CN118344494A - A fusion antigen of the N-terminus of IRP protein of Haemophilus parasuis and RLPB protein and its application - Google Patents

Abstract

The present invention belongs to the technical field of pathogenic bacterial disease prevention and treatment, and specifically relates to a fusion antigen of the N-terminus of IRP protein of Haemophilus parasuis and RLPB protein and its application. The amino acid sequence of the fusion antigen is shown in SEQ ID NO: 1. The reactivity of the fusion antigen is identified by Western-Blot, and it can be known that the fusion protein antigen can be recognized by the positive serum of Haemophilus parasuis antibody; and the indirect ELISA method constructed based on the fusion antigen has excellent specificity, sensitivity and repeatability for the positive serum of Haemophilus parasuis antibody; in addition, immunizing guinea pigs with the fusion antigen can obtain the protection effect against the attack of Haemophilus parasuis type 1 strain, type 5 strain and type 13 strain.

Description

A fusion antigen of the N-terminus of Haemophilus parasuis IRP protein and RLPB protein and its application

Technical Field

The invention belongs to the technical field of pathogenic bacteria disease prevention and treatment, and specifically relates to a fusion antigen of the N-terminus of an IRP protein of Haemophilus parasuis and an RLPB protein and an application thereof.

Background technique

Haemophilus parasuis (GPS) is a polymorphic, nicotinamide adenine dinucleotide (NAD)-dependent, non-motile Gram-negative bacillus belonging to the Pasteurellaceae family. When the pathogen was first isolated and identified, it was named Haemophilus influenzae. Later, it was found that the bacterium did not require blood factor X for growth, but only needed to add NAD, so the bacterium was renamed Haemophilus parasuis. Molecular biological information technology later identified that Haemophilus parasuis was a unique branch in the Pasteurellaceae family, different from other genera in the Pasteurellaceae family, so it was renamed Glaesserella parasuis. This pathogen is one of the important bacterial diseases that cause morbidity and mortality in piglets during the nursery stage; in clinical production, it can cause porcine Gram-negative disease (Glaser's disease) in piglets, which is mainly characterized by fibrinous polyserositis, pleurisy, polyarthritis and meningitis. Disease).

At present, the prevention and control of the disease mainly uses corresponding antimicrobial drugs. With the continuous addition and application of antibiotics in clinical production, the pathogen has developed strong drug resistance. The previous sensitive drugs have lost their ideal effect in the current clinical prevention and control, which not only causes drug waste, but also causes veterinary drugs to remain in pigs, affecting public health and safety, and delaying the best time to prevent and control Haemophilus parasuis. Immunization and prevention with safe, efficient and broad-spectrum Haemophilus parasuis vaccines can provide important help for the green prevention and control of Haemophilus parasuis. However, most of the existing vaccines for Haemophilus parasuis disease are monovalent or polyvalent inactivated vaccines. The serotypes of Haemophilus parasuis prevalent in clinical practice are complex and diverse. Currently, 15 serotypes have been identified using traditional serotype identification methods, but there are still clinically isolated strains that cannot be typed using traditional serotype identification methods, and there is a large heterogeneity between the serotypes. Even between strains of the same serotype, there are genotypic, phenotypic and regional differences, making it difficult for inactivated vaccines to provide ideal cross-protection. In addition, the current inactivated vaccines for Haemophilus parasuis disease contain a large number of non-protective immune antigens and endotoxins that cannot be inactivated by inactivators. Immunization with polyvalent antigen vaccines is likely to cause immune tolerance and stress responses in immunized pigs.

Therefore, screening out common protective antigens of different serotypes and preparing subunit vaccines of Haemophilus parasuis that can prevent multiple serotypes are of great significance for the prevention and control of the disease.

Summary of the invention

The present invention predicts the B cell epitopes, cytotoxic T cell epitopes and helper T cell epitopes of the surface immune protective antigen IRP protein and RLPB protein of Haemophilus parasuis by relevant biological techniques, and finds that the N terminus of the surface immune protective antigen IRP protein and the RLPB protein of Haemophilus parasuis contain abundant immune cell recognition sites. Therefore, the N terminus of the surface immune protective antigen IRP protein of Haemophilus parasuis and the RLPB protein gene sequence are spliced and fused to obtain a relatively stable immune protective fusion antigen, which can be used for the detection of Haemophilus parasuis and the preparation of recombinant subunit vaccines for Haemophilus parasuis infection diseases.

In order to achieve the above object, the present invention can adopt the following technical solutions:

In one aspect, the present invention provides a fusion antigen of the N-terminus of the IRP protein of Haemophilus parasuis (Glaesserella parasuis) and the RLPB protein, the amino acid sequence of which is shown in SEQ ID NO:1.

It should be noted that the reactivity of the fusion antigen was identified by Western-Blot, and it was found that the fusion protein antigen could be recognized by serum positive for Haemophilus parasuis antibodies.

Another aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen.

It should be noted that the nucleic acid molecules that can encode the above-mentioned amino acids as shown in SEQ ID NO: 1 are all protected in the present invention, for example, the nucleotide sequence can be a nucleic acid molecule as shown in SEQ ID NO: 2; of course, it also includes nucleic acid molecules optimized on the basis of the nucleotide sequence as shown in SEQ ID NO: 2, and the optimization can be performed according to methods known in the art.

In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

It should be noted that the above nucleic acid molecules can be expressed through a vector, and the vector is a vector known in the art, such as an expression vector.

In another aspect, the present invention provides an engineered cell, which comprises the above-mentioned expression vector.

It should be noted that the present invention can also express the above expression vector through engineered cells, and the engineered cells can be cells known in the art that can be used for protein expression.

In another aspect, the present invention provides a product for detecting Haemophilus parasuis or diagnosing Haemophilus parasuis infection diseases, which comprises the above-mentioned Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen.

It should be noted that, based on the good immunogenicity of the above fusion antigen, it can be prepared into a product for detecting Haemophilus parasuis or diagnosing Haemophilus parasuis infection. The product can be in any form known in the art, such as a reagent or a kit.

Preferably, the above-mentioned Haemophilus parasuis includes type 1 strain and/or type 5 strain and/or type 13 strain.

Preferably, the above product can be an indirect ELISA reagent or an indirect ELISA kit, wherein the N-terminus of the IRP protein of Haemophilus parasuis and the fusion antigen of the RLPB protein are the coating antigens.

In another aspect, the present invention provides a recombinant subunit vaccine for preventing and treating Haemophilus parasuis infection diseases, which comprises the above-mentioned Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen or the above-mentioned nucleic acid molecule or the above-mentioned expression vector or the above-mentioned engineered cell.

It should be noted that the fusion antigen of the N-terminus of the IRP protein of Haemophilus parasuis and the RLPB protein provided by the present invention was used to immunize guinea pigs to obtain a protective effect against Haemophilus parasuis type 1 strain, type 5 strain and type 13 strain. Therefore, the fusion antigen of the present invention can be prepared into a recombinant subunit vaccine to prevent and treat Haemophilus parasuis infection diseases.

In another aspect, the present invention provides an application of the above-mentioned Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen or the above-mentioned nucleic acid molecule or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of products for detecting Haemophilus parasuis or diagnosing porcine Gramsciosis.

Preferably, the product in the above application may be a reagent or a kit.

Preferably, the product in the above application may be an indirect ELISA reagent or an indirect ELISA kit, wherein the fusion antigen of Haemophilus parasuis is the coating antigen.

In another aspect, the present invention provides an application of the above-mentioned Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen or the above-mentioned nucleic acid molecule or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a recombinant subunit vaccine for preventing and treating Haemophilus parasuis infection diseases.

It should be noted that compared with traditional inactivated vaccines, recombinant subunit vaccines have the advantages of low toxicity, high effective antigen content and strong cross-protection.

Preferably, the above-mentioned Haemophilus parasuis includes type 1 strain and/or type 5 strain and/or type 13 strain.

Preferably, the above-mentioned disease for preventing and treating Haemophilus parasuis infection is porcine Gramsciosis.

In another aspect, the present invention provides a method for detecting antibodies against Haemophilus parasuis, comprising an indirect ELISA method constructed using the above-mentioned Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen as a coating antigen for detection.

The beneficial effects of the present invention include at least:

(1) The reactivity of the fusion antigen of the N-terminus of the IRP protein of Haemophilus parasuis and the RLPB protein provided by the present invention was identified by Western-Blot, and it was found that the fusion protein antigen could be recognized by serum positive for Haemophilus parasuis antibodies.

(2) The indirect ELISA method constructed based on the fusion antigen (as coating antigen) of the N-terminus of the IRP protein of Haemophilus parasuis provided by the present invention and the RLPB protein has excellent specificity, sensitivity and repeatability for Haemophilus parasuis antibodies.

(3) Immunizing guinea pigs with the fusion antigen of the N-terminus of the IRP protein of Haemophilus parasuis and the RLPB protein provided by the present invention obtained a protective effect against attack by Haemophilus parasuis type 1, type 5 and type 13 strains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the amplification and identification results of the N-terminal gene sequence of the GPS-IRP protein, wherein M: DL5000 DNA Mark, 1, 2: amplified N-terminal gene sequence of the GPS-IRP protein (1287 bp);

FIG2 is the amplification and identification results of the GPS-RLPB protein gene sequence, wherein M: DL5000 DNA Mark, 1, 2: amplified GPS-RLPB protein gene sequence (495 bp);

Figure 3 shows the results of overlapping PCR splicing of GPS-IRP protein N-terminal gene and RLPB protein gene sequences, where M: DL5000 DNA Mark, 1, 2: spliced GPS-IRP protein N-terminal gene + RLPB protein gene sequence;

Figure 4 is the result of PCR identification of constructed Pet28a-(IRP protein N terminus + RLPB) plasmid and strain, wherein, M: DL2000DNA Mark, 1: empty vector Pet28a plasmid, 2: constructed Pet28a-(IRP protein N terminus + RLPB) plasmid, 3, 4: screening of BL21(DE3) strain containing Pet28a-(IRP protein N terminus + RLPB) plasmid;

FIG5 is the result of SDS-PAGE identification of the expressed and purified GPS-IRP protein N-terminus and RLPB protein fusion antigen, wherein M: protein marker, 1: expressed and purified Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen;

Figure 6 shows the results of Western-blot identification of the reactivity of the GPS-IRP protein N-terminus and the RLPB protein fusion antigen, where M: pre-stained protein marker, 1: expressed and purified Haemophilus parasuis IRP protein N-terminus and RLPB protein fusion antigen.

Detailed ways

The examples are provided to better illustrate the present invention, but the content of the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above content of the invention, which still fall within the protection scope of the present invention.

The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. 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, parts, elements, materials or combinations thereof may exist or may be added. As used herein, "/" may be interpreted as "and" or "or", depending on the circumstances.

In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

1. Amplification and splicing of the N-terminal gene sequence of the IRP protein of Haemophilus parasuis and the RLPB gene sequence

(I) Primer design for the N-terminal gene sequence of IRP protein and RLPB gene sequence of Haemophilus parasuis

According to the complete gene sequence of the GPS-SH0165 strain logged in GenBank, with the sequence number CP-001321, a pair of primers IRP protein N-terminal-F and IRP protein N-terminal-R were designed to amplify the N-terminal gene sequence of the surface immune protective antigen IRP protein of Haemophilus parasuis, wherein the primer IRP protein N-terminal-F was added with a BamHⅠ restriction enzyme site (underlined) and protective bases (see Table 1 below); a pair of primers RLPB-F and RLPB-R were designed to amplify the immune protective antigen RLPB gene sequence of Haemophilus parasuis, wherein the primer RLPB-R was added with an XhoⅠ restriction enzyme site (underlined) and protective bases (see Table 1 below); wherein the primers IRP protein N-terminal-R and RLPB-F were reverse complementary sequences, which facilitated the splicing of the N-terminal gene and RLPB gene sequences of Haemophilus parasuis IRP protein by overlapping PCR.

Table 1 Primers for the N-terminal gene sequence of IRP protein of Haemophilus parasuis and the RLPB gene sequence

name sequence IRP protein N-terminal-F 5'-cgggatccatgattaaaaacaaaacaac-3' (BamHI restriction site) IRP protein N-terminal 5'-agagaagagtttttttaacattgtattagcgctgctttgca-3' RLPB-F 5'-tgcaaagcagcgctaatacaatgttaaaaaaactctctctct-3' RLPB-R 5'-ggctcgagttatttcgtatttgctaact-3'(XhoⅠ restriction site)

(II) PCR amplification of the N-terminal gene sequence of Haemophilus parasuis IRP protein

The wild-type strain of GPS serotype 5 was used as a template to PCR amplify a 1287 bp fragment of the N-terminal gene sequence of the IRP protein. After the amplification, it was identified using a 1% agarose gel, and the 1287 bp specific target fragment of the N-terminal gene sequence of the IRP protein was purified and recovered (see Figure 1). The gene sequence is shown in SEQ ID NO: 3; wherein, the PCR amplification conditions are shown in Table 2 below.

Table 2 PCR amplification conditions for the N-terminal gene sequence of Haemophilus parasuis IRP protein

(III) Amplification of RLPB gene sequence of Haemophilus parasuis

The 495bp fragment of the RLPB protein gene sequence was amplified using the GPS serotype 5 wild-type strain as a template. After the amplification, it was identified using a 1% agarose gel, and the 495bp specific target fragment of the RLPB gene sequence was purified and recovered (see Figure 2). The gene sequence is shown in SEQ ID NO: 4; wherein, the PCR amplification conditions are shown in Table 3 below.

Table 3 PCR amplification conditions for the RLPB protein gene sequence of Haemophilus parasuis

(IV) Overlapping PCR splicing of GPS-IRP protein N-terminal gene sequence and GPS-RLPB gene sequence

The GPS-IRP protein N-terminal gene sequence and the GPS-RLPB gene sequence amplified and purified and recovered above were spliced by overlapping PCR. After the overlapping PCR was completed, the fragment was identified by using a 1% agarose gel, and the fusion gene sequence fragment of the IRP protein N-terminal gene and the RLPB gene of Haemophilus parasuis was purified and recovered, with a size of 1782 bp (see Figure 3). The overlapping PCR specifically includes:

①The first step PCR reaction:

2 μL of the purified IRP protein N-terminal gene sequence and RLPB gene sequence were amplified, 2× PrimeSTAR maxDNA Polymerase 25 μL, ddH ₂ O 21 μL, and the total volume was 50 μL, and then divided equally into two PCR tubes, and the first step of PCR amplification was performed according to the system of 25 μL per tube. The PCR reaction conditions were: denaturation at 95°C for 5 min, and then entering the cycle, the cycle parameters were: 94°C for 40 s, 54°C for 20 s, 72°C for 40 s, and after 10 cycles, 72°C for 10 min;

②The second step PCR reaction:

After the first PCR reaction, the second PCR reaction was carried out. First, 50 μL of amplification system was prepared: 2 μL of 100 μmol/L IRP protein N-terminal-F primer, 2 μL of 100 μmol/L RLPB-R primer, 25 μL of 2× PrimeSTAR max DNA Polymerase, and 21 μL of ddH ₂ O. Then, 25 μL of the system in each tube was equally divided into the two tubes where the first PCR reaction was completed, and the second PCR amplification was carried out. The PCR reaction conditions were: denaturation at 95°C for 5 min before entering the cycle, and the cycle parameters were: 94°C for 40 s, 54°C for 20 s, 72°C for 1 min, and after 30 cycles, 72°C for 10 min.

2. Construction of the expression vector for the fusion sequence of the N-terminal and RLPB protein genes of Haemophilus parasuis IRP protein

(1) The N-terminal gene of the IRP protein and the RLPB protein gene splicing sequence purified and recovered above were double-digested with BamHⅠ and XhoⅠ, and the plasmid vector Pet28a was double-digested with BamHⅠ and XhoⅠ;

(2) Then the IRP protein N-terminal gene and the RLPB gene splicing sequence fragment were connected to the plasmid vector Pet28a at 4° C. for 16 hours; wherein the connection system is shown in Table 4 below;

Table 4 Connection system for connection with plasmid vector Pet28a

(3) After ligation, heat stress transformation was performed into BL21 (DE3) competent cells, and the cells were spread on LB plates supplemented with kanamycin at a final concentration of 50 μg/mL, cultured at 37°C for 16 h, and single colonies were picked and inoculated into LB liquid medium containing 50 μg/mL kanamycin, and the plasmids were extracted for identification;

(4) PCR screening was used to obtain the BL21 (DE3) strain containing the positive construction plasmid Pet28a-(IRP protein N terminus + RLPB) (see Figure 4 below).

3. Expression, purification and reactogenicity identification of the fusion antigen of the N-terminal of the IRP protein and the RLPB protein of Haemophilus parasuis

(I) Expression and purification of the fusion antigen of the N-terminal of the IRP protein and the RLPB protein of Haemophilus parasuis

(1) After overnight culture of a single E. coli BL21/pET28a-(IRP protein N terminus + RLPB), transfer the culture to a freshly prepared LB liquid medium containing kanamycin (50 μg/ml) at a 1% inoculum volume, culture at 37°C, 240 rpm for about 2 h, and add IPTG to a final concentration of 0.8 mmol/L. Transfer _the culture to 37°C, 240 rpm for 7 h, and collect the cells by centrifugation.

(2) Wash the cells three times with PBS buffer, collect the cells by centrifugation for the last time, and suspend the cells in Lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH=8.0);

(3) Add lysozyme to a final concentration of 1.5 mg/mL, place on ice for 30 min, sonicate the cells (400 W power, 6 s sonication time, 5 s interval, 35 min sonication), centrifuge at 10,000 rpm for 20 min at 4°C, and collect the supernatant;

(4) Filter the supernatant of the bacterial solution after centrifugation with a 0.45 μm filter, take 5 mL and add it to an affinity chromatography column containing 50% Ni-NTA, shake and mix at 200 r/min for 1 h, then pass it through the column, and after the liquid is drained, add 5 mL of Wash buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 50 mM imidazole, pH=8.0) and wash twice;

(5) Finally, 0.5 mL of Elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 300 mM Mimidazole, pH=8.0) was added to elute and collect the target protein. The concentration of the purified fusion protein antigen was determined to be 2.1 mg/mL, and SDS-PAGE electrophoresis was performed for identification. The results are shown in Figure 5 below. A specific protein band of approximately 83.8 KD was obtained, which is consistent with the expected size (Figure 5 below).

(II) Identification of the reactivity of the fusion antigen of the N-terminal of the IRP protein and the RLPB protein of Haemophilus parasuis

After the SDS-PAGE electrophoresis, Western-Blot was performed to identify the reactivity of the protein, which specifically included the following steps:

(1) Pre-cut PVDF membrane and filter paper of the same size as the gel strip and immerse them in electrotransfer buffer; remove the gel, balance it in transfer buffer, and then lay the membrane, gel and filter paper in order, i.e., filter paper - PVDF membrane - gel - filter paper; seal it tightly and place it in the electrotransfer tank, add electrotransfer solution, connect the cooling device, and transfer at 400mA constant current for 30min;

(2) After the transfer is completed, remove the PVDF membrane and wash it with PBST for 10 min × 3 times;

(3) Place the PVDF membrane in 5% skim milk and block it at 4°C overnight. Discard the blocking solution and wash the membrane with PBST for 10 min × 3 times.

(4) Add the primary antibody, i.e., Haemophilus parasuis antibody-positive pig serum diluted with PBS (1:50), shake steadily at room temperature for 1 h, discard the primary antibody, and wash the membrane with PBST for 10 min × 4 times;

(5) Add horseradish peroxidase-labeled goat anti-swine enzyme-labeled secondary antibody, dilute with PBS at a ratio of 1:5000, shake steadily at room temperature for 1 h, discard the secondary antibody, and wash the membrane with PBST for 10 min × 3 times;

(6) The PVDF membrane treated with the secondary antibody was placed in a color developing solution for color development. After the specific reaction band appeared, the reaction was terminated and photographed for preservation. The results of Western-blot analysis showed that the N-terminus of the IRP protein of Haemophilus parasuis and the RLPB fusion protein antigen could be recognized by the Haemophilus parasuis antibody-positive serum (as shown in Figure 6 below).

IV. Application of the fusion antigen of the N-terminal of IRP protein and RLPB protein of Haemophilus parasuis in serological detection

(I) Construction of indirect ELISA method

An indirect ELISA method for detecting Haemophilus parasuis antibodies was established using the expressed and purified N-terminal of the IRP protein of Haemophilus parasuis and the fusion antigen of the RLPB protein, which specifically includes the following steps:

(1) The expressed and purified IRP protein N-terminus and RLPB protein fusion antigen were diluted to 1.0 μg/mL with coating solution (0.05 mol/L carbonate buffer pH 9.6), added to the ELISA plate at a volume of 100 μL/well, and coated at 4°C overnight;

(2) Add 300 μL of PBST to each well, wash twice, pat dry, and block with 100 μL/well of blocking solution (0.01 g/mL BSA) at 37°C for 2 h;

(3) Add 300 μL of PBST to each well, wash twice, pat dry, add 100 μL of 1:100 diluted swine serum to be tested to each well, set up two wells (100 μL/well) for positive control samples of Haemophilus parasuis antibodies, and two wells (100 μL/well) for negative control samples, incubate at 37°C for 30 min, and then discard the liquid in the wells;

(4) Add 300 μL of PBST to each well, wash 5 times, pat dry, add 100 μL of 1:50,000 diluted HRP-labeled goat anti-pig IgG to each well, incubate at 37°C for 30 min, and then discard the liquid in the well;

(5) Add 300 μL of PBST to each well, wash 5 times, pat dry, add 100 μL of TMB single-component colorimetric solution to each well, and color for 10 min in the dark;

(6) Finally, 100 μL 2 mol/L H ₂ SO ₄ was added to each well to stop the color development, and the OD _450nm value was measured with an enzyme-labeled instrument. The result judgment criteria of the Haemophilus parasuis antibody detection method are: when the negative control OD _450nm average value (N) is less than 0.25, the positive control OD _450nm average value (P) is greater than 0.70, the sample OD _450nm value (S)/negative control OD _450nm average value (N) ≥ 2.1 is judged as positive, and the sample OD _450nm value (S)/negative control OD _450nm average value (N) < 2.1 is judged as negative.

(II) Determination of specificity, sensitivity and repeatability of the constructed indirect ELISA method

(1) Specificity determination of the constructed indirect ELISA method

The indirect ELISA method constructed as above was used to detect the positive pig sera for antibodies to porcine contagious pleuropneumonia, porcine pasteurellosis, porcine streptococcal disease, porcine blue ear virus, porcine circovirus, porcine foot-and-mouth disease virus, classical swine fever virus, porcine Japanese encephalitis virus, and porcine pseudorabies virus. The results were all negative, indicating that the detection method has good specificity. The results are shown in Table 5 below.

Table 5 Results of indirect ELISA for detection of positive serum for common swine diseases

Note: The average value of the positive control OD _450nm in this test is 1.168, and the average value of the negative control OD _450nm is 0.186. When S/N ≥ 2.1, it is positive, and when S/N ﹤ 2.1, it is negative. "+": the result is positive, "-": the result is negative.

(2) Determination of the sensitivity of the constructed indirect ELISA method

Six sera from pigs positive for Haemophilus parasuis antibodies were diluted at 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200, and tested using the above-mentioned indirect ELISA method and the BioChek GPS-OppA antibody ELISA detection kit. The results showed that the serum titer detected by this detection method was 1:800-1:1600: the highest titer of the serum detected by the BioChek GPS-OppA antibody kit was 1:400-1:800. The results are shown in Tables 6 and 7.

Table 6 Detection results of different dilution ratios of Haemophilus parasuis antibody-positive pig serum using the detection method established using the IRP protein N-terminus and RLPB protein fusion antigen

Note: The average value of the positive control OD _450nm in this test is 1.187, and the average value of the negative control OD _450nm is 0.189. When S/N ≥ 2.1, it is positive, and when S/N ﹤ 2.1, it is negative. "+": the result is positive, "-": the result is negative.

Table 7 Test results of different dilution ratios of HPS-OppA antibody positive pig serum by the Dutch 100Test HPS-OppA antibody kit

Note: The average value of the positive control OD _405nm in this test is 0.856, and the average value of the negative control OD _405nm is 0.264. The difference between the average value of the positive control and the average value of the negative control is greater than 0.15. The sample OD ₄₀₅ nm value/positive control OD _{405 nm} value ≥ 0.5 is judged as positive, otherwise it is judged as negative, "+": the result is positive, "-": the result is negative.

(3) Repeatability determination of the constructed indirect ELISA method

① Determination of intra-batch repeatability of the constructed indirect ELISA method

The detection method was used to detect 30 swine sera with known backgrounds from the same batch, including 15 sera positive for Haemophilus parasuis antibodies and 15 sera negative for Haemophilus parasuis antibodies; each of the 30 serum samples was tested three times, and the results showed that the intra-batch coefficient of variation was between 0.63% and 9.64% (see Table 8), indicating that the kit had good intra-batch repeatability.

Table 8 Results of testing pig serum samples using the same batch of testing methods

Note: The average value of positive control OD _450nm detected by different coated plates is 1.184, and the average value of negative control OD _450nm is 0.196. S/N ≥ 2.1 is positive, and S/N ﹤ 2.1 is negative. "+": the result is positive, "-": the result is negative.

②Batch reproducibility test

Different batches of this detection method were used to detect 30 swine sera with known background, including 15 sera positive for Haemophilus parasuis antibodies and 15 sera negative for Haemophilus parasuis antibodies; each of the 30 swine serum samples was tested simultaneously using three batches of the detection method, and the results showed that the batch-to-batch coefficient of variation was between 0.50% and 9.30% (see Table 9), indicating that the kit has good batch-to-batch repeatability.

Table 9 Results of different batches of detection methods for pig serum samples

Note: The average value of positive control OD _450nm of different batches tested is 1.084, and the average value of negative control OD _450nm is 0.186. S/N≥2.1 is positive, and S/N﹤2.1 is negative. "+": the result is positive, "-": the result is negative.

(III) Comparative study on clinical application of the constructed indirect ELISA method and similar detection methods

The above-constructed IRP protein N-terminal and RLPB protein fusion antigen detection method (the above-constructed indirect ELISA method) was compared with the kit produced by the Dutch Baice Company to detect 120 clinical pig sera; the results are shown in Table 10. Among the 120 sera detected by the antibody ELISA detection kit for the protein fusion antigen of Haemophilus parasuis constructed by the present invention, the positive rate was 41.67% (50/120) and the negative rate was 58.33% (70/120); the positive rate detected by the Haemophilus parasuis (GPS-OppA) antibody kit produced by the Dutch Baice Company was 38.33% (46/120) and the negative rate was 61.67% (74/120); the positive coincidence rate of the two was 92.00% (46/50), the negative coincidence rate was 94.59% (70/74), and the total coincidence rate was 96.67% (116/120).

Table 10 Comparative test of the constructed indirect ELISA method and similar products

5. The immune protection effect of the fusion antigen subunit vaccine of the N-terminal of IRP protein and RLPB protein of Haemophilus parasuis against different serotypes of Haemophilus parasuis

(1) A total of 54 clean-grade female guinea pigs (weighing 1.5-2 kg/guinea pig) were purchased and randomly divided into 9 groups, with 6 guinea pigs in each group. Each guinea pig in Groups 1, 2, and 3 was immunized with the N-terminal and RLPB protein fusion antigens of Haemophilus parasuis IRP protein. Groups 4, 5, and 6 were immunized with whole-bacterium inactivated vaccines prepared from Haemophilus parasuis type 1 G05 strain, type 5 G06 strain, and type 13 G08 strain, respectively, as positive controls. Groups 7, 8, and 9 were immunized with PBS+adjuvant as negative controls.

(2) Immunization was performed by multiple subcutaneous injections at the back. In the first, second and third groups, the expressed and purified fusion antigens of IRP protein N-terminal and RLPB protein of Haemophilus parasuis were mixed and emulsified with an equal amount of Freund's complete adjuvant, and each guinea pig was inoculated with 0.5 mL of the fusion antigen, with the content of the inoculated fusion antigen being 18 μg/guinea pig. The positive control groups of the fourth, fifth and sixth groups were immunized with 0.5 mL of the whole-bacterial inactivated vaccine of Haemophilus parasuis type 1 G05 strain, type 5 G06 strain and type 13 G08 strain, respectively. The negative control groups of the seventh, eighth and ninth groups were immunized with 0.5 mL of the mixture of PBS and Freund's complete adjuvant emulsified/guinea pig. In the subsequent immunizations, the corresponding antigens were mixed and emulsified with Freund's incomplete adjuvant, and the immunization was performed according to the same immunization dose and method. Each immunization was separated by 15 days. On the 10th day after the second immunization, blood was collected to separate the serum for the determination of antibody titer.

(3) The ELISA plate (100 μL/well) was coated with the expressed and purified N-terminal and RLPB protein fusion antigens of Haemophilus parasuis IRP protein (1.0 μg/mL), incubated at 4°C overnight, washed three times with PBST, blocked with 0.01 g/mL BSA at 100 μL/well, incubated at 37°C for 2 h, washed three times with PBST, guinea pig serum was diluted 1:1000 with PBS, 100 μL/well, incubated at 37°C for 30 min, washed three times with PBST; HRP-labeled goat anti-guinea pig enzyme-labeled secondary antibody was diluted 1:20000, 100 μL/well, incubated at 37°C for 30 min, washed three times with PBST; TMP single-component substrate was added at 100 μL/well, color developed at 37°C for 10 min, and the reaction was terminated with 2 M sulfuric acid stop solution at 100 μL/well; OD was read using an ELISA reader. _450nm value; the results are shown in Table 11.

Table 11 Antibody detection results of guinea pig IRP protein N-terminal and RLPB protein fusion antigen in each experimental group

Note: The average value of the positive control OD _450nm in this test is 1.168, and the average value of the negative control OD _450nm is 0.184. When S/N ≥ 2.1, it is positive, and when S/N ﹤ 2.1, it is negative. "+": the result is positive, "-": the result is negative.

It can be seen from Table 11 that the serum antibodies of guinea pigs immunized with the N-terminus of IRP protein and RLPB protein fusion antigens of Haemophilus parasuis were all positive, with an average OD _450nm of 2.461; the serum antibodies of guinea pigs immunized with the whole-bacterium inactivated vaccine of Haemophilus parasuis were also positive, with an average OD _450nm of 1.144; the antibody level of the group immunized with the N-terminus of IRP protein and RLPB protein fusion antigens was significantly higher than that of the whole-bacterium inactivated vaccine; the guinea pig IRP protein N-terminus and RLPB protein fusion antibodies in the negative control group were all negative, with an average OD _450nm of 0.211.

(4) Then, the corresponding immunized guinea pigs were challenged with Haemophilus parasuis type 1 G05 strain (3.45×10 ⁹ CFU/pig), type 5 G06 strain (1.98×10 ⁹ CFU/pig), and type 13 G08 strain (4.15×10 ⁹ CFU/pig) by intraperitoneal injection, and the protection rate of the guinea pigs was observed after the challenge. The results are shown in Table 12.

Table 12 Survival protection rate of guinea pigs in each immunization group

It can be seen from Table 12 above that the protection rate of guinea pigs immunized with the IRP protein N-terminal and RLPB protein fusion antigens against type 1 G05 strain, type 5 G06 strain, and type 13 G08 strain was 83.33%; the corresponding protection rate of guinea pigs immunized with the whole-bacterial inactivated vaccine of Haemophilus parasuis against type 1 G05 strain, type 5 G06 strain, and type 13 G08 strain was 66.67%. The protection rate of the group immunized with the IRP protein N-terminal and RLPB protein fusion antigens against different serotypes of Haemophilus parasuis immunization was higher than that of the group immunized with the whole-bacterial inactivated vaccine of Haemophilus parasuis.

In addition, the guinea pigs in the negative control immunization group showed obvious clinical symptoms after the virus attack, including accelerated breathing, depression, and rough fur, and all died during the observation period.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims (10)

1. The fusion antigen of the N-terminal of the haemophilus parasuis IRP protein and RLPB protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A nucleic acid molecule encoding the fusion antigen of the N-terminus of the IRP protein of haemophilus parasuis and the RLPB protein of claim 1.

3. The nucleic acid molecule according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 2.

4. A product for detecting haemophilus parasuis or diagnosing haemophilus parasuis infection diseases, comprising the haemophilus parasuis IRP protein N-terminal and RLPB protein fusion antigen as defined in claim 1.

5. The product according to claim 4, wherein the product is an indirect ELISA reagent or an indirect ELISA kit, and wherein the fusion antigen of the N-terminal of the IRP protein and RLPB protein of haemophilus parasuis is a coating antigen.

6. A recombinant subunit vaccine for preventing and treating haemophilus parasuis infection disease, comprising the haemophilus parasuis IRP protein N-terminal and RLPB protein fusion antigen of claim 1 or the nucleic acid molecule of claim 2 or 3.

7. Use of the fusion antigen of the N-terminal of the haemophilus parasuis IRP protein of claim 1 and RLPB protein or the nucleic acid molecule of claim 2 or 3 for preparing a product for detecting haemophilus parasuis or diagnosing haemophilus parasuis infection diseases.

8. Use of the fusion antigen of the N-terminal of the haemophilus parasuis IRP protein of claim 1 and RLPB protein or the nucleic acid molecule of claim 2 or 3 for preparing recombinant subunit vaccine for preventing and treating haemophilus parasuis infection diseases.

9. The use according to claim 8, characterized in that haemophilus parasuis comprises a strain of type 1 and/or a strain of type 5 and/or a strain of type 13.

10. The use according to claim 8 or 9, wherein the haemophilus parasuis infection disease is porcine leather RASER's disease.