sealing, application of sample and add monoclonal antibody, add enzymic-labelled antibody, colour developing and termination, measured value, data processing, in this competitive ELISA detection method:
Step 1) diluted sample: the dilution of serum sample is original volume 3 times, obtain test serum sample.
Step 2) antigen coated: envelope antigen used is the foot and mouth disease A type VP1 albumen 2 that expression plasmid pGEX-6p-1-VP1 induction obtains, and the coated concentration of envelope antigen is 0.5ug/mL.
Step 3) sealing: by 5% foetal calf serum solution confining liquid sealing step 2) coated antigen, be 60min off-period, the enzyme plate after being sealed.
Step 4) application of sample with add monoclonal antibody: by step 1) the test serum sample that obtains joins step 3) in enzyme plate after the sealing that obtains, serum antibody is combined with envelope antigen, adding extent of dilution is the foot and mouth disease A type VP1 albumen monoclonal antibody of 1:500 again, make foot and mouth disease A type VP1 albumen monoclonal antibody in conjunction with not with the residue envelope antigen of serum antibody response, obtain sample, the reacted enzyme plate of monoclonal antibody.
Step 5) adds enzymic-labelled antibody: after the sample obtaining in step 4), monoclonal antibody reaction, in enzyme plate, add enzyme labelled antibody, the weaker concn of enzymic-labelled antibody is 1:5000, enzymic-labelled antibody is combined with foot and mouth disease A type VP1 albumen monoclonal antibody, and be not combined with serum antibody, obtain adding the enzyme plate after enzyme labelling monoclonal antibody.The antibody of enzymic-labelled antibody used is goat anti-mouse IgG-HRP.
Step 6) colour developing with stop: adding of obtaining in step 5) adds freshly prepared substrate solution in the enzyme plate after enzymic-labelled antibody, the rear room temperature lucifuge of vibration mixing is hatched 15min and is developed the color, every hole adds stop buffer again, and blending incubation 7min termination reaction, obtains the solution that can measure.
Step 7) measured value: the solution of measuring determination step 6) obtaining is at OD 450nmthe light absorption value A at place 450nm;
Step 8) data processing: the light absorption value A that utilizes step 7) to measure 450nmcalculate testing sample inhibiting rate, described testing sample inhibiting rate PI%=(1-A 450sample/A 450negative control) * 100.Detected result is as shown in table 3, and accuracy rate and LPB-ELISA detection method are compared and calculated.
the competitive ELISA detection method of embodiment 6 sheep foot and mouth disease A type serum
Get the sheep serum of 6 doubtful infection foot and mouth disease A types, this serum is carried out to negative and positive judgement, the method comprise diluted sample, antigen coated, sealing, application of sample and add monoclonal antibody, add enzymic-labelled antibody, colour developing and termination, measured value, data processing, in this competitive ELISA detection method:
Step 1) diluted sample: the dilution of serum sample is original volume 4 times, obtain test serum sample.
Step 2) antigen coated: envelope antigen used is the foot and mouth disease A type VP1 albumen 2 that expression plasmid pGEX-6p-1-VP1 induction obtains, and the coated concentration of envelope antigen is 0.6ug/mL.
Step 3) sealing: by 5% skim-milk solution confining liquid sealing step 2) coated antigen, be 50min off-period, the enzyme plate after being sealed.
Step 4) application of sample with add monoclonal antibody: by step 1) the test serum sample that obtains joins step 3) in enzyme plate after the sealing that obtains, serum antibody is combined with envelope antigen, adding extent of dilution is the foot and mouth disease A type VP1 albumen monoclonal antibody of 1:500 again, make foot and mouth disease A type VP1 albumen monoclonal antibody in conjunction with not with the residue envelope antigen of serum antibody response, obtain sample, the reacted enzyme plate of monoclonal antibody.
Step 5) adds enzymic-labelled antibody: after the sample obtaining in step 4), monoclonal antibody reaction, in enzyme plate, add enzyme labelled antibody, the weaker concn of enzymic-labelled antibody is 1:5500, enzymic-labelled antibody is combined with foot and mouth disease A type VP1 albumen monoclonal antibody, and be not combined with serum antibody, obtain adding the enzyme plate after enzyme labelling monoclonal antibody.The antibody of enzymic-labelled antibody used is goat anti-mouse IgG-HRP.
Step 6) colour developing with stop: adding of obtaining in step 5) adds freshly prepared substrate solution in the enzyme plate after enzymic-labelled antibody, the rear room temperature lucifuge of vibration mixing is hatched 15min and is developed the color, every hole adds stop buffer again, and blending incubation 6min termination reaction, obtains the solution that can measure.
Step 7) measured value: the solution of measuring determination step 6) obtaining is at OD 450nmthe light absorption value A at place 450nm;
Step 8) data processing: the light absorption value A that utilizes step 7) to measure 450nmcalculate testing sample inhibiting rate, described testing sample inhibiting rate PI%=(1-A 450sample/A 450negative control) * 100.Detected result is as shown in table 3, and accuracy rate and LPB-ELISA detection method are compared and calculated.
The above embodiment of the present invention scheme is only can not limit the present invention to explanation of the present invention, in claims, pointed out scope of the present invention, and scope of the present invention is not pointed out in the explanation of above-mentioned specific embodiment, therefore, in the implication suitable with claims of the present invention and any change in scope, be all considered to be in the scope that is included in claims.
The present invention is through foot and mouth disease disease long term operation experience accumulation, and creates by creative work, and it is in actual tests detection and foot and mouth disease A type disease detection practical application journey, and the method detected result is reliable, and Detection accuracy is high.
The competitive ELISA detected result of the foot and mouth disease A type antibody in table 3 embodiment serum
Note: "+" represents testing sample PI% >=30%, and "-" represents testing sample PI% < 30%.
Sequence table
Animal epidemic prevention and control center, <110> Guangxi Zhuang Autonomous Region
The competitive ELISA method of <120> based on foot and mouth disease A type VP1 albumen and monoclonal antibody thereof
<130> 2013
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 213
<212> PRT
<213> foot and mouth disease virus (FMDV)
<400> 1
Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly Gly
5 10 15 20
Glu Thr Gln Val Gln Arg Arg Gln His Thr Asn Val Gly Phe Ile MET Asp Arg Phe Val
25 30 35 40
Lys Ile Pro Ser Gln Ser Pro Thr His Val Ile Asp Leu MET Gln Thr His Gln His Gly
45 50 55 60
Leu Val Gly Ala Leu Leu Arg Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val
65 70 75 80
Arg His Asp Asp Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Thr Ala Leu His Asn
85 90 95 100
Thr Ser Asn Pro Thr Ala Tyr His Lys Gly Pro Phe Thr Arg Leu Ala Leu Pro Tyr Thr
105 110 115 120
Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Thr Thr Lys Tyr Ser Thr Gly Asn
125 130 135 140
Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val Ala Ala Gln Leu Pro Ala
145 150 155 160
Ser Phe Asn Phe Gly Ala Ile Arg Ala Thr Val Ile His Glu Leu Leu Ala Arg MET Lys
165 170 175 180
Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Val Lys Val Thr Ser Gln Asp Arg
185 190 195 200
His Lys Gln Arg Ile Ile Ala Pro Ala Lys Gln Leu Leu
205 210 213
Sequence table
Animal epidemic prevention and control center, <110> Guangxi Zhuang Autonomous Region
The competitive ELISA method of <120> based on foot and mouth disease A type VP1 albumen and monoclonal antibody thereof
<130> 2013
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 213
<212> PRT
<213> foot and mouth disease virus (FMDV)
<400> 1
Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly Gly
5 10 15 20
Glu Thr Gln Val Gln Arg Arg Gln His Thr Asn Val Gly Phe Ile MET Asp Arg Phe Val
25 30 35 40
Lys Ile Pro Ser Gln Ser Pro Thr His Val Ile Asp Leu MET Gln Thr His Gln His Gly
45 50 55 60
Leu Val Gly Ala Leu Leu Arg Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val
65 70 75 80
Arg His Asp Asp Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Thr Ala Leu His Asn
85 90 95 100
Thr Ser Asn Pro Thr Ala Tyr His Lys Gly Pro Phe Thr Arg Leu Ala Leu Pro Tyr Thr
105 110 115 120
Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn Gly Thr Thr Lys Tyr Ser Thr Gly Asn
125 130 135 140
Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val Ala Ala Gln Leu Pro Ala
145 150 155 160
Ser Phe Asn Phe Gly Ala Ile Arg Ala Thr Val Ile His Glu Leu Leu Ala Arg MET Lys
165 170 175 180
Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Val Lys Val Thr Ser Gln Asp Arg
185 190 195 200
His Lys Gln Arg Ile Ile Ala Pro Ala Lys Gln Leu Leu
205 210 213

Claims (7)

1. a foot and mouth disease A type VP1 albumen, comprise foot and mouth disease A type VP1 albumen 1 and foot and mouth disease A type VP1 albumen 2 that two kinds of different phraseologies obtain, it is characterized in that, foot and mouth disease A type VP1 albumen 1 and foot and mouth disease A type VP1 albumen 2 aminoacid sequences are shown in SEQ ID NO:1.
2. a method of preparing foot and mouth disease A type VP1 albumen claimed in claim 1, it is characterized in that, the foot and mouth disease virus FMDV genome of take is template, utilize a pair of clone's primer and a pair of expression primer to carry out pcr amplification and obtain FMDV-A-VP1 gene, by FMDV-A-VP1 gene respectively with pET-32a, pGEX-6p-1 plasmid connects structure and obtains expression plasmid PET-32a-VP1, pGEX-6p-1-VP1, again expression plasmid PET-32a-VP1 and pGEX-6p-1-VP1 are proceeded to respectively to abduction delivering in prokaryotic expression host e. coli BL21 and obtain fusion rotein 1 and fusion rotein 2 respectively, fusion rotein 1 and the fusion rotein 2 respectively purified aminoacid sequence that obtains are the foot and mouth disease A type VP1 albumen 1 shown in SEQ ID NO:1 and foot and mouth disease A type VP1 albumen 2, described a pair of clone's primer comprises primer C1:5 '-TACCAAATTACACGGGAA-3 ', primer C2:5 '-GACATGTCCTCCTGCATCTG-3 ',
Described a pair of expression primer comprises primer E1:5 '-CCGGAATTCACCACCACTGTTGAGAACTACG-3 ', primer E2:5 '-CCGCTCGAGTCATTACAGGAGTTGCTTTGCAGGTGC-3 '.
3. the method for preparing foot and mouth disease A type VP1 albumen according to claim 2, is characterized in that, in described pcr amplification process, annealing temperature is 54.5 ~ 57.5 ℃; Described fusion protein purification is process His and GST column chromatography purification.
4. a method of preparing foot and mouth disease A type VP1 albumen monoclonal antibody, is characterized in that, this preparation method comprises the following steps:
Step is animal immune a): the BALB/c female mice in 5 ~ 7 week age of foot and mouth disease A type VP1 albumen 1 immunity of utilizing that expression plasmid PET-32a-VP1 induction obtains, after 3 immunity, filter out the tire mouse of > 1:12800 of immunoassay;
Step b) cytogamy: the splenocyte of getting the immune mouse that step a) obtains, carry out cytogamy with myeloma cell SP2/0, utilize expression plasmid pGEX-6p-1-VP1 to induce the foot and mouth disease A type VP1 albumen 2 obtaining as envelope antigen, the cell after merging is carried out to indirect ELISA screening and obtain hybridoma;
Step c) hybridoma a large amount of clones of monoclonal antibody: by step b) obtaining is expelled in mouse peritoneal, and the mouse after raising injection 1 ~ 3 week, collects the mouse ascites that belly expands, and ascites is carried out to purifying and obtain foot and mouth disease A type VP1 albumen monoclonal antibody;
The identical sequence albumen that described foot and mouth disease A type VP1 albumen 1, foot and mouth disease A type VP1 albumen 2 obtain for different phraseologies, both Argine Monohydrochloride sequences are shown in SEQ ID NO:1.
5. the competitive ELISA method of a foot and mouth disease A type VP1 albumen and monoclonal antibody thereof, the method comprise diluted sample, antigen coated, sealing, application of sample and add monoclonal antibody, add enzymic-labelled antibody, colour developing and termination, measured value, data processing, it is characterized in that, this competitive ELISA detection method step comprises:
Step 1) diluted sample: the dilution of serum sample is original volume 2 ~ 4 times, obtain test serum sample;
Step 2) antigen coated: envelope antigen used is the foot and mouth disease A type VP1 albumen 2 that expression plasmid pGEX-6p-1-VP1 induction obtains, and the coated concentration of envelope antigen is 0.5 ~ 0.7ug/mL;
Step 3) sealing: by confining liquid sealing step 2) coated antigen, be 40 ~ 60min off-period, the enzyme plate after being sealed;
Step 4) application of sample with add monoclonal antibody: by step 1) the test serum sample that obtains joins step 3) in enzyme plate after the sealing that obtains, serum antibody is combined with envelope antigen, adding extent of dilution is the foot and mouth disease A type VP1 albumen monoclonal antibody of 1:300 ~ 1:500 again, make foot and mouth disease A type VP1 albumen monoclonal antibody in conjunction with not with the residue envelope antigen of serum antibody response, obtain sample, the reacted enzyme plate of monoclonal antibody;
Step 5) adds enzymic-labelled antibody: after the sample obtaining in step 4), monoclonal antibody reaction, in enzyme plate, add enzyme labelled antibody, the weaker concn of enzymic-labelled antibody is 1:4500 ~ 6000, enzymic-labelled antibody is combined with foot and mouth disease A type VP1 albumen monoclonal antibody, and be not combined with serum antibody, obtain adding the enzyme plate after enzymic-labelled antibody;
Step 6) colour developing with stop: adding of obtaining in step 5) adds freshly prepared substrate solution in the enzyme plate after enzymic-labelled antibody, the rear room temperature lucifuge of vibration mixing is hatched 10 ~ 20min and is developed the color, every hole adds stop buffer again, mixes termination reaction, obtains the solution that can measure;
Step 7) measured value: the solution of measuring determination step 6) obtaining is at OD 450nmthe light absorption value A at place 450nm;
Step 8) data processing: the light absorption value A that utilizes step 7) to measure 450nmcalculate testing sample inhibiting rate, described testing sample inhibiting rate PI%=(1-A 450sample/A 450negative control) * 100;
Described foot and mouth disease A type VP1 albumen 2 its aminoacid sequences are shown in SEQ ID NO:1.
6. the competitive ELISA method based on foot and mouth disease A type VP1 albumen monoclonal antibody according to claim 5, is characterized in that, described confining liquid be in 1%BSA solution, 1% gelatin solution, 5% foetal calf serum solution, 5% skim-milk solution any.
7. according to the competitive ELISA method based on foot and mouth disease A type VP1 albumen monoclonal antibody described in claim 5 or 6 any one, it is characterized in that, the antibody of described enzymic-labelled antibody is goat anti-mouse IgG-HRP; Described A 450negative control is the light absorption value A out of the determination of serum with not infecting FMDV virus 450.
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CN109655623A (en) * 2019-01-31 2019-04-19 中国农业科学院兰州兽医研究所 A kind of the visualization quick detection kit and its application of A type antibodies against foot-and-mouth disease virus
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KR102614002B1 (en) * 2021-04-12 2023-12-15 주식회사 바이오노트 Antibody exhibiting immunoreactivity to foot and mouth disease virus type A, composition for detecting foot and mouth disease virus type A antibody comprising the same, and method for detecting foot and mouth disease virus type A antibody using the same

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