CN107356754A - A kind of porcine reproductive and respiratory syndrome virus Nsp2 expression and its ELISA kit - Google Patents
A kind of porcine reproductive and respiratory syndrome virus Nsp2 expression and its ELISA kit Download PDFInfo
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Abstract
The invention provides a kind of porcine reproductive and respiratory syndrome virus Nsp2 expression and its ELISA kit, using passing through clonal expression Guizhou separation strains PT strain Nsp2 recombinant proteins, establish the indirect ELISA method of detection Nsp2 antibody, the method is quick, effective, sensitive, easy, there is good application prospect.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of porcine reproductive and respiratory syndrome virus Nsp2 expression and its
ELISA kit.
Background technology
(PRRS is by porcine reproductive and respiratory syndrome virus (porcine to porcine reproductive and respiratory syndrome
Reproductive and respiratory syndrome virus, PRRSV) caused by a boar high degree in contact
Viral infectious, the pig at any age can infect the virus, and main clinical manifestation is to be flowed after in-pig infects
The breeding difficulty diseases such as production, premature labor and stillborn foetus, respiratory symptom is mainly showed after growing and fattening pigs and Infection in Piglets.Wherein, it is high to cause a disease
Property PRRSV can cause the piglet incidence of disease 100%, the death rate more than 50%, Sow abortion rate more than 30%.2006, China was sudden and violent
Send out PRRS epidemic situations highly pathogenic, involve individual province more than 20.Current popular present situation is sufficiently complex, popular PRRSV strains in swinery
Species is various, and different genotype or the popular strains of pathogenic of gene hypotype and distribution have differences, and China swinery is universal in addition
Using different types of vaccine, diagnosis sick to this brings extreme difficulties.PRRSV is non-segmented negative, polyadenylation, has capsule
The single strand plus RNA virus of film.Its genome length is about 15Kb, contains 8 ORFs (ORFs) frames.Its gene base is suitable
Sequence is followed successively by：5′-ORF1a-ORF1b-ORF(2-6)-ORF7-3′.The 6 kinds of structural proteins of coding of ORF2~7, ORF1 (including
ORF1a and ORF1b) coding polymeric protein cracking produce 12 non-structural proteins (Nsp1~12).Nsp2 is PRRSV genomes
In be easiest to the region morphed, therefore can be ground by understanding Nsp2 variation detection the situations of PRRSV genetic mutations
The normal target gene using Nsp2 as PRRSV Molecular Epidemic Hygienic monitoring on hands of childhood of the person of studying carefully.Nsp2 is the maximum non-structural protein of variation, and is had
There are multiple B cell epitopes, body can be stimulated to produce specific antibody in the course of infection of virus, and this antibody is at least
Continue 202d, prompt Nsp2 to have in the diagnosis of PRRSV persistent infections and be of great significance.Domestic and international multidigit scholar grinds
Study carefully discovery, non-structural protein Nsp2 has extremely strong immunogenicity, and body can be induced to produce very high antibody, it might even be possible to super
Crossing nucleocapsid protein (N protein) stimulates antibody level caused by body, and Nsp2 plays during host immune response is adjusted
Highly important role.The PRRSV NSP2 fusion proteins of vivoexpression also have good immunogenicity.This research success gram
The grand region of the Main Antigenic of highly pathogenic PRRSV Guizhou separation strains PT strain Nsp2 genes, and it is high in Escherichia coli
Effect expression, fusion protein can be identified by PRRS positive serums.The Nsp2- tentatively established as envelope antigen using purifying protein
ELISA antibody detection methods sensitiveness is high, high specificity, routine diagnosis and epidemiology survey available for PRRS.ELISA is
One of the most frequently used method of PRRS antibody is detected, there is the advantages of quick, economic, sensitive and semi-automatic, knot can be shown automatically
Fruit, convenient and easy in extensive checkout and diagnosis high-volume blood serum sample in the short time, operating process and result judge energy making the grade
Change, and specificity and sensitiveness with height, suitable for basic unit's Veterinary office and pig farm to the sick diagnosis and serological surveillance,
It is that one kind uses widest method.The ELISA detection method established at present is mostly using PRRSV totivirus or the core of expression
Capsid (N) albumen establishes inspection as antigen, this research and utilization by clonal expression Guizhou separation strains PT strain Nsp2 recombinant proteins
The indirect ELISA method of Nsp2 antibody is surveyed, the method is quick, effective, sensitive, easy, there is good application prospect.
The content of the invention：
Specifically, it is an object of the invention to provide a kind of porcine reproductive and respiratory syndrome virus Nsp2 expressions, including
Following steps：PCR reaction system 50L, by 10 × PCR Buffer 2-4L, MgCl2 (25mM) 1-5L, dNTP (10mM) 1-3L,
Each 1-3L of Nsp2-1/Nsp2-2 primers, the appropriate 1-3L of Taq enzyme 0.5-2L, cDNA, deionized water complement to 50 and formed；PCR reacts
Parameter is：90-98 DEG C of 2-4min pre-degeneration, then carry out 30 and circulate, 90-98 DEG C, 25-35s, 52-60 DEG C, 25-35s, 68-
76 DEG C of 40-60, last 68-76 DEG C of extension 4-6min；Primer be Nsp2-15 '-
CGCGGATCCGACACCTCCTTTGATTGGAAT-3 ', restriction enzyme site BamH I, Nsp2-25-
CCGGAATTCCGAACCGTTAGGGACATTGTC-3, restriction enzyme site EcoR I, pre- amplification length：606bp.
Further, a kind of porcine reproductive and respiratory syndrome virus Nsp2 expressions of the present invention, comprise the following steps：
PCR reaction system 50L, i.e. 10 × PCR Buffer 3L, MgCl2 (25mM) 2.5L, dNTP (10mM) 1L, Nsp2-1/Nsp2-2
Each 2L of primer, Taq enzyme 1L, appropriate cDNA, deionized water complement to 50L；PCR response parameters are：94 DEG C of 3min pre-degenerations, then
Carry out 30 circulations, 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C of 50s, last 72 DEG C of extensions 5min；Primer be Nsp2-15 '-
CGCGGATCCGACACCTCCTTTGATTGGAAT-3 ', restriction enzyme site BamH I, Nsp2-25 '-
CCGGAATTCCGAACCGTTAGGGACATTGTC-3, restriction enzyme site EcoR I, pre- amplification length：606bp.
It is yet another object of the invention to provide a kind of ELISA kit of Nsp2 albumen in PRRSV, it includes 80-120
μ L recombinant proteins coating, 80-120 μ L positive controls, 80-120 μ L negative controls, 80-120 ELIAS secondary antibodies (working concentration 1:
1000), 80-120 μ L μ L substrate solutions and 80-120 μ L μ L (2mol/LH2SO4) terminate liquid.
Further, in a kind of PRRSV Nsp2 albumen ELISA kit, it include 100 μ L recombinant proteins coating, 100
μ L positive controls, 100 μ L negative controls, 100 ELIAS secondary antibodies (working concentration 1:1000), 100 μ L μ L substrate solutions and 100 μ L μ L
(2mol/L H2SO4) terminate liquid.
The coated concentration of recombinant protein described above is 8.123 μ g/mL, positive and negative control sera dilution ratio
For 1:40th, the dilution factor of ELIAS secondary antibody is 1:1000.
Positive control described above is that PRRSV separation poison is injected in positive serum prepared by the pig not being immunized, negative and positive
Property its critical value be 0.3；Negative control is to be uninfected by the serum with porcine reproductive and respiratory syndrome vaccine rather, dilution factor 1:
40。
The concentration that serum dilution described above is BSA is 1g/100mL.
The concentration that confining liquid described above is BSA is 1g/100mL.
Substrate solution described above is tetramethyl benzidine.
Terminate liquid described above is the sulfuric acid that concentration is 2mol/L.
The specific formula of tetramethyl benzidine (TMB) substrate solution described above is：Chromogenic Substrate Solution：First by TMB with
0.1mol/L concentration is dissolved in dimethyl sulfoxide (DMSO), then, then it is dissolved in 1mmol/L and H202 with 3.0mmol/L concentration
0.2mol/L sodium acetates/citrate buffer (pH4.0), you can application；Terminate liquid：2mol/L sulfuric acid；Determine wavelength：450nm.
It is also another object of the present invention to provide a kind of ELISA kit preparation method of Nsp2 albumen in PRRSV, bag
Include following steps：
(1) blank ELISA Plate is taken, 100 μ L Nsp2 protein solutions are added in every hole, in being mixed on oscillator, capping is sealed
4 DEG C of coatings of membrana oralis are overnight；
(2) remaining liq in hole is discarded, elisa plate is washed 5 times with cleaning solution (1 × PBST), is patted dry in blotting paper；
(3) confining liquid 100 μ L, 37 DEG C of closing 1h are added per hole；
(4) repeat step (2)；
(5) primary antibody is diluted to working concentration, 100 μ L is added per hole, after gently vibrating, 37 DEG C of effect 1h；
(6) repeat step (2)；
(7) ELIAS secondary antibody goat-anti pig IgG-HRP is diluted to working concentration (1:1000) 100 μ L, are added per hole, are gently shaken
After swinging, 37 DEG C of effect 1h；
(8) repeat step (2)；
(9) 100 μ L substrate solutions, 37 DEG C of Incubation in dark 15min are added per hole；
(10) terminate liquid (2mol/L H2SO4) 100 μ L terminating reactions are added per hole, immediately under ELIASA 450nm wavelength
Read absorbance value.
Beneficial effects of the present invention are：
This research and utilization establishes detection Nsp2 antibody by clonal expression Guizhou separation strains PT strain Nsp2 recombinant proteins
Indirect ELISA method, the method is quick, effective, sensitive, easy, there is good application prospect.
Brief description of the drawings：
Accompanying drawing 1：Recombinant plasmid PCR and digestion identification：(M1.-HindⅢ digest；2. I/EcoR of recombinant plasmid BamH
I double digestion；The digested plasmids of 3.BamH I；4. non-digested plasmid；5. the PCR amplifications of recombinant plasmid；6.PCR positive controls
M2.DL200 DNA Marker；)；
Accompanying drawing 2：Overnight expressing protein result (the Lane A of 16 DEG C of Rosetta pLysS cells:Product before not inducing；
Lane B:Product after induction；Lane C:Induced product supernatant；Lane D:Induced product precipitates；MK:Molecule protein
Marker)；
Accompanying drawing 3：Overnight expressing protein soluble analysis (the Lane A of 16 DEG C of Rosetta pLysS cells:The production of uncracked bacterium
Thing；Lane B:Precipitated after cracking；Lane C:Supernatant product after cracking；MK:Molecule protein Marker)；
Accompanying drawing 4：Expressing protein Western-Blotting analyzes (Lane A:Recombinant bacterium is not induced；Lane B:After induction
Whole bacterial protein；Lane C:Induction bacterium supernatant protein；LaneD:Induction bacterium protein precipitation；MK:Molecule protein Marker)；
Accompanying drawing 5：Expressing protein purification result (MK：Albumen Marker；A：The destination protein of purifying)；
Embodiment
In order to deepen the understanding of the present invention, the present invention is specifically described below by embodiment and experimental example.Have
It is necessary it is pointed out here that be that the present embodiment is served only for being described further invention, it is impossible to be interpreted as to the scope of the present invention
Limitation, if the person skilled in the art in the field makes some nonessential improvement and tune according to the invention described above content to the present invention
It is whole, still fall within the scope of the present invention.
Embodiment 1：The expression of Nsp2 recombinant proteins：
1 materials and methods
1.1 bacterial strains and carrier
Bacillus coli DH 5 alpha, Rosetta pLysS are that this laboratory preserves；Prokaryotic expression carrier pET-32a, purchased from U.S.
Novagen companies of state；PMD18-T-Nsp2/JL recombinant plasmids are completed by this research and establishment.
1.2 toolenzymes and various reagents
Gel Extraction Kit, plasmid extraction kit are purchased from OMEGA companies；T4DNA ligase, restriction enzyme
Enzyme (BamH I and Hind III) is purchased from MBI Fermentas companies；Pvdf membrane, IPTG are purchased from SIGMA companies；PRRSV positive bloods
Prepared clearly and preserved by this laboratory；Goat-anti pig IgG-HRP is purchased from Beijing Suo Laibao Science and Technology Ltd；Protein Marker is purchased from
Beijing Tiangeng biology Co., Ltd；Protein purification kit (HisBind Purification Kit, model 70239-3) is purchased
From Novagen companies of the U.S.；DAB colour reagents box is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；Other is analysis
It is pure.
The amplification of 1.3 Nsp2 genes
According to the PRRSV JXA1 pnca gene sequences on GenBank, by analysis, one is designed and synthesized using Primer5
To Nsp2 gene-specific primers, synthesized by precious bioengineering Co., Ltd.Primer sequence is shown in Table 1.
The primer nucleotide sequences of table 1
μ L of PCR reaction systems 50 μ L, i.e. 10 × PCR Buffer 3, μ L of MgCl2 (25mM) 2.5, the μ L of dNTP (10mM) 1,
Each 2 μ L of Nsp2-1/Nsp2-2 primers, μ L of Taq enzyme 1, appropriate cDNA, deionized water complement to 50 μ L；PCR response parameters are：94
DEG C 3min pre-degenerations, then carry out 30 circulations, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 50s, last 72 DEG C of extensions 5min.
The structure of 1.4 Nsp2 gene RT-PCR expression plasmids and identification
Double digestion, production are carried out to target gene fragment and carrier pMD18-T using restriction enzyme BamH I and Hind III
Thing electrophoresis reclaims.The target gene of recovery purifying is connected overnight for 4 DEG C with carrier pET-32a, transformed competence colibacillus cell
Rosetta pLysS, are coated on the LB agar plates containing Amp (50mg/L), 37 DEG C of incubated 12~16h, and picking is single
Positive bacterium colony is inoculated in the LB fluid nutrient mediums containing Amp (50mg/L), 37 DEG C of 180r/min shaken cultivations, extracts matter in a small amount
Grain, after PCR and the identification of the double digestions of III/BamH of Hind I, by the sequencing identification of positive plasmid Song Bao bioengineering Co., Ltd.
The prokaryotic expression of 1.5 Nsp2 recombinant plasmids
The single restructuring bacterium colony for being accredited as the positive is inoculated into 5mL LB nutrient solutions (containing 50 μ g/mL Amp), 37 DEG C are shaken
Swing overnight.Take and be incubated overnight bacterium solution 1mL and be inoculated in 100mL LB culture mediums (containing 50 μ g/mL Amp), 37 DEG C of shaken cultivations are extremely
When bacterium solution OD600 is 0.4~1.0, IPTG to final concentration of 0.1mM is added, 16 DEG C of induced expressions are stayed overnight, and take bacterium solution to carry out SDS-
PAGE is analyzed, while sets bacterial strain conduct control before induction.
The soluble analysis of 1.6 recombinant proteins
Recombinant bacterium is induced by optimum condition, the bacterium solution after induction is centrifuged into 10min through 10 000g, supernatant is abandoned and collects thalline,
Add 1 × Binding Buffer and thalline is resuspended, centrifuged after ultrasonic treatment, take the loading of supernatant precipitation plus equivalent respectively
Buffer solution carries out SDS-PAGE electrophoresis, to determine the expression way of albumen.
1.7 recombinant protein N sp2 Western-Blotting analyses
After the recombinant protein of expression carries out SDS-PAGE, by destination protein in 4 DEG C, pvdf membrane is transferred under conditions of 30V,
Then film is put into trace bag, 3% BSA/PBST closing 3h, by film and the pig source PRRSV positives after PBST is fully washed
Serum (diluting according to a certain percentage) is incubated 3h in 37 DEG C of wet box, goat-anti pig IgG-HRP reaction 3h is added after PBST washings, most
Transfer film is developed the color with DAB colour reagents box afterwards, reacted after outstanding obvious band with distilled water color development stopping, analysis result.
Embodiment 2：The foundation of indirect ELISA method
1.8.1 recombinant protein N sp2 purifying
Albumen application HisBind Purification Kit after filtering are purified, method is entered to specifications
OK.A small amount of albumen after purification is taken to carry out SDS-PAGE analyses.
1.8.2 the step of indirect ELISA method
(1) blank ELISA Plate is taken, 100 μ L Nsp2 protein solutions are added in every hole, in being mixed on oscillator, capping is sealed
4 DEG C of coatings of membrana oralis are overnight；
(2) remaining liq in hole is discarded, elisa plate is washed 5 times with cleaning solution (1 × PBST), is patted dry in blotting paper；
(3) confining liquid 100 μ L, 37 DEG C of closing 1h are added per hole；
(4) repeat step (2)；
(5) primary antibody is diluted to working concentration, 100 μ L is added per hole, after gently vibrating, 37 DEG C of effect 1h；
(6) repeat step (2)；
(7) ELIAS secondary antibody goat-anti pig IgG-HRP is diluted to working concentration (1:1000) 100 μ L, are added per hole, are gently shaken
After swinging, 37 DEG C of effect 1h；
(8) repeat step (2)；
(9) 100 μ L substrate solutions, 37 DEG C of Incubation in dark 15min are added per hole；
(10) terminate liquid (2mol/L H2SO4) 100 μ L terminating reactions are added per hole, immediately under ELIASA 450nm wavelength
Read absorbance value.
1.8.3 the best effort concentration mensuration of antigen and serum
Albumen is diluted to by 65 μ g/mL, 32.5 μ g/ with coating buffer solution according to the concentration of the recombinant protein measured successively
ML, 16.25 μ g/mL, 8.125 μ g/mL, 4.063 μ g/mL, 2.032 μ g/mL, 1.016 μ g/mL levels, per the μ L of hole 100
ELISA reaction plates are coated with, 4 DEG C of coatings are overnight.According to matrix method, it is coated with the coated reacting hole of concentration and divides for same antigen
The positive and negative serum of different dilution factors is not added.Standard positive and negative serum is diluted to 1 respectively:10、1:20、1:40、1:80、
1:160 and 1:320.Other steps are carried out by indirect ELISA operating procedure, determine OD450nm, average value is calculated, selects positive value
1.0 or so, and the small antigen concentration of negative control background value and positive and negative serum dilution are as the optimal of antigen and serum
Working concentration.
1.8.4 the optimization of confining liquid and serum dilution
Using the concentration of optimal antigen and serum, change confining liquid species, while set up the control do not closed.Wherein close
1% gelatin, 5%FBS, 1%BSA and 5% skimmed milk is respectively adopted in liquid, is sealed simultaneously under 37 DEG C of temperature conditionss with matrix method
1.0h is closed, other are carried out by ELISA conventional steps.OD450nm values are determined, and calculate P/N values, with the closed group that P/N values are maximum
It is combined into optimal confining liquid.
1.8.5 the optimization of optimal ELIAS secondary antibody working concentration：
Using the concentration of optimal antigen and standard serum, ELISA Plate is closed with optimal confining liquid, changes enzyme mark two
Anti- working concentration, to detect optimal ELIAS secondary antibody concentration.By ELIAS secondary antibody respectively with 1:500、1:800、1:1000、1:1500 Hes
1:2000 dilutions, other are carried out by ELISA conventional steps.OD450nm values are determined, and calculate P/N values, it is determined that optimal ELIAS secondary antibody
Working concentration.
1.8.6 the determination of criterion
Known 20 parts of the porcine reproductive and respiratory syndrome negative antibody serum of laboratory preservation is randomly selected, according to having optimized
Optimal indirect ELISA condition detection serum antibody OD450nm values.Statistical analysis is carried out to result, calculates sample OD450nm
The average value (X) and standard deviation (S) of value.Therefore, the critical value using the value as PRRSV Nsp2 albumen indirect ELISA methods.
1.8.7 specific test
Using the ELISA method optimum reaction condition of foundation, the swine fever, pseudorabies, pig for causing pig reproductive diseases are justified
Circovirus virus disease II types, pig Japanese B encephalitis and swine foot-and-mouth disease virus positive serum and negative serum are detected, and identify the party
The specificity of method.
1.8.8 replica test
Elisa plate is coated with the Nsp2 expressing proteins of a collection of preparation, by the ELISA program determinations established, takes 3 parts of positives
Replica test is carried out on ELISA Plate with 1 part of negative serum, blank control wells is concurrently set, as a result carries out statistical analysis,
Average (X), standard deviation (S) and the coefficient of variation (CV) of each group are calculated respectively.
1.8.9 sensitivity tests
Expression recombinant protein is coated with by optimal coating concentration, positive serum presses 1:100,1:200,1:400,1:
800,1:1600,1:3200 and l:6400 this 7 gradient dilutions, remaining condition carry out indirect ELISA behaviour by optimum reaction condition
Make.
Experimental example：The Preliminary Applications of indirect ELISA method
1.9 applicating expedition methods：
221 parts of immune PRRS attenuated vaccine Swine serums are gathered respectively from the regional pig farm in 5, Guizhou Province,
Pig PRRSV non-structural protein Nsp2 antibody in the indirect ELISA method detection and analysis serum sample established using this experiment
Production, while application IDEXX porcine reproductive and respiratory syndrome virus ELISA antibody assay kits detect these serum
The antibody situation of sample, compares both results.
2 results
The identification of 2.1 recombinant expression plasmids
Target gene is connected with expression vector pET-32a, is built into recombinant expression plasmid pET-32a/Nsp2, it is inverted
Rosetta pLysS, recombinant expression plasmid is extracted, using recombinant expression plasmid as template, uses Nsp2-1/Nsp2-2 primer PCRs
Identified with double digestion.As a result Fig. 1 is seen.
By PCR in Fig. 1 and digestion qualification result, tentatively judge that target gene has been connected into expression vector pET-32a, will
The recombinant plasmid that PCR and double digestion are accredited as the positive send precious bioengineering (Dalian) Co., Ltd to carry out sequencing.As a result with
Cloning and sequencing result is identical.
2.2 are expressed using expressive host bacterium Escherichia coli Rosetta pLysS
The single restructuring bacterium colony for being accredited as the positive is inoculated into 5mL LB nutrient solutions (containing 50 μ g/mL Amp), 37 DEG C
Shaken overnight.Take and be incubated overnight bacterium solution 1mL and be inoculated in 100mL LB culture mediums (containing 50 μ g/mL Amp), 37 DEG C of shaken cultivations
When to bacterium solution OD600 being 0.4~1.0, IPTG to final concentration of 0.1mM is added, 16 DEG C of induced expressions stay overnight, and take bacterium solution to carry out
SDS-PAGE is analyzed, while sets bacterial strain conduct control before induction.As a result Fig. 2 is seen.
As seen from Figure 2, in Rosetta pLysS expressive host bacterium, 16 DEG C of induced expressions are stayed overnight, with phase before induction
Than the recombinant bacterium expression Nsp2 molecular weight of albumen after induction is about 41.9kDa, is consistent with the theoretical molecular of the albumen, Nsp2
Recombinant protein is expressed.
2.3 recombinant protein soluble analysis results
Substantial amounts of induced expression is carried out to recombinant bacterium according to above expression condition, supernatant is taken respectively after ultrasonic treatment centrifugation
SDS-PAGE analyses, the solubility of testing goal albumen are carried out with precipitation.As a result show, expression product is primarily present in supernatant
In, albumen is mainly based on solubility expression.See Fig. 3.
2.4 expression product immunoreactivities are analyzed
The recombinant protein of expression is transferred on pvdf membrane, after being reacted with PRRSV positive serums, with the goat-anti of HRP marks
Pig is that secondary antibody carries out Western-Blotting identifications, as a result sees Fig. 4.
As seen from Figure 4, about at 41.9kDa occur one, show expression restructuring whole bacterial protein, supernatant protein and
Protein precipitation can be identified there is good immunologic competence by PRRSV positive serums, and the recombinant bacterium not induced does not go out
Now obvious specific reaction band.
The foundation of 2.5 recombinant protein indirect ELISA methods and Preliminary Applications
2.5.1 recombinant protein purification interpretation of result
Destination protein is eluted from post.A small amount of albumen after purification is taken to carry out SDS-PAGE analyses.As a result figure is seen
5。
As seen from Figure 5, after Elute Buffer elutions, most of foreign protein is eliminated, has been obtained purer
Destination protein.
2.5.2 the foundation of indirect ELISA method
2.5.2.1 the best effort concentration mensuration of antigen and serum
Using matrix method design different dilution factors (65 μ g/mL, 32.5 μ g/mL, 16.25 μ g/mL, 8.125 μ g/mL,
4.063 μ g/mL, 2.032 μ g/mL, 1.016 μ g/mL) Nsp2 albumen and different dilution factors (1:10、1:20、1:40、1:80、1:
160 and 1:320) positive, negative serum reaction, determines its OD450nmValue, and its P/N value (being shown in Table 4) is calculated.Select P/N values
Best effort of the maximum and small negative control background value antigen concentration and positive and negative serum dilution as antigen and serum
Concentration.It the results are shown in Table 2.
The determination of the most suitable coating concentration of the antigen of table 2 and serum dilution
From table 2 it can be seen that comprehensive negative and positive serum OD450nmBe worth height, P/N ratios size, serum dilution and
The factors such as antigen coat concentration, determine that antigen coat amount is 8.123 μ g/mL, serum dilution 1:When 40, negative control background
It is worth relatively low, and P/N values are maximum, are 9.817.
2.5.2.2 the optimization of confining liquid and serum dilution
With most suitable antigen concentration coating elisa plate, after 4 DEG C are stayed overnight, with different confining liquid (1% gelatin, 5%FBS, 1%
BSA, 5% skimmed milk, do not close) closed, 37 DEG C of closing 1h, determine the OD in each hole450It is worth, and calculates P/N values, as a result table
It is bright with 1%BSA as confining liquid and serum dilution when, negative control OD450 values are minimum, and P/N value highests.
2.5.2.3 the optimization of optimal ELIAS secondary antibody working concentration
By ELIAS secondary antibody respectively with 1:500、1:800、1:1000、1:1500 and 1:2000 dilutions, other are conventional by ELISA
Step is carried out.Determine OD450nmValue.As a result when ELIAS secondary antibody dilution factor is 1：When 1000, and negative serum OD values are relatively low, now P/
N values are maximum.
2.5.2.4 the determination of criterion is by the optimal ELISA reaction conditions established, determine 20 parts of pigs breedings with
Respiration syndrome negative serum sample, the results are shown in Table 3.
The ELISA measurement results of table 3
It is computed, 20 parts of negative serum average value X=0.16005 of the above, standard deviation S=0.04705, ELISA negative and positive are faced
Dividing value=X+3S=0.3012, for the ease of identification, it is yin and yang attribute serum critical value to take 0.3.
2.5.2.5 specific test is using the ELISA method optimum reaction condition established, to causing pig reproductive diseases
Swine fever, pseudorabies, Porcine circovirus desease II types, pig Japanese B encephalitis and swine foot-and-mouth disease virus positive serum and negative serum
Detected, identify the specificity of this method.It the results are shown in Table 4.
The specific detection result of table 4
Shown by the result of table 4, porcine reproductive and respiratory syndrome is positive and negative serum control meets standard, and other are sick
Viral disease yin and yang attribute blood serum values are negative (＜ 0.32), show this indirect ELISA method established using Nsp2 albumen and pig
Pest, porcine pseudorabies viral disease, Porcine circovirus desease II types, Japanese B encephalitis and the equal no cross reaction of aftosa serum, tentatively
Demonstrate the specificity of this method.
2.5.2.6 replica test is coated with elisa plate with the Nsp2 expressing proteins of a collection of preparation, by the ELISA established
Program determination, 3 parts of positives and 1 part of negative serum is taken to carry out replica test on ELISA Plate.
It the results are shown in Table 5.
The replica test result of table 5
As shown in Table 5, to test data carry out statistical analysis, its coefficient of variation between 2.24%~8.11%,
Respectively less than 15%, illustrate that same sample degree of variation in different batches experiment is small, repeatability is preferably.
2.5.2.7 sensitivity tests will be expressed recombinant protein and is coated with by optimal coating concentration, and positive serum presses 1:
100,1:200,1:400,1:800,1:1600,1:3200 and l:6400 this 7 gradient dilutions, remaining condition press optimum response bar
Part carries out indirect ELISA operation.It the results are shown in Table 6.
The sensitivity Detection result of table 6
As shown in Table 6, when positive serum is diluted to 1:When 800, sample OD is detected450>0.3, it is still the positive, it was demonstrated that the party
Method sensitiveness is preferable.
The Preliminary Applications of 2.6 indirect ELISA methods
From 221 parts of immune PRRS attenuated vaccine Swine serums of the 5 regional pig farm collections in Guizhou Province, profit
The Antibody Results for indirect ELISA and IDEXX kits the detection serum sample established with this experiment recombinant protein show positive rate
Respectively 59.73% (132/221) and 61.09% (135/221), it is as a result basically identical.Examined through t, the not notable (P of difference>
0.5)。
Claims (9)
- A kind of 1. porcine reproductive and respiratory syndrome virus Nsp2 expressions, it is characterised in that：Comprise the following steps：PCR reacts System 50L, drawn by 10 × PCR Buffer 2-4L, MgCl2 (25mM) 1-5L, dNTP (10mM) 1-3L, Nsp2-1/Nsp2-2 Each 1-3L of thing, the appropriate 1-3L of Taq enzyme 0.5-2L, cDNA, deionized water complement to 50 and formed；PCR response parameters are：90-98℃ 2-4min pre-degenerations, then carry out 30 and circulate, 90-98 DEG C, 25-35s, 52-60 DEG C, 25-35s, 68-76 DEG C 40-60, finally 68-76 DEG C of extension 4-6min；Primer is Nsp2-15 '-CGCGGATCCGACACCTCCTTTGATTGGAAT-3 ', restriction enzyme site BamH I, Nsp2-25-CCGGAATTCCGAACCGTTAGGGACATTGTC-3, restriction enzyme site EcoR I, pre- amplification length： 606bp。
- 2. porcine reproductive and respiratory syndrome virus Nsp2 expressions according to claim 1, it is characterised in that：Including with Lower step：PCR reaction system 50L, i.e. 10 × PCR Buffer 3L, MgCl2 (25mM) 2.5L, dNTP (10mM) 1L, Nsp2- Each 2L of 1/Nsp2-2 primers, Taq enzyme 1L, appropriate cDNA, deionized water complement to 50L；PCR response parameters are：94 DEG C of 3min are pre- Denaturation, then carries out 30 circulations, 94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 50s, last 72 DEG C of extensions 5min；Primer is Nsp2- 15 '-CGCGGATCCGACACCTCCTTTGATTGGAAT-3 ', restriction enzyme site BamH I, Nsp2-25 '- CCGGAATTCCGAACCGTTAGGGACATTGTC-3, restriction enzyme site EcoR I, pre- amplification length：606bp.
- A kind of 3. ELISA kit of Nsp2 albumen in PRRSV, it is characterised in that：Be coated with including 80-120 μ L recombinant proteins, 80-120 μ L positive controls, 80-120 μ L negative controls, 80-120 ELIAS secondary antibodies, 80-120 μ L substrate solutions and 80-120 μ L are terminated Liquid.
- 4. the ELISA kit of Nsp2 albumen in PRRSV according to claim 3, it is characterised in that：Including 100 μ L weights Histone coating, 100 μ L positive controls, 100 μ L negative controls, 100 ELIAS secondary antibodies, 100 μ L substrate solutions and 100 μ L terminate liquids.
- 5. the ELISA kit of Nsp2 albumen in the PRRSV according to right 3 or 4, it is characterised in that：Described restructuring egg White coated concentration is 8.123 μ g/mL, and positive and negative control sera dilution ratio is 1:40th, the dilution factor of ELIAS secondary antibody For 1:1000.
- 6. the ELISA kit of Nsp2 albumen in the PRRSV according to right 3 or 4, it is characterised in that：Described is positive right According to the positive serum that the pig not being immunized preparation is injected in for PRRSV separation poison, its critical value of yin and yang attribute is 0.3；Negative control To be uninfected by the serum with porcine reproductive and respiratory syndrome vaccine rather, dilution factor 1:40.
- 7. the ELISA kit of Nsp2 albumen in the PRRSV according to right 3 or 4, it is characterised in that：Described serum is dilute It is 1g/100mL to release the concentration that liquid is BSA, and the concentration that confining liquid is BSA is 1g/100mL, and described substrate solution is tetramethyl connection Aniline, terminate liquid are the sulfuric acid that concentration is 2mol/L.
- 8. the ELISA kit of Nsp2 albumen in PRRSV according to claim 7, it is characterised in that：Described tetramethyl The formula of benzidine (TMB) substrate solution is：Chromogenic Substrate Solution：TMB is first dissolved in dimethyl sulfoxide (DMSO) with 0.1mol/L concentration, Then, 0.2mol/L sodium acetates/citrate buffer then by it with 1mmol/L and H202 is dissolved in 3.0mmol/L concentration (pH4.0), you can application；Terminate liquid：2mol/L sulfuric acid；Determine wavelength：450nm.
- A kind of 9. ELISA kit preparation method of Nsp2 albumen in PRRSV, it is characterised in that：Comprise the following steps：(1) blank ELISA Plate is taken, 100 μ L Nsp2 protein solutions are added in every hole, in being mixed on oscillator, is capped sealed membrane 4 DEG C of coatings are overnight；(2) remaining liq in hole is discarded, elisa plate is washed 5 times with cleaning solution (1 × PBST), is patted dry in blotting paper；(3) confining liquid 100 μ L, 37 DEG C of closing 1h are added per hole；(4) repeat step (2)；(5) primary antibody is diluted to working concentration, 100 μ L is added per hole, after gently vibrating, 37 DEG C of effect 1h；(6) repeat step (2)；(7) ELIAS secondary antibody goat-anti pig IgG-HRP is diluted to working concentration (1:1000) 100 μ L, are added per hole, are gently vibrated Afterwards, 37 DEG C of effect 1h；(8) repeat step (2)；(9) 100 μ L substrate solutions, 37 DEG C of Incubation in dark 15min are added per hole；(10) terminate liquid (2mol/L H2SO4) 100 μ L terminating reactions are added per hole, are read immediately under ELIASA 450nm wavelength Absorbance value.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057145A (en) * | 2019-11-22 | 2020-04-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof |
| CN113321712A (en) * | 2021-07-20 | 2021-08-31 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Antigen polypeptides of porcine reproductive and respiratory syndrome virus and application thereof |
| CN114778852A (en) * | 2022-04-26 | 2022-07-22 | 中国农业大学 | Indirect ELISA (enzyme-linked immuno sorbent assay) method for detecting PRRSV PLP2 antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645712A (en) * | 2016-09-21 | 2017-05-10 | 福建省农业科学院畜牧兽医研究所 | Kit for PRRSV (porcine reproductive and respiratory syndrome virus) serology differential diagnosis |
-
2017
- 2017-07-24 CN CN201710605175.9A patent/CN107356754A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645712A (en) * | 2016-09-21 | 2017-05-10 | 福建省农业科学院畜牧兽医研究所 | Kit for PRRSV (porcine reproductive and respiratory syndrome virus) serology differential diagnosis |
Non-Patent Citations (2)
| Title |
|---|
| 周思旋等: "猪繁殖与呼吸综合征病毒贵州PT分离株Nsp2基因克隆及序列变异分析", 《中国畜牧兽医》 * |
| 罗弦: "PRRSV变异株Nsp2基因的克隆、原核表达及其ELISA方法的初建", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057145A (en) * | 2019-11-22 | 2020-04-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof |
| CN111057145B (en) * | 2019-11-22 | 2021-10-08 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Porcine reproductive and respiratory syndrome virus Nsp2 protein nano antibody and application thereof |
| CN113321712A (en) * | 2021-07-20 | 2021-08-31 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Antigen polypeptides of porcine reproductive and respiratory syndrome virus and application thereof |
| CN113321712B (en) * | 2021-07-20 | 2023-02-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Antigen polypeptides of porcine reproductive and respiratory syndrome virus and application thereof |
| CN114778852A (en) * | 2022-04-26 | 2022-07-22 | 中国农业大学 | Indirect ELISA (enzyme-linked immuno sorbent assay) method for detecting PRRSV PLP2 antibody |
| CN114778852B (en) * | 2022-04-26 | 2023-09-01 | 中国农业大学 | Indirect ELISA method for detecting PRRSV PLP2 antibody |
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