CN110221064A - Pig Seneca Valley virus ELISA antibody detection method and application - Google Patents
Pig Seneca Valley virus ELISA antibody detection method and application Download PDFInfo
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Abstract
The present invention provides a boar Seneca Valley virus ELISA antibody detection method and application, this method is using SVV 3AB recombinant protein as envelope antigen, 3AB ELISA antibody detection method of the invention is as a kind of detection instrument, it can be used for clinical serum detection, lay a good foundation for the diagnosis of epidemic disease, the research and development of SVV antibody assay kit.
Description
Technical field
The invention belongs to immunological detection method fields, and in particular to pig Seneca Valley virus ELISA antibody detection method
And application.
Background technique
Seneca Valley virus (Senecavirus, SVV) belongs to Picornaviridae, Seneca Valley virus category, with heart disease
It is closely similar that poison belongs to member, is a kind of no cyst membrane, single strand plus RNA virus.In recent years, the U.S., Brazil, Thailand, Australia,
Italy, Canada and China Report SVV infection show that SVV evolves and propagates rapidly.Infect swinery cause hydroa and
Popular of short duration newborn loses (ETNL).The disease clinical symptoms are similar to aftosa (FMD), pig blisters (SVD), blister mouth
The inflammation blisters Animal diseases such as (VS) and vesicle (VE) endanger pig breeding industry sound development.
SVV has the typical genome signature of other picornavirus, it then follows the L-4-3-4 of standard is laid out.Its gene group leader
Spend about 7280 nucleotide, including 5 ' UTR (666bp), single long open reading frame (6543bp) and 3 ' UTR (71bp).Individually
Long open reading frame (ORF) can encode 2181 amino acid polyproteins, and 12 are cut under the action of virus protease
Maturation protein, sequence are as follows: L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D.Wherein, VP1, VP2, VP3 can be in diseases
The expression of malicious capsid outer surface, have been demonstrated can with anthrax toxin acceptor 1 interaction is gone forward side by side an one-step inducing with cause infection jointly
Host cell antibody response;3A be considered as the anchorin that picornavirus replication complex and membrane structure combine and with virus
The cytopathic effect of induction is related with the decomposition of intracellular protein is blocked;3B is referred to as viral genome connexin (VPg),
It can hold and be covalently attached to newborn picornavirus 5 ', may have the function of RNA synthetic primer.
Common SVV method of detecting infection specifically includes that virus neutralization tests (VNT), indirect immunofluorescence assay
(IFA), reverse transcriptase-polymerase chain reaction (RT-PCR), real-time fluorescence quantitative PCR (qRT-PCR), the immune survey of enzyme-linked absorption
Fixed (ELISA) and virus isolation and Identification.Currently, foreign scholar has built up SVV VP1ELISA antibody detection method, it is used for
Whether measurement swinery is infected by SVV, but its specificity and sensibility are still undesirable, and China is also without commercialization detection reagent
Box.The antibody that this research compares infection several virus-encoded proteinaceous of piglet (VP1,2C, 3A, 3AB, 3C, 3D, L) generates dynamic,
It was found that the antigenicity of SVV 3AB is preferably.In view of this, we establish 3AB indirect ELISA antibody detection method, and are used for SVV serum
Epidemiological survey.
Summary of the invention
The technical issues of proposing for background technique, the purpose of the present invention is to provide a boar Seneca Valley virus
ELISA antibody detection method and application.
The purpose of the present invention can be achieved through the following technical solutions:
One boar Seneca Valley virus ELISA antibody assay kit, the kit is using SVV 3AB recombinant protein as packet
By antigen.
The SVV 3AB recombinant protein the preparation method comprises the following steps: design specific primer:
SVV-3AB-F:5'GAGGAATTCAGCCCTAACGA 3'
SVV-3AB-R:5'GCGCTCGAGTCACTGTTGCATTTC 3',
SVV 3AB gene fragment clone to pET30a (+) expression vector is obtained into recombinant plasmid, extremely by recombinant plasmid transformed
Inducing expression is carried out in the expression bacterium E.coli BL21 (DE3) of competence, is collected supernatant and is purified, is obtained after purification
Recombinant protein.
As a kind of optimal technical scheme, which further includes confining liquid, coating buffer, serum dilution and enzyme mark
Secondary antibody further comprises TMB developing solution and terminate liquid.
It is further preferred that the confining liquid is 5% (g/100ml) skimmed milk, the coating buffer is pH
9.6 carbonate buffer solution (weighs 0.17g anhydrous Na2CO3、0.28gNaHCO3In distilled water and it is settled to 100ml), it is described
Serum dilution be PBST, the ELIAS secondary antibody be HRP-SPA.
Application of the above-mentioned kit in detection pig Seneca Valley virus, the application is not with the diagnosing and treating of disease
For the purpose of.
A kind of pig Seneca Valley virus indirect ELISA antibody detection method of non-diagnosis and treatment purpose, the method steps are as follows:
(1) SVV 3AB recombinant protein described in claims 1 or 2 after purification coated elisa plate: is coated with micropore
Plate;Peridium concentration is 1.0 μ g/mL;After coating condition is 37 DEG C of placement 2h, 4 DEG C of placement 12h;
(2) ELISA Plate is closed;With 5% skimmed milk, 37 DEG C of closing 3h;
(3) serum to be checked after dilution, 37 DEG C of incubation 45min are added;It is described to be diluted to blood to be checked with serum dilution
It is clear to make 100 times of dilutions;
(4) it is added with the diluted ELIAS secondary antibody HRP-SPA of 1:10000,37 DEG C of effect 30min;
(5) it develops the color, terminates, and read OD in microplate reader450Value.Preferably, developing solution colour developing 15min is added.
SVV 3AB recombinant protein is as envelope antigen in preparation pig Seneca Valley virus ELISA antibody assay kit
Application.
Beneficial effects of the present invention:
Seneca Valley virus (SVV) is a kind of new cause of disease for endangering China's pig breeding industry, clinical symptoms and aftosa etc.
Blister Animal diseases are difficult to differentiate between, and China there is no antibody assay kit at present.This research uses escherichia expression system
SVV 3AB, 2C, 3C, 3D, L and VP1 recombinant protein are prepared, above-mentioned virus protein antibody produces in measurement SVV infection Swine serum
Raw rule, discovery SVV 3AB protein antibodies generation time is most fast, antibody level is higher and the duration is longer.By reacting item
Piece optimization, establishes 3AB-ELISA antibody detection method, sensibility 91.3%, and specificity is 92.59%, with PRRSV,
Without intercrossing, the coefficient of variation is respectively less than 15% for CSFV, PRV, PCV2 and O-shaped FMDV antibody.This method and virucidin try
Testing coincidence rate is 92.0%.3249 parts of 2014-2018 clinical health swinery serum is measured using this method, antibody positive rate is
84.0%, for China, the prevention and control provide important method and epidemic data.In short, 3AB ELISA antibody detection method is made
It for a kind of detection instrument, can be used for clinical serum detection, established for the diagnosis of epidemic disease, the research and development of SVV antibody assay kit
Basis.
Detailed description of the invention
Fig. 1 is the identification of SVV recombinant protein
Wherein, M, protein standard;1, pET-32a (m) unloaded control;2, VP1 recombinant proteins;3, pET-
28a zero load control;4, L recombinant proteins;5,2C recombinant proteins;6,3AB recombinant proteins;7,3C recombinant proteins;8,3D recombinant proteins
Fig. 2 is that SVV infects piglet different virus coding protein antibodies generation dynamic analysis
Fig. 3 is the optimization of ELISA reaction condition
Fig. 4 is the retrospective analysis of East China SVV infection.
Specific embodiment
1 material and method
1.1 viruses, serum, plasmid and reagent
It SVV SD strain, neutralizing antibody positive Swine serum, neutralizing antibody negative serum and attacks malicious serum and is protected by this laboratory
It deposits.3249 parts of clinical pig anteserum sample, 126 pig farms from Shandong, Jiangsu Province.pET-28a(+),pET-32a(m)
E.coli BL21 (DE3) is saved by this laboratory.SVV SD strain involved in experimentation is known to the skilled person
Conventional strain, carrier be can pass through market buy conventional carrier.
Staphylococcal protein A (HRP-SPA) antibody of HRP label is purchased from doctor's moral Bioisystech Co., Ltd,
TMB developing solution is purchased from the green skies Bioisystech Co., Ltd in Shanghai, and restriction enzyme, T4 ligase are purchased from TaKaRa biology section
Skill Co., Ltd, hypersensitive ECL chemical luminescence reagent kit are Pierce Products, and plasmid extraction kit is purchased from Biomiga
Biotechnology Co., Ltd, other reagents are that domestic analysis is pure.
The synthesis of 1.2 primers
According to SVVSD strain (GenBank:MH779611) sequence, using the artificial synthesized VP1,2C of Primer 5.0,3AB,
The specific primer of six genetic fragments of 3C, 3D, L, is shown in Table 1.
The synthesis of 1 primer of table
After virus infected cell, extract RNA and through reverse transcription obtain cDNA, as template amplification obtain 2C, 3AB, 3C,
3D, L genetic fragment.By 2C, 3AB, 3C, 3D, L gene cloning to pET-28a (+), VP1 gene cloning to pET-32a (m) is carried
The recombinant plasmid trust money Si Rui Bioisystech Co., Ltd of body, acquisition is sequenced.
The inducing expression and purifying of 1.3 recombinant proteins
Accurate recombinant plasmid transformed will be sequenced to expression bacterium E.coli BL21 (DE3), picking single bacterium falls within 3ml LB training
It supports overnight, gives over to strain;The optimization of expression condition is induced and carried out in a small amount;60ul bacterium solution is taken to be inoculated in 200mL LB;When
OD600When=1.0, final concentration of 1.0mmol/L IPTG induction 6-10h is added;Bacterium solution 8000r/min is centrifuged 10min, thallus
After PBS is washed 3 times, it is resuspended with 20mL PBS;After ultrasonication centrifugation, supernatant is sucked out, and precipitating PBS is resuspended, SDS-PAGE identification
The expressivity form of recombinant protein, Western blotting identify the specificity of recombinant protein.
1.4 SVV infect piglet different virus protein antibodies and generate dynamic
Using indirect ELISA method, the antibody for comparing different coding albumen in 0-5 weeks SVV infection Swine serum generates rule.
Recombination SVV albumen to peridium concentration with the dilution purifying of the carbonate buffer solution of pH 9.6 is 2.0ug/ml, 100 holes μ L/, 37 DEG C
It is incubated for 2h, is then washed 3 times with PBST, each 5min.Using 5% (g/100ml) skimmed milk as sealer, 200 holes μ L/, 37 DEG C of envelopes
3h is closed, washing is same as above.It is added 0-5 weeks and attacks malicious serum (1:100PBST dilution), 100 holes μ L/, 37 DEG C of effect 1h are added after washing
Tmb substrate developing solution, 100 μ are added after washing 3 times in HRP-SPA (1:10000 dilution), 100 holes μ L/, 37 DEG C of effects 45min, PBST
The hole L/, 37 DEG C of colour developing 10min.50 hole μ L/ of 2M sulfuric acid is added and terminates reaction, reads each hole OD450nm value with microplate reader, and calculate
P/N value.
1.5 indirect ELISA reaction condition optimizations
Protein antibodies are encoded by analysis infection piglet different virus and generate dynamic, and it is anti-to determine that immunogenicity is preferably coated with
It is former.Serum optimum diluting multiple and optimal peridium concentration are screened using square matrix titration;Again respectively to the coating time, sealer kind
Nine class, off-period, serum incubation time, HRP-SPA dilution, HRP-SPA incubation time and developing time conditions carry out
Optimization.
The determination of 1.6 critical values
30 parts of SVV viruses are determined using SVV 3AB indirect ELISA method and neutralize negative serum, calculate separately OD450With
The average value of S/PWith standard deviation (SD), criterion is calculated.When be determined as feminine gender,When be determined as the positive;It is judged between the two suspicious;When be determined as feminine gender, When be determined as the positive;It is between the two suspicious.
1.7 specificity and repetitive test
PRRSV, CSFV, PRV, PCV are detected using SVV 3AB indirect ELISA method2, O-shaped FMDV positive serum, carry out
Specific test.Using the coated ELISA Plate of same batch, 6 parts of clinical serums of random detection, every part of sample is repeated 3 times, and is carried out
Repetitive test in batch;Simultaneously with 3 pieces of coated ELISA Plates of different batches, 6 parts of clinical serums of random detection, every part of sample repetition
3 times, carry out repetitive test between criticizing.
1.8 sensibility and coincidence rate analysis
46 parts of viruses, which are detected, using the ELISA method of foundation neutralizes positive serum and 54 parts of virus neutralization negative serums, fortune
With relative sensitivity (%)=[number positive/(number positive+false negative number)] × 100%;Relative specificity (%)=[negative number/
(negative number+false positive number)] × 100%;Total coincidence rate (%)=[(number positive+feminine gender number)/detection sum] × 100%3
Formula analyzes the sensibility, specificity and coincidence rate of this method respectively.
1.9 retrospective analysis
Using the SVV 3AB ELISA method of foundation, 3249 parts for detecting 126 pig farms such as nearly five Nian Shandong, Jiangsu face
Bed serum counts positive number, calculates positive rate.
2 results
2.1 SVV encode protein expression, purifying and identification
Using PCR amplification SVV 2C, 3AB, 3C, 3D, L, VP1 gene, expression of recombinant e. coli plasmid is constructed.3AB egg
It is white to be expressed in supernatant, it is purified using affinity chromatography method;2C, 3C, 3D, L, VP1 recombinant protein are expressed with inclusion bodies, warp
Washing, denaturation, the purifying of three step of renaturation.SDS-PAGE electrophoresis showed: destination protein band after purification is single, and purification effect is preferable
(Figure 1A);Western Blotting the result shows that, six kinds of recombinant proteins can with His monoclonal antibody occur specific reaction (figure
1B)。
2.2 SVV infect piglet different virus protein antibodies and generate dynamic
Antibody level of several virus-encoded proteinaceous of SVV in infection swinery is compared, discovery is attacked after poison for SVV 3AB
The antibody level that albumen generates is higher, and the antibody level that SVV 3D, 2C, 3C albumen generate is suitable, and is directed to SVV L, VP1 albumen
Antibody level it is lower (Fig. 2).
The optimization of 2.3 ELISA reaction conditions
It is screened using square matrix burette test, while to two antigen coat concentration, serum dilution reaction conditions.Such as
Table 2, when antigen coat concentration is 1.0ug/ml and serum makees 100 times of dilutions, P/N is maximum, is determined as optimal conditions.
The screening of table 2 peridium concentration and serum dilution
In addition, other reaction conditions are successively optimised.4 DEG C are relayed in short, being coated with after good ELISA Plate places 37 DEG C of 2h
12h closes 3h (Fig. 3 A) after washing with 5% skimmed milk;Serum to be checked is added and is incubated for 45min (Fig. 3 B), combines antigen-antibody
Sufficiently;ELIAS secondary antibody HRP-SPA (1:10000) effect 30min (Fig. 3 C, D) is added after abandoning liquid washing;It is eventually adding TMB colour developing
15min, through 2M H2SO4OD450 is read after termination.
The determination of 2.4 ELISA critical values
30 parts of negative serum OD are detected using the indirect ELISA method of foundation450Average valueIt is 0.178;Standard deviation
Poor (SD) is 0.065;According to Principle of Statistics, yin-yang critical value C is determinedP=0.373, CN=0.308, i.e. measuring samples detect
Value OD450 >=0.373 for the positive;OD450 < 0.308 is feminine gender;It is suspicious for falling between.In order to reduce human factor,
The influence of environmental factor, we introduce S/P value, the average value of 30 parts of negative serum S/P valuesIt is 0.066;Standard deviation
It (SD) is 0.075;Similarly, yin-yang critical value C is calculatedP=0.291, CN=0.216, i.e. measuring samples detected value S/P >=0.30
For the positive;S/P < 0.20 is feminine gender;It is suspicious for falling between.
2.5 ELISA specificity and repetitive test
PRRSV, CSFV, PRV, PCV are detected using this method2, O-shaped FMDV positive serum, result is feminine gender;Weight in batch
Retrial tests the coefficient of variation between 0.84%-8.57%, and interassay coefficient of variation is between 2.78%-13.10%, the two variation lines
Number is respectively less than 15%, illustrates this method specificity, repeatability preferably.
2.6 ELISA sensibility and coincidence rate analysis
Detecting 100 parts of clinical serums using indirect ELISA method, (46 parts positive for neutralization test, and 54 parts are neutralization test
It is negative), there are 42 parts of positive serums, 50 parts of negative serums, 4 parts of false positive, 4 parts of false negative.According to above data, this method is opposite
Sensibility is 91.30%, relative specificity 92.59%, and total coincidence rate is 92.00%.
3 sensibility of table and coincidence rate analyze result s
2.7 retrospective serum antibody investigation
3249 parts of clinical serums from provinces such as Shandong, Jiangsu, Zhejiang, Jiangxi are detected with this method, compare the past
5 years SVV antibody positive rates (Fig. 4 A).In 2014, SVV infection rate was 80.3%, 90% was broken through by 2015 and in 2016
Reach infection peak value year.Testing result further confirms: being within 2015 the turning point that SVV expands and 2016 are then considered as
The turning point of China's SVV epidemiology.Statistical result showed (Fig. 4 B) based on different swinerys, suckling pig and growing and fattening pigs resist
Body positive rate is lower, respectively 59.2%, 69.2%.Furthermore it has been found that: infection rate is lower (Fig. 4 C) in the fall for the disease.
3 discuss
Since 2015, SVV cases of infection have been made a definite diagnosis by multiple countries including China, and new strain is not
It is disconnected to be separated.The long-term propagation of different SVV strains and presence will further facilitate the evolution and mutation of SVV.It is severe, China
Not yet develop propagable vaccine preparation.Therefore, diagnostic tool is developed, identifies SVV and infects spreading to for the prevention and control disease
It closes important.
The level of specific antibody is to identify the useful parameter of virus infection in serum.Currently, SVV rVP1 ELISA and
VP2ELISA has been reported for the antibody level of SVV in detection clinical serum, but VP1 specific IgG antibodies are after disease regression
Decline, the duration is shorter, and VP2 is primarily involved in the neutralizing antibody reaction of early stage after virus infection, and two methods all have centainly
Limitation.This research is by comparing several virus proteins (2C, 3A, 3AB, 3C, 3D, L, VP1) of SVV in infection population
Antibody generates rule, and discovery body is higher for the antibody level of SVV 3AB and the duration is longer.Therefore, we establish
SVV 3AB ELISA, sensibility 91.30%, specificity are 92.59%, and this method and virus neutralization tests show
Higher coincidence rate out.Seroepidemiological survey is carried out to East China using this method, it has been found that: after 2016,
Fashion trend of the SVV in Shandong, Jiangsu, Jiangxi San Sheng slows down;It is worth noting that, SVV is in the anti-of Zhejiang Province after 2017
Body positive rate is gone up again;In addition, the infection rate of kind swinery is higher, more concerns should be given.In short, 3AB ELISA is anti-
Body detecting method can be used for clinical serum detection as a kind of detection instrument, be diagnosis, the SVV antibody assay kit of epidemic disease
Research and development lay a good foundation.
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Claims (8)
1. a boar Seneca Valley virus ELISA antibody assay kit, which is characterized in that the kit is with SVV 3AB recombination
Albumen is as envelope antigen.
2. pig Seneca Valley virus ELISA antibody assay kit according to claim 1, which is characterized in that the SVV
3AB recombinant protein the preparation method comprises the following steps: design specific primer:
SVV-3AB-F:5'GAGGAATTCAGCCCTAACGA 3'
SVV-3AB-R:5'GCGCTCGAGTCACTGTTGCATTTC 3',
SVV 3AB gene fragment clone to pET30a (+) expression vector is obtained into recombinant plasmid, by recombinant plasmid transformed to impression
Inducing expression is carried out in the expression bacterium E.coli BL21 (DE3) of state, is collected supernatant and is purified, obtains recombination after purification
Albumen.
3. pig Seneca Valley virus ELISA antibody assay kit according to claim 1, which is characterized in that the reagent
Box further includes confining liquid, coating buffer, serum dilution and ELIAS secondary antibody.
4. pig Seneca Valley virus ELISA antibody assay kit according to claim 3, which is characterized in that described
Confining liquid is 5% skimmed milk, the carbonate buffer solution that the coating buffer is pH 9.6, and the serum dilution is
PBST, the ELIAS secondary antibody are HRP-SPA.
5. application of the kit of any of claims 1-4 in detection pig Seneca Valley virus, the application is not
For the purpose of the diagnosing and treating of disease.
6. a kind of pig Seneca Valley virus indirect ELISA antibody detection method of non-diagnosis and treatment purpose, it is characterised in that: this method step
It is rapid as follows:
(1) SVV 3AB recombinant protein described in claims 1 or 2 after purification coated elisa plate: is coated with microwell plate;Packet
It is 1.0 μ g/mL by concentration;After coating condition is 37 DEG C of placement 2h, 4 DEG C of placement 12h;
(2) ELISA Plate is closed;With 5% skimmed milk, 37 DEG C of closing 3h;
(3) serum to be checked after dilution, 37 DEG C of incubation 45min are added;It is described to be diluted to be made serum to be checked with serum dilution
100 times of dilutions;
(4) it is added with the diluted ELIAS secondary antibody HRP-SPA of 1:10000,37 DEG C of effect 30min;
(5) it develops the color, terminates, and read OD in microplate reader450Value.
7.SVV 3AB recombinant protein is as envelope antigen in preparation pig Seneca Valley virus ELISA antibody assay kit
Using.
8. application according to claim 7, which is characterized in that the SVV 3AB recombinant protein the preparation method comprises the following steps: design
Specific primer:
SVV-3AB-F:5'GAGGAATTCAGCCCTAACGA 3'
SVV-3AB-R:5'GCGCTCGAGTCACTGTTGCATTTC 3',
SVV 3AB gene fragment clone to pET30a (+) expression vector is obtained into recombinant plasmid, by recombinant plasmid transformed to impression
Inducing expression is carried out in the expression bacterium E.coli BL21 (DE3) of state, is collected supernatant and is purified, obtains recombination after purification
Albumen.
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Citations (10)
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