A protective subunit antigen Ptsg of Haemophilus parasuis, antigen combination, subunit vaccine and application thereof

Abstract

The present invention is applicable to the technical field of veterinary vaccines, and provides a subunit antigen Ptsg, an antigen combination, a subunit vaccine and an application thereof for Haemophilus parasuis immune protection. The amino acid sequences of the antigens Ptsg, Vacj, GAPDH and PalA are shown in SEQ ID NO.1 to SEQ ID NO.4, respectively, and the nucleotide sequences encoding the antigens Ptsg, Vacj, GAPDH and PalA are shown in SEQ ID NO.5 to SEQ ID NO.8, respectively. The subunit vaccine comprises an antigen combination and an adjuvant MontanideTM Gel 01. The subunit vaccine can be used in the preparation of drugs for preventing and treating Haemophilus parasuis. The present invention lays a foundation for the development of an efficient, low-cost and broad-spectrum subunit vaccine for Haemophilus parasuis, provides an effective technical means for the prevention and treatment of the disease, and has important application value and promotion prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to an immune protective subunit antigen Ptsg of Haemophilus parasuis, an antigen combination, a subunit vaccine and applications thereof. Background Art

[0002] Haemophilus parasuis disease, also known as polyfibrinous serositis and arthritis, can cause Glasser's disease in pigs, and its clinical features include fever, joint swelling, dyspnea, polyserositis, arthritis and high mortality. This disease specifically infects pigs, mainly affecting young pigs from 2 weeks to 4 months old, especially common in pigs around weaning and during the nursery stage, that is, pigs aged 5 to 8 weeks. Its incidence rate is generally 10-15%, and the mortality rate can reach 50% in severe cases, which seriously endangers the health of piglets and fattening pigs. In recent years, the disease has been widely prevalent in my country, and Haemophilus parasuis is often mixed with other viruses and bacteria, especially some respiratory pathogens, which leads to increased morbidity and mortality in pigs, causing huge economic losses to the pig industry and seriously hindering the development of my country's pig industry.

[0003] At present, Haemophilus parasuis is divided into 15 serotypes, and the prevalent serotypes in different countries are slightly different. In my country, types 4, 5, 12, and 13 are the main types. Although antibiotics are an effective means of preventing and treating bacterial diseases in pigs, the abuse of antibiotics has led to the gradual increase in bacterial resistance, and even the emergence of multidrug resistance, which has turned the effective method of preventing Haemophilus parasuis disease to vaccination. At present, the research on vaccines related to Haemophilus parasuis disease mainly includes inactivated vaccines and subunit vaccines. Among them, inactivated vaccines have been commercialized and have good protection against the same serotype, but the cross-protection effect between different serotypes is poor, or even no protection, and there are many problems such as insufficient safety, short protection time, and poor immune effect. Subunit vaccines are vaccines with immune protective components made from secretory proteins or outer membrane proteins of microorganisms. They have the advantages of safety and few side effects, and the body can produce high-level specific antibodies against subunit vaccines. The glucose transporter gene (ptsg) can encode a glucose-specific transporter (enzyme EIIBC) to transport glucose into cells and provide cells with the required carbon source. It has an important influence on the formation of biofilm and oxidative stress tolerance of Haemophilus parasuis, but there is no relevant research on its immune protection effect. At the same time, due to the weak immunogenicity of subunit vaccines, immunization with a single protein may not provide comprehensive protection. Therefore, a protective subunit antigen Ptsg of Haemophilus parasuis, an antigen combination, a subunit vaccine and its application are proposed. Summary of the invention

[0004] The purpose of the present invention is to provide an immunoprotective subunit antigen Ptsg of Haemophilus parasuis, an antigen combination, a subunit vaccine and applications thereof, aiming to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A combination of Haemophilus parasuis antigens, comprising antigen Ptsg, antigen Vacj and antigen GAPDH; wherein:

[0007] The amino acid sequence of the antigen Ptsg is shown in SEQ ID NO.1, and the nucleotide sequence encoding the antigen Ptsg is shown in SEQ ID NO.5;

[0008] The amino acid sequence of antigen Vacj is shown in SEQ ID NO.2, and the nucleotide sequence encoding antigen Vacj is shown in SEQ ID NO.6;

[0009] The amino acid sequence of the antigen GAPDH is shown in SEQ ID NO.3, and the nucleotide sequence encoding the antigen GAPDH is shown in SEQ ID NO.7.

[0010] Furthermore, the antigens Vacj, Ptsg and GAPDH are all obtained by prokaryotic expression, the expression host is E. coli BL21 (DE3), the expression vectors of the antigens Vacj and Ptsg are pET-32a vectors, and the expression vector of the antigen GAPDH is pET-28a vectors.

[0011] A subunit vaccine of Haemophilus parasuis, comprising the above-mentioned Haemophilus parasuis antigen combination and adjuvant MontanideTM Gel 01; in each dose of the vaccine, the contents of antigens Vacj, Ptsg and GAPDH are each within the range of 20-40 mg, and the added amount of adjuvant MontanideTM Gel 01 is 10% of the total volume.

[0012] A use of the above-mentioned Haemophilus parasuis subunit vaccine in the preparation of a drug for preventing and treating Haemophilus parasuis.

[0013] Application of an antigen Ptsg in the preparation of a subunit vaccine of Haemophilus parasuis.

[0014] The invention discloses an application of an antigen PalA in the preparation of a subunit vaccine of Haemophilus parasuis.

[0015] Application of an antigen GAPDH in the preparation of a subunit vaccine against Haemophilus parasuis.

[0016] Application of antigen Vacj and antigen Ptsg in the preparation of Haemophilus parasuis subunit vaccine.

[0017] The invention discloses an application of antigen Vacj, antigen GAPDH and antigen PalA in preparing a subunit vaccine of Haemophilus parasuis. The amino acid sequence of antigen PalA is shown in SEQ ID NO.4, and the nucleotide sequence encoding antigen PalA is shown in SEQ ID NO.8.

[0018] The invention discloses an application of antigen Vacj, antigen Ptsg and antigen PalA in preparing a subunit vaccine of Haemophilus parasuis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention explores the immune protection effect of the antigen Ptsg. As a newly discovered immunogenic protein, it can provide effective immune protection after mice are infected with Haemophilus parasuis, and can be used as a candidate antigen for the development of Haemophilus parasuis subunit vaccines.

[0021] 2. The present invention evaluates the immune protection of single antigen proteins, combines antigen proteins with strong immune protection and cross-protection, determines the antigen combination scheme with the best protection effect (Vacj+Ptsg+GAPDH), and prepares it into a subunit vaccine with the adjuvant MontanideTM Gel 01, which can provide cross-protection against different serotypes of Haemophilus parasuis, and the protection rates against HPS4 and HPS5 strains are as high as 80% and 100%, respectively.

[0022] 3. The recombinant proteins selected in the antigen protein combination of the present invention have been optimized to successfully achieve soluble expression, are easy to express and purify in large quantities, and reduce production costs.

[0023] 4. The present invention lays a foundation for the development of a highly effective, low-cost, broad-spectrum subunit vaccine for Haemophilus parasuis, and provides an effective technical means for the prevention and treatment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 PCR amplification results, where: M-Marker; 1-Vacj; 2-Ptsg; 3-PalA; 4-GAPDH.

[0025] Figure 2 The results are from double enzyme digestion.

[0026] Figure 3 This is a diagram of recombinant protein purification, where: M-Marker; 1-before induction; 2-after induction; 3-supernatant; 4-precipitate; 5-outflow; 6-20mM imidazole eluent; 7-50mM imidazole eluent; 8-500mM imidazole eluent.

[0027] Figure 4 The immunoprotective effect of each protein group on HPS5.

[0028] Figure 5 The immunoprotective effect of each protein group on HPS4.

[0029] Figure 6 is the antibody level in mouse serum of each protein group.

[0030] Figure 7 This is the immune protection effect of each protein mixture group on HPS4.

[0031] Figure 8 This is the immune protection effect of each protein mixture group on HPS5.

[0032] Fig. 9 The antibody levels in the mouse serum of the multi-component protein group. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0035] The amino acid sequence of protein PalA is shown in SEQ ID NO.4, and the nucleotide sequence encoding protein PalA is shown in SEQ ID NO.8.

[0036] In the following experiments, HPS4 (serotype 4 of Haemophilus parasuis) and HPS5 (serotype 5 of Haemophilus parasuis) were isolated and stored in our laboratory.

[0037] Example 1, Construction of recombinant protein expression vector;

[0038] Primers were designed according to the gene sequence of the target protein in GenBank (primer sequences are shown in Table 1). The gene fragments of the target protein were amplified by PCR using the genome of the strain preserved in the laboratory as a template. The target fragments in the PCR product were recovered using a gel recovery kit, and 1 μl was taken to determine its concentration using an ultra-micro spectrophotometer. The target gene and vector pET-32a and pET-28a were double-digested with BamHI and XhoI (double-digestion system is shown in Table 2), and digested in a water bath at 37°C for 30 minutes. After completion, the product purification kit was used for purification. The target protein gene fragment obtained by gel recovery and purification was connected with pET-28a (pET-32a) at 4°C overnight to construct a recombinant plasmid. The ligation product was transformed into E. coli DH5a competent cells and cultured overnight at 37°C. The next day, several single colonies were picked from the plate and inoculated into LB resistance culture medium for expansion culture. Positive clones were identified by bacterial solution PCR, plasmids were extracted using a plasmid extraction kit, and PCR products were sent to Changchun Kumei Company for sequencing.

[0039]

[0040]

[0041] When HPS5 genome was used as template, the gene fragments of Vacj, Ptsg, PalA, and GAPDH target proteins were amplified using the primer sequences shown in Table 1. After PCR amplification, the results of nucleic acid electrophoresis showed that ( Figure 1 ), the band sizes of Vacj, Ptsg, PalA, and GAPDH were 687bp, 306bp, 450bp, and 1020bp, respectively, which were in line with expectations. Subsequently, the vector was constructed, and the amplified target gene fragments were cloned into pET-32a and pET-28a expression vectors, respectively. The sequencing results also showed that the target gene was cloned into the vector, and there was no mutation or frameshift in the sequence. In order to further verify the correctness of the recombinant plasmid, the four recombinant plasmids constructed were double-enzyme-cut and verified. Figure 2 The results showed that the fragments separated after double enzyme digestion were consistent with the gene fragment size, further proving that the gene fragments of the four target proteins, Vacj, Ptsg, PalA, and GAPDH, were correctly cloned into the corresponding vectors, indicating that the pET-32a-Vacj, pET-28a-Ptsg, pET-32a-PalA, and pET-32a-Vacj prokaryotic expression plasmids were correctly constructed.

[0042] Example 2: Induced expression and purification of recombinant protein;

[0043] The positive plasmid identified correctly was transferred into E.coli BL21(DE3) competent cells. A single colony was selected on the plate and inoculated into a liquid medium containing resistance. The culture was cultured overnight at 37°C. Then, the overnight culture solution was transferred to a new culture medium at a ratio of 1%, and cultured in a 37°C shaker until the OD of the culture solution was 0.6-0.8. The pre-induction culture solution was retained. Then, 1 / 1000 IPTG was added to induce expression (cultured at 16°C for 16h). After the induction, the post-induction culture solution was retained. The bacteria were then collected by centrifugation at 8000r for 10min. After the centrifugation, the supernatant was discarded and resuspended with 30-50ml buffer. The resuspended culture solution was placed in ice and ultrasonically broken (200W, 4s / time) until the culture solution became clear. The culture solution was then centrifuged at 12000r for 10min. After the centrifugation, the supernatant was collected and the precipitate was retained. Finally, the above four proteins (PalA, Vacj, GAPDH, and Ptsg) were purified using Ni column resin according to the instructions, and the purified proteins were analyzed by SDS-PAGE electrophoresis. The results are as follows Figure 3 As shown, proteins with sizes of 34.5 KDa, 41 KDa, 39.8 KDa and 30 KDa were obtained, respectively, which was in line with expectations.

[0044] Example 3, immune protection experiment of recombinant protein;

[0045] The four purified proteins were used for mouse immune protection experiments, and blank control group, adjuvant control group and commercial inactivated vaccine group were set up. The inactivated vaccine used was the inactivated vaccine of Haemophilus parasuis disease of Kofuning. There were 10 mice in each group. The prepared proteins were used to immunize the mice subcutaneously at multiple points on the back, and the operation was performed according to Table 3. The immunization was performed once every two weeks, for a total of two immunizations. The antibody levels in the serum of mice at 0, 14 and 28 days after immunization were detected by indirect ELISA. And 14 days after the second immunization, 5 mice in each group were infected with HPS4 (containing 3.4*10^9CFU / mouse), and the other 5 mice were infected with HPS5 (containing 2.4*10^9CFU / mouse), and the incidence and mortality of mice in each group were observed.

[0046]

[0047] Figure 5 The results showed that after HPS4 infection, all the adjuvant group died, while the survival rates of the control group and the commercial inactivated vaccine group were both 40%. The immune protection effects of PalA, GAPDH, Ptsg and Vacj were all good, with survival rates of 75%, 60%, 100% and 75% respectively. Figure 4The results showed that after HPS5 infection, all the inactivated vaccine group died, and the survival rate of the control group and adjuvant group was only 20%. The protection rates of the PalA, GAPDH, Ptsg and Vacj immunization groups against HPS5 were 50%, 40%, 75% and 100%, respectively. It can be seen that each group of proteins can provide a certain protective effect against HPS type 4 and type 5. The serum antibody level of mice before infection was detected by indirect ELISA. Figure 6 The results showed that as time went by, the serum antibody levels of mice in each immunization group gradually increased, significantly higher than those in the control group and adjuvant group. It can also be seen that the specific antibody levels of Ptsg and Vacj were significantly higher than those in other groups, which is consistent with the results of the immune protection experiment. Among them, Ptsg, as a new antigen protein of Haemophilus parasuis, showed a good immune protection effect and had good immunogenicity.

[0048] Example 4: Immune protection experiment of multi-component recombinant protein;

[0049] During the research, it was found that a specific combination of virulence proteins of Haemophilus parasuis can achieve significantly improved immunogenicity. In order to seek a more stable and better immune protection effect, the above-screened proteins with a certain protective effect on HPS were mixed in a certain proportion in this example, and the next round of immune protection effect screening experiments were carried out on these mixed groups. The specific grouping is shown in Table 4, the infection dose is the same as in Example 3, and the survival rate and serum antibody titer of each group of mice are monitored after infection, and the specific method is the same as in 3.

[0050]

[0051] Figure 7 The results showed that after HPS4 infection, all the mice in the control group and the adjuvant group died, the survival rate of the commercial inactivated vaccine group was 20%, and the protection rates of Vacj+Ptsg, Vacj+Ptsg+PalA, Ptsg+GAPDH+PalA, Vacj+Ptsg+GAPDH, and Vacj+GAPDH+PalA for the immunized mice were 60%, 60%, 40%, 80%, and 50%, respectively. Figure 8 The results showed that after HPS5 challenge, all the mice in the control group and the commercial inactivated vaccine group died, the survival rate of the adjuvant group was 40%, and the protection rates of Vacj+Ptsg, Vacj+Ptsg+PalA, Ptsg+GAPDH+PalA, Vacj+Ptsg+GAPDH, and Vacj+GAPDH+PalA to the immunized mice were 50%, 80%, 40%, 100%, and 60%, respectively. In addition, the serum antibody levels of mice before challenge were detected by indirect ELISA. Fig. 9The results showed that each component in the immunization group could produce high levels of specific antibodies. Compared with the antibody level after the first immunization for 14 days, the antibody level after the second immunization for 14 days was significantly increased, indicating that each immunization group had good immunogenicity and could induce high levels of specific antibodies. Overall, the Vacj+Ptsg+GAPDH group had the best immune protection effect, providing 80% and 100% protection rates for HPS4 and HPS5, respectively. Therefore, Vacj+Ptsg+GAPDH was the best candidate combination antigen.

[0052] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A combination of Haemophilus parasuis antigens, characterized in that: It contains antigen Ptsg, antigen Vacj and antigen GAPDH; wherein: The amino acid sequence of the antigen Ptsg is shown in SEQ ID NO.1, and the nucleotide sequence encoding the antigen Ptsg is shown in SEQ ID NO.5; The amino acid sequence of antigen Vacj is shown in SEQ ID NO.2, and the nucleotide sequence encoding antigen Vacj is shown in SEQ ID NO.6; The amino acid sequence of the antigen GAPDH is shown in SEQ ID NO.3, and the nucleotide sequence encoding the antigen GAPDH is shown in SEQ ID NO.

7.

2. A subunit vaccine of Haemophilus parasuis, comprising the Haemophilus parasuis antigen combination as claimed in claim 1 and an adjuvant MontanideTM Gel 01.

3. The subunit vaccine of Haemophilus parasuis according to claim 2, characterized in that In each dose of vaccine, the content of antigens Vacj, Ptsg and GAPDH is within the range of 20-40 mg respectively, and the added amount of adjuvant MontanideTM Gel 01 is 10% of the total volume.

4. Use of the Haemophilus parasuis subunit vaccine according to claim 3 in the preparation of drugs for preventing and treating Haemophilus parasuis.

5. Use of an antigen Ptsg in the preparation of a subunit vaccine of Haemophilus parasuis, characterized in that: The amino acid sequence of the antigen Ptsg is shown in SEQ ID NO.1, and the nucleotide sequence encoding the antigen Ptsg is shown in SEQ ID NO.

5.

6. An application of antigen Vacj and antigen Ptsg in the preparation of a subunit vaccine of Haemophilus parasuis, characterized in that: The amino acid sequence of antigen Vacj is shown in SEQ ID NO.2, and the nucleotide sequence encoding antigen Vacj is shown in SEQ ID NO.6; the amino acid sequence of antigen Ptsg is shown in SEQ ID NO.1, and the nucleotide sequence encoding antigen Ptsg is shown in SEQ ID NO.

5.

7. Use of antigen Vacj, antigen Ptsg and antigen PalA in preparing a subunit vaccine of Haemophilus parasuis, characterized in that: The amino acid sequence of antigen Vacj is shown in SEQ ID NO.2, and the nucleotide sequence encoding antigen Vacj is shown in SEQ ID NO.6; the amino acid sequence of antigen Ptsg is shown in SEQ ID NO.1, and the nucleotide sequence encoding antigen Ptsg is shown in SEQ ID NO.5; the amino acid sequence of antigen PalA is shown in SEQ ID NO.4, and the nucleotide sequence encoding antigen PalA is shown in SEQ ID NO.8.

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