CN116041549A - Haemophilus parasuis HAPS0901 and WAZ fusion protein and application thereof - Google Patents
Haemophilus parasuis HAPS0901 and WAZ fusion protein and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of vaccine preparation, and particularly relates to haemophilus parasuis HAPS0901 and WAZ fusion proteins and application thereof. The fusion protein comprises a sequence shown as SEQ ID NO. 4. The fusion protein is used as subunit vaccine of haemophilus parasuis for immunization, the antibody titer can reach 1:800-1:1600, and the survival protection rate is higher than that of the inactivated vaccine of haemophilus parasuis. The fusion protein can be used as a coating antigen of an ELISA detection method for detecting the haemophilus parasuis antibody, and has the advantages of strong specificity, high sensitivity, good repeatability and high accuracy.
Description
Technical Field
The invention belongs to the technical field of vaccine preparation, and particularly relates to haemophilus parasuis HAPS0901 and WAZ fusion proteins and application thereof.
Background
Haemophilus parasuis (Haemophilus parasuis, HPS) can cause pig's multiple serositis, arthritis and meningitis as main characteristics of pig's Coriolis disease
Break). The pathogenic bacterium is a polymorphic, nicotinamide Adenine Dinucleotide (NAD) -dependent, motionless gram-negative bacillus parvus belonging to the genus Haemophilus of the family Pasteureidae. The disease is widely existed and popular in a plurality of countries and regions, is a primary cause of morbidity and mortality of weaned pigs of 2-4 months of age, and seriously jeopardizes the development of pig industry. The prevention and control of the disease are mainly applied to corresponding antibacterial drugs and detection auxiliary prevention and control of haemophilus parasuis at present. However, the phenomenon of misuse and abuse of the antibacterial drugs in clinical application is extremely serious, so that the extremely serious drug resistance phenomenon of pathogenic bacteria is caused, and the ideal effect is not obtained by utilizing the antibiotic control. Vaccine control of the disease is the most cost effective control approach.
The current haemophilus parasuis inactivated vaccine is most widely applied, but the haemophilus parasuis serotypes are numerous, and the safe and effective cross immune protection is lacking among the serotypes, and the current commercial inactivated vaccine only comprises a plurality of limited serotype antigens, so that the immune protection range is limited; in addition, a large amount of endotoxin which cannot be inactivated by an inactivating agent exists in the commercial haemophilus parasuis inactivated vaccine, and the stress response of immunized piglets is easy to cause in clinical application. The subunit vaccine can realize targeted high-efficiency expression of immune protective antigen with low haemophilus parasuis content, can generate immune protective antibody with higher titer after immunization of animals, and can efficiently remove the influence of endotoxin in the preparation process.
Therefore, the development of a safe, efficient and broad-spectrum haemophilus parasuis subunit vaccine has important significance for prevention and control of haemophilus parasuis.
Disclosure of Invention
Aiming at the problems, one of the purposes of the invention is to provide a haemophilus parasuis subunit vaccine which is obtained by splicing and fusion expression of haemophilus parasuis immunoprotection antigens HAPS0901 and WAZ gene sequences, the antibody titer can reach 1:800-1:1600, and the survival protection rate is far higher than that of the haemophilus parasuis inactivated vaccine.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
in one aspect, the invention provides a haemophilus parasuis HAPS0901 and WAZ fusion protein comprising a sequence as shown in SEQ id No. 4.
In another aspect, the invention provides a haemophilus parasuis vaccine comprising the haemophilus parasuis HAPS0901 and WAZ fusion proteins described above.
In a further aspect, the invention provides an application of the haemophilus parasuis HAPS0901 and WAZ fusion protein in preparing haemophilus parasuis vaccine.
In still another aspect, the invention provides a haemophilus parasuis antibody detection reagent comprising the haemophilus parasuis HAPS0901 and WAZ fusion proteins described above.
The invention also provides a haemophilus parasuis antibody detection kit which comprises the haemophilus parasuis HAPS0901 and WAZ fusion proteins or the haemophilus parasuis antibody detection reagent.
In still another aspect, the invention provides an application of the haemophilus parasuis HAPS0901 and WAZ fusion protein in detection of haemophilus parasuis antibodies.
In yet another aspect, the invention provides a method for detecting haemophilus parasuis antibodies comprising: the fusion proteins of Haemophilus parasuis HAPS0901 and WAZ described above were used as coating antigens for detection using ELISA detection methods.
The beneficial effects of the invention include:
(1) The fusion protein of haemophilus parasuis HAPS0901 and WAZ provided by the invention is used as subunit vaccine of haemophilus parasuis for immunization, the antibody titer can reach 1:800-1:1600, the survival protection rate is high (83.33%), and under the same immunization condition, the survival protection rate is higher than that of the haemophilus parasuis inactivated vaccine (66.67%).
(2) ELISA detection method based on the haemophilus parasuis HAPS0901 and WAZ fusion protein provided by the invention as coating antigen can not generate cross reaction of other swine virus antibodies (porcine reproductive and respiratory syndrome virus antibody, porcine circovirus antibody, classical swine fever virus antibody, porcine foot and mouth disease virus antibody, porcine pseudorabies virus antibody, porcine epidemic disease virus antibody, porcine infectious pleuropneumonia antibody, porcine Pasteurella antibody and porcine type 2 streptococcus antibody) when detecting haemophilus parasuis antibody, and has extremely strong specificity; in addition, the serum titer of the detection method can reach 1:800-1:1600, namely the sensitivity is high; in addition, the intra-batch variation coefficient of the detection method is 1.14-9.06%, and the inter-batch variation coefficient is 1.11-9.39%, namely, the repeatability of both the intra-batch and inter-batch is good; and the detection method has high accuracy.
Drawings
FIG. 1 is a diagram showing the identification of amplified products of HAPS-0901 gene sequences;
FIG. 2 is an identification of the HPS-WZA gene sequence amplification product;
FIG. 3 is an overlapping PCR splice HAPS0901 gene and WAZ gene sequence identification;
FIG. 4 shows PCR identification of constructed Pet28a- (HAPS 0901+ WAZ) plasmid and strain;
FIG. 5 shows SDS-PAGE to identify expression of purified HAPS0901 and WAZ fusion proteins;
FIG. 6 is a Western blot to identify immunoreactivity of HAPS0901 and WAZ fusion proteins.
Wherein, in fig. 1, M: DL5000 DNA Mark; 1. 2: amplified HAPS-0901 gene sequence; in fig. 2, M: DL5000 DNA Mark; 1. 2: amplified HPS-WZA gene sequence; in fig. 3, M: DL5000 DNA Mark,1, 2: spliced HAPS0901+ WAZ gene sequence; in fig. 4, M: DL5000 DNA Mark;1: constructed Pet28a- (HAPS 0901+ WAZ) plasmid; 2. 3: screening BL21 (DE 3) strain containing the Pet28a- (HAPS 0901+ WAZ) plasmid; in FIG. 5, M is a protein marker;1: expressing purified haemophilus parasuis HAPS0901 and WAZ fusion proteins; in fig. 6, M: pre-dyeing a protein marker;1: purified haemophilus parasuis HAPS0901 and WAZ fusion proteins were expressed.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides a haemophilus parasuis HAPS0901 and WAZ fusion protein which comprises a sequence shown in SEQ ID NO. 4.
The gene sequence amplification primers of haemophilus parasuis HAPS0901 and WAZ refer to the whole gene sequence of HPS-SH0165 strain in GenBank gene library, and the sequence number is CP-001321. The haemophilus parasuis HAPS0901 and WAZ fusion proteins are obtained by respectively carrying out PCR amplification on the HAPS0901 and WAZ gene sequences, and then carrying out overlapping PCR reaction on the two PCR amplification products to obtain the HAPS0901 and WAZ fusion gene sequences (shown as SEQ ID NO. 3); then the expression vector is used for expression, and the haemophilus parasuis HAPS0901 and WAZ fusion proteins are obtained through screening. SDS-PAGE electrophoresis identified the fusion protein band was approximately 86KD.
Another embodiment of the present invention provides a haemophilus parasuis vaccine comprising the haemophilus parasuis HAPS0901 and WAZ fusion proteins described above.
It should be noted that, the fusion protein combined vaccine adjuvant of haemophilus parasuis HAPS0901 and WAZ in the present invention may be prepared into haemophilus parasuis vaccine, and the vaccine adjuvant is known in the art, such as complete Freund's adjuvant. In addition, the prepared haemophilus parasuis vaccine is subunit vaccine, has higher safety, and in some specific embodiments, the survival protection rate of the guinea pigs immunized by the haemophilus parasuis vaccine is far higher than that of the guinea pigs immunized by the haemophilus parasuis inactivated vaccine; and the serum antibody titer of the immunized guinea pigs reaches 1:800-1:1600, which is far higher than that of the immunized guinea pigs of the haemophilus parasuis inactivated vaccine (1:200-1:400). In addition, the haemophilus parasuis HAPS0901 and WAZ fusion proteins can be combined with other components to immunize a human body, so that the effect is improved.
The invention also provides an application of the haemophilus parasuis HAPS0901 and WAZ fusion protein in preparing haemophilus parasuis vaccine.
The haemophilus parasuis HAPS0901 and WAZ fusion proteins can be used as vaccines to immunize organisms, and adjuvants or other components can be added to prepare the haemophilus parasuis vaccine.
Still another embodiment of the present invention provides a haemophilus parasuis antibody detection reagent, which includes the haemophilus parasuis HAPS0901 and WAZ fusion proteins described above.
It should be noted that, the fusion proteins of haemophilus parasuis HAPS0901 and WAZ in the present invention can be used as vaccines, and also can be used for detecting haemophilus parasuis antibodies, and the fusion proteins of haemophilus parasuis HAPS0901 and WAZ in the present invention are used as coating antigens, and combined with reagents such as a color reagent, a buffer solution, an enzyme-labeled antibody, etc. to form a haemophilus parasuis antibody detection reagent for detecting haemophilus parasuis antibodies.
The invention also provides a haemophilus parasuis antibody detection kit which comprises the haemophilus parasuis HAPS0901 and WAZ fusion proteins or the haemophilus parasuis antibody detection reagent.
It should be noted that, the fusion proteins of haemophilus parasuis HAPS0901 and WAZ or the detection reagent of the haemophilus parasuis antibodies can also be prepared into a kit, and the kit comprises some conventional components, such as a test tube, a test paper or a dropper, besides the fusion proteins or the detection reagents.
The invention further provides an application of the haemophilus parasuis HAPS0901 and WAZ fusion proteins in detection of haemophilus parasuis antibodies.
It should be noted that, the fusion proteins of haemophilus parasuis HAPS0901 and WAZ are applied in the detection of haemophilus parasuis antibodies, which have extremely strong specificity to haemophilus parasuis antibodies, do not cross react with other porcine virus antibodies (porcine reproductive and respiratory syndrome virus antibodies, porcine circovirus antibodies, classical swine fever virus antibodies, porcine foot and mouth disease virus antibodies, porcine pseudorabies virus antibodies, porcine epidemic pleuropneumonia antibodies, porcine pasteurellosis antibodies and porcine type 2 streptococcus antibodies), and have relatively strong specificity; the titer of the antibody to serum can reach 1:800-1:1600, namely the sensitivity is high; in addition, the repeatability in the batch and the batch is good, and the accuracy is high.
In yet another embodiment, the present invention provides a method for detecting haemophilus parasuis antibodies, comprising: the fusion proteins of Haemophilus parasuis HAPS0901 and WAZ were used as coating antigens for detection by ELISA detection.
The haemophilus parasuis HAPS0901 and WAZ fusion proteins may be used as coating antigens to detect haemophilus parasuis antibodies using ELISA detection methods, which are known in the art. In some embodiments, a method of detecting a haemophilus parasuis antibody may comprise: diluting the haemophilus parasuis HAPS0901 and WAZ fusion protein with coating liquid, adding into an ELISA plate, and coating overnight; washing, and sealing by using sealing liquid after beating to dry; washing, beating, adding diluted pig serum to be detected, setting an antibody positive control sample and an antibody negative control sample respectively, and incubating at 37 ℃ respectively to discard liquid in the hole; adding 300 mu L of PBST, washing for 5 times, and drying by beating; adding HRP-labeled rabbit anti-pig IgG, and discarding the liquid in the hole after 30min of action at 37 ℃; adding 300 mu L of PBST into each hole, washing and drying; adding a color development liquid, and developing for 10min in dark; adding H 2 SO 4 Terminating the color development, and measuring the OD by using an enzyme-labeled instrument 450nm Values. In some embodiments, the determination criteria for the method for detecting haemophilus parasuis antibodies may be: when antibody negative control OD 450nm Average value (N) less than 0.20, antibody positive control OD 450nm When the average value (P) is greater than 0.60, the OD of the sample is 450nm Value (S)/negative control OD 450nm When the average value (N) is more than or equal to 2.1, the sample OD is positive 450nm Value (S)/negative control OD 450nm When the average value (N) was less than 2.1, the test was negative.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
Example 1 amplification and Concatenation of the Gene sequences of Haemophilus parasuis HAPS0901, WAZ
(1) HAPS0901 gene sequence amplification, purification and recovery
Based on the complete gene sequence of HPS-SH0165 strain registered in GenBank, the sequence number is CP-001321, a pair of primers HAPS0901-F and HAPS0901-R for amplifying the gene sequence of the immune protective antigen HAPS0901 of haemophilus parasuis are designed, wherein the primers HAPS0901-F are added with BamHI restriction enzyme cutting sites (underlined) and protective bases.
HAPS 0901-F5'-cgggatccatggcaaattacgcattact-3' (BamHI cleavage site)
HAPS0901-R:5'-gctttagttaacttacacatctcatcaacattctccttcag-3'
The 939bp fragment of the HAPS0901 gene sequence is amplified by PCR using the HPS serum 5 type wild strain as a template. The PCR amplification system for the HAPS0901 gene sequence was 50. Mu.L: 2. Mu.L of bacterial liquid template, 1. Mu.L of 100. Mu. Mol/L HAPS0901-F/R primer each, 2X PrimeSTAR max DNA Polymerase. Mu.L, ddH 2 O21.0. Mu.L. The PCR reaction conditions were: after denaturation at 95 ℃ for 5min, the mixture enters a cycle with the cycle parameters of 94 ℃ for 30s,58 ℃ for 20s,72 ℃ for 40s, and after 30 cycles, the mixture is extended for 10min at 72 ℃. After the completion, the specific target fragment 939bp in HAPS0901 gene sequence (see FIG. 1) was purified and recovered by agarose gel with a concentration of 1%, as shown in SEQ ID NO. 1.
(2) WAZ gene sequence amplification, purification and recovery
A pair of primers WZA-F and WZA-R for amplifying the gene sequence of the haemophilus parasuis immunoprotection antigen WAZ was also designed based on the whole gene sequence of the HPS-SH0165 strain (GenBank accession number: CP-001321), wherein the primers WZA-R are added with XhoI restriction sites (underlined) and protective bases. Wherein the primers HAPS0901-R and WZA-F are reverse complementary sequences, so that the sequences of the Haemophilus parasuis HAPS0901 gene and WAZ gene can be spliced conveniently by overlapping PCR.
WZA-F:5'-ctgaaggagaatgttgatgagatgtgtaagttaactaaagc-3'；
WZA-R5'-ggctcgagccaattcctcactcttaagat-3' (XhoI cleavage site);
the 1152bp fragment of the WAZ gene sequence is amplified by PCR by taking the HPS serum 5 type wild strain as a template. The PCR amplification system of WAZ gene sequence was 50. Mu.L: 2 mu L of bacterial liquid template, 1 mu L of 100 mu mol/L WAZ-F/R primer, 2X PrimeSTAR max DNA Polymerase mu L of each WAZ-F/R primer and ddH 2 O21.0. Mu.L. The PCR reaction conditions were: after denaturation at 95 ℃ for 5min, the mixture enters a cycle with the cycle parameters of 94 ℃ for 30s,57 ℃ for 20s,72 ℃ for 50s, and after 30 cycles, the mixture is extended for 10min at 72 ℃. After the completion, the specific target fragment of 1152bp of WAZ gene sequence (see FIG. 2) is purified and recovered by agarose gel with the concentration of 1%, as shown in SEQ ID NO.2Shown.
(3) HAPS0901 and WAZ gene sequence splicing
And splicing the HAPS0901 gene sequence and the WAZ gene sequence which are amplified, purified and recovered by adopting an overlap PCR method. The method comprises the following steps:
overlapping PCR reaction first step: HAPS0901 gene sequence and WAZ gene sequence each 2. Mu.L, 2X PrimeSTAR max DNA Polymerase. Mu.L, ddH 2 O21. Mu.L, total volume 50. Mu.L, and then aliquoted into two PCR tubes, and the first PCR amplification was performed in a system of 25. Mu.L per tube. The PCR reaction conditions were: after denaturation at 95 ℃ for 5min, the mixture enters a cycle with the cycle parameters of 94 ℃ 40s,58 ℃ 20s,72 ℃ 30s, and after 5 cycles, the mixture is extended for 10min at 72 ℃.
After amplification, carrying out an overlapping second-step PCR reaction, and firstly preparing a 50 mu L amplification system: 100. Mu. Mol/L HAPS0901-F primer 2. Mu.L, 100. Mu. Mol/L WZA-R primer 2. Mu.L, 2X PrimeSTAR max DNA Polymerase. Mu.L, ddH 2 O21. Mu.L was then aliquoted into two tubes after the end of the first-step overlap PCR reaction in 25. Mu.L per tube, and a second-step PCR amplification was performed with 50. Mu.L per tube. The PCR reaction conditions were: after denaturation at 95 ℃ for 5min, the mixture enters a cycle with the cycle parameters of 94 ℃ 40s,57 ℃ 20s,72 ℃ 60s and 20 cycles, and then the mixture is extended for 10min at 72 ℃.
After the completion, the identification is carried out by agarose gel with the concentration of 1 percent, and fusion gene sequence fragments spliced by the HAPS0901 gene and the WAZ gene of the haemophilus parasuis are purified and recovered, wherein the size of the fusion gene sequence fragments is 2091bp (see figure 3), and the fusion gene fragments are shown as SEQ ID NO. 3.
EXAMPLE 2 construction of expression vectors for Haemophilus parasuis HAPS0901 and WAZ Gene fusion sequences
The purified and recovered Haemophilus parasuis HAPS0901 gene and WAZ gene splice sequences were digested with BamHI and XhoI, and simultaneously the plasmid vector Pet28a was digested with BamHI and XhoI, and then the HAPS0901 gene and WAZ gene splice sequence fragments were ligated to the plasmid vector Pet28 a. The reaction system is as follows: the HAPS0901 gene and WAZ gene splice sequences were 4. Mu.L, pet28a 4. Mu.L, 10 Xligation buffer 1. Mu.L, T4 DNA ligase 1. Mu.L, and ligation at 4℃for 16 hours, then heat-stressed transformed into BL21 (DE 3) competence, plated on LB plates supplemented with 50. Mu.g/mL kanamycin, incubated at 37℃for 16 hours, single colonies were picked up and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and plasmids were extracted for identification. PCR screening gave BL21 (DE 3) strain containing positive construction plasmid Pet28a- (HAPS 0901+ WAZ) (see FIG. 4).
EXAMPLE 3 expression, purification and identification of the reactogenicity of the fusion proteins of Haemophilus parasuis HAPS0901 and WAZ
(1) Expression and purification of HAPS0901 and WAZ fusion proteins
After picking E.ColiBL 21/pET28a- (HAPS 0901+ WAZ) single colony, transferring to fresh LB liquid medium containing kanamycin (50 μg/ml) at 1% inoculum size, shake culturing at 37deg.C at 240rpm for about 2 hr, OD 600nm To 0.8-1.0, IPTG was added to a final concentration of 1.0mmol/L, the mixture was transferred to 37℃and cultured with shaking at 240rpm for 6 hours, and the cells were collected by centrifugation. The cells were washed three times with PBS buffer and collected by centrifugation at the last time with Lysis buffer (50 mM NaH) 2 PO 4 300mM NaCl,10mM imidazole,pH =8.0) suspended cells. Adding lysozyme with final concentration of 1mg/mL, standing on ice for 30min, ultrasonic lysing and breaking cells (400W power, ultrasonic breaking time 5s, interval time 5s, ultrasonic 30 min), centrifuging at 4deg.C and 10000rpm for 15min, and collecting supernatant. Filtering the centrifuged bacterial liquid supernatant with 0.45 μm filter, adding 5mL into an affinity chromatography column containing 50% Ni-NTA, mixing at 200r/min for 1 hr, passing through the column, and adding 5mL of Wash buffer (50 mM NaH) 2 PO 4 Wash 2 times 300mM NaCl,50mM imidazole,pH =8.0 and finally add 0.5mL of Elution buffer (50 mM NaH) 2 PO 4 300mM NaCl,300mM imidazole,pH =8.0) and the target protein was collected by elution, the purified protein concentration was determined to be 1.8mg/mL, and SDS-PAGE electrophoresis was performed. SDS-PAGE identification result is shown in FIG. 5, a specific protein band of about 86KD is obtained, the predicted fusion protein has an amino acid sequence shown as SEQ ID NO.4, and the predicted fusion protein has the expected size.
(2) Immunogenicity identification of HAPS0901 and WAZ fusion proteins
After SDS-PAGE electrophoresis, western-Blot was performed to identify the immunogenicity of the protein, and nitrocellulose membrane (NC) and filter paper of the same size as the gel strip were cut in advance and immersed in an electrotransport buffer. The gel was removed, after equilibration in transfer buffer, the membrane, gel and filter were laid down in sequence, i.e., filter paper-NC membrane-gel-filter paper, and gently rolled through the sandwich with a clean glass rod to eliminate air bubbles between the layers. And (3) placing the sealed materials into an electrotransfer tank, adding electrotransfer liquid, and then connecting a cooling device, and transferring the 200mA constant current for 1h. Taking off NC membrane after transfer, washing membrane with TBST for 5min×3 times; placing NC membrane into 5% skimmed milk, sealing overnight at 4deg.C, discarding sealing solution, and washing membrane with TBST for 5min×3 times; adding primary antibody, namely TBST, diluting (1:100) positive swine serum of haemophilus parasuis antibody, shaking steadily, discarding the primary antibody at room temperature for 1h, and washing the membrane with TBST for 5min multiplied by 4 times; adding horseradish peroxidase-labeled rabbit anti-pig enzyme-labeled secondary antibody, diluting with TBST at ratio of 1:50000, shaking smoothly at room temperature for 1 hr, discarding the secondary antibody, and washing the membrane with TBST for 5min×3 times. And placing the NC film acted by the secondary antibody into a color development liquid for color development, stopping the reaction after a specific reaction band appears, and taking a picture for storage. The Western-blot analysis results are shown in FIG. 6, and the HAPS0901 and WAZ fusion proteins can be identified by the H.parasuis antibody positive serum.
Example 4 application of Haemophilus parasuis HAPS0901 and WAZ fusion proteins in serological detection
An indirect ELISA method for detecting the haemophilus parasuis antibody is established by utilizing the expressed and purified haemophilus parasuis HAPS0901 and WAZ fusion protein antigen. The fusion proteins expressing purified HAPS0901 and WAZ were diluted to 2.0. Mu.g/mL with coating solution (0.05 mol/L carbonate buffer pH 9.6), added to the ELISA plate at 100. Mu.L/well, and coated overnight at 4 ℃; PBST 300. Mu.L was added to each well, washed 2 times, and after drying, blocked with blocking solution (0.01 g/mL BSA) at 100. Mu.L/well for 2h at 37 ℃; adding 300 mu L of PBST into each hole, washing for 2 times, beating, adding 100 mu L of 1:100 diluted pig serum to be detected into each hole, simultaneously setting two holes (100 mu L/hole) of a haemophilus parasuis antibody positive control sample and two holes (100 mu L/hole) of a negative control sample, and removing liquid in the holes after incubation at 37 ℃ for 30 min; 300 μl of PBST was added to each well, washed 5 times, and patted dry, 100 μl of 1:50000 dilution of HRP-labeled rabbit anti-pig IgG was added to each well, and the mixture was allowed to act at 37deg.C for 30min and then discardedRemoving liquid in the holes; adding 300 mu L of PBST into each hole, washing for 5 times, beating to dry, adding 100 mu L of TMB single-component color development liquid into each hole, and developing for 10min in a dark place; finally, 100 mu L of 2mol/L H are added to each well 2 SO 4 Terminating the color development, and measuring the OD by using an enzyme-labeled instrument 450nm Values. The result judgment standard of the haemophilus parasuis antibody detection method is as follows: when negative control OD 450nm Average value (N) less than 0.20, positive control OD 450nm When the average value (P) is greater than 0.60, the OD of the sample is 450nm Value (S)/negative control OD 450nm When the average value (N) is more than or equal to 2.1, the sample OD is positive 450nm Value (S)/negative control OD 450nm When the average value (N) was less than 2.1, the test was negative.
(1) Determination of the specificity of the detection method
The detection method is used for detecting the positive pig serum of the porcine reproductive and respiratory syndrome virus antibody, the porcine circovirus antibody, the classical swine fever virus antibody, the porcine foot-and-mouth disease virus antibody, the porcine pseudorabies virus antibody, the porcine epidemic pleuropneumonia antibody, the porcine Pasteurella antibody and the porcine type 2 streptococcus antibody, and the results are negative, so that the detection method has good specificity, and the results are shown in table 1.
TABLE 1 results of the test on common porcine positive serum
Note that: positive control OD was detected at this time 450nm Average value of 1.257, negative control OD 450nm The average value of (2) is 0.189, positive when S/N is more than or equal to 2.1, negative when S/N is less than 2.1, "+": results were positive, "-": the result was negative.
(2) Determination of sensitivity of the detection method
6 parts of haemophilus parasuis antibody positive pig serum are taken and diluted according to 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3.200, and the serum titer detected by the detection method is found to be 1:800-1:1:600 by respectively detecting the method and the Dutch hundred test (BioChek) haemophilus parasuis (HPS-OppA) antibody ELISA detection kit established above: the highest serum titer of the haemophilus parasuis antibody kit produced by Holland Bai Jib is 1:400-1:800, and the results are shown in tables 2-3.
TABLE 2 detection results of different dilution ratios of Haemophilus parasuis antibody positive porcine serum using the detection method established by HAPS0901 and WAZ fusion antigen
Note that: positive control OD was detected at this time 450 nm Average value of 1.184, negative control OD 450 nm The average value of (2) is 0.187, positive when S/N is not less than 2.1, negative when S/N is less than 2.1, "+": results were positive, "-": the result was negative.
TABLE 3 detection results of different dilution ratios of Haemophilus parasuis antibody positive porcine serum by using Holland hundred-test Haemophilus parasuis (HPS-OppA) antibody kit
Note that: positive control OD was detected at this time 405nm Average value of 0.756, negative control OD 405 nm The average value of (2) is 0.237, the difference between the average value of the positive control and the average value of the negative control is more than 0.15, and the OD of the sample is greater than that of the negative control 405 nm value/positive control OD 405 nm The value is more than or equal to 0.5, and is judged as positive, otherwise, is judged as negative, "+": results were positive, "-": the result was negative.
(3) The detection method is used for repeatedly measuring
1) The detection method is used for measuring the repeatability in batch
30 parts of known background pig serum are detected by using the detection method of the same batch, wherein 18 parts of haemophilus parasuis antibody positive serum and 12 parts of haemophilus parasuis antibody negative serum. 3 replicates were performed on each of the 30 serum samples, and the results showed that the intra-batch coefficient of variation was between 1.14% and 9.06% (see table 4), indicating that the kit was good in intra-batch reproducibility.
TABLE 4 results of detection of porcine serum samples by the same batch detection method
Note that: positive control OD detected by different coated plates 450nm Average value of 1.185, negative control OD 450nm The average value of (2) is 0.185, the S/N is positive when not less than 2.1, and the S/N is negative when less than 2.1, "+": results were positive, "-": the result was negative.
2) Batch-to-batch reproducibility test
30 parts of known background pig serum are detected by using the detection methods of different batches, wherein 18 parts of haemophilus parasuis antibody positive serum and 12 parts of haemophilus parasuis antibody negative serum. The results of 3 batch detection methods carried out simultaneously on each serum in 30 pig serum samples show that the batch variation coefficient is between 1.11% and 9.39% (see table 5), which shows that the batch repeatability of the kit is good.
TABLE 5 results of detection of porcine serum samples by different batch detection methods
Note that: positive control OD for different batch detection 450nm Average value of 1.186, negative control OD 450nm The average value of (2) is 0.190, the S/N is positive when the S/N is more than or equal to 2.1, the S/N is negative when the S/N is less than 2.1, "+": results were positive, "-": the result was negative.
(4) Comparing test with clinical application of similar detection method
At present, no commercial haemophilus parasuis antibody detection kit is marketed in China, and the foreign similar products comprise Holland hundred-test (BioChek) haemophilus parasuis (HPS-OppA) antibody ELISA detection kit. Comparing the self-established detection method with a kit produced by Holland hundred-test company, detecting 100 parts of clinical pig serum, and detecting 100 parts of serum by using an established antibody ELISA detection kit of haemophilus parasuis fusion protein antigen, wherein the positive rate is 56.00% (56/100) and the negative rate is 44.00% (44/100); the positive rate of the test of the haemophilus parasuis (HPS-OppA) antibody kit produced by Holland hundred-testing company is 51.00 percent (51/100), and the negative rate is 49.00 percent (49/100); the positive coincidence rate of the two is 91.07% (51/56), the negative coincidence rate is 89.80% (44/49), and the total coincidence rate is 95.00% (95/100).
Example 5 application of Haemophilus parasuis HAPS0901 and WAZ fusion proteins in subunit vaccine immunoprotection
Clean guinea pigs 18 were purchased from Guangdong province medical laboratory animal center, females, weighing 1.5kg-2kg, were randomly divided into three groups, the first group immunized with Haemophilus parasuis HAPS0901 and WAZ fusion proteins, the second group immunized with whole-cell inactivated Haemophilus parasuis vaccine as positive control, and the third group immunized with PBS+ adjuvant as negative control. Immunization is carried out by adopting a back subcutaneous multipoint injection mode, in the first group of immunization, the first immunization mixes and emulsifies the expressed and purified haemophilus parasuis HAPS0901 and WAZ fusion protein antigen with equivalent Freund's complete adjuvant, each guinea pig is inoculated with 0.5mL, the inoculated fusion protein content is 20 mug/item, the positive control group immunizes each guinea pig with the haemophilus parasuis complete inactivated vaccine with 0.5mL, and the negative control group immunizes PBS and Freund's complete adjuvant emulsified mixture with 0.5 mL/item. In the subsequent immunization, the corresponding antigen is mixed and emulsified with Freund's incomplete adjuvant, and the mixture is treated in the same immunization dose and method, and the immunization interval is 15 days, and the blood is taken and serum is separated for measuring the antibody titer on the 10 th day after the second immunization. ELISA plates (100. Mu.L/well) were coated with antigen expressing purified H.parasuis HAPS0901 and WAZ fusion proteins (2.0. Mu.g/mL)) Washing 3 times with PBST overnight at 4 ℃, blocking with 0.01g/mL BSA at 100. Mu.L/well, washing 3 times with PBST after 2h action at 37 ℃, and washing guinea pig serum with PBS from 1: sequentially performing multiple dilution at 1000, 100 μl/well, standing at 37deg.C for 30min, and washing with PBST for 3 times; HRP-labeled goat anti-guinea pig enzyme-labeled secondary antibody 1: diluting with 50000, 100 μl/well, allowing to act at 37deg.C for 30min, and washing with PBST 3 times; TMP monocomponent substrate was added at 100. Mu.L/well, developed at 37℃for 10min, and the reaction was stopped with 2M sulfuric acid stop solution at 100. Mu.L/well. Reading OD with an enzyme-labeled detector 450nm Values. The results show that the serum antibody titer of the guinea pigs immunized with the haemophilus parasuis HAPS0901 and WAZ fusion protein antigens reaches 1:800-1:1.600, the serum antibody titer of the guinea pigs immunized with the haemophilus parasuis whole-cell inactivated vaccine reaches 1:200-1:400, and the guinea pig HAPS0901 and WAZ fusion protein antibodies in the negative control group are all negative (see table 6).
TABLE 6 HAPS0901 and WAZ fusion protein antibody titers of guinea pigs in each experimental group
Note that: positive control OD was detected at this time 450nm Average value of 1.157, negative control OD 450nm The average value of (2) is 0.195, positive when S/N is more than or equal to 2.1, negative when S/N is less than 2.1, "+": results were positive, "-": the result was negative.
Each guinea pig of the test group was then challenged by intraperitoneal injection with the HPS wild-type strain (2.15X10) 9 CFU/dose), observing the protection rate of guinea pigs after toxin attack; as shown in Table 7, the survival rate of the guinea pigs in the HAPS0901 and WAZ fusion antigen immune group was 83.33%, the survival rate of the guinea pigs in the haemophilus parasuis inactivated vaccine immune group was 66.67%, and the immune protection rate of the HAPS0901 and WAZ fusion antigen immune group on the guinea pigs was higher than that of the haemophilus parasuis whole bacteria inactivated vaccine immune group. Control guinea pigsObvious clinical symptoms are shown after the toxin is attacked, the respiration is quickened, the spirit is depressed, the fur is rough and disordered, and all the patients die in the observation period.
TABLE 7 protection against challenge survival of guinea pigs in each immunized group
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (8)
1. A haemophilus parasuis HAPS0901 and WAZ fusion protein is characterized by comprising a sequence as shown in SEQ ID NO. 4.
2. A haemophilus parasuis vaccine comprising haemophilus parasuis HAPS0901 and WAZ fusion proteins according to claim 1.
3. Use of the haemophilus parasuis HAPS0901 and WAZ fusion protein of claim 1 as or for the preparation of a haemophilus parasuis vaccine.
4. The haemophilus parasuis antibody detection reagent is characterized by comprising the haemophilus parasuis HAPS0901 and WAZ fusion protein of claim 1.
5. The haemophilus parasuis antibody detection kit is characterized by comprising the haemophilus parasuis HAPS0901 and WAZ fusion protein of claim 1 or the haemophilus parasuis antibody detection reagent of claim 4.
6. Use of the haemophilus parasuis HAPS0901 and WAZ fusion protein of claim 1 for detecting haemophilus parasuis antibodies.
7. A method for detecting haemophilus parasuis antibodies, comprising: the fusion proteins of Haemophilus parasuis HAPS0901 and WAZ according to claim 1 are used as coating antigen for detection by ELISA detection.
8. The method for detecting a haemophilus parasuis antibody according to claim 7, wherein an antibody negative control and an antibody positive control are provided when the antibody negative control OD 450nm Average value less than 0.20, antibody positive control OD 450nm At average values greater than 0.60, the sample OD 450nm Value/negative control OD 450nm When the average value is more than or equal to 2.1, the sample OD is positive 450nm Value/negative control OD 450nm When the average value is less than 2.1, the test is negative.
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