CN109030830B

CN109030830B - Adhesin albumin A pd and its preparing the application in haemophilus parasuis indirect ELISA antibody assay kit

Info

Publication number: CN109030830B
Application number: CN201810730559.8A
Authority: CN
Inventors: 徐晓娟; 蔡旭旺; 刘云宝; 齐毅; 杜钰娇; 张勤学; 王湘如; 何启盖; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2018-07-05
Filing date: 2018-07-05
Publication date: 2019-09-10
Anticipated expiration: 2038-07-05
Also published as: CN109030830A

Abstract

The invention discloses a kind of adhesin albumin A pd and its preparing the application in haemophilus parasuis indirect ELISA antibody assay kit.The invention demonstrates that adhesin albumin A pd is located at the outer membrane of haemophilus parasuis, there is expression in known 15 serotype, but in the not no albumen of other bacterial pathogens of pig, uses it that there is high sensibility and specificity as diagnostic antigen.Optimum combination sticks the method for expression and the purifying of fibroin, obtains immunogenicity and the good recombinant protein of reactionogenicity.Optimize the component and dosage, reaction condition, result judgement standard to recombinate the indirect ELISA method for sticking fibroin as envelope antigen, is prepared for the indirect ELISA antibody assay kit of haemophilus parasuis.Animal experiment confirms the antibody test that the kit can be used for after haemophilus parasuis vaccine immunity and bacterium infection.In the Synthetical prevention and seroepidemiological survey of Haemophilus parasuis, which will have broad application prospects.

Description

Adhesin albumin A pd and its preparing haemophilus parasuis indirect ELISA antibody test Application in kit

Technical field

The present invention relates to biotechnologys and zoonosis field, and in particular to a kind of adhesin albumin A pd and its is making Application in standby haemophilus parasuis indirect ELISA antibody assay kit.

Background technique

Haemophilus parasuis (Haemophilus parasuis, HPS) is the normal in bacterium of the pig upper respiratory tract, belongs to Pasteur Bacteriaceae hemophilus, for tiny spherical, club-shaped to long Filamentous Gram-negative bacteria.Pig immunity it is low or by To stress when HPS can cause pig that Haemophilus parasuis (also known as Ge Laseshi disease) occurs.The acute infection of HPS can lead to more Hair property scrositis, arthritis and meningitis, septicemia and death；Chronic infection can cause suppurative rhinotracheitis and pneumonia. HPS would generally be such that disease complicates with Escherichia coli, Streptococcus suis, pasteurella multocida mixed infection.The disease is in the world There are generation and prevalence in the countries and regions of upper pig raising, can lead to 30% death rate in weanling pig.Due to China pig Inhibitive ability of immunity disease is multiple, and the generation of Haemophilus parasuis and prevalence are especially serious.In the blood of Haemophilus parasuis It is clear to learn in the antibody detection and seroepidemiological survey diagnosed, after vaccine immunity, a kind of special, sensitive antibody test Method is essential.

The antibody detection method of bacterial pathogen is generally difficult to establish, and especially HPS has 15 serotypes and 27% not Can parting bacterial strain (Oliveira et al., 2003).Though plate agglutination test is simple and easy, can use thallus suspension as It is aggregated antigen, but this method specificity and sensibility are low, and limited by agglutination antigen serotype, i.e. particular serotype Antigen can only make detection to the antibody of corresponding serotype.Indirect ELISA is carried out using thallus as envelope antigen, and because thin The structure and composition of bacterium is complicated, antigen type and quantity are more, envelope antigen would generally it is similar with other bacteriums in tested serum at Cross reaction occurs for the antibody of part, leads to false positive.

The HPS antibody detection method of early stage has complement fixation test (CFT), indirect hemagglutination test and ELISA (Oliveira et al.,2004).Complement fixation test (CFT) needs sheep red blood cell (SRBC) and guinea pig complement, because these materials are not easy to obtain and prepare immediately, And experimental implementation is cumbersome, is rarely employed now.There are two types of the sensitising antigens of indirect hemagglutination and the envelope antigen of ELISA, bacterium The LPS that supernatant (polysaccharide and albumen) or hot phenol-water method after ultrasonic disruption extract.It is established with both antigenic components Not only indirect hemagglutination and ELISA antibody detection method are unstable, are also easy to false negative occur, to the pig blood of adaptive immune protection Being detected clearly also can so (Oliveira et al., 2004).

Oligopeptides permease (OppA) (vehicle is bravely good etc., 2015), the lethal poison of cell expansion having using HPS domestic in recent years The albumen of the element Bacillus coli expressions such as (CDT) (Liu Shuanhong, 2016) and outer membrane protein (OMP2, OMP5) (Jia Aiqing etc., 2011) The antibody detection method of HPS indirect ELISA is established as diagnostic antigen.Domestic market also has BioChek company, Holland Haemophilus parasuis indirect ELISA antibody assay kit, using oligopeptides permease (OppA) as diagnostic antigen.But because Oligopeptides permease gene (98% nucleosides highly conserved in HPS, Escherichia coli, salmonella typhi and pasteurella multocida Sour consistency), cell expansion lethal toxin has structure and function similar homologous protein (44% amino acid in Escherichia coli Consistency), and outer membrane protein OMP2, OMP5 also have homologous albumen (42% and 71% amino in Actinobacillus pleuropneumoniae Sour consistency).So such antibody detection method or kit can inevitably be sent out with the cause of disease with comm on antigen ingredient Raw cross reaction, leads to false positive.

Summary of the invention

It is an object of the invention to overcome the deficiencies of the prior art and provide one kind to stick fibroin (Aida passenger Domain, Apd) and its preparing the application in haemophilus parasuis ELISA antibody assay kit.Due to sticking fibroin Apd is a kind of albumen specific to haemophilus parasuis, is all not present in other associated bacterial cause of diseases of pig.Mouse Immunity test and immunoblotting confirm that recombination Apd albumen has good immunogenicity and reactionogenicity.And recombination Apd Albumen can be reacted with the antiserum of 13 serological type strains, antiserum can with 15 serotype bacterial reactions, show with Apd will not be limited by serotype is different as diagnostic antigen.To recombinate Apd albumen as the bloodthirsty bar of secondary pig of envelope antigen Bacterium ELISA antibody assay kit can all be examined from the Swine serum that haemophilus parasuis inactivated vaccine is immune and viable bacteria infects Measure its antibody, and the positive rate of antibody and pig to attack malicious protective rate related.Serum of the kit in haemophilus parasuis Learning will have broad application prospects in diagnosis, the evaluation of immune effect of vaccine and seroepidemiological survey.

To achieve the above object, the present invention devises a kind of adhesin albumin A pd, the feature of amino acid sequence are as follows:

(1) protein that the amino acid sequence shown in SEQ ID No.2 forms；Or

(2) amino acid sequence homology limited with sequence SEQ ID No.2 encodes identical function egg 90~100% The amino acid sequence of white matter；Or

(3) amino acid sequence shown in SEQ ID No.2 through increase, missing or replaces one or more amino acid and tool There is the same active albumen as derived from (1)；

The gene apd of above-mentioned adhesin is encoded, nucleotide sequence is as shown in SEQ ID No.1.

The present invention also provides a kind of for obtaining the primer pair of above-mentioned adhesin gene apd, the primer pair are as follows:

P1,5 '-CGGAATTC(EcoRI) GGAAACTTATATTGTTACTGGTGAA-3 ',

P2,5 '-CCCTCGAG(XhoI)GGTGATTGTGATTTTATTGTGGT-3’。

The present invention also provides a kind of recombinant plasmid pET-Apd, the recombinant expression carrier is contains claim 3 institute State the expression vector of adhesin gene apd, wherein the expression vector is pET-25b.

The present invention also provides a kind of host cell of above-mentioned recombinant expression carrier, the host cell is Escherichia coli BL21(DE3)。

The present invention also provides a kind of preparation methods for recombinating adhesin albumin A pd: the following steps are included:

(1) by the competent escherichia coli cell BL21 (DE3) containing recombinant plasmid pET-Apd, overnight incubation, culture Bacterium solution centrifugation, precipitating are resuspended with buffer, obtain suspension bacteria liquid；

(2) bacterial cell disruption is carried out to suspension bacteria liquid under cryogenic, supernatant is collected by centrifugation, supernatant is purified, obtains Stick fibroin rApd to recombination.

The present invention also provides a kind of haemophilus parasuis indirect ELISA antibody assay kits, including adhesin to be coated with ELISA Plate, sample diluting liquid, yin and yang attribute control serum, 20 times of concentrated cleaning solutions, ELIAS secondary antibody, developing solution A, developing solution B, Terminate liquid；

The coated ELISA Plate of fibroin is sticked in the preparation: preparation method is as follows: using the rApd albumen of purifying as packet By antigen, rApd is diluted to by 0.5 μ g/mL with carbonate buffer solution, 4 DEG C of coatings are stayed overnight, with the skimmed milk containing 0.05g/mL PBS buffer solution (pH7.4) is in 37 DEG C of closing 2h.

The sample diluting liquid: PBS buffer solution (pH7.4) 50mL, 25 μ L of bovine serum albumin(BSA) 0.25g, Tween-20；

The positive control serum: the preparation method is as follows: 1 monthly age sodium selenite is immune twice with haemophilus parasuis, two Serum is collected in blood sampling in two weeks after exempting from, and detects its OD₆₃₀The serum being worth between 1.00- 1.20, takes 4 parts of mixing, is diluted with sample Liquid does 1:200 dilution and is used as positive control serum；

The negative control sera: the healthy Swine serum raised under equal conditions in positive control serum preparation, detection Its OD₆₃₀The serum being worth between 0.15-0.25, takes 4 parts of mixing, does 1:200 dilution with sample diluting liquid and is used as negative control Serum；

20 times of concentrated cleaning solutions: NaCl 160.00g, KCl 4.00g, Na₂HPO₄·12H₂O 72.60g, KH₂PO₄ 4.80g is dissolved in 900mL deionized water, with concentrated hydrochloric acid tune pH to 7.4,10mL Tween-20 is added, is settled to 1L, filter Degerming, room temperature preservation；

The ELIAS secondary antibody: the goat-anti pig secondary antibody of horseradish peroxidase-labeled, being purchased from Wuhan An Tejie biotechnology has Limit company (import packing), does 1:15000 dilution with sample diluting liquid；

The developing solution A:Na₂HPO₄·12H₂O 14.60g, citric acid 9.33g, 30% hydrogen peroxide 2ml add 800ml to go Ionized water dissolution, and adjust pH value to 5.0~5.4, it is settled to 1000ml；

The developing solution B: tetramethyl benzidine (TMB) 0.02g, dehydrated alcohol 10.00ml add deionized water dissolving simultaneously It is settled to 1000ml；The terminate liquid: 25 μ L hydrofluoric acid are added in the deionized water of 10mL.

The method of mentioned reagent box detection haemophilus parasuis antibody, comprising the following steps:

(1) serum to be checked is done into 1:200 dilution with sample diluting liquid, every part of sample takes 100 μ L that ELISA Plate is added, simultaneously Take 100 μ L of yin and yang attribute control serum that ELISA Plate is also added；It is discarded after 37 DEG C of incubator effect 45min, it is dense by 20 times with deionized water 200 μ L are added in every hole after contracting cleaning solution does 1:19 dilution, stand 5min, discard and pat dry, and wash 3 times；

(2) 100 μ L of ELIAS secondary antibody is added in every hole, discards after 37 DEG C of effect 30min, and 200 μ L cleaning solutions are added in every hole, 5min is stood, discards and pats dry, is washed 3 times；

(3) 50 μ L substrate solution A are added in every hole, add 50 μ L substrate solution B, and room temperature is protected from light 15min colour developing after mixing, most After be added 50 μ L terminate liquids, the OD value of wavelength 630nm is measured in 10min in microplate reader；

(4) result judgement: when serum S/N >=2.60 to be checked, it is determined as the positive；S/N < 2.6 are determined as feminine gender.S is represented Serum OD to be checked₆₃₀Value, N represent negative control sera OD₆₃₀Value, the condition for testing establishment is yin and yang attribute control serum OD₆₃₀Difference More than or equal to 0.5, wherein the Swine serum to be checked: porcine vein separates serum.

Beneficial effects of the present invention:

It is disclosed in this invention to stick that fibroin is peculiar for haemophilus parasuis, disclosed antibody assay kit Detection can be made to the antibody after haemophilus parasuis vaccine immunity or pathogen infection, be not present and other associated bacterials The cross reaction of cause of disease will not be limited by haemophilus parasuis different serotypes.

Detailed description of the invention

Fig. 1 is that the Bacillus coli expression of three kinds of recombinant proteins detects figure；

Fig. 2 is the purifying figure of three kinds of recombinant proteins；

Fig. 3 is that three kinds of recombinant proteins detect haemophilus parasuis yin and yang attribute serum result figure；

Fig. 4 is the reactionogenicity detection figure for recombinating adhesin albumen；

Fig. 5 is the detection and localization figure for sticking fibroin in haemophilus parasuis；

Fig. 6 is the serum titer figure for recombinating adhesin protein immunization mouse；

Fig. 7 is to detect 15 bloodthirsty bars of serotype pair pig by Western blot with recombination adhesin antibodies antiserum Bacterium result figure；

Fig. 8 is to pass through Western blot detection recombination adhesin egg with 15 serotype haemophilus parasuis antiserums White result figure；

Fig. 9 is detection yin and yang attribute serum result figure after ELISA reaction condition optimization；

Figure 10 is that supernatant detects yin and yang attribute serum result figure after haemophilus parasuis is broken；

Figure 11 is that haemophilus parasuis thallus detects yin and yang attribute serum result figure；

Figure 12 is that kit detects Swine serum result figure after vaccine immunity and bacterium infection；

Figure 13 is that kit detects antibody dynamic regularity result figure after vaccine immunity and bacterium infection.

Specific embodiment

The present invention is described in further detail in the following with reference to the drawings and specific embodiments, so as to those skilled in the art Member understands.

The synthesis of 1 adhesin gene apd of embodiment or clone

(1) synthesis of adhesin gene apd

According to nucleotide sequence as shown in SEQ ID No.1, synthesis obtains gene apd.

(2) clone of adhesin gene apd

Drawn with haemophilus parasuis separation strains CF7066 (by Hua Zhong Agriculture University's teacher's Xu Xiaojuan preservation) genome design Object pair:

P1,5 '-CGGAATTC(EcoRI) GGAAACTTATATTGTTACTGGTGAA-3 ',

P2,5 '-CCCTCGAG(Xho I)GGTGATTGTGATTTTATTGTGGT- 3'；

Using haemophilus parasuis separation strains CF7066 genome as template, PCR amplification is carried out, PCR system is as follows: 31.5 μ L deionized water, 5 μ 10 × Pyrobest of L Buffer II, 4 μ L dNTP Mixture (2.5mM), 2 μ L P1 (10 μM/L), 2 μ L P2 (10 μM/L), 0.5 μ L Pyrobest DNA Polymerase (Takara), 5 μ L haemophilus parasuis genomic DNAs (150ng/μL)；PCR response procedures: 94 DEG C of initial denaturation 5min, into circulation, 94 DEG C of denaturation 30s, 60 DEG C of renaturation 30s, 72 DEG C Extend 90s, 30 circulations, last 72 DEG C of extensions 10min, 4 DEG C save, and obtain PCR product；PCR product sequencing, thuja acid sequence Column are named as " gene apd " as shown in SEQ ID No.1.

Embodiment 2 sticks the preparation of fibroin

(1) acquisition of adhesin gene

The present invention synthesizes the DNA fragmentation for obtaining 1577 nucleotide by DNA；Or pass through PCR described in embodiment 1 Primer, system and response procedures, amplification obtains the DNA fragmentation of 1577bp from haemophilus parasuis separation strains CF7066.It should DNA fragmentation is named as apd, and nucleotide sequence is as shown in SEQ ID No.1.

(2) building of adhesin expression plasmid

The DNA fragmentation synthesized with deionized water dissolving；Or recycling pcr amplified fragment.By DNA fragmentation and carrier pET-25b (Novagen) EcoR I, Xho I double digestion are used, recycling digestion products are connected with carrier, and connection product is converted Escherichia coli DH5 α, 37 DEG C of overnight incubations are to growing single colonie.Picking, which is individually cloned, extracts plasmid, and correct pET-Apd plasmid will be sequenced Be stored in -20 DEG C it is spare.

(3) stick the expression of fibroin

Plasmid pET-Apd is converted into competent escherichia coli cell BL21 (DE3), 37 DEG C of overnight incubations.Picking single colonie In the LB liquid medium of 5mL, 37 DEG C of shaken cultivations are stayed overnight.It transfers in 1:100 ratio in the LB liquid medium of 1000mL In, 37 DEG C of shake cultures to OD₆₀₀About 0.6, IPTG to final concentration of 0.8mM, the shake culture 4h in 37 DEG C is then added. Thallus is resuspended with 4 DEG C of centrifugation 1min of 3200g, with the combination buffer of 1/10 bacterium solution volume in bacterium solution.With low-temperature ultrahigh-pressure cell Broken instrument repeats to be crushed 3~5 times, then with 3200g refrigerated centrifuge 10min, it is spare to collect supernatant in 4 DEG C of broken thallus.

The IPTG (0.8M): dissolving 2g IPTG in 8mL distilled water, be settled to 10mL, is filtered with 0.22 μm of filter Degerming, -20 DEG C of preservations.

(4) stick the purifying of fibroin

Broken thallus supernatant is harvested, is filled out with the 6 Fast Flow of Ni Sepharose of GE Healthcare company Material (filler is stored in 20% dehydrated alcohol) purifies broken thallus supernatant, obtains recombination adhesin antibodies RApd, amino acid sequence be SEQ ID No.2 shown in,

The purifying process is as follows:

Dress column: covering the lid of purification column lower end, and 2mL filler is taken to be slowly added to along chromatography post jamb, avoids generating bubble And uniform filling distribution is kept as far as possible, 4 DEG C upright to stand overnight；

Balance: the lid of purification column lower end is opened, flows out liquid in column naturally.The distillation washing of 10~20mL is added 20mL 500mM imidazole buffer washing nickel ion packed column is added after liquid outflow and (newly fills out for ethyl alcohol when filler being gone to save Charge can save this operation), then the nickel ion that 10~20mL combination buffer balance has loaded is added after liquid outflow and fills out Fill column；

Loading: after liquid flows completely out, the 80~100mL of broken thallus supernatant placed on ice is slow by several times It adds in purification column.

Washing: after protein sample completes dress column, 10~20mL combination buffer balance is added, then with 20mL washing buffer Liquid washes away foreign protein.

Elution: being slowly added to 10mL elution buffer by several times and wash lower destination protein sample, is collected with 1.5mL EP pipe, uses BCA Kit (Biosharp company) measurement protein concentration is placed on -80 DEG C of preservations.The amino acid sequence of the purifying protein such as SEQ Shown in ID No.2.

The combination buffer: NaCl 29.22g, Tris-base 2.42g, imidazoles 0.34g are dissolved in 800mL deionization In water, with concentrated hydrochloric acid tune pH to 7.4, it is dissolved to 1L, 0.22 μm of filter filtering, 4 DEG C save backup.

The washing buffer: NaCl 29.22g, Tris-base 2.42g, imidazoles 1.36g are dissolved in 800mL deionization In water, with concentrated hydrochloric acid tune pH to 7.4, it is dissolved to 1L, 0.22 μm of filter filtering, 4 DEG C save backup.

The elution buffer: NaCl 29.22g, Tris-base 2.42g, imidazoles 6.8g are dissolved in 800mL deionization In water, with concentrated hydrochloric acid tune pH to 7.4, it is dissolved to 1L, 0.22 μm of filter filtering, 4 DEG C save backup.

The 500mM imidazole buffer: weighing NaCl 29.22g, Tris-base 2.42g, and imidazoles 34.04g is dissolved in In 800mL deionized water, with concentrated hydrochloric acid tune pH to 7.4, it is dissolved to 1L, 0.22 μm of filter filtering, 4 DEG C save backup

20% ethyl alcohol: 200mL dehydrated alcohol is dissolved in 800mL deionized water, 0.22 μm of filter filtering, 4 DEG C of preservations It is spare.

The recombination of embodiment 3 sticks fibroin as haemophilus parasuis indirect ELISA antibody assay kit envelope antigen Evaluation

(1) expression of three kinds of recombinant proteins of haemophilus parasuis

Select haemophilus parasuis 3 outer membrane proteins extracellular region and construct its prokaryotic expression plasmid pET-Ag2, PET-Ag3 and pET-Apd.E. coli bl21 (DE3) is converted with 3 plasmids, in different temperatures and various concentration IPTG condition Lower induction different time determines that the optimum condition of solubility expression occurs for destination protein.The result shows that rAg2 The most suitable expression condition of (recombinant Ag2) albumen is 16 DEG C, and the IPTG of 0.2mM induces 12h；rAg3 (recombinant Ag3) albumen is 16 DEG C, and the IPTG of 0.2mM induces 12h；Albumen is most by rApd (recombinant Apd) Suitable expression condition is 37 DEG C, and the IPTG of 0.8mM induces 4h (Fig. 1).

(2) purifying of three kinds of recombinant proteins of haemophilus parasuis

With the Escherichia coli after the broken induction of low-temperature ultrahigh-pressure cell crushing instrument, centrifuging and taking supernatant passes through the affine layer of nickel column Analysis purifying destination protein, respectively elutes albumen with the imidazole concentration of 5mM~300mM.It is final to determine rAg2 washing buffer Liquid imidazoles containing 50mM, elution buffer imidazoles containing 150mM；R Ag3 washing buffer imidazoles containing 20mM, elution buffer contain 150mM imidazoles；RApd washing buffer imidazoles containing 20mM, elution buffer imidazoles containing 100mM.After obtaining purifying protein, use It is 706 μ g/mL, rAg3 protein concentrations is 325 μ g/mL, rApd that BCA determination of protein concentration kit, which measures rAg2 protein concentration, Protein concentration is 450 μ g/mL, carries out SDS-PAGE electrophoresis (Fig. 2) to purifying protein.

(3) three kinds of recombinant proteins detect haemophilus parasuis yin and yang attribute serum

Three kinds of recombinant proteins of expression and purification are diluted to 1 μ g/mL coated elisa plate, the yin and yang attribute of haemophilus parasuis Each 4 parts of Swine serum are done 1:100 dilution, carry out indirect ELISA according to conventional program.The result shows that when rApd albumen is coated with, yin Property serum OD₆₃₀Average value is 0.297, positive serum OD₆₃₀Average value is 2.201 (P < 0.001), and the two difference is extremely significant；And After rAg2 and rAg3 albumen coating, negative and positive serum OD₆₃₀Value is both dispersed in the range of 1.0~2.0, is not gathered Class (Fig. 3).Thus choose the envelope antigen that rApd albumen is haemophilus parasuis antibody assay kit.

(4) reactionogenicity of fibroin is sticked in recombination

PET-Apd is converted into competent escherichia coli cell BL21 (DE3), the recombination adherency for inducing and obtaining after purification Fibroin carries out Western blot with the His monoclonal antibody of source of mouse, haemophilus parasuis yin and yang attribute Swine serum.Since rApd is big His label has been merged when expressing in enterobacteria, has occurred reaction zone, the secondary bloodthirsty bar of pig at 85kDa when detecting using His monoclonal antibody Also there is detection band at 85kDa size in the positive serum of bacterium, and negative serum does not have, and converts BL21 (DE3) with empty carrier Also reaction zone (Fig. 4) is not detected in extract afterwards.This is the result shows that the rApd of expression and purification can be with haemophilus parasuis Pig source antibody react, it was demonstrated that it is with good reactionogenicity.

(5) stick positioning of the fibroin in haemophilus parasuis

Haemophilus parasuis late log phase culture bacterium solution 1000mL is centrifuged 15min in 4 DEG C of 3200g, abandons supernatant.Use 60mL Bacterial precipitation is resuspended in 50mM Tis-HCl (pH7.2), and 300 μ l protease inhibitors PMSF (10mg/mL) are added, and ultrasonic wave is broken Broken, each 25s is spaced 1min.Until bacterium solution becomes limpid.It is centrifuged 15min in 3200g, the 0.1M for taking supernatant and being pre-chilled on ice Na₂CO₃(pH11) it is mixed according to the ratio of 1:100,4 DEG C of stirring 1h.Solution is centrifuged 1h, precipitating 1mL TE in 4 DEG C of 15000g Dissolution, gained is outer membrane protein.Haemophilus parasuis late log phase culture bacterium solution 1000mL is centrifuged in 4 DEG C of 3200g 30min collects supernatant and is filtered with 0.22 μm of filter.Isometric saturation (NH is added to filtrate₄)₂SO₄It saltouts in 4 DEG C of stirrings Overnight, 3200g is centrifuged 15min, is precipitated with TE buffer solution, gained is secretory protein.It is carried out with the monoclonal antibody of Apd albumen Western blot detection.The result shows that there is test strip at about 85kD size in rApd, haemophilus parasuis it is complete Also there is an equal amount of detection band in mycoprotein and outer membrane protein, and secretory protein does not have, and illustrate that Apd albumen is located at secondary pig Haemophilus outer membrane (Fig. 5).

(6) the antiserum Western blot for recombinating adhesin antibodies detects 15 serotype haemophilus parasuis

Antiserum is prepared with purifying 4 BALB/c mouses of rApd protein immunization.It is immune that blood sampling separates serum afterwards three times, with The coated ELISA Plate of rApd detects its potency, and the serum titer of all mouse all reaches 1:204800 (Fig. 6).By 15 serum The culture of type reference culture and separation strains CF7066 transferring film after SDS-PAGE electrophoresis, it is anti-clear with the mouse blood of rApd albumen Immunoblotting analysis is carried out, as a result about 85kDa reaction zone all occur in all bacterial strains, and negative control sera does not detect reaction Band (Fig. 7).As it can be seen that in 15 serotype reference cultures of HPS, it is all anti-in the presence of what can be reacted with rApd antiserum Former composition illustrates that rApd can be used for detecting the antibody of 15 serotype of haemophilus parasuis as diagnostic antigen.

Fibroin is sticked in (7) 15 serotype haemophilus parasuis antiserum Western blot detection recombinations

Prepare 15 kinds of serotype reference cultures of haemophilus parasuis and separation strains CF7066 and the pathogenic large intestine bar of pig Bacterium, actinobacillus pleuropneumoniae, pasteurella multocida, Streptococcus suis, B.bronchisepticai BALB/c mouse antiserum.The preparation method is as follows: being washed carefully overnight incubation on TSA plate is spread evenly across with PBS (pH7.4) Under, bacterium amount is adjusted to 10¹⁰Then CFU/mL is emulsified with the Freund's adjuvant of same volume；Every 300 μ L of mouse immune (1.5 × 10⁹CFU/mL), immunization ways are the subcutaneous multi-point injection of the nape of the neck；Head exempts to use Freund's complete adjuvant, incomplete with Freund later Adjuvant.Three exempt to pass through Western blot detection detection rApd after blood sampling separation serum in latter 5 days.In addition to the anti-blood of 6 types and 14 types Clear outer, the antiserum of remaining 13 serotype reference culture and separation strains CF7066 are all reacted with rApd, and swine escherichia coli, Actinobacillus pleuropneumoniae, Streptococcus suis, B.bronchisepticai antiserum do not reacted with rApd (Fig. 8).The result confirms that the adhesin antibodies immunogenicity of haemophilus parasuis is strong, can stimulate animal body generation can The antibody of detection, and the albumen is relatively conservative in haemophilus parasuis different serotypes.So using it anti-as diagnosis The antibody detection method that original is established is not limited not only by serotype, but also will not bacterial pathogen generation relevant to other Cross reaction.

The selection of 4 haemophilus parasuis indirect ELISA antibody assay kit component of embodiment and dosage and reaction item The determination of part

(1) determination of antigen coat concentration and ELIAS secondary antibody working concentration

RApd is diluted to 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL, enzyme mark with coating buffer Secondary antibody is diluted to 1:5000,1:10000,1:15000,1:20000 times with sample diluting liquid and dilutes.The sun of haemophilus parasuis Property and negative serum do 1:100 dilution with sample diluting liquid.The results show that it is dilute for 0.5 μ g/mL, secondary antibody to work as antigen coat concentration When degree of releasing is 1:15000, positive and negative serum OD₆₃₀Ratio (P/N) it is maximum, therefore with 0.5 μ g/mL and 1:15000 work For the best peridium concentration of antigen and secondary antibody optimum dilution degree (table 1).

The determination of table 1. best antigen coat concentration and ELIAS secondary antibody working concentration

(2) determination of antigen coat condition

With the antigen concentration coated elisa plate of 0.5 μ g/mL, coating condition is respectively as follows: 4 DEG C and is coated with overnight, 37 DEG C of coating 1h 4 DEG C of coatings are overnight overnight, after 37 DEG C of coating 2h, 37 DEG C of coating 2h for 4 DEG C of coatings afterwards.The result shows that 4 DEG C are coated with P/N value overnight and are 12.097, it is all higher than other coating conditions, therefore it is 4 DEG C overnight (tables 2) that antigen, which is most preferably coated with condition,.

2. antigen of table is most preferably coated with the determination of condition

(3) determination of serum diluting multiple and reaction time

Yin and yang attribute serum is diluted with 1:50,1:100,1:200,1:400 times, 100 μ L is taken to be added to coated enzyme mark Plate acts on 30min, 45min, 60min, 75min respectively in 37 DEG C of incubators and carries out indirect ELISA.The results show that working as serum When with 1:200 times of dilution, reaction time being 45min, P/N value is maximum, therefore serum optimum diluting multiple to be checked is 1:200, most The good reaction time is 45min (table 3).

The determination of table 3. serum optimum diluting multiple and reaction time

(4) selection of confining liquid

After albumen coating, closed using different confining liquids.The PBS buffer solution that confining liquid is respectively as follows: contains 0.1g/L degreasing Cream, 0.5g/L skimmed milk, 0.05g/L BSA, 0.2g/L BSA.The results show that when confining liquid contains 5g/L skimmed milk, P/N value Maximum, therefore best confining liquid is the PBS buffer solution (pH7.4) (table 4) containing 5g/L skimmed milk.

The determination of the best confining liquid of table 4.

(5) determination of off-period

3 different off-periods: 37 DEG C of closing 1h, 37 DEG C of closing 2h, 37 DEG C of closing 3h are set.Each off-period Detect OD₆₃₀Value, the results show that P/N value is maximum when being 2h between when closed, therefore best off-period is 2h (table 5).

The determination of the best off-period of table 5.

(6) determination of ELIAS secondary antibody action time

Using the ELISA reaction condition of above-mentioned optimization, 100 μ L goat-anti pig secondary antibodies are added in every hole, in 37 DEG C of incubators respectively Act on 30min, 45min, 60min, 75min.The results show that P/N value is maximum, therefore secondary antibody most fine piece of writing when secondary antibody acts on 30min It is 30min (table 6) with the time.

The determination of 6. ELIAS secondary antibody the best use time of table

(7) determination of substrate developing time

It is protected from light respectively at room temperature aobvious after substrate solution A and substrate solution B is added using the ELISA reaction condition of above-mentioned optimization Color 5min, 10min, 15min, 20min.The results show that when develop the color 15min when, P/N value is maximum, therefore the best developing time of substrate For 15min (table 7).

The determination of the best developing time of 7. substrate of table

(8) yin and yang attribute Swine serum is detected after ELISA reaction condition optimization

With rApd peridium concentration, confining liquid and the off-period of above-mentioned optimization, the extension rate of serum to be checked, enzyme mark two Anti- diluted concentration and action time and substrate developing time, 15 parts each to haemophilus parasuis yin and yang attribute Swine serum are examined It surveys.The result shows that after condition optimizing

The background of ELISA testing result significantly reduces.Positive serumIt is fallen to from 2.514

1.438 negative serumIt is reduced to 0.452 from 1.400, standard deviation also reduced 0.14 (Fig. 9), explanation The optimization of ELISA component dosage and reaction condition effectively increases the detection effect of kit

5 haemophilus parasuis indirect ELISA antibody assay kit positive critical value of embodiment determines

(1) critical value determines formula

Indirect ELISA Positive judgement standards are generally using the mean OD value of a large amount of negative samples plus one times or several times yin The standard deviation of property sample OD value, i.e., So indirect After every experimental condition of ELISA operation is fixed, the determination of positive critical value in three steps: 1. in critical value calculation formula N value；2. the standard deviation of more parts of negative samples, Serum Number is the more then worth more reliable；3. the negative control in actually detected It is worth (Wu Baocheng, 1994).

(2) the n value measurement in critical value

Referring to the method that Wu Baocheng etc. (1994) is used, the measuring method of n value is as follows.Take the haemophilus parasuis positive and Each portion of negative serum carries out doubling dilution to 1:204800 since 1:100, sequentially adds the coated ELISA Plate of rApd, often A dilution respectively adds 8 holes to measure the OD in every hole₆₃₀Value, and calculate each dilution positive and negative serum OD₆₃₀Be averaged Value and corresponding t value (table 8).It is for statistical analysis to its it is found that when dilution be 1:12800 when, t=2.571 > 2.145 {t_(0.05,14), yin and yang attribute blood serum sampleSignificant difference；When dilution continues to increase,Sun is less thanYin illustrates that this method cannot distinguish between yin and yang attribute serum.As it can be seen that t value 2.571 is when dilution is 1:12800 N value in critical value.When the result also indicates that serum to be checked does 1:12800 dilution, this method can be made yin and yang attribute serum It detects out, shows that its sensibility is very high.

The determination of 8. critical value Plays difference multiple of table

(3) haemophilus parasuis negative serum screening technique

By the supernatant after the haemophilus parasuis of culture and bacterial cell disruption centrifugation, multiple proportions after bacterium amount and protein concentration is measured Coated elisa plate is diluted, each portion of haemophilus parasuis yin and yang attribute Swine serum is detected.The results show that when on broken When being used as envelope antigen clearly, negative and positive serum OD₆₃₀It is worth all higher, P/N value between 1~2 (Fig. 9)；And with complete bacterium When body is as envelope antigen, yin and yang attribute serum OD₆₃₀The difference of value significantly increases, and P/N value is greater than 2.Especially when the coating of thallus Concentration is 0.5 × 10⁸When CFU/mL, P/N value is 2.47, positive serum OD₆₃₀Value is 1.153, negative serum OD₆₃₀Value is 0.467 (Figure 10).

(4) in critical value negative serum standard deviation measurement

With 0.5 × 10⁸CFU/mL haemophilus parasuis thallus suspension coated elisa plate is fed by indirect ELISA from clinic Haemophilus parasuis negative serum, OD are screened in suckling piglet, child care pig, growing and fattening pigs and 350 parts of sow serum₆₃₀Value is less than 0.467 judgement is negative serum.As a result 40 parts of feminine genders have been obtained, wherein the 4 of suckling pig part, 23 parts of child care pig, fattening 13 parts of pig.Above-mentioned 40 parts of serum is carried out with the ELISA antibody detection method of the invention

Detection, OD₆₃₀Average valueIt is 0.189, standard deviation (SD) is 0.118.

Table 9. detects the result of 40 parts of haemophilus parasuis negative serums

(5) judgement of positive critical value

It is 2.571,40 parts of negative serum sample OD according to n value₆₃₀ It is 0.189, standard deviation (SD) is 0.118, with AndCritical value=0.492 can be obtained, that is, works as OD₆₃₀≥ When 0.492, blood serum sample is the positive；Work as OD₆₃₀When < 0.492, blood serum sample is feminine gender.

In view of the error of the personal operation in the differences between batches and use process in ELISA kit preparation process, originally Invention is under the premise of stringent preparation and control yin and yang attribute control serum, positive critical value judgment basis S/N value (0.492/ 0.189=2.60), i.e. when serum S/N >=2.60 to be checked, it is determined as the positive；S/N < 2.6 are determined as feminine gender.S represents blood to be checked Clear OD₆₃₀Value, N represent negative control sera OD₆₃₀Value, the condition for testing establishment is yin and yang attribute control serum OD₆₃₀Difference is greater than Or it is equal to 0.5.

The composition of 6 haemophilus parasuis indirect ELISA antibody assay kit of embodiment and preparation

(1) recombination adhesin albumin A pd coated elisa plate preparation

It is 0.5 μ g/mL that the rApd albumen of purifying, which is diluted to concentration with coating buffer, and every 100 μ L of hole is added in ELISA Plate, 4 DEG C refrigerator overnight；Coating buffer is discarded, 200 μ L cleaning solutions are added in every hole, stand 5min, abandon supernatant and pat dry, wash 3 times；

Then the confining liquid of 200 μ L is added in every hole, and 37 DEG C of incubators act on 2h；Confining liquid is discarded, every hole is added 200 μ L and washes Liquid is washed, 5min is stood, supernatant is abandoned and pats dry, wash 3 times；Freeze-drying, vacuum and low temperature save.

The coating buffer: carbonate buffer solution (pH9.6), Na₂CO₃ 1.59g、NaHCO₃2.93g adds appropriate deionization Water dissolution, with hydrochloric acid tune pH to 9.6, is settled to 1L, 4 DEG C of high pressure sterilization save backup；

The cleaning solution: 50 μ L Tween-20 are added in 100mL PBS buffer solution (pH7.4)；

The confining liquid: the PBS buffer solution (pH7.4) containing 5g/L skimmed milk.

(2) stick fibroin indirect ELISA antibody assay kit composition

Kit contain coated 2 pieces of 96 hole ELISA detection plate of adhesin, 1 bottle of sample diluting liquid (50mL/ bottles), yin and yang attribute Control serum each 1 pipe (1.5mL/ pipe), 20 times of 1 bottle of concentrated cleaning solutions (30mL/ bottles), 1 bottle of ELIAS secondary antibody (20mL/ bottles), colour developing Each 1 bottle of liquid A, developing solution B, terminate liquid (10mL/ bottles).

The developing solution A:Na₂HPO₄·12H₂O 14.60g, citric acid 9.33g, 30% hydrogen peroxide 2ml add 800ml to go Ionized water dissolution, adjusts pH value to 5.0~5.4, is settled to 1000ml；

The developing solution B: tetramethyl benzidine (TMB) 20.00mg, dehydrated alcohol 10ml add deionized water dissolving and determine Hold to 1000ml；

The terminate liquid: 25 μ L hydrofluoric acid are added in the deionized water of 10mL.

(3) application method of kit

Blood serum sample to be checked is done into 1:200 dilution with sample diluting liquid, every part of sample takes 100 μ L that ELISA Plate is added, simultaneously Take 100 μ L of yin and yang attribute control serum that ELISA Plate is also added；It is discarded after 37 DEG C of incubator effect 45min, it is dense by 20 times with deionized water 200 μ L are added in every hole after contracting cleaning solution does 1:19 dilution, stand 5min, discard and pat dry, and wash 3 times；

100 μ L of ELIAS secondary antibody is added in every hole, discards after 37 DEG C of effect 30min, and 200 μ L cleaning solutions are added in every hole, stands 5min is discarded and is patted dry, and is washed 3 times；

50 μ L substrate solution A are added in every hole, add 50 μ L substrate solution B, and room temperature is protected from light 15min colour developing after mixing, finally plus Enter 50 μ L terminate liquids, measures the OD value of wavelength 630nm in 10min in microplate reader；

Result judgement: when serum S/N >=2.60 to be checked, it is determined as the positive；S/N < 2.6 are determined as feminine gender.S represents to be checked Serum OD₆₃₀Value, N represent negative control sera OD₆₃₀Value, the condition for testing establishment is yin and yang attribute control serum OD₆₃₀Difference is greater than Or it is equal to 0.5.

The Swine serum to be checked: porcine vein separates serum.

The repeatability of 7 haemophilus parasuis indirect ELISA antibody assay kit of embodiment

4 parts of positive serums of haemophilus parasuis and 4 parts of negative serums are carried out respectively with the coated ELISA Plate of same batch Repeated experiment in batch.Detection 4 times is repeated, the coefficient of variation is between 1.9%~9.5%；With the coated enzyme mark of different batches Plate to same 8 parts of serum criticize between repeated experiment, the coefficient of variation (table 10) between 1.4%~9.4% shows ELISA detection kit of the invention has good repeatability.

10. batches of interior repetitions of table and batch between repeat test

The application of 8 haemophilus parasuis indirect ELISA antibody assay kit of embodiment

(1) detection of vaccine immunity and weak poison infection Swine serum

The Swine serum of haemophilus parasuis vaccine immunity and low virulent strain infection is had detected with the ELISA kit, wherein epidemic disease 15 parts of seedling immune serum, low virulent strain 4 parts of serum of infection and 20 parts of PBS control serum determine mark using S/N >=2.60 as the positive It is quasi-.The result shows that vaccine immunity group has 93% (14/15) test positive, low virulent strain A infected group has 100% (4/4) to be The positive, PBS control group have 75% (15/20) negative (Figure 12).It is worth noting that, PBS control group has 5 parts of serum to be detected as Antibody positive, autogenous infection or cross-infection may has occurred because of pig during test in this, because secondary haemophilus is normal In bacterium, animal experiment continue for 6 weeks.However, the result of challenge test has show PBS control group to have 8 not occur clinic Symptom, there may be the generations of actual infection and antibody for indication PBS pig.In short, the above results show that the kit can be with It detects haemophilus parasuis vaccine immunity and the metainfective serum antibody of viable bacteria, can be applied to haemophilus parasuis vaccine and exempt from The evaluation and the antibody test after pathogen infection of epidemic disease protecting effect.

(2) evaluation of antibody dynamic regularity after vaccine immunity and weak poison infect

Lasting antibody test has been carried out to virus-shedding Pattern with the ELISA antibody assay kit.One monthly age piglet 16 Head divides 4 groups, every group 4, is spaced immune/infection in two weeks twice, carries out inactivated vaccine A respectively and be immunized, gene-deleted strain B and C Infection, using S/N >=2.60 as Positive judgement standards.The result shows that one week ELISA antibody goes out after infection by low virulent strain B and C Now switch to the positive, persistently rises after two weeks；Vaccine immunity group exempts from latter Zhou Kangti and significantly rises to switch to the positive two.After immune Two weeks vaccine immunities are identical with the antibody level of low virulent strain infected group, and the antibody test of PBS control group is continuously negative (figure 13).It is evaluated as it can be seen that kit detection can also generate rule to the antibody after vaccine immunity and pathogen infection.

(3) detection of clinical Swine serum

With the ELISA antibody kit have detected 3 pig farms collection 222 parts of serum, including suckling pig, child care pig, Growing and fattening pigs, sow serum.The result shows that positive rate be followed successively by suckling pig 12%, child care pig 16%, growing and fattening pigs 36%, Sow 68%, total positives recall rate 35%.With 0.5 × 10⁸The coated ELISA Plate of CFU/mL haemophilus parasuis is detected, The positive rate of detection be suckling pig be 84%, child care pig is 74%, growing and fattening pigs 86%, sow 100%, total positives rate For 86% (table 11).Two kinds of detection methods are all shown, with the increase of pig age in days, antibody positive rate also constantly increases.But with bacterium When body is as envelope antigen, the positive rate of detection is obviously higher.May be more because of the type and quantity of somatic antigen component, with it The cross reaction that his bacterium generates antibody is related, and use and recombinate adhesin Apd albumen as envelope antigen, can be to avoid this Class cross reaction.Importantly, the total positives rate detected using the kit in 222 parts of serum samples is 35%, this and pair Separation rate 30% of the haemophilus suis in swinery is almost the same, more can truly reflect infection and the stream of haemophilus parasuis Capable trend.

11. kit of table and thallus ELISA detect clinical serum

Other unspecified parts are the prior art.Although above-described embodiment is made that in detail the present invention Description, but it is only a part of the embodiment of the present invention, rather than whole embodiments, people can also exist according to the present embodiment Without other embodiments are obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.

Sequence table

<110>Hua Zhong Agriculture University

<120>adhesin albumin A pd and its in preparing haemophilus parasuis indirect ELISA antibody assay kit Using

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1577

<212> DNA

<213>haemophilus parasuis (Haemophilus parasuis)

<400> 1

cggaattcgg aaacttatat tgttactggt gaaaataaca atgtgcgcgg cgaacaattc 60

caacgtttac ttaaccaagc caaagatggc gatacaattc gcttagacgg agatactcaa 120

ttattgcgtg gaagtggttt tttcaatgtt gataaacaag taacaattga tggacaagga 180

aatcgcttac accttgaagg ttataatttg tccttaggac aagatgttac tttttcgaat 240

gtaaatttat ccttaaaacc tagtcgagat ttaaacaata tttttggttc tggatttaat 300

aatttaaaag cagcatctac ctatattgtg accaatggaa ataaattgat cttggataat 360

gttagaacga atgttaatgg tgaacctgca gatactcgcc caatgatttt acttggaaat 420

attgatccaa caaaaaataa taatggacac gatgctcttg tagtgacaaa tgctcaccca 480

acagaaacaa tattatcttc agtggttgtt ataggcaata acaattcaaa tactgacacc 540

ccagtaacga ttagtttagg ggaaaatgtc agctttgtta atcaggtaat atttgagaga 600

gaaggagaag aggatatata tagtggtgcg gtgtatctag ctggaatgaa taatgatcaa 660

gaacataaca gagctattaa tttctctagt tcatcaaatg ctatcactaa aatttatacg 720

ggagaagcta caaatgtttc tgttacgtta aataacatca atcctgaaac tgctatcact 780

ttacaagatg ttaaagattt aacattaaat aatagccgta ttactttgga taagaattta 840

tctgtttcag aaaccttaac tctgaataat caatccgcaa tcactacagt ggcgagtaaa 900

gatgcttttg gtgacatcgc taagactagt ttgatgttaa ataatatcca taccaatggt 960

aataatagta atatcactat cgttcgggac aatgctcttt tgattaatgg taatattact 1020

ggtgaattga ctattaatac agaagataaa gataataacg ttagtcttcc taatggcagc 1080

actttagtag gagaaaatgg cagctatgtt gtgaagctaa aaacagaagt aactcaacct 1140

gtatcagaaa atacaactac tgacagcagt aatactgatg taacccctgc taaacctacg 1200

gatagcacac cagcaacaac aggcgaacaa actggttcgt cagatacaac atcaccaaca 1260

aatacagctg caactagcaa tgaaactaca ccgcctgcta caacgggaga tacagcgact 1320

aacagcagta atactgatgt accccctgct acacctacgg atagcacacc agcaacaaca 1380

ggcgaacaaa ctggttcgtc agatataaca tcgccaacag atacagctca aacagacgat 1440

caagcggagg cttctagctc tgacactgca acatctacac cagcacaacc aacgggcgat 1500

gtagcttcaa caagtgaaca agcgacaacg tcgagtacat ctacaaacca caataaaatc 1560

acaatcaccc tcgaggg 1577

<210> 2

<211> 557

<212> PRT

<213>haemophilus parasuis (Haemophilus parasuis)

<400> 2

Met Asp Ile Gly Ile Asn Ser Asp Pro Asn Ser Glu Thr Tyr Ile Val

1 5 10 15

Thr Gly Glu Asn Asn Asn Val Arg Gly Glu Gln Phe Gln Arg Leu Leu

20 25 30

Asn Gln Ala Lys Asp Gly Asp Thr Ile Arg Leu Asp Gly Asp Thr Gln

35 40 45

Leu Leu Arg Gly Ser Gly Phe Phe Asn Val Asp Lys Gln Val Thr Ile

50 55 60

Asp Gly Gln Gly Asn Arg Leu His Leu Glu Gly Tyr Asn Leu Ser Leu

65 70 75 80

Gly Gln Asp Val Thr Phe Ser Asn Val Asn Leu Ser Leu Lys Pro Ser

85 90 95

Arg Asp Leu Asn Asn Ile Phe Gly Ser Gly Phe Asn Asn Leu Lys Ala

100 105 110

Ala Ser Thr Tyr Ile Val Thr Asn Gly Asn Lys Leu Ile Leu Asp Asn

115 120 125

Val Arg Thr Asn Val Asn Gly Glu Pro Ala Asp Thr Arg Pro Met Ile

130 135 140

Leu Leu Gly Asn Ile Asp Pro Thr Lys Asn Asn Asn Gly His Asp Ala

145 150 155 160

Leu Val Val Thr Asn Ala His Pro Thr Glu Thr Ile Leu Ser Ser Val

165 170 175

Val Val Ile Gly Asn Asn Asn Ser Asn Thr Asp Thr Pro Val Thr Ile

180 185 190

Ser Leu Gly Glu Asn Val Ser Phe Val Asn Gln Val Ile Phe Glu Arg

195 200 205

Glu Gly Glu Glu Asp Ile Tyr Ser Gly Ala Val Tyr Leu Ala Gly Met

210 215 220

Asn Asn Asp Gln Glu His Asn Arg Ala Ile Asn Phe Ser Ser Ser Ser

225 230 235 240

Asn Ala Ile Thr Lys Ile Tyr Thr Gly Glu Ala Thr Asn Val Ser Val

245 250 255

Thr Leu Asn Asn Ile Asn Pro Glu Thr Ala Ile Thr Leu Gln Asp Val

260 265 270

Lys Asp Leu Thr Leu Asn Asn Ser Arg Ile Thr Leu Asp Lys Asn Leu

275 280 285

Ser Val Ser Glu Thr Leu Thr Leu Asn Asn Gln Ser Ala Ile Thr Thr

290 295 300

Val Ala Ser Lys Asp Ala Phe Gly Asp Ile Ala Lys Thr Ser Leu Met

305 310 315 320

Leu Asn Asn Ile His Thr Asn Gly Asn Asn Ser Asn Ile Thr Ile Val

325 330 335

Arg Asp Asn Ala Leu Leu Ile Asn Gly Asn Ile Thr Gly Glu Leu Thr

340 345 350

Ile Asn Thr Glu Asp Lys Asp Asn Asn Val Ser Leu Pro Asn Gly Ser

355 360 365

Thr Leu Val Gly Glu Asn Gly Ser Tyr Val Val Lys Leu Lys Thr Glu

370 375 380

Val Thr Gln Pro Val Ser Glu Asn Thr Thr Thr Asp Ser Ser Asn Thr

385 390 395 400

Asp Val Thr Pro Ala Lys Pro Thr Asp Ser Thr Pro Ala Thr Thr Gly

405 410 415

Glu Gln Thr Gly Ser Ser Asp Thr Thr Ser Pro Thr Asn Thr Ala Ala

420 425 430

Thr Ser Asn Glu Thr Thr Pro Pro Ala Thr Thr Gly Asp Thr Ala Thr

435 440 445

Asn Ser Ser Asn Thr Asp Val Pro Pro Ala Thr Pro Thr Asp Ser Thr

450 455 460

Pro Ala Thr Thr Gly Glu Gln Thr Gly Ser Ser Asp Ile Thr Ser Pro

465 470 475 480

Thr Asp Thr Ala Gln Thr Asp Asp Gln Ala Glu Ala Ser Ser Ser Asp

485 490 495

Thr Ala Thr Ser Thr Pro Ala Gln Pro Thr Gly Asp Val Ala Ser Thr

500 505 510

Ser Glu Gln Ala Thr Thr Ser Ser Thr Ser Thr Asn His Asn Lys Ile

515 520 525

Thr Ile Thr Leu Glu Ile Lys Arg Ala Ser Gln Pro Glu Leu Ala Pro

530 535 540

Glu Asp Pro Glu Asp Val Glu His His His His His His

545 550 555

Claims

1. a kind of adhesin gene apd, nucleotide sequence is as shown in SEQ ID No.1.

2. the primer pair for obtaining adhesin gene apd described in claim 1, it is characterised in that: the primer pair are as follows:

P1,5 '-CGGAATTCGGAAACTTATATTGTTACTGGTGAA-3 ',

P2,5 '-CCCTCGAGGGTGATTGTGATTTTATTGTGGT-3’。

3. a kind of recombinant expression carrier pET-Apd, it is characterised in that: the recombinant expression carrier is containing described in claim 1 The expression vector of adhesin gene apd, wherein the expression vector is pET-25b.

4. the host cell containing recombinant expression carrier described in claim 3, it is characterised in that: the host cell is large intestine bar Bacterium BL21 (DE3).

5. utilizing the method for host cell preparation and reorganization adhesin albumin A pd described in claim 4: it is characterized by comprising with Lower step:

(1) by the competent escherichia coli cell BL21 (DE3) containing recombinant expression carrier pET-Apd, overnight incubation cultivates bacterium Liquid centrifugation, precipitating are resuspended with buffer, obtain suspension bacteria liquid；

(2) bacterial cell disruption is carried out to suspension bacteria liquid under cryogenic, supernatant is collected by centrifugation, supernatant is purified, obtain weight Group sticks fibroin rApd.

6. a kind of haemophilus parasuis indirect ELISA antibody assay kit, it is characterised in that: it includes claim 5 preparation The coated ELISA Plate of adhesin, sample diluting liquid, yin and yang attribute control serum, 20 times of concentrated cleaning solutions, ELIAS secondary antibody, developing solutions A, developing solution B, terminate liquid；Wherein,

The sample diluting liquid: pH7.4 is PBS buffer solution 50mL, 25 μ L of bovine serum albumin(BSA) 0.25g, Tween-20；

The positive control serum: the preparation method is as follows: 1 monthly age sodium selenite is immune twice with haemophilus parasuis, after two exempt from Serum is collected in blood sampling in two weeks, detects its OD₆₃₀The serum being worth between 1.00~1.20, takes 4 parts of mixing, makes of sample diluting liquid 1:200 dilution is used as positive control serum；

The negative control sera: the healthy Swine serum raised under equal conditions in positive control serum preparation detects it OD₆₃₀The serum being worth between 0.15~0.25, takes 4 parts of mixing, does 1:200 dilution with sample diluting liquid and is used as negative control blood Clearly；

20 times of concentrated cleaning solutions: NaCl 160.00g, KCl 4.00g, Na₂HPO₄·12H₂O 72.60g, KH₂PO₄ 4.80g is dissolved in 900mL deionized water, and with concentrated hydrochloric acid tune pH to 7.4,10mL Tween-20 is added, is settled to 1L, crosses and filters out Bacterium, room temperature preservation；

The ELIAS secondary antibody: the goat-anti pig secondary antibody of horseradish peroxidase-labeled does 1:15000 dilution with sample diluting liquid；

The developing solution A:Na₂HPO₄·12H₂O 14.60g, citric acid 9.33g, 30% hydrogen peroxide 2ml add 800ml deionization Water dissolution, adjusts pH value to 5.0~5.4, is settled to 1000ml；

The developing solution B: tetramethyl benzidine 20.00mg, dehydrated alcohol 10.00ml, add deionized water dissolving and are settled to 1000ml；

7. haemophilus parasuis indirect ELISA antibody assay kit according to claim 6, it is characterised in that: described glutinous The attached coated ELISA Plate of fibroin the preparation method is as follows: using the rApd albumen of purifying as envelope antigen；With 0.10M, pH9.6 Carbonate buffer solution as coating buffer；RApd is diluted to 0.50 μ g/mL, 4 DEG C of coatings are overnight；With the degreasing containing 0.05g/mL The PBS buffer solution of the pH7.4 of cream is in 37 DEG C of closing 2h.