CN101968490A

CN101968490A - Indirect ELISA (Enzyme-Linked Immunosorbent Assay) method and kit for detecting haemophilus parasuis antibodies

Info

Publication number: CN101968490A
Application number: CN2010102644999A
Authority: CN
Inventors: 李春玲; 王贵平; 陈善真; 宋帅; 贾爱卿
Original assignee: INSTITUTE OF VETERINARY MEDICINE GUANGDONG ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: INSTITUTE OF VETERINARY MEDICINE GUANGDONG ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2010-08-26
Filing date: 2010-08-26
Publication date: 2011-02-09

Abstract

The invention discloses indirect ELISA (Enzyme-Linked Immunosorbent Assay) method and kit for detecting haemophilus parasuis antibodies. The indirect ELISA method comprises the steps of: taking a haemophilus parasuis OMPP5 protein as an envelope antigen; wherein the judgment standard is as followed: OD450 value of to-be-detected serum is greater than 0.375, OD450 value of the to-be-detected serum/OD450 value of standard negative serum is greater than or equal to 2.1. The kit for realizing the method comprises an envelope buffer solution, confining liquid, a washing buffer solution, an antibody diluent, a chromogenic substrate solution and a stop solution, wherein the envelop antigen used in the method is the haemophilus parasuis OMPP5 protein. Proved by a specificity test, a blocking-up experiment, a repeatability test and clinic application detection, the invention has the characteristics of excellent specificity, high sensitiveness, good repeatability, rapidness, simpleness and accuracy, and can be used in clinic large-scale detection of haemophilus parasuis antibodies and epidemiological survey.

Description

Detect the indirect ELISA method and the kit of haemophilus parasuis antibody

Technical field

The present invention relates to molecular biology, animal bacteria and epizootiology detection technique field, particularly a kind of indirect ELISA method and kit that detects haemophilus parasuis antibody.

Background technology

The haemophilus parasuis disease is one of important cause of disease that causes weanling pig morbidity and death.Haemophilus parasuis (Haemophilus parasuis, HPS) as a kind of bacterium of being everlasting of the pig upper respiratory tract, invading body under given conditions and can cause serious systemic disease (Glasser ' s disease), is principal character with fiber disposition polyserositis, arthritis and meningitis.This sick a kind of sporadic, stress disease that always is considered to piglet in the past.But, in recent years, along with the development of the industrialization of raising pigs, the appearance of increasing high healthy swinery or SPF swinery is if these pigs are in a kind of not immune state, in case HPS invasion, will be subjected to severe infections, in addition dead, and the pig of various ages in days all can infect, and the incidence of disease can reach 50～70%, and mortality ratio can reach more than 10%.Cause that at present the pathogenesis that haemophilus parasuis infects also is not very clear, some researchs think that haemophilus parasuis can be concurrent or be secondary to diseases such as blue otopathy, pseudoabies, parvovirus, PCV-II disease and swine fever.

In recent years, the haemophilus parasuis disease has become the main bacteria disease that influences pig industry in the global range, endangers day by day seriously, brings massive losses to pig industry.Making diagnosis in early days, exactly and carrying out vaccine prevention is this sick important means of control, but haemophilus parasuis serotype is numerous, and popular haemophilus parasuis serotype is generally the stronger serotype of virulence, as serum 5 types, 4 types and 13 types etc.

The detection method of the HPS that has reported both at home and abroad has 16SrRNA PCR, Southern hybridization, complement fixation test (CFT) (CF), indirect hemagglutination test (IHA) and ELISA etc. at present.Yet, be difficult to be applied to practice based on PCR that detects its DNA and Southern hybridizing method, and CF also has many, the shortcomings such as influence factor is complicated, complex operation step of composition of the reaction of participating in, IHA and ELISA method are the main HPS antibody detection methods that present Chinese scholars is explored, that different is the preparation method of antigen, and Feng Xiaoming (2009), the green usefulness ultrasonication antigens such as (2007) of stone and capsular polysaccharide prove that as the indirect ELISA method result of the foundation of envelope antigen the susceptibility of ELISA is than IHA height.The whole cell of lipopolysaccharides at present both at home and abroad commonly used, LPS, deactivation carries out ELISA as envelope antigen, testing result less stable and false negative often occurs, or also do not verify its coincidence rate and susceptibility.Therefore, still need to set up a kind of method of quick, the easy and detection haemophilus parasuis antibody that specificity is high.

Summary of the invention

The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of indirect ELISA method that detects haemophilus parasuis antibody with not enough.Quick, easy, recall rate advantages of higher that this method has.

Another purpose of the present invention is to provide the kit of realizing above-mentioned indirect ELISA method.

Purpose of the present invention is achieved through the following technical solutions: a kind of indirect ELISA method that detects haemophilus parasuis antibody is as envelope antigen with haemophilus parasuis (Haemophilus parasuis) OMP P5 albumen;

The amino acid sequence of described haemophilus parasuis OMP P5 albumen is as follows: MKKSLIALAVSGLAVASVAQAAPQANTFYVGAKAGWATFHNDINQIDSKYANDARY DATNLKYGISRNSVTYGVFGGYQIIDNLAVELGYDYFGRVRGNKQEFRAFKHSAHG THLSLKPSYEVLNGLDVYGKVGAALVRNDYKRYSQTAGVQTQKAHNLKTSLVLGAG VEYAILPELAFRVEYQWLSRVGNANKAAQKRGDTAMFGPGSTYSPDAHSVSAGISY RFGQGAAPVVAAPEVVTKNFAFSSDVLFDFGKANLKPAAAQTLDAVHTEIVNLGLA NPAVQVNGYTDRIGKDAANLTLSQKRAETVANYIVSKGVNPANVTAVGYGEANPVT GNTCDAVKGRKALITCLAPDRRVEIQVQGSKEVSM

Described haemophilus parasuis OMP P5 albumen preferably obtains by gene engineering method, and is specific as follows:

(1) haemophilus parasuis OMP P5 nucleotide sequence as follows multiple clone site by expression vector pET-32a (+) is cloned among the expression vector pET-32a (+), obtains pET-32a (+)-OMP P5 plasmid; Wherein, 5 ' of this haemophilus parasuis OMP P5 nucleotide sequence end is arranged in 3 ' end of the Trx Taq coded sequence of expression vector pET-32a (+);

(haemophilus parasuis OMP P5 nucleotide sequence

atgaaaaaatctttaattgcattagcagtatcaggtttagcagttgcttctgtagctcaagctgctccacaagctaacactttctatgtaggtgctaaagctggttgggcaacattccacaatgatattaaccaaattgactctaagtatgctaatgatgcacgttatgatgctactaatttaaaatatggcattagccgtaactctgtaacttacggtgtgtttggtggttaccaaattattgacaacttagcagttgaattaggttacgattatttcggtcgtgttcgtggtaacaaacaagaattccgtgcattcaaacactctgcacacggtacacacttaagcttaaaaccaagctatgaagtattaaacggtttagatgtttacggtaaagttggtgctgcattagttcgtaacgattacaaacgttatagccaaacagctggagtgcaaactcaaaaagctcataacttaaaaacctctttagtattaggtgcaggtgttgagtatgcaattcttccagaattagcattccgtgttgagtaccaatggttaagccgtgtgggtaatgctaacaaagcagctcaaaaacgtggcgacacagctatgtttggtccaggttctacatatagccctgatgcacactcagtatctgcaggtatttcataccgcttcggtcaaggtgcagcaccagttgtagcagcaccagaagttgtaactaaaaacttcgcgttcagctcagacgtattatttgacttcggtaaagcaaacttaaaaccagctgcagcacaaacattagacgcagtacacactgagatcgtaaatttaggtttagcaaaccctgctgtacaagtaaacggttacacagaccgtat?tggtaaagatgctgccaacttaactctttcacaaaaacgtgcagaaacagtagcaaactacatcgtttctaaaggtgttaacccagctaacgtaacagcggtaggttacggtgaagctaacccagtaactggtaacacatgtgacgcagttaaaggtcgtaaagcattaatcacttgcttagcaccagatcgccgtgttgaaatccaagttcaaggttcaaaagaagtttctatgtaa

(2) according to the operating guidance of expression vector pET-32a (+), pET-32a (+)-OMP P5 plasmid is converted in the expressive host, expresses, purifying and renaturation, obtain haemophilus parasuis OMP P5 albumen;

Described step (2) is more preferably: with pET-32a-OMP P5 plasmid transformation escherichia coli (Escherichia coli) BL21 (DE3), then, the single bacterium colony of picking is 37 ℃ of shaken cultivation in containing the LB fluid nutrient medium of Amp (final concentration of ampicillin is at least 50 μ g/mL), and (final concentration is 0.2mmolL to treat the OD600 value to reach 0.6 o'clock adding IPTG ^-1) induced 5～6 hours, collect thalline, ultrasonic degradation is collected the thalline that obtains, and with the HisTag fusion purification kit of Novagen, carries out the extracting and the purifying of inclusion body according to the kit instructions; With the urea renaturation of dialysing, obtain haemophilus parasuis OMP P5 albumen behind the purifying.

The indirect ELISA method of described detection haemophilus parasuis antibody, the package amount that is preferably described haemophilus parasuis OMP P5 albumen is 0.02～0.05 μ g;

The indirect ELISA method of described detection haemophilus parasuis antibody, more preferably (S is that test serum is measured the OD obtain in S/N 〉=2.1 ₄₅₀, N is the OD of standard female serum ₄₅₀), and OD ₄₅₀＞0.375;

Realize the kit of the indirect ELISA method of described detection haemophilus parasuis antibody, comprise and be cushioned liquid, confining liquid, lavation buffer solution, antibody diluent, substrate colour developing liquid and stop buffer that wherein, envelope antigen is a haemophilus parasuis OMP P5 albumen.

The present invention has following advantage and effect with respect to prior art:

(1) detects by specificity test, blocking experiment, replica test and clinical practice, the indirect ELISA method that proves detection haemophilus parasuis antibody of the present invention has that specificity is good, susceptibility is high, good reproducibility and quick, easy, characteristic of accurate, can be used for the clinical extensive detection and the epidemiology survey of haemophilus parasuis antibody.

(2) the used envelope antigen of the present invention, promptly haemophilus parasuis OMP P5 albumen can obtain by gene engineering method, the biological safety height.At present both at home and abroad this albumen also of no use is set up the relevant report of HPS antibody indirect ELISA detection method as antigen, has broad application prospects.

Description of drawings

Fig. 1 is the pcr amplification figure of haemophilus parasuis OMP P5 gene;

Wherein: swimming lane M is DL2000DNA Marker; Swimming lane 1 positive contrast; Swimming lane 11 negative contrasts; Swimming lane 2～10 and 12～17 is a pcr amplification product.

Fig. 2 is that the enzyme of pET-32a (+)-OMP P5 plasmid is cut evaluation figure;

Wherein: swimming lane M is wide region DNA Marker (100～6000bp);

Swimming lane

2 and 3 is respectively the product that EcoRV enzyme and BamH I enzyme carry out double digestion; Swimming lane 1 is the PCR product; Swimming lane 4 is pET-32a (+) plasmid single endonuclease digestion.

Fig. 3 is the SDS-PAGE figure of HPS OMP P5 albumen;

Wherein: swimming lane M is high-molecular-weight protein Marker; Swimming lane 1 is the empty carrier contrast; Swimming lane 2 is not for inducing contrast; Swimming lane 3 is the nutrient culture media contrast;

Swimming lane

4 and 9 is the whole cell expression product;

Swimming lane

5,6 and 10 is the ultrasonication bacterial sediment;

Swimming lane

7,8 and 11 is ultrasonication thalline supernatant.

Fig. 4 is the SDS-PAGE figure of HPS OMP P5 protein purification;

Wherein: swimming lane 1～3 is for passing the peak; Swimming lane 4～8 is component under the rinsing; Swimming lane M is high-molecular-weight protein Marker; Swimming lane 9～14 is a component under the wash-out.

Embodiment

The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.

Embodiment 1

One, clone, expression and the purifying of haemophilus parasuis OMP P5 protein gene

1, the PCR design of primers is with synthetic

(1) according to HPS OMP P5 gene order (the gene accession number is CP001321), designed 1 pair of Auele Specific Primer, the full gene of amplification HPS OMP P5 with biosoftware Premier Primer5.0.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The sequence of upstream primer HPS P1: 5 '-GCC GATATCGCTCCACAAGCTAACACT-3 ' (underscore partly is the EcoRV restriction enzyme site); The sequence of downstream primer HPS P2: 5 '-CGC GGATCCTTACATAGAAACTTCTTTTGAACCT-3 ' (underscore partly is the BamHI restriction enzyme site).

2, the clone and the expression vector establishment of HPS OMP P5 gene

With clinical isolates strain HPS bacterium liquid is template, with the Auele Specific Primer P1/P2 that designs (the 95 ℃ of pre-sex change 5min that increase; 94 ℃ of sex change 45s, 50 ℃ of annealing 45s, 72 ℃ are extended 1min, carry out 30 circulations; Last 72 ℃ are extended 10min) (see figure 1).Amplified production reclaims kit recovery purpose fragment (1116bp) with the Bio-tek E.Z.N.A.GelExtraction Kit dna gel of Omega company.After the purpose fragment reclaims, be connected with the pMD18-T carrier, connect back (according to molecular cloning) and be transformed into bacillus coli DH 5 alpha (Guangzhou is closed and reached bio tech ltd), coat and contain ampicillin (concentration of ammonia benzyl is 100 μ g/mL) LB solid medium, 37 ℃, after 230rpm cultivates 12～16h, the positive colony recon that picking grows is inoculated in and increases the bacterium cultivation in the LB nutrient solution that is added with ampicillin, from cloned plasmids pMD18-T-OMP P5, amplify haemophilus parasuis OMP P5 genetic fragment with PCR method then, behind EcoR V and BamHI double digestion, with the gene subclone in prokaryotic expression carrier pET-32a (+), make up recombinant expression carrier, the performing PCR of going forward side by side is identified and enzyme is cut the evaluation (see figure 2).Positive plasmid carries out sequencing (as follows) by Shanghai Ying Jun Bioisystech Co., Ltd, positive plasmid called after pET-32a (+)-OMP P5.

atgaaaaaatctttaattgcattagcagtatcaggtttagcagttgcttctgtagctcaagctgctccacaagctaacactttctatgtaggtgctaaagctggttgggcaacattccacaatgatattaaccaaattgactctaagtatgctaatgatgcacgttatgatgctactaatttaaaatatggcattagccgtaactctgtaacttacggtgtgtttggtggttaccaaattattgacaacttagcagttgaattaggttacgattatttcggtcgtgttcgtggtaacaaacaagaattccgtgcattcaaacactctgcacacggtacacacttaagcttaaaaccaagctatgaagtattaaacggtttagatgtttacggtaaagttggtgctgcattagttcgtaacgattacaaacgttatagccaaacagctggagtgcaaactcaaaaagctcataacttaaaaacctctttagtattaggtgcaggtgttgagtatgcaattcttccagaattagcattccgtgttgagtaccaatggttaagccgtgtgggtaatgctaacaaagcagctcaaaaacgtggcgacacagctatgtttggtccaggttctacatatagccctgatgcacactcagtatctgcaggtatttcataccgcttcggtcaaggtgcagcaccagttgtagcagcaccagaagttgtaactaaaaacttcgcgttcagctcagacgtattatttgacttcggtaaagcaaacttaaaaccagctgcagcacaaacattagacgcagtacacactgagatcgtaaatttaggtttagcaaaccctgctgtacaagtaaacggttacacagaccgtattggtaaagatgctgccaacttaactctttcacaaaaacgtgcagaaacagtagcaaactacatcgtttctaaaggtgttaacccagctaacgtaacagcggtaggttacggtgaagctaacccagtaactggtaacacatgtgacgcagttaaaggtcgtaaagcattaatcacttgcttagcaccagatcgccgtgttgaaatccaagttcaaggttcaaaagaagtttctatgtaa

3, abduction delivering, purifying and the detection of reorganization pET-32a-OMP P5 fusion

According to molecular cloning method, with positive recombinant expression plasmid pET-32a-OMP P5 transformed into escherichia coli BL21 (DE3) (Guangzhou is closed and reached bio tech ltd), the single bacterium colony of picking is 37 ℃ of shaken cultivation in containing the LB fluid nutrient medium of Amp.Isopropylthio-β-(final concentration is 0.2mmolL to D-galactose nucleosides IPTG to treat the OD600 value to reach adding in 0.6 o'clock ^-1) induce.Behind the abduction delivering 6h, centrifugal collection bacterium liquid is used lysate (the 50mM NaH2PO4 of an amount of pH8.0 then, pH 8.0,300mM NaCl, 10mM imidazoles) resuspended above-mentioned bacterial sediment, adding lysozyme to final concentration in the resuspended liquid of bacterium is 1mg/mL, and places ice bath to hatch 30min.The broken bacterium of ice-bath ultrasonic, working method: 400W power, ultrasonication time 2s, interval time 5s, ultrasonic 20min, until liquid become bright till.Behind the ultrasonic degradation, 4 ℃ of centrifugal 30min of 9000r/min collect bacterium liquid supernatant and precipitation respectively, are used for purifying (going up the preceding membrane filtration with 0.45 μ m of sample); Last cleer and peaceful precipitation does not keep sample and does the SDS-PAGE check and analysis.SDS-PAGE electrophoresis detection result shows (see figure 3), and recombination bacillus coli can specific expressed HPS OMP P5 albumen, and mainly express with the inclusion body form.

The albumen of collecting carries out the extracting and the purifying of inclusion body to specifications with the Ni-NTA HisBind fusion affinity purification post of Novagen company.The extraction steps of inclusion body is as follows: 1) the centrifugal 10min of 9000rpm collects thalline, abandons supernatant, removes nutrient culture media as far as possible, adds the resuspended thalline of 40mL 1 * binding buffer liquid proportional by every 100mL nutrient culture media gained thalline, and this step is not with denaturant; 2) undertaken by preceding method ultrasonic, resuspended bacterium liquid, shear degradation nucleic acid; 3) 9000rpm is centrifugal 20 minutes, and inclusion body and cell fragment are arranged in precipitation, and other soluble proteins partly are arranged in supernatant; 4) remove supernatant, the precipitation of every 100ml culture is resuspended in 20mL 1 * binding buffer liquid, repeats for the 3rd step.May need ultrasonic with thorough resuspended precipitation; 5) remove supernatant, add 5mL damping fluid ratio, add the resuspended precipitation of 1 * binding buffer liquid that contains 6M urea in every 100mL culture; 6) ice bath was hatched 1 hour, thoroughly dissolved inclusion body, and 9000rpm removed insoluble composition in centrifugal 30 minutes, and Ni-NTA HisBind purifying is before with 0.45 μ m membrane filtration supernatant.Purification step is as follows: 1) 1ml 50%Ni-NTA HisBind resin suspension is added in the 4ml cell pyrolysis liquid, and soft mixing (as impeller 200rpm), room temperature is in conjunction with 15-60min; 2) potpourri with lysate and Ni-NTAHisBind resin carefully adds in the empty chromatographic column of lower end closed; 3) remove post lower end closed lid, collect effluent (passing the peak), preservation is used for the SDS-PAGE electrophoretic analysis; 4) with 4ml buffer C rinsing foreign protein 2 times.Preserve the rinsing component and be used for the SDS-PAGE electrophoretic analysis; 5) with 0.5ml buffer D wash-out destination protein 4 times, wash 4 times with 0.5ml buffer E again, collect component, analyze (as shown in Figure 4) with SDS-PAGE; Protein monomer gets final product wash-out with buffer D usually, and polymer, polymkeric substance and the albumen that contains two HisTag labels are usually by buffer E wash-out.Behind the destination protein wash-out, with the level pad balance pillar of 5mL, and then fill 20% ethanol, sealing is in order to using next time.

Purifying protein adds behind the reduced glutathione that final concentration is 1mg/mL respectively with the PBS damping fluid of the pH8.5 that contains 4mol/L, 3mol/L, 2mol/L, 1mol/L, 0mol/L urea, carries out gradient dialysis renaturation, obtains activated destination protein.

Two, haemophilus parasuis OMP P5 albumen is as the evaluation of the indirect ELISA method of antigen

1, haemophilus parasuis OMP P5 albumen is as the prescription of the indirect ELISA detection method related reagent of antigen

Bag is cushioned liquid: (0.05M carbonate buffer solution pH9.6): 1.59g Na ₂CO ₃, 2.93g NaHCO ₃Be dissolved in the 800mL distilled water, regulate pH value to 9.6, and be settled to 1000mL.

Confining liquid: 1%BSA, promptly 1g bovine serum albumin(BSA) (BSA) adds in the coating buffer of 100mL, and mixing is now with the current.

Lavation buffer solution (pH7.4PBST): 0.2g KH ₂PO4,2.9g Na ₂HPO ₄12H ₂O, 8.0g NaCl, 0.2g KCl, after the above accurate weighing of each material of 0.5mLTween-20 (0.05%V/V), after adding distilled water and dissolving fully, adjust pH to 7.4 is settled to 1000mL.

Antibody diluent: 0.5g BSA is dissolved among the 100mLPBST (lavation buffer solution).

TMB (tetramethyl benzidine) substrate colour developing liquid: get A liquid 1mL+B liquid 50mL (ratio of A liquid and B liquid is 1: 50).

A liquid: 5mg/mL TMB 50mg is dissolved among the 10mL DMSO 4 ℃ and keeps in Dark Place.

B liquid: phosphoric acid-citrate buffer solution (PH 5.0) first liquid: 0.2M (71.6g/L) Na ₂HPO ₄.12H ₂O; Second liquid: 0.1M (19.2/L) citric acid.Compound method: 51.4mL first liquid+48.6mL second liquid+100mL ultrapure water, add 3,0%H,2O2 112 μ L as in the brown bottle 4 ℃ keep in Dark Place.

Stop buffer: (2M H ₂SO ₄): distilled water 178.3mL dropwise adds the concentrated sulphuric acid (98%) 21.7mL.

2, HPS OMP P5 albumen is as the running program of the indirect EL1SA method of antigen

As antigen coated polystyrene reactant plate, set up indirect ELISA method with pET-32a (+)-OMP P5 albumen of purifying, step is as follows:

(1) bag quilt: behind coating buffer dilution antigen liquid, 100 μ L/ holes add 96 hole polystyrene enzyme-linked reaction plates, and 4 ℃ are spent the night.Dry the back washing, every hole adds 250 μ L cleansing solutions, and each 3 minutes, dry, wash 3 times.

(2) sealing: every hole adds confining liquid 100 μ L, and 37 ℃ of effect sealing 2h dry, and every hole adds 250 μ L cleansing solutions washs, and each 3 minutes, dry, wash 3 times.

(3) application of sample: add 100 μ L with every hole, antibody diluent dilute serum (1: 160) back, 37 ℃ of effect 1h dry.Every hole add 250 μ L cleansing solutions wash, each 3 minutes, dry, wash 3 times.

(4) the goat-anti rabbit ELIAS secondary antibody of adding horseradish peroxidase-labeled: every hole adds two of the 50 the suitableeest extension rates of μ L (1: 5000) and resists, and 37 ℃ of effect 30min dry.Every hole adds 250 μ L cleansing solutions washs, and each 3 minutes, dry, wash 3 times.

(5) add colour developing liquid: every hole adds the colour developing liquid 50 μ L of new preparation, room temperature lucifuge colour developing 10min.

(6) add stop buffer: every hole adds 100 μ L stop buffer cessation reactions.

(7) survey the OD value: enzyme connection detector is measured each hole OD450nm value automatically.

3, HPS OMP P5 albumen is as the specificity test of the indirect EL1SA method of antigen

Utilizing the preliminary ELISA that sets up to detect porcine contagious pleuropneumonia Actinobacillus (App) positive serum, Streptococcus suis (SS) positive serum, pig pasteurella multocida (PM) positive serum, swine escherichia coli (E.coli) positive serum, PRV (ADV) positive serum, porcine circovirus 2 type (PCV2) positive serum and pig breeding detects with respiratory disorder syndrome (PRRSV) positive serum, and establish HPS positive and negative serum in contrast, to detect its specificity; Observe the cross reaction of above-mentioned each serum and HPS OMP P5 albumen.The results are shown in Table 1.

Table 1 specificity test findings

As shown in Table 1, remove OD450＞0.375 (critical value) of HPS standard positive serum, be judged to outside the positive, the OD450 value of the standard positive serum of other bacteriums and viral disease is all less than critical value, and is negative.The result shows that indirect ELISA detection method of the present invention has good specificity.

4, HPS OMP P5 albumen is as the blocking test of the indirect EL1SA method of antigen

With each 4 parts of positive serum and negative serum is that the recombinant protein HPSOMP P5 solution of 1.319mg/mL was by 1: 4 (v/v) mixing with concentration respectively, behind 37 ℃ of effect 1h, the centrifugal 20min of 12000r/min, get supernatant and do not add negative serum and the positive serum that antigen is handled, measure the relatively variation of the OD450nm value of serum before and after the blocking-up according to the indirect ELISA method of having set up.The result shows: the OD value of the positive serum of handling with HPS OMP P5 albumen is significantly less than the positive serum OD value that does not add the antigen processing, and significant difference (P＜0.05), and also be significantly less than the OD value (the serum while that does not add OMP P5 albumen is in contrast centrifugal) of the negative serum that does not add antigen with the negative serum OD value that HPS OMP P5 albumen is handled, illustrate that this albumen can well block the antibody in the serum.

5, HPS OMP P5 albumen is as the replica test of the indirect EL1SA method of antigen

(1) criticizes interior the repetition

With antigen coated ELISA Plate with a collection of preparation, get 4 parts of known positive serums, 4 parts of negative serums, serum to be checked detects for 2 parts, and every part of serum repeats 3 holes.The results are shown in Table 2, the analysis showed that through the SSPS statistical analysis software, the replica test result does not have significant difference in batch, illustrates that the repeatability of this ELISA detection method is good.Operation steps is the same.

Reproducible results in the table 2 batch

(2) repeat between criticizing

With the antigen coated ELISA Plate of different batches, get 4 parts of known positive serums, 4 parts of negative serums, serum to be checked is examined side for 2 parts, and every part of serum detects 3 holes, duplicate detection 3 times.Compare its OD _450nmValue and S/N calculate each sample standard deviation and the coefficient of variation.The results are shown in Table 3, the analysis showed that through the SSPS statistical analysis software,, the revision test coefficient of variation shows that less than 8.8% the indirect ELISA method of foundation has good repeatability between batch, the infected pigs that can diagnose haemophilus parasuis fast and accurately is only.

Reproducible results between table 3 batch

6, HPS OMP P5 albumen is as the clinical practice of the indirect EL1SA method of antigen

317 parts of the porcine blood serum that does not carry out the haemophilus parasuis immunity of the pig farm collection from different places from Guangdong, are used the indirect ELISA method of having set up clinical collection serum are carried out the HPS antibody test as envelope antigen with purifying protein.The result detects 11 parts of positive, recall rate is 3.47%, this indirect ELISA detection method that shows that the present invention sets up is suitable for a large amount of samples of clinical collection are carried out fast detecting, can accurately assess the infection state of swinery haemophilus parasuis, has broad application prospects.

The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims

1. indirect ELISA method that detects haemophilus parasuis antibody, it is characterized in that: this method is as envelope antigen with haemophilus parasuis (Haemophilus parasuis) OMP P5 albumen; And criterion is as follows: the OD of test serum ₄₅₀Value＞0.375, and the OD of test serum ₄₅₀The OD of value/standard female serum ₄₅₀Value 〉=2.1.

2. according to the indirect ELISA method of the described detection haemophilus parasuis of claim 1 antibody, it is characterized in that: the amino acid sequence of described haemophilus parasuis OMP P5 albumen is shown in SEQ ID NO.1.

3. according to the indirect ELISA method of the described detection haemophilus parasuis of claim 2 antibody, it is characterized in that: described haemophilus parasuis OMP P5 albumen obtains by gene engineering method, and is specific as follows:

(1) with nucleotide sequence shown in SEQ ID NO.2 the multiple clone site of haemophilus parasuis OMP P5 nucleotide sequence by expression vector pET-32a (+) be cloned among the expression vector pET-32a (+), obtain pET-32a (+)-OMP P5 plasmid; Wherein, 5 ' of this haemophilus parasuis OMP P5 nucleotide sequence end is arranged in 3 ' end of the Trx Taq coded sequence of expression vector pET-32a (+);

(2) according to the operating guidance of expression vector pET-32a (+), pET-32a (+)-OMP P5 plasmid is converted in the expressive host, expresses, purifying and renaturation, obtain haemophilus parasuis OMP P5 albumen.

4. according to the indirect ELISA method of the described detection haemophilus parasuis of claim 3 antibody, it is characterized in that: described step (2) is: with pET-32a-OMP P5 plasmid transformation escherichia coli (Escherichiacoli) BL21 (DE3), then, the single bacterium colony of picking in containing the LB fluid nutrient medium of Amp in 37 ℃ of shaken cultivation, treat that the OD600 value reaches 0.6 o'clock adding IPTG and induces, collect thalline, ultrasonic degradation is collected the thalline that obtains, with the HisTag fusion purification kit of Novagen, carry out the extracting and the purifying of inclusion body according to the kit instructions; With the urea renaturation of dialysing, obtain haemophilus parasuis OMP P5 albumen behind the purifying.

5. according to the indirect ELISA method of the described detection haemophilus parasuis of claim 1 antibody, it is characterized in that: the package amount of described haemophilus parasuis OMP P5 albumen is 0.02～0.05 μ g.

6. realize the kit of the indirect ELISA method of each described detection haemophilus parasuis antibody of claim 1～5, comprise and be cushioned liquid, confining liquid, lavation buffer solution, antibody diluent, substrate colour developing liquid and stop buffer, it is characterized in that: used envelope antigen is a haemophilus parasuis OMP P5 albumen.