CN104165997B

CN104165997B - PRRSV genetic marker vaccine strain ELISA differential diagnosis kit and methods and applications

Info

Publication number: CN104165997B
Application number: CN201310217805.7A
Authority: CN
Inventors: 童光志; 周艳君; 孙晶; 姜峰; 姜一峰; 童武
Original assignee: Shanghai Veterinary Research Institute CAAS
Current assignee: Shanghai Veterinary Research Institute CAAS
Priority date: 2013-06-03
Filing date: 2013-06-03
Publication date: 2016-09-14
Anticipated expiration: 2033-06-03
Also published as: CN104165997A

Abstract

The open a kind of PRRSV genetic marker vaccine strain ELISA differential diagnosis kit of the present invention, described test kit comprises NP49 polypeptide antigen, and the aminoacid sequence of this NP49 polypeptide is as shown in SEQ ID NO:1.PRRSV genetic marker vaccine strain ELISA differential diagnosis kit of the present invention, using NP49 polypeptide as envelope antigen, there is high specificity, highly sensitive, reproducible feature, the serum antibody of tradition PRRS vaccine immunity and marker vaccine immune swine can be distinguished quickly and accurately, Differential Diagnosis quickly and accurately can go out PRRSV genetic marker vaccine strain and natural infection strain, the wide clinical application for PRRS genetic marker vaccine strain is significant simultaneously.

Description

PRRSV genetic marker vaccine strain ELISA differential diagnosis kit and methods and applications

Technical field

The present invention relates to porcine reproductive and respiratory syndrome virus (PRRSV) detection technique field, be specifically related to a kind of PRRSV base Because of marker vaccine strain ELISA differential diagnosis kit and methods and applications.

Background technology

Porcine reproductive and respiratory syndrome (PRRS) is serious harm in the global range caused by porcine reproductive and respiratory syndrome virus The infectious disease of pig industry.Within 1987, first find that this disease, China isolate PRRSV for 1996 first in the U.S., but 2006 Year high-pathogenicity porcine reproductive breaks out on a large scale with respiration syndrome (HP-PRRS), brings massive losses, epidemic disease to China's aquaculture Seedling immunity is the Main Means of current anti-PRRSV processed, the most domestic for preventing the commercialized vaccine of PRRSV mainly to have high cause Characteristic of disease reproductive and respiratory syndrome inactivated vaccine, classical CH-1R attenuated vaccine, 3 kinds of high-pathogenicity blue ear disease attenuated vaccines and external A little commercialization attenuated vaccines etc., the PRRS vaccine of above-mentioned numerous kinds is widely used at home, although the use of vaccine can be controlled PRRS's processed is popular, also increases the difficulty to pig blue-ear disease pathogen infection Differential Diagnosis simultaneously.It is therefore desirable to development of new Marker vaccine exists to solve current all multi-vaccine Parallel runnings and highly pathogenic PRRSV simultaneously with classical PRRSV and is difficult to The trouble waters distinguished.The most in this context, this laboratory is at PRRSV attenuated vaccine HuN4-F112 strain infection clones On the basis of, utilize reverse genetics manipulation technology, using 49 aminoacid (NP49) of Newcastle disease virus NP albumen end as mark Note, inserts the nonessential absent region (Δ 508-532) of replicating of Nsp2, successfully builds double labelling genetic engineering attenuated vaccine strain (rHN4-Δ 25+NP49 strain), and experiments prove that this genetic marker vaccine strain is not only to high-pathogenicity porcine reproductive and respiratory syndrome virus Infection has preferable immune protective effect, simultaneously because introduce marker gene inside vaccine strain, for realizing marker vaccine Differential Diagnosis is laid a good foundation.

Summary of the invention

The invention solves the problems that being widely used due to numerous kinds PRRS vaccine at present, increase and pig blue-ear disease pathogen infection is reflected Do not diagnose the technical problem of difficulty, it is provided that a kind of PRRSV genetic marker vaccine strain ELISA differential diagnosis kit, this test kit Using NP49 as diagnostic antigen, can effectively distinguish the serum antibody of tradition PRRS vaccine immunity and marker vaccine immune swine, its spirit Sensitivity height, high specificity, reproducible.

In order to solve above-mentioned technical problem, the present invention is achieved through the following technical solutions:

In one aspect of the invention, it is provided that a kind of PRRSV genetic marker vaccine strain ELISA differential diagnosis kit, institute Stating test kit and comprise NP49 polypeptide antigen, the aminoacid sequence of this NP49 polypeptide is as shown in SEQ ID NO:1.

Described test kit also comprise ELISA ELISA Plate, confining liquid, serum dilution, ELIAS secondary antibody, display liquid, stop buffer, Standard positive serum, standard female serum.

Preferably, the concentration that is coated of NP49 polypeptide antigen is 500ng/ hole.

Preferably, described ELIAS secondary antibody is the goat anti-pig antibody of horseradish peroxidase-labeled, and its dilution factor is 1: 10000.

Described standard positive serum is to prepare, this restructuring NDVNP49 antigen with restructuring NDVNP49 antigen protein immune swine Albumen by the recombinant expression carrier comprising NP49 polypeptide coding genes through abduction delivering, prepare after purification.

Preferably, described confining liquid and bovine serum albumin that serum dilution is 2% weight concentration.

In another aspect of this invention, the PRRSV genetic marker vaccine strain ELISA additionally providing a kind of non-diagnostic purpose differentiates Method, comprises the following steps:

It is coated ELISA ELISA Plate with NP49 polypeptide antigen；

After test serum and envelope antigen effect, it is sequentially added into ELIAS secondary antibody, nitrite ion and stop buffer；

Microplate reader is utilized to read absorbance OD₄₅₀Nm, and press formula: S/P=(test serum Sample OD-Negative comparison OD)/(sun Property comparison OD-negative control OD), calculate test serum sample S/P value；

Test serum sample S/P value >=marginal value be judged to the positive, S/P value ＜ marginal value be judged to feminine gender, described marginal value is logical Cross the S/P value corresponding when sensitivity is with specificity numerical value sum maximum that ROC statistical analysis method obtains.

The dilution factor of described test serum is 1: 40.

In another aspect of this invention, additionally provide mentioned reagent box to exempt from preparation Differential Diagnosis PRRS genetic engineering marker vaccine Application in the product of epidemic disease pig.

PRRSV genetic marker vaccine strain ELISA differential diagnosis kit of the present invention, uses specific marker position section Avian pneumo-encephalitis virus 49 aminoacid (NP49) polypeptide of NP albumen end, as antigen, have high specificity, highly sensitive, reproducible spy Point, can distinguish the serum antibody of tradition PRRS vaccine immunity and marker vaccine immune swine quickly and accurately, simultaneously can be quick, accurate The Differential Diagnosis of true ground goes out PRRSV genetic marker vaccine strain and natural infection strain, for the clinic of PRRS genetic marker vaccine strain Extensively application is significant.

Accompanying drawing explanation

The present invention is further detailed explanation with detailed description of the invention below in conjunction with the accompanying drawings.

Fig. 1 is that structure, expression, purification and the antigenicity of the NDV NP49 prokaryotic expression vector of the embodiment of the present invention 1 are divided Analysis result figure；

Fig. 2 is that the embodiment of the present invention 2 determines that antigen is most preferably coated the result figure of condition；

Fig. 3 is the result figure that the embodiment of the present invention 2 determines confining liquid；

Fig. 4 is the result figure that the embodiment of the present invention 2 determines off-period；

Fig. 5 is the result figure that the embodiment of the present invention 2 determines serum dilution；

Fig. 6 is the result figure that the embodiment of the present invention 2 determines the seroreaction time；

Fig. 7 is that the embodiment of the present invention 2 determines enzyme labelled antibody dilution result figure；

Fig. 8 is the result figure that the embodiment of the present invention 2 determines enzyme labelled antibody action time；

Fig. 9 is the result figure that the embodiment of the present invention 2 determines the substrate reactions time；

Figure 10 is 679 parts of clinical serum sample NP49-ELISA testing result figures of the embodiment of the present invention 5；

Figure 11 is the embodiment of the present invention 5 negative serum and positive serum samples NP49-ELISA testing result figure；

Figure 12 is the S/P value result figure of the embodiment of the present invention 5 negative serum and positive serum samples；

Figure 13 is the NP49-ELISAROC curve chart of the embodiment of the present invention 5.

Detailed description of the invention

In the following example, the experimental technique of unreceipted actual conditions, the most routinely condition, such as " Molecular Cloning: A Laboratory guide " (Pehanorm Brooker J, Russell DW write, Huang Peitang, Wang Jiaxi, Zhu Houchu, wait and translate. Molecular Cloning: A Laboratory guide, 3rd edition, Beijing: Science Press, 2002) method described in is carried out.

Porcine reproductive and respiratory syndrome virus (PRRSV) rHN4-Δ 25+NP₄₉Strain is a kind of for preventing high-pathogenicity blue ear disease Novel gene markers attenuated vaccine Candidate Strain, in order to coordinate the Efficacy evaluation of this genetic marker vaccine strain, the present invention with mark Note gene NP49 (Newcastle disease virus NP PROTEIN C end 49 aminoacid, i.e. NP49) polypeptide (SEQ ID NO:1) is as bag By antigen, set up and can be used for detecting rHN4-Δ 25+NP₄₉For the specific ELISA of marker gene in vaccine strain immune swine serum Antibody.By the optimization to a series of ELISA working condition such as antigen coated concentration, antibody dilutions, result shows The suitableeest concentration that is coated of the polypeptide antigen of NP49-ELISA is 500ng/ hole, and tested serum optimum dilution degree is 1: 40, ELIAS secondary antibody Optimum dilution degree is 1: 10000.S/P marginal value is 0.2 to utilize ROC curve method to determine, to sample clinical known to 200 parts of backgrounds In product are carried out batch and batch between its coefficient of variation of replica test be respectively less than 10%, show that this detection method has preferable repeatability. Specific detection result of the test shows, the method and other several important swine diseases serum do not have cross reaction；A large amount of clinical sample inspections Survey result shows, the method testing result is 87.9% with total coincidence rate of IDEXX test kit testing result, can effectively distinguish Tradition PRRS vaccine immunity and the serum antibody of marker vaccine immune swine, the NP that the present invention sets up₄₉-ELISA antibody detection method and Test kit is to coordinate PRRSV novel gene markers attenuated vaccine rHN4-Δ 25+NP further₄₉The clinical practice of strain has provided Try hard to keep barrier.

The preparation of embodiment 1 standard serum and the acquisition of clinical serum

1. preparation restructuring NDV NP49 antigen protein

(1) structure of recombinant expressed NDV NP49 antigen prokaryotic expression carrier

NDV La Sota strain (GenBank, NO.AF077761) NP protein gene sequence design specificity according to having announced draws Thing, forward primer (NP49-UP): 5 '-TTAGAATTCGGGGATGGGGAGACCCAAT-3 ' (SEQ ID NO:2), Its 5 ' end is designed with EcoR I restriction enzyme site；Downstream primer (NP49-DP): 5 '-ATACTCG4GATACCCCCAGTCG GTGTCGT-3 ' (SEQ ID NO:3), its 5 ' end is designed with Xho I restriction enzyme site.Expection amplification length is 165bp. Primer is synthesized by Invitrogen company (Shanghai), and the primer sterilizing distilled water of synthesis is diluted to final concentration 10mmol/L, is placed in -20 DEG C standby.

NDV La Sota strain total serum IgE is extracted by the description of RNeasy Plus Mini Kit test kit.Say with reference to AMV product Bright book, with U12 random primer as reverse transcription primer, synthesizes the first chain cDNA.Reverse transcription reaction system and transcriptive process,reversed are such as Under.

Reverse transcription system is 20uL:

Reverse transcription reaction condition is: 42 DEG C of water-bath 60min, 70 DEG C of 5min.After cDNA synthesis ,-20 DEG C save backup.

Then with cDNA as template, NP49-UP/NP49-DP primer is used to carry out PCR amplification.PCR reacts cumulative volume 50 μ L, include 10 × PCR buffer 5 μ L, dNTP (10mmol/L) 1.5 μ L, each 1 μ L of upstream and downstream primer, rTaq DNA poly- Synthase 1 μ L, cDNA1 μ L, add water to 39.5 μ L.PCR program is: 95 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 60 DEG C of 30s, 72 DEG C extend 30s, carry out 35 circulations altogether；Last 72 DEG C extend 10min.After amplification, take 5 μ L Product carries out agarose gel electrophoresis the observed result of 1%.Reclaim the electrophoretic band being accredited as the positive.

By reclaim purpose PCR primer and prokaryotic expression carrier pGEX-6P-1 respectively through EcoR I and Xho I double digestion, after enzyme action Two kinds of products respectively with glue reclaim test kit be purified recovery.Subsequently the genes of interest of enzyme action is pressed suitable volume with carrier Than mixing, under the effect of T4DNA ligase, 16 DEG C connect overnight, will connect product and convert to JM109 competent cell, Overnight incubation on the LB flat board containing ampicillin.After resistance screening, picking positive colony expands, and uses PCR respectively Method, double digestion method (EcoR I and Xho I) are identified, and the bacterium solution being initially identified as the positive is sent to Shanghai Invitrogen Bioisystech Co., Ltd's order-checking is identified.The named pGEX-NP49 of plasmid of the positive will be accredited as.

(2) the restructuring expression of NDVNP49 antigen, purification

PGEX-NP49 Plastid transformation Escherichia coli BL21 (DE3) of the positive will be accredited as with PCR method, double digestion method and order-checking Competent cell, chooses bacterium and is inoculated in the LB culture medium containing ampicillin, 37 DEG C of incubated overnight, and next day is by 1: 100 inoculation Amount be inoculated in the LB culture medium containing ampicillin, 37 DEG C, 220rpm cultivate about 4h, when bacterium solution OD₆₀₀When value reaches 0.6～0.8, Adding IPTG to final concentration of 1mmol/L, 37 DEG C of abduction deliverings, collect bacterium solution 1mL after 12h, 12000r/min is centrifuged 3min, Suspending with the PBS of 40 μ L pH7.4, add 10 μ L5 × SDS sample-loading buffer, mixing, water-bath is boiled 10min, is carried out SDS-PAGE electrophoretic analysis, through coomassie brilliant blue staining, methanol and glacial acetic acid decolouring observed result (setting empty carrier to compare).With Time expressing protein carried out Western blot detection.The detection method of Western Blot is as follows: protein band uses half-dried transfer Method is transferred in cellulose acetate membrane, overnight closes with 10% defatted milk powder (mass/volume ratio) 4 DEG C；After PBST washs three times, Add NDV polyvalent antibody IgG, room temperature reaction 60min；With uncombined in PBST three abundant removing cellulose acetate membrane of washing Serum IgG；Anti-chicken lgG (goat) antibody that cellulose acetate membrane immersion is diluted with confining liquidThe anti-solution of Conjugated bis-, room temperature lucifuge reaction 60min；Three abundant removal acetic acid are washed fine with PBST Dimension element film unconjugated two resists；Finally, by Dual band IR laser imaging system (odyssey company, the U.S.) analytical reactions result.

Being enlarged cultivating at Escherichia coli BL21 (DE3) by the plasmid containing pGEX-NP49, next day is by 1: 100 inoculation Amount is inoculated in the LB culture medium of the 200mL containing ampicillin, and 37 DEG C of 220rpm cultivate about 4h, when bacterium solution OD₆₀₀Value reaches When 0.6～0.8, add IPTG to final concentration of 1mmol/L, 37 DEG C of abduction deliverings, collect bacterium solution after 12h, 12000rpm from Heart 10min collects thalline.Add in the thalline collected 20mL excusing from death disruption buffer (20mM Tris/HCL, pH8.0, 0.5mM EDTA), the most repeatedly blow and beat mixing, use ultrasonic method to crush thalline.Ultrasonic condition is: 200w, and super 5s stops 5s, altogether 5min.4 DEG C, 1,2000rpm is centrifuged 10min, stays supernatant standby.Egg is carried out according to the description of GST affinity column Bai Chunhua.The destination protein being recovered to is carried out WesternBlot detection simultaneously.

(3) the expression identification result of restructuring NDVNP49 albumen

NDV NP49 gene is cloned in pGEX-6P-1 prokaryotic expression carrier structure pGEX-NP49, positive colony warp After PCR, enzyme action and order-checking are accredited as the positive (Figure 1A), convert DE3 escherichia coli and express, and use GST affinity chromatograph Column purification obtains the NP49 albumen (Figure 1B) of amalgamation and expression, and period expresses with Western Blot detection and purification obtains NP49 egg White reactionogenicity (Fig. 1 C).

2. prepare standard serum

By the restructuring NDVNP49 antigen protein for preparing after purification, immunity market pig one, the PRRS of market pig in three times Antigen and antibody test are all negative.

Immunization method and dosage are as follows: immunity for the first time, and not formula Freund's complete adjuvant and purifying protein mix and fully emulsified by 1: 1, 500 μ g purifying proteins, the immunity of subcutaneous multiple spot.Carrying out two after two weeks to exempt from, not formula Freund's incomplete adjuvant and purifying protein are by 1: 1 mixing And fully emulsified, 500 μ g purifying proteins, the immunity of subcutaneous multiple spot.Same dose and method, carry out three after two weeks and exempt from.3rd After secondary immune one week, market pig using throat caval vein blood sampling, separates serum stand-by, this serum is standard serum.

3. the acquisition of clinical serum

PRRSV HuN4-F112 positive serum, PRRSV negative serum, pig parvoviral (PPV) positive serum, pig circle Ring 2 type virus (PCV-2) positive serum provides for veterinary institute swine diseases research department, Harbin；PRRSV rHN4-Δ25+NP49 Strain positive serum, pseudorabies (PRV) positive serum, swine fever (CSFV) positive serum and porcine epizootic diarrhea (PEDV) Positive serum, is this laboratory and preserves.

Embodiment 2NP49-ELISA experimental condition optimization

1. antigen is most preferably coated concentration and the determination of optimal serum dilution

(1) 10 times of gradients are diluted with 0.05M carbonate buffer solution (pH9.6), by NP49 polypeptide antigen (10 μ g/mL to 0.125 μ g/mL) it is coated on 96 orifice plates, every hole 100 μ L；

(2) condition of being coated is 4 DEG C and is coated overnight, and closing is to use 5% skimmed milk, under the conditions of 37 DEG C, closes 2h.

(3) PRRSV rHN4-Δ 25+NP49 strain positive serum, negative serum are respectively adopted 1: 20,1: 40,1: 80,1: 160 gradient dilutions, every hole 100 μ L diluent, hatch 1h under the conditions of 37 DEG C and carry out ELISA square formation test.

(4) 1: 10000 times of dilution of goat anti-pig IgG antibody of horseradish peroxidase (HRP) labelling, every hole 100 μ L, Hatch 1h for 37 DEG C.

(5) 50 μ L TMD chromogenic substrates, room temperature lucifuge colour developing 10min are added.

(6)50μL2mol/L H₂SO₄Terminate reaction.Measure each hole OD₄₅₀Nm value, determines optimal antigen coated concentration and serum Diluted concentration.

Result:

The initial concentration of antigen is 1mg/ml, determines optimal antigen coated concentration and serum-dilution concentration by chessboard method.By table 1 Understanding, antigen and the increase of antibody extension rate, there is downward trend in the OD value of positive serum, the OD value change of negative serum The most notable.Select the OD of negative serum₄₅₀Nm value≤0.100, the OD of positive serum₄₅₀Nm value >=0.200, positive serum and Negative serum OD₄₅₀The greatest dilution of nm ratio P/N >=2, the i.e. dilution factor of serum are 1: 40, and the diluted concentration of antigen is 1: 200, every hole package amount be 500ng antigen be optimal package amount.

Table 1 chessboard method measures positive serum and the OD450nm value of negative serum

2. antigen is most preferably coated the determination of condition

Use optimal antigen coated concentration and optimal serum dilution, respectively with 37 DEG C of 1h, 37 DEG C of 2h, 37 DEG C of 4h and 4 DEG C Overnight 4 kinds of different conditions are coated polypeptide protein, and other conditions are constant, by PRRS rHN4-Δ 25+NP49 positive serum and feminine gender Serum carries out indirect ELISA mensuration, determines the condition that is most preferably coated of antigen.

Result, as in figure 2 it is shown, 4 DEG C of P/N values the most coated are apparently higher than other, is coated effect preferably, and selecting 4 DEG C is overnight Antigen is most preferably coated condition.

3. the determination of confining liquid

With the suitableeest antigen concentration, 4 DEG C of overnight coated elisa plates, after washing, respectively by BSA (bovine serum albumin), calf serum, Defatted milk powder, PBST, IDEXX diluent etc. make different confining liquids.Other conditions are constant, read with enzyme connection detector OD₄₅₀Nm value, respectively organizes the OD of positive and negative serum₄₅₀Nm value and P/N value, to select suitable confining liquid.

Result: select PBST, 1%BSA, 2%BSA, 5% defatted milk, 10% hyclone, IDEXX diluent as envelope Close liquid.Respectively organize the OD of positive and negative serum₄₅₀Nm value and P/N value, result is as it is shown on figure 3, with 2%BSA as confining liquid Time, negative serum OD₄₅₀Nm value is minimum, and P/N value is maximum, and selecting this condition is suitable confining liquid.

4. the determination of off-period

With the suitableeest antigen concentration and be coated condition coated elisa plate, add confining liquid, 200 μ L/ holes after washing, be divided into 4 groups, point It is not 37 DEG C and closes 1h, 2h, 4h and 4 DEG C overnight.Other conditions are constant, respectively organize the OD of positive and negative serum₄₅₀Nm value With P/N value, to determine off-period.

Result: during off-period determines, closes, different time under the conditions of 37 DEG C, and P/N value does not has a notable difference, and 4 DEG C Under the conditions of overnight, P/N value is apparently higher than other conditions (see Fig. 4), and selecting 4 DEG C is overnight optimal off-period.

5. the selection of serum dilution

1%BSA, 2%BSA, 10% hyclone it is separately added into dilute as serum dilution and IDEXX serum in PBST Release liquid and carry out ELISA mensuration, calculate each P/N value, to select main component in serum dilution.

Result shows, uses PBST and IDEXX diluent, records the OD of negative serum₄₅₀Nm is the most higher；2%BSA is dilute When releasing liquid, the OD of negative serum₄₅₀Nm value is less than 0.100, and P/N value is maximum (see Fig. 5), and selection 2%BSA is serum-dilution Liquid.

6. the determination of serum optimum reacting time

With selected serum dilution, known positive and negative serum is done 1: 40 dilution, act on respectively at 37 DEG C 30min, 60min, 120min, carry out ELISA detection, determines serum optimum reacting time.

Found that positive and negative OD₄₅₀Nm value the most substantially changes, and selecting short reaction time 30min is Best Times (see Fig. 6).

7. the dilution factor of ELIAS secondary antibody determines

By the goat anti-pig antibody of HRP labelling respectively according to 1: 1000,1: 2000,1: 4000,1: 8000,1: 10000, 1: 20000 dilution, reacts 1h at 37 DEG C, after washing, add the substrate solution 50 μ L/ hole newly joined, color development at room temperature 10min, use Enzyme connection instrument measures OD₄₅₀Nm value, respectively organizes the OD of positive and negative serum₄₅₀Nm value and P/N value, to determine the dilution of ELIAS secondary antibody Degree.

Result: other conditions are constant, ELIAS secondary antibody, under different dilution factors, measures OD₄₅₀There were significant differences for nm value, positive blood Clear OD₄₅₀Nm value scope is 1.221 to 0.199, negative serum OD₄₅₀Nm value scope is 0.282 to 0.043, selects negative blood Clear OD₄₅₀Nm value is less than 0.100, and P/N value the greater, and i.e. 1: 10000 is two anti-optimum dilution degrees (see Fig. 7).

8. the action time of ELIAS secondary antibody

The goat anti-pig antibody of HRP labelling is diluted to working concentration, 100ul/ hole, act on the most at room temperature 30min, 45min, 60min, 90min, compare the OD of positive and negative serum₄₅₀Nm value and P/N value, to determine the working time of ELIAS secondary antibody.

Result: other conditions are constant, only changes the action time of ELIAS secondary antibody, measures OD₄₅₀Nm value, action time is 90min Time feminine gender value substantially rise, other P/N do not have notable difference, selection 30min to be action time (see Fig. 8).

9. the determination of substrate reactions time

The substrate-function time is divided into 4 groups, be respectively room temperature lucifuge develop the color 5,7.5,10,15min.Relatively positive and negative blood Clear OD value and P/N value, to determine the developing time of substrate.

Result: other conditions are constant, along with substrate reactions time lengthening, negative serum and positive serum OD₄₅₀Nm value be all by Gradual change is big, compares the P/N value of each point, selects 10min as response time (see Fig. 9).

Embodiment 3 specificity experiments

With the indirect NP49-ELISA method set up, detect CSFV, PRV, PPV, PCV-2, PEDV, PRRS clinically HuN4-F112 positive serum, sets up PRRSV rHN4-Δ 25+NP49 strain positive serum and negative serum for compareing, really simultaneously Determine NP49 polypeptide antigen whether with other Prevention of Common Occurrence Porcine Disease positive serum generation cross reactions.

Result: by detection CSFV, PRV, PPV, PCV-2, PEDV, PRRS HuN4-F112 positive serum, measure OD₄₅₀Nm value is 0.049,0.075,0.099,0.044,0.061,0.068, determines NP49 polypeptide antigen and other common pigs Sick positive serum does not has cross reaction.

Embodiment 4 repeated experiment

Choose in 6 parts of different porcine blood serums of PRRS rHN4-Δ 25+NP49 antibody horizontal carry out criticizing and repeat test, in identical test Under the conditions of detect, every part of serum is parallel does 4 repetitions, and testing result is carried out statistical analysis.

Repeat between Pi to test and choose 6 parts of different porcine blood serums of PRRS rHN4-Δ 25+NP49 antibody horizontal, use 3 different time Between coated ELISA detect plate, detect under same experimental conditions, every part of serum is parallel does 2 repetition, ties detection Fruit carries out statistical analysis.

Result (table 2) shows, in batch, the coefficient of variation of replica test is less than 8%, and between batch, the coefficient of variation of replica test is little In 9%, demonstrate good repeatability.

Table 2NP49-ELISA repeats result of the test between repeating test in criticizing and criticizing

The detection of the composition of embodiment 5NP49-ELISA differential diagnosis kit, NP49-ELISA method and NP49-ELISA Marginal value defines

1.NP49-ELISA the composition of differential diagnosis kit

By NP49 polypeptide antigen (envelope antigen), ELISA ELISA Plate to be coated, it is coated buffer, confining liquid, serum-dilution Liquid, ELIAS secondary antibody (in the embodiment of the present invention, the goat anti-pig IgG antibody of preferred HRP labelling is ELIAS secondary antibody), display liquid, Stop buffer, cleaning mixture, standard positive serum, standard female serum, be assembled into test kit, assembles and is placed on appropraite condition preservation.

Wherein, when selecting cleaning mixture, NP49-ELISA experiment selects PBS to add non-ionic detergent Tween 20, its concentration The NP49 antigen desorption being coated in solid phase can be made to lower the sensitivity of test higher than 0.2% between 0.05%-0.2%. By increasing cleaning mixture salt ionic concentration, can effectively remove non-specific influences.During washing, PBST is used to need 40min Can effectively remove non-specific, after increasing salt ionic concentration, wash time can foreshorten to 15min.

2. carry out NP49-ELISA method detection with above-mentioned test kit

With test kit of the present invention, 679 parts of clinical samples are carried out NP49-ELISA method detection, simultaneously with presently commercially available IDEXX The testing result of detection PRRS test kit compares.2%BSA conduct is used in specifically comprising the following steps that of NP49-ELISA method detection Serum to be checked is made 1: 40 times of dilution, ELISA Plate every hole 100ul, 37 DEG C of effect 30min by serum dilution, discards liquid. Each hole is fully cleaned 4 times with the cleaning mixture of about 300ul, washs 5min every time, after last 1 washing, discard liquid and Pat in absorbent paper to noresidue liquid；The anti-pig of goat two of HRP labelling is anti-carries out 1: 10000 dilution, and every hole adds 100ul, 37 DEG C of effect 30min, wash 4 times；Every hole adds TMB nitrite ion 50ul, room temperature lucifuge effect 10min；Every Kong Zhongjia Enter 50ul H₂SO₄, terminate reaction.Under the wavelength of 450nm, each hole OD value is measured by microplate reader.The self-control of above-described embodiment 1 Positive serum is standard positive serum, and SPF porcine blood serum is standard female serum, detects serum to be checked simultaneously.

3.NP49-ELISA marginal value defines

(1) standard of yin and yang attribute serum

Detect clinical acquisitions serum with IDEXX PRRS test kit, carry out operation according to the description of test kit and data process, Clinical serum used is divided into negative serum and positive serum.For this experiment, it is known that the PRRSV HuN4-F112 of background Positive serum is considered as negative serum to NP49-ELISA.So, the negative serum of this experiment includes PRRS negative serum and Know the HuN4-F112 positive serum of background.

(2) clinical sample OD value detection

With self-control NDV NP49 polypeptide antigen ELISA Plate detection clinical sample, set up positive standard serum and the moon in each ELISA Plate Property standard serum, as comparison, records the OD of clinical serum₄₅₀Value.

(3) clinical sample S/P calculates

S/P=(Sample OD-Negative comparison OD)/(positive control OD-negative control OD)

According to the OD value recorded, calculate the S/P value of blood serum sample to be checked.Testing sample S/P value >=marginal value be judged to the positive, S/P value < marginal value be judged to feminine gender.

(4) ROC statistical analysis marginal value

By SPSS (Statistical Product and Service Solutions) Software on Drawing ROC curve, draw NP49-ELISA The sensitivity (Sensitivity) of detection method and specificity (Specificity).Data Management Analysis, when sensitivity (Se)+special During degree (Sp) numerical value maximum, corresponding S/P is yin and yang attribute serum marginal value (Cut off value).

4. result

(1) clinical sample IDEXX testing result

679 parts of clinical serum sample, judges with IDEXX detection PRRS test kit detection, negative serum has 102 parts, positive Serum is 577 parts；The PRRS negative serum of known background totally 120 parts, detects with IDEXX, coincidence rate 100%

(2) NP49-ELISA detection clinical serum OD value is analyzed

With self-control NDVNP49 polypeptide antigen ELISA Plate detection clinical sample 679 parts, the OD450 numeric distribution of detection, OD450 Distribution value feature is that numerical value has seriality, and numerical value is concentrated mainly between 0.150-0.300, does not has obvious separation (see Figure 10).

Select negative serum (222 parts) and HuN4-F112 positive serum (80 parts) and the positive serum (577 of IDEXX detection Part), detect its OD Distribution value (see Figure 11) with NP49-ELISA.The OD450 value of negative serum concentrates interval to be 0.10-0.20, 94.04% is accounted for below 0.2, and positive serum OD₄₅₀Value concentrates interval to be 0.25-0.35, and more than 0.2, ratio is 87.50%.Both data intervals do not have segmentation peak value, merely from OD₄₅₀Value cannot determine yin and yang attribute marginal value.

(3) blood serum sample S/P value is analyzed

With self-control standard positive serum for positive reference, calculating the S/P value of sample, distribution results such as Figure 12, the intersection point of curve falls In 0.15-0.20 interval, the S/P value 11 parts of negative samples more than 0.20, it is positioned at 0.20-0.30 section；And 577 parts of positives Blood serum sample has 21 parts less than 0.15.

(4) ROC methods analyst yin and yang attribute marginal value

The testing result of blood serum sample is inputted Excel, then imports SPSS software analysis, draw ROC curve, such as Figure 13. Under ROC curve, area is 0.973 (95%CI: 0.964～0.982).

ROC curve: i.e. with sensitivity as vertical coordinate, the curve drawn by with 1-specificity as abscissa.Sensitivity and specificity with The value of diagnostic variable (index) separation and change.

ROC analyzes and has listed file names with the sensitivity and 1-specificity that continuous 378 point of contacts are corresponding from-0.947 to 2.052, should List exports to Excel from SPSS, sues for peace the sensitivity of each point of contact and specificity, and the numerical value maximum obtained is 1.870, about Mounting index 0.870, corresponding S/P value is 0.200, and this value is yin and yang attribute marginal value, and now, sensitivity is 88.9% and special Degree 98.0% (table 3).

Partial sensitivity, special angle value and respective calculated in table 3ROC analysis

(5) NP49-ELISA Yu IDEXX testing result contrast

The decision method of the NP49-ELISA method that application is set up compares with IDEXX testing result, enters 679 parts of blood serum samples Row judges, the results are shown in Table 4, both positive coincidence rate are 87.5%, and negative match-rate is 90.1%, and total coincidence rate reaches 87.9%.

IDEXX with the NP49-ELISA testing result of table 4 blood serum sample compares

Embodiment described above only have expressed embodiments of the present invention, and it describes more concrete and detailed, but can not therefore manage Solve as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, without departing from On the premise of present inventive concept, it is also possible to make some deformation and improvement, these broadly fall into protection scope of the present invention.Therefore, The protection domain of patent of the present invention should be as the criterion with claims.

Sequence table

<110>China Agriculture Academe Shanghai Veterinary Institute

<120>PRRSV genetic marker vaccine strain ELISA differential diagnosis kit and methods and applications

<160>3

<170>PatentIn version3.3

<210>1

<211>49

<212>PRT

<213>Newcastle disease virus NP 49

<400>1

Gly Asp Gly Glu Thr Gln Phe Leu Asp Leu Met Arg Ala Val Ala Asn

1 5 10 15

Ser Met Arg Glu Ala Pro Asn Ser Ala Gln Gly Thr Pro Gln Ser Gly

20 25 30

Pro Pro Pro Thr Pro Gly Pro Ser Gln Asp Asn Asp Thr Asp Trp Gly

35 40 45

Tyr

<210>2

<211>28

<212>DNA

<213>artificial sequence

<220>

<221>misc_feature

<222>(1)..(28)

<223>primer

<400>2

ttagaattcg gggatgggga gacccaat 28

<210>3

<211>29

<212>DNA

<213>artificial sequence

<220>

<221>misc_feature

<222>(1)..(29)

<223>primer

<400>3

atactcgaga tacccccagt cggtgtcgt 29

Claims

1. a PRRSV genetic marker vaccine strain ELISA differential diagnosis kit, it is characterised in that described test kit comprises NP49 Polypeptide antigen, the aminoacid sequence of this NP49 polypeptide is as shown in SEQ ID NO:1, and this NP49 polypeptide antigen is by comprising NP49 The recombinant expression carrier of polypeptide coding genes is through abduction delivering, restructuring NDV NP49 antigen protein prepared after purification；Described examination Agent box also comprises ELISA ELISA Plate, confining liquid, serum dilution, ELIAS secondary antibody, display liquid, stop buffer, standard positive blood Clearly, standard female serum；The concentration that is coated of described NP49 polypeptide antigen is 500ng/ hole；Described ELIAS secondary antibody is Radix Cochleariae officinalis peroxide The goat anti-pig antibody of compound enzyme labelling, its dilution factor is 1:10000；Described standard positive serum is with restructuring NDV NP49 Antigen protein immune swine and prepare；Described restructuring NDV NP49 antigen protein is by the restructuring table comprising NP49 polypeptide coding genes Reach carrier through abduction delivering, prepare after purification；Described confining liquid and the bovine serum albumin that serum dilution is 2% weight concentration； Wherein, the preparation method of NP49 polypeptide antigen is:

According to the GenBank NO.AF077761NDV La Sota strain NP protein gene sequence design specific primer announced, Forward primer NP49-UP:5 '-TTAGAATTCGGGGATGGGGAGACCCAAT-3 ', SEQ ID NO:2, its 5 ' ends are designed with EcoR I restriction enzyme site；Downstream primer NP49-DP:5 '-ATACTCGAGATACCCCCAGTCG GTGTCGT-3 ', SEQ ID NO:3, its 5 ' end is designed with Xho I restriction enzyme site；Expection amplification length is 165bp；Draw Thing is synthesized by Shanghai Invitrogen company, and the primer sterilizing distilled water of synthesis is diluted to final concentration 10mmol/L, is placed in-20 DEG C Standby；

NDV La Sota strain total serum IgE is extracted by the description of RNeasy Plus Mini Kit test kit；Say with reference to AMV product Bright book, with U12 random primer as reverse transcription primer, synthesizes the first chain cDNA；

Then with cDNA as template, NP49-UP and NP49-DP primer is used to carry out PCR amplification；PCR reacts cumulative volume 50 μ L, include 10 × PCR buffer 5 μ L, 10mmol/L dNTP 1.5 μ L, each 1 μ L of upstream and downstream primer, rTaq DNA Polymerase 1 μ L, cDNA1 μ L, add water to 39.5 μ L；PCR program is: 95 DEG C of denaturations 5min；94 DEG C of degeneration 1min, 60 DEG C of 30s, 72 DEG C extend 30s, carry out 35 circulations altogether；Last 72 DEG C extend 10min；After amplification, take 5 μ L anti- Product is answered to carry out agarose gel electrophoresis the observed result of 1%；Reclaim the electrophoretic band being accredited as the positive；

By reclaim purpose PCR primer and prokaryotic expression carrier pGEX-6P-1 respectively through EcoR I and Xho I double digestion, enzyme action After two kinds of products respectively with glue reclaim test kit be purified recovery；Subsequently the genes of interest of enzyme action is pressed suitable body with carrier The mixing of long-pending ratio, under the effect of T4DNA ligase, 16 DEG C connect overnight, will connect product and convert to JM109 competent cell, Overnight incubation on the LB flat board containing ampicillin；After resistance screening, picking positive colony expands, and uses PCR respectively Method, EcoR I and Xho I double digestion method are identified, and it is raw that the bacterium solution being initially identified as the positive is sent to Shanghai Invitrogen The order-checking of thing Technology Co., Ltd. is identified；The named pGEX-NP49 of plasmid of the positive will be accredited as；

(2) the restructuring expression of NDVNP49 antigen, purification

PGEX-NP49 Plastid transformation Escherichia coli BL21 (DE3) of the positive will be accredited as with PCR method, double digestion method and order-checking Competent cell, chooses bacterium and is inoculated in the LB culture medium containing ampicillin, 37 DEG C of incubated overnight, and next day is by 1: 100 inoculation Amount be inoculated in the LB culture medium containing ampicillin, 37 DEG C, 220rpm cultivate about 4h, when bacterium solution OD₆₀₀Value reach 0.6～ When 0.8, add IPTG to final concentration of 1mmol/L, 37 DEG C of abduction deliverings, after 12h, collect bacterium solution 1mL, 12000r/min Centrifugal 3min, suspends with the PBS of 40 μ L pH7.4, adds 10 μ L5 × SDS sample-loading buffer, and mixing, water-bath is boiled 10min, carries out SDS-PAGE electrophoretic analysis, through coomassie brilliant blue staining, methanol and glacial acetic acid decolouring observed result, if empty Carrier compares；Expressing protein is carried out Western blot detection simultaneously；The detection method of Western Blot is as follows: by albumen Band uses half-dried transfer printing to be transferred in cellulose acetate membrane, is that 10% defatted milk powder 4 DEG C is overnight closed with mass/volume ratio； After PBST washs three times, add NDV polyvalent antibody IgG, room temperature reaction 60min；Fully remove for three times with PBST washing Remove unconjugated serum IgG in cellulose acetate membrane；The Anti-chicken lgG that cellulose acetate membrane immersion is diluted with confining liquid goat antibodyThe anti-solution of Conjugated bis-, room temperature lucifuge reaction 60min；Three are washed with PBST Secondary abundant removal cellulose acetate membrane unconjugated two resists；Finally, with the Dual band IR laser imaging system of odyssey company of the U.S. System analytical reactions result；

Being enlarged cultivating at Escherichia coli BL21 (DE3) by the plasmid containing pGEX-NP49, next day is by 1: 100 inoculation Amount is inoculated in the LB culture medium of the 200mL containing ampicillin, and 37 DEG C of 220rpm cultivate about 4h, when bacterium solution OD₆₀₀Value reaches When 0.6～0.8, add IPTG to final concentration of 1mmol/L, 37 DEG C of abduction deliverings, collect bacterium solution after 12h, 12000rpm from Heart 10min collects thalline；The excusing from death disruption buffer of 20mL is added: 20mM Tris-HCl, pH8.0 in the thalline collected, 0.5mM EDTA, blows and beats mixing the most repeatedly, uses ultrasonic method to crush thalline；Ultrasonic condition is: 200w, and super 5s stops 5s, altogether 5min；4 DEG C, 1,2000rpm is centrifuged 10min, stays supernatant standby；Carry out according to the description of GST affinity column Protein purification；The destination protein being recovered to is carried out WesternBlot detection simultaneously.

2. the use of non-diagnostic purpose test kit as claimed in claim 1 carries out PRRSV genetic marker vaccine strain ELISA mirror Other method, it is characterised in that comprise the following steps:

Being coated ELISA ELISA Plate with NP49 polypeptide antigen, this NP49 polypeptide antigen is by the weight comprising NP49 polypeptide coding genes Group expression vector is through abduction delivering, restructuring NDV NP49 antigen protein prepared after purification, the aminoacid sequence of this NP49 polypeptide Row are as shown in SEQ ID NO:1；

After test serum and envelope antigen effect, it is sequentially added into ELIAS secondary antibody, display liquid and stop buffer；

Microplate reader is utilized to read absorbance OD₄₅₀Nm, and press formula: S/P=(test serum Sample OD-Negative comparison OD)/(positive Comparison OD-negative control OD), calculate the S/P value of test serum sample；

Test serum sample S/P value>=marginal value be judged to the positive, S/P value<marginal value be judged to feminine gender, described marginal value is to pass through ROC The S/P value corresponding when sensitivity is with specificity numerical value sum maximum that statistical analysis method obtains；Described test serum dilute Degree of releasing is 1:40.

3. the application in the product of preparation Differential Diagnosis PRRSV genetic engineering marker vaccine immune swine of the test kit described in claim 1.