CN110221064A - Pig Seneca Valley virus ELISA antibody detection method and application - Google Patents

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

Pig Seneca Valley virus ELISA antibody detection method and application

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 determined_P=0.373, C_N=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 calculated_P=0.291, C_N=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.