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

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 protein Apd and application thereof in preparation of indirect ELISA (enzyme-linked immunosorbent assay) antibody detection kit for haemophilus parasuis

Technical Field

The invention relates to the fields of biotechnology and animal infectious diseases, in particular to an adhesin protein Apd and application thereof in preparation of an indirect ELISA antibody detection kit for haemophilus parasuis.

Background

Haemophilus Parasuis (HPS) is a common bacterium of the upper respiratory tract of pigs, belongs to the family Pasteurellaceae, the genus Haemophilus, and is a fine spherical, coccobate-to-filamentous gram-negative bacterium. HPS can cause haemophilus parasuis (also called Gratherse disease) in pigs when the immunity of the pigs is low or the pigs are stressed. Acute infection with HPS can lead to multiple serositis, arthritis and meningitis, septicemia and death; chronic infection can cause suppurative rhinotracheitis and pneumonia. HPS is often co-infected with E.coli, S.suis, and Pasteurella multocida, complicating the disease. The disease occurs and is prevalent in countries and regions of the world where pigs are raised, and can result in 30% mortality in weaned piglets. Because the immunosuppressive diseases of pigs in China are multiple, the occurrence and prevalence of haemophilus parasuis are particularly serious. A specific and sensitive antibody detection method is indispensable in serological diagnosis of haemophilus parasuis, antibody monitoring after vaccine immunization and serum epidemiological investigation.

Antibody detection methods for bacterial pathogens are often difficult to establish, especially for HPS with 15 serotypes and 27% of untypible strains (Oliveira et al, 2003). The plate agglutination test is simple and easy to implement, and can use thallus suspension as agglutination antigen, but the method has low specificity and sensitivity and is limited by the serum type of the agglutination antigen, namely, the specific serotype antigen can only detect the corresponding serotype antibody. The indirect ELISA is carried out by taking thalli as a coating antigen, and because the structure and the composition of bacteria are complex and the types and the quantity of antigens are large, the coating antigen usually has cross reaction with antibodies of similar components of other bacteria in detected serum, thereby causing false positive.

Early methods for detection of HPS antibodies were complement fixation assays, indirect hemagglutination assays and ELISA (Oliveira et al, 2004). The complement fixation test requires sheep red blood cells and guinea pig complement, and these materials are not readily available and prepared in a timely manner, and are cumbersome to perform and are rarely used today. There are two kinds of antigens, namely, an antigen for indirect hemagglutination and an antigen for ELISA, and the supernatant (polysaccharide and protein) after bacterial ultrasonication or LPS extracted by a hot phenol water method. The indirect hemagglutination and ELISA antibody detection methods established with these two antigen components are not only unstable, but also prone to false negatives, as well as detection of immunoprotected porcine sera (Oliveira et al, 2004).

In recent years, there has been established an antibody detection method for HPS indirect ELISA using, as a diagnostic antigen, a protein expressed by escherichia coli, such as HPS oligopeptide permease (OppA) (plantarenaline et al, 2015), cytoswelling lethal toxin (CDT) (liushuanghong, 2016) and outer membrane protein (OMP2, OMP5) (gazelle et al, 2011). In the domestic market, an indirect ELISA antibody detection kit for haemophilus parasuis of BioChek company, Netherlands, takes oligopeptide permease (OppA) as a diagnostic antigen. However, because the oligopeptide permease gene is highly conserved in HPS, E.coli, Salmonella typhi and Pasteurella multocida (98% nucleotide identity), the cytodistending lethal toxin has structurally and functionally similar homologous proteins in E.coli (44% amino acid identity), while the outer membrane proteins OMP2, OMP5 also have homologous proteins in A. pleuropneumoniae (42% and 71% amino acid identity). Such antibody detection methods or kits inevitably cross-react with pathogens having similar antigenic components, resulting in false positives.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an adhesin protein (Aida passagermain, Apd) and application thereof in preparation of a haemophilus parasuis ELISA antibody detection kit. Since the adhesin protein Apd is a protein specific to haemophilus parasuis, it is not present in other relevant bacterial pathogens in pigs. The mouse immune test and immunoblotting confirm that the recombinant Apd protein has good immunogenicity and reactivity. Moreover, the recombinant Apd protein was reactive with antisera from 13 serotype strains, which were reactive with 15 serotype bacteria, indicating that Apd as a diagnostic antigen was not restricted to serotype differences. The haemophilus parasuis ELISA antibody detection kit using the recombinant Apd protein as a coating antigen can detect the antibody from the pig serum immunized by the haemophilus parasuis inactivated vaccine and infected by live bacteria, and the positive rate of the antibody is related to the toxic attack protection rate of pigs. The kit has wide application prospect in serological diagnosis of haemophilus parasuis, evaluation of vaccine immunity effect and seroepidemiological investigation.

To achieve the above object, the present invention provides an adhesin protein Apd, which has an amino acid sequence characterized by:

(1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2; or

(2) An amino acid sequence which has 90-100% homology with the amino acid sequence defined by the sequence SEQ ID No.2 and encodes the protein with the same function; or

(3) Protein which is derived from the protein (1) and has the same activity and is obtained by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No. 2;

the nucleotide sequence of the gene apd for coding the adhesin is shown as SEQ ID No. 1.

The invention also provides a primer pair for obtaining the adhesin gene apd, which comprises the following components in parts by weight:

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

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

the present invention also provides a recombinant plasmid pET-Apd, wherein the recombinant expression vector is an expression vector containing the adhesin gene apd of claim 3, and the expression vector is pET-25 b.

The invention also provides a host cell of the recombinant expression vector, and the host cell is escherichia coli BL21(DE 3).

The invention also provides a preparation method of the recombinant adhesin protein Apd, which comprises the following steps: comprises the following steps:

(1) culturing escherichia coli competent cells BL21(DE3) containing recombinant plasmid pET-Apd overnight, centrifuging culture bacterial liquid, and resuspending a precipitate by using a buffer solution to obtain suspension bacterial liquid;

(2) and (3) carrying out thallus crushing on the suspension liquid at a low temperature, centrifugally collecting a supernatant, and purifying the supernatant to obtain the recombinant adhesin protein rApd.

The invention also provides an indirect ELISA antibody detection kit for haemophilus parasuis, which comprises an adhesin-coated ELISA plate, a sample diluent, negative and positive control serum, 20-time concentrated washing liquid, an enzyme-labeled secondary antibody, a developing liquid A, a developing liquid B and a stop solution;

the preparation of the adhesin protein coated ELISA plate comprises the following steps: the preparation method comprises the following steps: purified rApd protein was used as the coating antigen, rApd was diluted to 0.5. mu.g/mL with carbonate buffer, coated overnight at 4 ℃ and blocked with 0.05g/mL skim milk in PBS buffer (pH7.4) for 2h at 37 ℃.

The sample diluent is: 50mL of PBS buffer solution (pH7.4), 0.25g of bovine serum albumin and 0.5 μ L of Tween-2025;

the positive control serum: the preparation method comprises the following steps: immunizing 1-month-old healthy piglet with Haemophilus parasuis twice, collecting blood serum two weeks after two-time immunization, and detecting OD₆₃₀Taking 4 parts of serum with the value of 1.00-1.20, mixing, and diluting with a sample diluent at a ratio of 1:200 to serve as positive control serum;

the negative control serum: detecting OD of healthy pig serum raised under the same condition during positive control serum preparation₆₃₀Mixing 4 parts of serum with a value of 0.15-0.25, and diluting with sample diluent at a ratio of 1:200 to obtain negative control serum;

the 20-fold concentrationWashing liquid: 160.00g of NaCl, 4.00g of KCl and Na₂HPO₄·12H₂O 72.60g，KH₂PO₄4.80g of the extract is dissolved in 900mL of deionized water, the pH value is adjusted to 7.4 by concentrated hydrochloric acid, 10mL of Tween-20 is added, the volume is fixed to 1L, and the extract is filtered, sterilized and stored at room temperature;

the enzyme-labeled secondary antibody: horse radish peroxidase-labeled goat anti-pig secondary antibody purchased from Wuhan Ante & Tejie biotechnology Limited (imported subpackaging) and diluted by sample diluent at a ratio of 1: 15000;

the color developing liquid A: na (Na)₂HPO₄·12H₂Adding 800ml of deionized water into 14.60g of O, 9.33g of citric acid and 2ml of 30% hydrogen peroxide for dissolving, adjusting the pH value to 5.0-5.4, and setting the volume to 1000 ml;

the color developing liquid B: 0.02g of Tetramethylbenzidine (TMB) and 10.00ml of absolute ethyl alcohol, adding deionized water for dissolving and fixing the volume to 1000 ml; the stop solution is as follows: to 10mL of deionized water was added 25. mu.L of hydrofluoric acid.

The method for detecting the haemophilus parasuis antibody by using the kit comprises the following steps:

(1) diluting the serum to be detected by sample diluent at a ratio of 1:200, adding 100 mu L of each sample into an enzyme label plate, and simultaneously adding 100 mu L of negative and positive control serum into the enzyme label plate; performing incubator at 37 deg.C for 45min, diluting 20 times of concentrated washing solution with deionized water at a ratio of 1:19, adding 200 μ L per well, standing for 5min, discarding, drying, and washing for 3 times;

(2) adding 100 μ L of enzyme-labeled secondary antibody into each well, reacting at 37 deg.C for 30min, discarding, adding 200 μ L of washing solution into each well, standing for 5min, discarding, drying, and washing for 3 times;

(3) adding 50 mu L of substrate solution A into each hole, adding 50 mu L of substrate solution B, uniformly mixing, keeping out of the sun at room temperature for 15min, developing, adding 50 mu L of stop solution, and measuring the OD value with the wavelength of 630nm on an enzyme-linked immunosorbent assay instrument within 10 min;

(4) and (4) judging a result: when the S/N of the serum to be detected is more than or equal to 2.60, the serum is judged to be positive; S/N<2.6, the result is judged to be negative. S generationSerum OD to be examined₆₃₀Value, N represents the negative control serum OD₆₃₀Value, the condition for the test to be established is the negative and positive control serum OD₆₃₀The difference value is more than or equal to 0.5, wherein the serum of the pig to be detected: porcine venous blood serum was isolated.

The invention has the beneficial effects that:

the adhesion protein disclosed by the invention is specific to haemophilus parasuis, and the antibody detection kit disclosed by the invention can be used for detecting antibodies after haemophilus parasuis vaccine immunization or pathogen infection, does not have cross reaction with other related bacterial pathogens, and is not limited by different serotypes of haemophilus parasuis.

Drawings

FIG. 1 is a diagram showing the detection of E.coli expression of three recombinant proteins;

FIG. 2 is a diagram of the purification of three recombinant proteins;

FIG. 3 is a result chart of three recombinant proteins for detecting Haemophilus parasuis negative and positive serum;

FIG. 4 is a graph showing the immunogenicity of recombinant adhesin proteins;

FIG. 5 is a map of the localization of adhesin protein in Haemophilus parasuis;

FIG. 6 is a graph showing the serum titer of mice immunized with recombinant adhesin protein;

FIG. 7 is a graph showing the results of detection of 15 serological Haemophilus parasuis by Western blot using recombinant adhesin protein antiserum;

FIG. 8 is a graph showing the results of detection of recombinant adhesin protein by Western blot using 15 serotype Haemophilus parasuis antiserums;

FIG. 9 is a result chart of detection of negative and positive sera after optimization of ELISA reaction conditions;

FIG. 10 is a graph showing the results of negative and positive serum detection of the supernatant after the disruption of Haemophilus parasuis;

FIG. 11 is a graph showing the results of detection of positive and negative serum by Haemophilus parasuis;

FIG. 12 is a graph showing the results of the kit for detecting the sera of pigs after vaccine immunization and bacterial infection;

FIG. 13 is a result diagram of the antibody growth and death law of the kit after detecting vaccine immunity and bacterial infection.

Detailed Description

The invention is described in further detail below with reference to the figures and specific embodiments for the understanding of those skilled in the art.

EXAMPLE 1 Synthesis or cloning of the adhesin Gene apd

(1) Synthesis of adhesin Gene apd

The gene apd is synthesized according to the nucleotide sequence shown as SEQ ID No. 1.

(2) Cloning of the adhesin Gene apd

Primer pairs were designed with the genome of haemophilus parasuis isolate CF7066 (held by the dawn teacher, university of china agriculture):

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

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

taking a haemophilus parasuis isolate CF7066 genome as a template to carry out PCR amplification, wherein a PCR system comprises the following steps: 31.5. mu.L of deionized water, 5. mu.L of 10 XProbest Buffer II, 4. mu.L of dNTP mix (2.5mM), 2. mu. L P1 (10. mu.M/L), 2. mu. L P2 (10. mu.M/L), 0.5. mu.L of Pyrobest DNA Polymerase (Takara), 5. mu.L of Haemophilus parasuis genomic DNA (150 ng/. mu.L); PCR reaction procedure: pre-denaturation at 94 ℃ for 5min, cycling, denaturation at 94 ℃ for 30s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 90s, 30 cycles, extension at 72 ℃ for 10min, and storing at 4 ℃ to obtain a PCR product; sequencing the PCR product, wherein the nucleotide sequence of the PCR product is shown as SEQ ID No.1, and the PCR product is named as 'gene apd'.

EXAMPLE 2 preparation of adhesin proteins

(1) Obtaining of adhesin Gene

The invention obtains DNA fragment of 1577 nucleotides by DNA synthesis; or a 1577bp DNA fragment was amplified from the Haemophilus parasuis isolate CF7066 by the PCR primers, system and reaction procedure described in example 1. The DNA fragment is named as apd, and the nucleotide sequence of the DNA fragment is shown as SEQ ID No. 1.

(2) Construction of adhesin expression plasmid

Dissolving the synthesized DNA fragment with deionized water, or recovering PCR amplified fragment, double enzyme cutting the DNA fragment and a vector pET-25b (Novagen) by EcoR I and Xho I, recovering the enzyme cutting product and connecting the vector, transforming the connecting product into Escherichia coli DH5 α, culturing overnight at 37 ℃ until a single colony grows out, picking up a single clone to extract plasmid, and storing the pET-Apd plasmid with correct sequencing at-20 ℃ for later use.

(3) Expression of adhesin proteins

Escherichia coli competent cells BL21(DE3) were transformed with the plasmid pET-Apd and cultured overnight at 37 ℃. Single colonies were picked in 5mL of LB liquid medium and cultured overnight with shaking at 37 ℃. Transferring into 1000mL LB liquid culture medium at a ratio of 1:100, and shake culturing at 37 deg.C to OD₆₀₀About 0.6, IPTG was then added to a final concentration of 0.8mM and shake-cultured at 37 ℃ for 4 h. The bacterial suspension was centrifuged at 3200g at 4 ℃ for 1min, and the bacterial suspension was resuspended in 1/10 volumes of binding buffer. And (3) crushing the thalli at 4 ℃ by using a low-temperature ultrahigh-pressure cell crusher, repeatedly crushing for 3-5 times, then freezing and centrifuging for 10min at 3200g, and collecting the supernatant for later use.

The IPTG (0.8M): 2g of IPTG was dissolved in 8mL of distilled water to a volume of 10mL, and the solution was sterilized by filtration through a 0.22 μm filter and stored at-20 ℃.

(4) Purification of adhesin proteins

The supernatant of the disrupted cells was harvested, and the supernatant of the disrupted cells was purified with a Ni Sepharose 6 Fast Flow packing (the packing was stored in 20% absolute ethanol) of GE Healthcare to obtain a recombinant adhesin protein rApd whose amino acid sequence is shown in SEQ ID No.2,

the purification process is as follows:

column assembling: covering a cover at the lower end of the purification column, slowly adding 2mL of filler along the wall of the chromatography column to avoid generating bubbles and keep the filler uniformly distributed as much as possible, and standing at 4 ℃ right side for overnight;

balancing: the cap at the lower end of the purification column is opened to allow the liquid in the column to flow out naturally. Adding 10-20 mL of distilled water to wash off ethanol during storage of the filler, adding 20mL of 500mM imidazole buffer solution to wash the nickel ion packed column after the liquid flows out (the operation can be omitted for the new filler), and adding 10-20 mL of combined buffer solution to balance the packed nickel ion packed column after the liquid flows out;

loading: and after the liquid completely flows out, slowly adding 80-100 mL of the crushed thallus supernatant placed on ice into a purification column in a fractional manner.

Washing: and after the protein sample is filled into the column, adding 10-20 mL of binding buffer solution for balancing, and then washing with 20mL of washing buffer solution to remove the foreign protein.

And (3) elution: the target protein sample was washed by adding 10mL of elution buffer slowly in portions, collected in 1.5mL of EP tubes, measured for protein concentration with BCA kit (Biosharp Co.), and stored at-80 ℃. The amino acid sequence of the purified protein is shown as SEQID No. 2.

The binding buffer: NaCl 29.22g, Tris-base 2.42g and imidazole 0.34g, dissolved in 800mL deionized water, adjusted to pH7.4 with concentrated hydrochloric acid, fixed dissolved to 1L, filtered through a 0.22 μm filter, and stored at 4 ℃ for later use.

The washing buffer: NaCl 29.22g, Tris-base 2.42g and imidazole 1.36g, dissolved in 800mL deionized water, adjusted to pH7.4 with concentrated hydrochloric acid, fixed dissolved to 1L, filtered through a 0.22 μm filter, and stored at 4 ℃ for later use.

The elution buffer: NaCl 29.22g, Tris-base 2.42g and imidazole 6.8g are dissolved in 800mL deionized water, the pH value is adjusted to 7.4 by concentrated hydrochloric acid, the solution is fixed to 1L, and the solution is filtered by a 0.22 mu m filter and stored at 4 ℃ for later use.

The 500mM imidazole buffer: weighing 29.22g of NaCl, 2.42g of Tris-base and 34.04g of imidazole, dissolving in 800mL of deionized water, adjusting the pH to 7.4 by using concentrated hydrochloric acid, dissolving to 1L, filtering by using a 0.22 mu m filter, and storing at 4 ℃ for later use

The content of the 20% ethanol: 200mL of absolute ethanol was dissolved in 800mL of deionized water, filtered through a 0.22 μm filter, and stored at 4 ℃ for further use.

Example 3 evaluation of recombinant adhesin protein as coating antigen of Haemophilus parasuis Indirect ELISA antibody detection kit

(1) Expression of three recombinant proteins of haemophilus parasuis

The extracellular regions of 3 outer membrane proteins of Haemophilus parasuis are selected and prokaryotic expression plasmids pET-Ag2, pET-Ag3 and pET-Apd are constructed. Coli BL21(DE3) was transformed with 3 plasmids and induced for different times at different temperatures and different concentrations of IPTG to determine the optimal conditions for soluble expression of the target protein. The result shows that the optimal expression condition of rAg2 (recombiant Ag2) protein is 16 ℃, and 0.2mM IPTG induces for 12 h; rAg3 (recombiant Ag3) protein was induced at 16 ℃ for 12h with 0.2mM IPTG; the optimal expression conditions for the rApd (recombinant Apd) protein were 37 ℃ and 0.8mM IPTG induction for 4h (FIG. 1).

(2) Purification of three recombinant proteins of haemophilus parasuis

And (3) crushing the induced escherichia coli by using a low-temperature ultrahigh-pressure cell crusher, centrifuging to take supernatant, purifying target protein by using nickel column affinity chromatography, and eluting the protein with the concentration of 5 mM-300 mM of imidazole respectively. Finally, rAg2 washing buffer containing 50mM imidazole and elution buffer containing 150mM imidazole; the Ag3 wash buffer contained 20mM imidazole and the elution buffer contained 150mM imidazole; the rApd wash buffer contained 20mM imidazole and the elution buffer contained 100mM imidazole. After obtaining the purified protein, the protein concentration of rAg2 was 706. mu.g/mL, the protein concentration of rAg3 was 325. mu.g/mL, and the protein concentration of rApd was 450. mu.g/mL, as determined by BCA protein concentration assay kit, and the purified protein was subjected to SDS-PAGE (FIG. 2).

(3) Three recombinant proteins for detecting Haemophilus parasuis negative positive serum

The three recombinant proteins expressed and purified are diluted to 1 microgram/mL coated enzyme label plate, 4 parts of negative and positive pig serum of haemophilus parasuis are diluted by 1:100 respectively, and indirect ELISA is carried out according to a conventional procedure. The results show that negative serum OD is present when the rApd protein is coated₆₃₀The mean value was 0.297, positive serum OD₆₃₀The average value was 2.201 (P)<0.001), the difference between the two is extremely obvious; while OD of negative and positive sera after rAg2 and rAg3 protein coating₆₃₀The values were all scattered within the range of 1.0 to 2.0, with no clustering (FIG. 3). Therefore, the rApd protein is selected as the coating antigen of the haemophilus parasuis antibody detection kit.

(4) Reactogenicity of recombinant adhesin proteins

Escherichia coli competent cells BL21(DE3) were transformed with pET-Apd, and Western blot was performed using murine His monoclonal antibody and Haemophilus parasuis negative positive pig serum, and the recombinant adhesin protein obtained after induction and purification. Since rApd fused a His tag when expressed in E.coli, a band was observed at 85kDa in the case of detection using His-monoclonal antibody, and a band was observed at 85kDa in positive serum of Haemophilus parasuis, whereas no band was observed in the extract after BL21(DE3) transformation with empty vector (FIG. 4). The result shows that the rApd expressed and purified can react with the porcine antibody of haemophilus parasuis, and the good reactogenicity of the rApd is proved.

(5) Localization of adhesin proteins in haemophilus parasuis

1000mL of haemophilus parasuis logarithmic phase late culture bacterial liquid, centrifuging for 15min at 4 ℃ of 3200g, and discarding the supernatant. The bacterial pellet was resuspended in 60mL of 50mM Tis-HCl (pH7.2), 300. mu.l of PMSF (10mg/mL) as a protease inhibitor was added, and the pellet was sonicated for 25 seconds each at 1min intervals. Until the bacterial fluid becomes clear. Centrifuging at 3200g for 15min, collecting supernatant and 0.1MNa pre-cooled on ice₂CO₃(pH11) was mixed at a ratio of 1:100 and stirred at 4 ℃ for 1 hour. The solution was centrifuged at 15000g at 4 ℃ for 1h and the precipitate was dissolved in 1mL TE to give the outer membrane protein. 1000mL of Haemophilus parasuis logarithmic phase late phase culture solution, centrifuging at 4 ℃ of 3200g for 30min, collecting supernatant, and filtering with a 0.22 μm filter. To the filtrate was added an equal volume of saturated (NH)₄)₂SO₄Stirring and salting out at 4 ℃ overnight, centrifuging 3200g for 15min, and dissolving the precipitate with TE buffer solution to obtain the secreted protein. Western blot detection was performed with a monoclonal antibody to Apd protein. The results showed that rApd showed a detection band at about 85kD, and that the whole bacterial protein and the outer membrane protein of Haemophilus parasuis showed detection bands of the same size, while the secreted protein did not, indicating that Apd protein is located on the outer membrane of Haemophilus parasuis (FIG. 5).

(6) Antiserum Western blot of recombinant adhesin protein for detecting 15 serotype haemophilus parasuis

Antiserum was prepared by immunizing 4 BALB/c mice with the purified rApd protein. Serum was isolated after three immunizations and titer was measured on an rApd-coated ELISA plate, and serum titer reached 1:204800 for all mice (FIG. 6). Cultures of the 15 serotype standard and isolate CF7066 were subjected to SDS-PAGE followed by membrane transfer and immunoblotting with mouse serum antiserum to the rApd protein, resulting in the appearance of an approximately 85kDa band for all strains, whereas no band was detected for the negative control serum (fig. 7). As can be seen, in the 15 serotype standard strains of HPS, there are antigenic components that can react with the rApd antiserum, indicating that rApd as a diagnostic antigen can be used to detect antibodies of 15 serotypes of Haemophilus parasuis.

(7) Western blot detection of recombinant adhesin protein by 15 serotype haemophilus parasuis antiserum

Preparing 15 serotype standard strains and isolates of haemophilus parasuis CF7066 and BALB/c mouse antiserum of porcine pathogenic escherichia coli, infectious actinobacillus pleuropneumoniae, pasteurella multocida, streptococcus suis and bordetella bronchiseptica. The preparation method comprises the following steps: the cells were spread evenly on TSA plates and cultured overnight, washed with PBS (pH7.4) and the cell content was adjusted to 10¹⁰CFU/mL, then emulsified with Freund's adjuvant in the same volume; each mouse was immunized with 300. mu.L (1.5X 10)⁹CFU/mL), the immunization mode is subcutaneous multipoint injection of the neck and the back; freund's complete adjuvant was used for the first immunization, followed by Freund's incomplete adjuvant. Serum was separated 5 days after the three-immunization and detected by Western blot detection rApd. Except for the antisera of type 6 and type 14, the antisera of the 13 serotype standard and isolate CF7066 reacted with rApd, whereas the antisera of porcine escherichia coli, infectious actinobacillus pleuropneumoniae, streptococcus suis, bordetella bronchiseptica did not react with rApd (fig. 8). The results confirm that the adhesin protein of the haemophilus parasuis has strong immunogenicity, can stimulate animal bodies to produce detectable antibodies, and is relatively conserved in different serotypes of haemophilus parasuis. Therefore, the antibody detection method established by using the antibody as a diagnostic antigen is not limited by serotype, and can not generate cross reaction with other related bacterial pathogens.

Example 4 selection of Components and amounts of kit for detecting Haemophilus parasuis Indirect ELISA antibody and determination of reaction conditions

(1) Determination of antigen coating concentration and enzyme-labeled secondary antibody working concentration

rApd was diluted with the coating solution to 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125 mu g/mL, and diluting the enzyme-labeled secondary antibody with a sample diluent to 1:5000, 1:10000, 1:15000 and 1:20000 times. The positive and negative sera of Haemophilus parasuis were diluted 1:100 with the sample diluent. The results showed that positive and negative serum OD were obtained when the antigen coating concentration was 0.5. mu.g/mL and the secondary antibody dilution was 1:15000₆₃₀The ratio (P/N) of (A) was the largest, and thus 0.5. mu.g/mL and 1:15000 were used as the antigen optimum coating concentration and the secondary antibody optimum dilution (Table 1).

TABLE 1 determination of optimal antigen coating concentration and enzyme-labeled secondary antibody working concentration

(2) Determination of antigen coating conditions

Coating the enzyme label plate with an antigen concentration of 0.5 mu g/mL, wherein the coating conditions are as follows: coating at 4 ℃ overnight, coating at 37 ℃ for 1h, then coating at 4 ℃ overnight, coating at 37 ℃ for 2h, then coating at 4 ℃ overnight, and coating at 37 ℃ for 2 h. The results showed that the overnight P/N value at 4 ℃ was 12.097, which is higher than that of the other coating conditions, so that the optimal antigen coating condition was 4 ℃ overnight (Table 2).

TABLE 2 determination of optimal conditions for antigen coating

(3) Determination of serum dilution factor and reaction time

Diluting the positive and negative serum by 1:50, 1:100, 1:200 and 1:400 times, adding 100 μ L into the coated enzyme label plate, and performing indirect ELISA in 37 deg.C incubator for 30min, 45min, 60min and 75min respectively. As a result, the P/N value was the largest when the serum was diluted 1:200 times and the reaction time was 45min, and therefore the optimal dilution ratio of the serum to be tested was 1:200 and the optimal reaction time was 45min (Table 3).

TABLE 3 determination of optimal dilution times and reaction times for sera

(4) Selection of confining liquids

After coating, the protein is blocked by different blocking liquids. The confining liquid is respectively as follows: the PBS buffer contained 0.1g/L skim milk, 0.5g/L skim milk, 0.05g/L BSA, 0.2g/L BSA. As a result, it was found that the P/N value was the largest when the blocking solution contained 5g/L of skim milk, and therefore the most preferable blocking solution was PBS buffer (pH7.4) containing 5g/L of skim milk (Table 4).

TABLE 4 determination of optimal blocking solution

(5) Determination of the closing time

3 different closure times were set: sealing at 37 ℃ for 1h, sealing at 37 ℃ for 2h, and sealing at 37 ℃ for 3 h. Detecting OD per closure time₆₃₀As a result, it was found that the P/N value was the largest when the blocking time was 2 hours, and the optimum blocking time was 2 hours (Table 5).

TABLE 5 determination of optimal seal time

(6) Determination of enzyme-labeled Secondary antibody action time

By adopting the optimized ELISA reaction conditions, 100 mu L of goat anti-pig secondary antibody is added into each hole, and the mixture is respectively acted for 30min, 45min, 60min and 75min in a 37 ℃ incubator. The results showed that the secondary antibody had the greatest P/N value at 30min, so the optimal action time was 30min (Table 6).

TABLE 6 determination of optimal action time of enzyme-labeled secondary antibody

(7) Determination of the development time of a substrate

And (3) adopting the optimized ELISA reaction conditions, adding the substrate solution A and the substrate solution B, and then performing color development at room temperature in a dark place for 5min, 10min, 15min and 20min respectively. The results showed that the P/N value was the greatest when the color was developed for 15min, so the optimal color development time for the substrate was 15min (Table 7).

TABLE 7 determination of optimal coloration time of the substrate

(8) Detection of negative and positive pig serum after optimization of ELISA reaction conditions

And (3) detecting 15 parts of each of the Haemophilus parasuis negative and positive pig serum by using the optimized rApd coating concentration, the sealing solution and the sealing time, the dilution multiple of the serum to be detected, the dilution concentration and the action time of the enzyme-labeled secondary antibody and the substrate development time. The results show that the conditions are optimized

The background of the ELISA test results was significantly reduced. Of positive seraFrom 2.514 to

1.438, negative serumA decrease from 1.400 to 0.452 and a decrease in standard deviation of 0.14 (FIG. 9) indicate ELThe detection effect of the kit is effectively improved by optimizing the use amount of ISA components and reaction conditions

Example 5 determination of Positive threshold value of Indirect ELISA antibody detection kit for Haemophilus parasuis

(1) Formula for determining critical value

The indirect ELISA positive determination standard generally adopts the average OD value of a large number of negative samples plus one or more times of the standard deviation of the OD values of the negative samples, namely Therefore, after the test conditions of the indirect ELISA operation are fixed, the positive critical value is determined in three steps, namely n value in ① critical value calculation formula, more reliable value when the standard deviation of ② negative samples is larger and the serum number is larger, and negative control value in ③ actual detection (Wu Bao Cheng, 1994).

(2) Determination of the value of n in the threshold value

The method for measuring the n-value is as follows, with reference to the method used by Wu Baocheng et al (1994). Taking positive and negative serums of haemophilus parasuis, diluting to 1:204800 from 1:100 in a multiple ratio, sequentially adding an rApd coated enzyme label plate, and adding 8 holes at each dilution to determine OD of each hole₆₃₀The OD of the positive and negative serum was calculated for each dilution₆₃₀And the corresponding t value (table 8). The statistical analysis shows that when the dilution is 1:12800, t is 2.571 > 2.145{ t ═ t_(0.05,14)Of samples of negative and positive seraThe difference is obvious; as the dilution continues to increase in size,yang is less thanNegative, indicating that this method has been unable to distinguish between the yin-yang sera. It can be seen that at a dilution of 1:12800, the t value 2.571 is the value of n in the threshold. The result also shows that when the serum to be detected is diluted at a rate of 1:12800, the method can detect the yin-yang serum, and shows that the sensitivity is very high.

TABLE 8 determination of multiple of standard deviation in threshold values

(3) Method for screening haemophilus parasuis negative serum

And (3) crushing and centrifuging the cultured haemophilus parasuis and the thallus, measuring the bacterial load and the protein concentration, diluting the obtained product in a multiple ratio, coating an enzyme label plate, and detecting one portion of each of the haemophilus parasuis negative and positive pig serum. The results showed that when the supernatant after disruption was used as the coating antigen, negative and positive serum OD₆₃₀The values are all higher, and the P/N value is between 1 and 2 (figure 9); while when the whole bacteria is used as the coating antigen, the negative and positive serum OD₆₃₀The difference in values increases significantly, with P/N values greater than 2. Especially when the coating concentration of the bacteria is 0.5X 10⁸The P/N value is 2.47 and the positive serum OD is at CFU/mL₆₃₀Value 1.153, negative serum OD₆₃₀The value was 0.467 (fig. 10).

(4) Determination of negative serum standard deviation in cutoff values

By 0.5X 10⁸CFU/mL haemophilus parasuis thallus suspension coating ELISA plate, screening haemophilus parasuis negative serum, OD from 350 parts of clinical suckling piglet, nursery pig, fattening pig and sow serum by indirect ELISA₆₃₀A value less than 0.467 is judged as negative serum. The result was 40 negative, 4 for suckling piglets, 23 for nursery pigs, and 13 for fattening pigs.The ELISA antibody detection method of the present invention was used to detect 40 sera

Detection, OD₆₃₀Average value of (2)It was 0.189 and the Standard Deviation (SD) was 0.118.

TABLE 9 results of detecting 40 portions of Haemophilus parasuis negative serum

(5) Determination of Positive Critical value

According to the n value of 2.571, the OD of 40 negative serum samples₆₃₀ Is 0.189, the Standard Deviation (SD) is 0.118, andavailable threshold value of 0.492, i.e. when OD₆₃₀When the content is more than or equal to 0.492, the serum sample is positive; when OD is reached₆₃₀<At 0.492, the serum sample was negative.

Considering the batch difference in the preparation process of the ELISA kit and the personal operation error in the use process, under the premise of strictly preparing and controlling the negative and positive control serum, the positive critical value is judged to be positive according to the S/N value (0.492/0.189-2.60), namely when the S/N value of the serum to be detected is more than or equal to 2.60; S/N<2.6, the result is judged to be negative. S represents the OD of the serum to be tested₆₃₀Value, N represents the negative control serum OD₆₃₀Value, test was established as negative and positive control serum OD₆₃₀The difference is greater than or equal to 0.5.

Example 6 composition and preparation of Indirect ELISA antibody detection kit for Haemophilus parasuis

(1) Preparation of recombinant adhesin protein Apd coated ELISA plate

Diluting the purified rApd protein with a coating solution to a concentration of 0.5 mu g/mL, adding 100 mu L of the protein into an ELISA plate per hole, and keeping the temperature in a refrigerator at 4 ℃ overnight; discarding the coating solution, adding 200 μ L of washing solution into each hole, standing for 5min, discarding the supernatant, and washing for 3 times;

then adding 200 mu L of sealing liquid into each hole, and acting for 2 hours at 37 ℃ in an incubator; removing the confining liquid, adding 200 μ L of washing liquid into each well, standing for 5min, removing the supernatant, drying, and washing for 3 times; freeze drying, and vacuum preserving at low temperature.

The coating liquid comprises: carbonate buffer (pH9.6), Na₂CO₃ 1.59g、NaHCO₃2.93g, adding a proper amount of deionized water for dissolution, adjusting the pH to 9.6 by hydrochloric acid, fixing the volume to 1L, sterilizing at high pressure and storing at 4 ℃ for later use;

the washing solution comprises: 50. mu.L of Tween-20 was added to 100mL of PBS buffer (pH7.4);

the sealing liquid is prepared by the following steps: PBS buffer (pH7.4) containing 5g/L skim milk.

(2) Kit composition for detecting adhesin protein indirect ELISA antibody

The kit comprises 2 blocks of an adhesin-coated 96-hole ELISA detection plate, 1 bottle (50 mL/bottle) of sample diluent, 1 tube (1.5 mL/tube) of negative and positive control serum, 1 bottle (30 mL/bottle) of 20-time concentrated washing solution, 1 bottle (20 mL/bottle) of enzyme-labeled secondary antibody, 1 bottle (10 mL/bottle) of developing solution A, developing solution B and stop solution.

The sample diluent is: 50mL of PBS buffer solution (pH7.4), 0.25g of bovine serum albumin and 0.5 μ L of Tween-2025;

the positive control serum: the preparation method comprises the following steps: immunizing 1-month-old healthy piglet with Haemophilus parasuis twice, collecting blood serum two weeks after two-time immunization, and detecting OD₆₃₀Mixing 4 parts of serum with value of 1.00-1.20, diluting with sample diluent at a ratio of 1:200Used as a positive control serum;

the negative control serum: detecting OD of healthy pig serum raised under the same condition during positive control serum preparation₆₃₀Mixing 4 parts of serum with a value of 0.15-0.25, and diluting with sample diluent at a ratio of 1:200 to obtain negative control serum;

the 20-fold concentrated washing solution: 160.00g of NaCl, 4.00g of KCl and Na₂HPO₄·12H₂O 72.60g，KH₂PO₄4.80g of the extract is dissolved in 900mL of deionized water, the pH value is adjusted to 7.4 by concentrated hydrochloric acid, 10mL of Tween-20 is added, the volume is fixed to 1L, and the extract is filtered, sterilized and stored at room temperature;

the enzyme-labeled secondary antibody: horse radish peroxidase-labeled goat anti-pig secondary antibody purchased from Wuhan Ante & Tejie biotechnology Limited (imported subpackaging) and diluted by sample diluent at a ratio of 1: 15000;

the color developing liquid A: na (Na)₂HPO₄·12H₂Adding 800ml of deionized water into 14.60g of O, 9.33g of citric acid and 2ml of 30% hydrogen peroxide for dissolving, adjusting the pH value to 5.0-5.4, and fixing the volume to 1000 ml;

the color developing liquid B: 20.00mg of Tetramethylbenzidine (TMB) and 10ml of absolute ethyl alcohol, adding deionized water for dissolving and fixing the volume to 1000 ml;

the stop solution is as follows: to 10mL of deionized water was added 25. mu.L of hydrofluoric acid.

(3) Method of using kit

Diluting a serum sample to be detected by a sample diluent at a ratio of 1:200, taking 100 mu L of each sample, adding the sample into an enzyme label plate, and simultaneously taking 100 mu L of negative and positive control serum and adding the negative and positive control serum into the enzyme label plate; performing incubator at 37 deg.C for 45min, diluting 20 times of concentrated washing solution with deionized water at a ratio of 1:19, adding 200 μ L per well, standing for 5min, discarding, drying, and washing for 3 times;

adding 100 μ L of enzyme-labeled secondary antibody into each well, reacting at 37 deg.C for 30min, discarding, adding 200 μ L of washing solution into each well, standing for 5min, discarding, patting dry, and washing for 3 times;

adding 50 mu L of substrate solution A into each hole, adding 50 mu L of substrate solution B, uniformly mixing, keeping out of the sun at room temperature for 15min, developing, adding 50 mu L of stop solution, and measuring the OD value with the wavelength of 630nm on an enzyme-linked immunosorbent assay instrument within 10 min;

and (4) judging a result: when the S/N of the serum to be detected is more than or equal to 2.60, the serum is judged to be positive; S/N<2.6, the result is judged to be negative. S represents the OD of the serum to be tested₆₃₀Value, N represents the negative control serum OD₆₃₀Value, the condition for the test to be established is the negative and positive control serum OD₆₃₀The difference is greater than or equal to 0.5.

The pig serum to be detected is as follows: porcine venous blood serum was isolated.

Example 7 reproducibility of the Haemophilus parasuis Indirect ELISA antibody detection kit

And respectively carrying out batch repeatability experiments on 4 parts of positive serum and 4 parts of negative serum of the haemophilus parasuis by using the enzyme label plates coated in the same batch. Repeating the detection for 4 times, wherein the variation coefficient is between 1.9 and 9.5 percent; the ELISA plates coated in different batches are used for carrying out batch-to-batch repeatability experiments on the same 8 parts of serum, and the variation coefficient of the ELISA plates is between 1.4 and 9.4 percent (shown in a table 10), which shows that the ELISA detection kit has good repeatability.

TABLE 10 Intra-and inter-batch repeat tests

Example 8 application of Haemophilus parasuis Indirect ELISA antibody detection kit

(1) Detection of vaccine immunity and attenuated pig serum

The ELISA kit is used for detecting pig serum immunized by a haemophilus parasuis vaccine and infected by a low virulent strain, wherein 15 parts of the vaccine immune serum, 4 parts of low virulent strain infected serum and 20 parts of PBS control serum are used as positive judgment standards, and S/N is more than or equal to 2.60. The results showed that 93% (14/15) of the vaccine immunization groups were positive, 100% (4/4) of the attenuated strain a infection group was positive, and 75% (15/20) of the PBS control group was negative (fig. 12). Notably, the PBS control group tested positive for antibodies with 5 sera, which may be due to endogenous or cross-infection of pigs during the test, since haemophilus parasuis is a common bacterium and the animal test lasted for 6 weeks. However, the results of the challenge test showed that there were 8 cases of the PBS control group showing no clinical symptoms, suggesting that PBS pigs may have a substantial infection and antibody production. In sum, the results show that the kit can detect the serum antibody of the haemophilus parasuis vaccine after immunization and live bacteria infection, and can be applied to the evaluation of the haemophilus parasuis vaccine immune protection effect and the antibody detection after pathogen infection.

(2) Evaluation of antibody growth and elimination rules after vaccine immunization and attenuated infection

The ELISA antibody detection kit is used for carrying out continuous antibody detection on experimental infected pigs. 16 piglets of one month old are divided into 4 groups, 4 piglets in each group are immunized/infected twice at intervals of two weeks, inactivated vaccine A immunization and gene deletion strain B and C infection are respectively carried out, and S/N is more than or equal to 2.60 and is taken as a positive judgment standard. The results show that the appearance of the ELISA antibodies of the low virulent strains B and C turns positive one week after infection, and the ELISA antibodies continuously rise two weeks later; the vaccine immunization group turned positive for a week of secondary immunization with a significant increase in antibody. The antibody levels of the vaccine immunization and the attenuated strain-infected group were the same two weeks after immunization, while the antibody detection of the PBS control group continued to be negative (fig. 13). Therefore, the kit can also be used for evaluating the antibody generation rule after vaccine immunization and pathogen infection.

(3) Detection of clinical pig serum

The ELISA antibody kit is used for detecting 222 parts of serum collected by 3 pig farms, including suckling piglet, nursery pig, fattening pig and sow serum. The results show that the method has the advantages of high yield,the positive detection rate is 12% of suckling piglets, 16% of nursery pigs, 36% of fattening pigs and 68% of sows, and the total positive detection rate is 35%. By 0.5X 10⁸The CFU/mL haemophilus parasuis coated ELISA plate was tested, and the positive rates detected were 84% for suckling piglets, 74% for nursery pigs, 86% for fattening pigs, 100% for sows and 86% for total positive rate (Table 11). Both tests showed that the antibody positive rate increased with the increase of the day-old pig. However, when the bacterial cells were used as the coating antigen, the positive rate was significantly high. The cross reaction of antibody produced by other bacteria can be related to the variety and quantity of the antigen components of the thallus, and the cross reaction can be avoided by using the recombinant adhesin Apd protein as the coating antigen. More importantly, the total positive rate detected by using the kit in 222 serum samples is 35%, which is basically consistent with the separation rate of the haemophilus parasuis in a swinery of 30%, and can reflect the infection and epidemic trend of the haemophilus parasuis more truly.

TABLE 11 kit and ELISA for detection of clinical sera by bacteria

Other parts not described in detail are prior art. Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> adhesin protein Apd and application thereof in preparation of indirect ELISA antibody detection kit for haemophilus parasuis

<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 (7)

1. An adhesin gene apd, the nucleotide sequence of which is shown in SEQ ID No. 1.

2. Primer pair for obtaining the adhesin gene apd of claim 1, characterized in that: the primer pair is as follows:

P1，5’-CGGAATTCGGAAACTTATATTGTTACTGGTGAA-3’，

P2，5’-CCCTCGAGGGTGATTGTGATTTTATTGTGGT-3’。

3. a recombinant expression vector pET-Apd, comprising: the recombinant expression vector is an expression vector containing the adhesin gene apd of claim 1, wherein the expression vector is pET-25 b.

4. A host cell comprising the recombinant expression vector of claim 3, wherein: the host cell is Escherichia coli BL21(DE 3).

5. A method of producing recombinant adhesin protein Apd using the host cell of claim 4: the method is characterized in that: the method comprises the following steps:

(1) culturing escherichia coli competent cells BL21(DE3) containing a recombinant expression vector pET-Apd overnight, centrifuging culture bacterial liquid, and resuspending a precipitate by using a buffer solution to obtain suspension bacterial liquid;

(2) and (3) carrying out thallus crushing on the suspension liquid at a low temperature, centrifugally collecting a supernatant, and purifying the supernatant to obtain the recombinant adhesin protein rApd.

6. An indirect ELISA antibody detection kit for haemophilus parasuis is characterized in that: the kit comprises an adhesin coated ELISA plate prepared by claim 5, sample diluent, negative and positive control serum, 20-time concentrated washing solution, an ELISA secondary antibody, a developing solution A, a developing solution B and a stop solution; wherein,

the sample diluent is: pH7.4 is 50mL of PBS buffer solution, 0.25g of bovine serum albumin and Tween-2025 mu L;

the positive control serum: the preparation method comprises the following steps: immunizing 1-month-old healthy piglet with Haemophilus parasuis twice, collecting blood serum two weeks after two-time immunization, and detecting OD₆₃₀Taking 4 parts of serum with the value of 1.00-1.20, mixing, and diluting with a sample diluent at a ratio of 1:200 to serve as positive control serum;

the negative control serum: detecting OD of serum of healthy pig fed under the same condition during preparation of positive control serum₆₃₀Mixing 4 parts of serum with value of 0.15-0.25, and diluting with sampleThe solution was diluted 1:200 to serve as negative control serum;

the 20-fold concentrated washing solution: 160.00g of NaCl, 4.00g of KCl and Na₂HPO₄·12H₂O 72.60g，KH₂PO₄4.80g of the extract is dissolved in 900mL of deionized water, the pH value is adjusted to 7.4 by concentrated hydrochloric acid, 10mL of Tween-20 is added, the volume is fixed to 1L, and the extract is filtered, sterilized and stored at room temperature;

the enzyme-labeled secondary antibody: diluting a goat anti-pig secondary antibody marked by horseradish peroxidase by using a sample diluent at a ratio of 1: 15000;

the color developing liquid A: na (Na)₂HPO₄·12H₂Adding 800ml of deionized water into 14.60g of O, 9.33g of citric acid and 2ml of 30% hydrogen peroxide for dissolving, adjusting the pH value to 5.0-5.4, and fixing the volume to 1000 ml;

the color developing liquid B: 20.00mg of tetramethyl benzidine and 10.00ml of absolute ethyl alcohol, adding deionized water for dissolving and fixing the volume to 1000 ml;

the stop solution is as follows: to 10mL of deionized water was added 25. mu.L of hydrofluoric acid.

7. The indirect ELISA antibody detection kit for Haemophilus parasuis according to claim 6, wherein: the preparation method of the elisa plate coated by the adhesin protein comprises the following steps: taking the purified rApd protein as a coating antigen; taking 0.10M carbonate buffer solution with pH of 9.6 as coating solution; rApd was diluted to 0.50. mu.g/mL and coated overnight at 4 ℃; blocking was performed with 0.05g/mL skim milk in PBS buffer pH7.4 at 37 ℃ for 2 h.