Poultry pasteurella multocida bivalent subunit vaccine as well as preparation method and application thereof

Abstract

The invention discloses a poultry pasteurella multocida bivalent subunit vaccine as well as a preparation method and application thereof. The poultry pasteurella multocida bivalent subunit vaccine is prepared by mixing and emulsifying a protein mixed solution consisting of PpdD-C1 protein and PpdD-C2 protein and a Freund's complete adjuvant. It is found for the first time that avian pasteurella multocida IV type pili of different serotypes are only divided into two types, and the bivalent subunit vaccine prepared based on the two types of IV type pili protein can provide a good protection rate for all the serotypes of avian pasteurella multocida. Compared with the existing vaccine, the divalent subunit vaccine prepared by the invention has the advantages of good cross protection, high safety and no endotoxin, and the stress reaction on the organism is obviously reduced.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a bivalent subunit vaccine of avian Pasteurella multocida and a preparation method and application thereof. Background Art

[0002] Pasteurella multocida ( Pasteurella multocida Pasteurella multocida (PM) is a zoonotic pathogen with a wide range of hosts, including poultry, pigs, cattle, sheep, rabbits, dogs, cats, marine mammals, etc. (Pak). After animals are infected with Pasteurella multocida, the main sources of infection are the infected animals, their excrement, secretions, and contaminated feed. It is mainly transmitted through the respiratory and digestive tracts, as well as damaged mucous membranes and skin. It can cause infection in animals and humans through bites, scratches, or licking of wounds by infected dogs or cats. Clinical symptoms mainly include atrophic rhinitis, pneumonia, hemorrhagic sepsis, or chronic upper respiratory tract inflammation. However, when the host's immunity is weakened due to harsh living environment, seasonal changes, climate change, long-distance transportation, feed changes, nutritional deficiencies, parasitic infections, etc., Pasteurella multocida can enter the host's lower respiratory tract and cause respiratory diseases or even systemic infections. Avian Pasteurella multocida has multiple serotypes, which can be divided into 5 serotypes (A, B, D, E, F) based on capsular antigens and 16 serotypes (1-16) based on lipopolysaccharide antigens. The cross-protection between different serotypes is poor.

[0003] my country's prevention and control of avian Pasteurella multocida primarily relies on vaccines and antibiotic treatment. The frequent and indiscriminate use of antibiotics has led to multidrug resistance in clinical isolates of avian Pasteurella multocida. Furthermore, in an environment characterized by antibiotic restrictions and prohibitions, vaccines are currently the most effective means of prevention and control. Consequently, vaccines with strong cross-protection and high safety are urgently needed. Existing vaccines primarily include inactivated, attenuated, and subunit vaccines. The poor cross-protection between different serotypes of inactivated vaccines, their short protection period, and the stress response caused by endotoxins, as well as the safety concern of attenuated vaccines with potential virulence reversion, have limited their clinical application. With technological advancements, the advantages of subunit vaccines are becoming increasingly prominent.

[0004] Fimbriae are filamentous protein appendages on the surface of bacteria that enable bacteria to act on host cells and cause infection. The pili of pathogenic bacteria play an important role in adhesion to host cells, tissue colonization and infection. Among them, type IV pili are the most widely distributed. More and more studies have shown that type IV pili are important adhesins and virulence factors on the cell surface, and play an important role in the pathogenic process of pathogens. During the bacterial infection process, the expression of pili is beneficial to the survival and colonization of bacteria on the one hand. On the other hand, because the pili carry a large number of copies of subunits, they will also become the target of the host immune response, and the candidate protein for the development of subunit vaccines.

[0005] Therefore, there is an urgent need to develop a bivalent subunit vaccine for avian Pasteurella multocida. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies in the prior art and provides a bivalent subunit vaccine which is protective against all serotypes of avian Pasteurella multocida and has low toxic and side effects, as well as a preparation method and application thereof.

[0007] The technical solutions of the present invention are as follows: The invention discloses a bivalent subunit vaccine for avian Pasteurella multocida, which is prepared by emulsifying a protein mixture consisting of PpdD-C1 protein and PpdD-C2 protein and Freund's complete adjuvant.

[0008] In the above scheme, the mass ratio of the PpdD-C1 protein to the PpdD-C2 protein is 1:0.8-1.2.

[0009] In the above scheme, the concentrations of PpdD-C1 protein and PpdD-C2 protein in the subunit vaccine are both 90-110 μg / mL.

[0010] The method for preparing the avian Pasteurella multocida bivalent subunit vaccine comprises the following steps: synthesis ppdD-C1 The gene was cloned into the pET-32a vector to construct the recombinant plasmid pET-32a- ppdD-C1 , the recombinant plasmid pET-32a- ppdD-C1 The protein was transformed into BL21 strain and purified to obtain PpdD-C1 protein; synthesis ppdD-C2 The gene was cloned into the pET-32a vector to construct the recombinant plasmid pET-32a- ppdD-C2 , the recombinant plasmid pET-32a- ppdD-C2 The protein was transformed into BL21 strain and purified to obtain PpdD-C2 protein; The PpdD-C1 protein and the PpdD-C2 protein are diluted with a solvent to prepare a protein mixture, which is then mixed and emulsified with Freund's complete adjuvant to obtain the avian Pasteurella multocida bivalent subunit vaccine.

[0011] In the above scheme, ppdD-C1 The nucleotide sequence of the gene is SEQ ID No.1.

[0012] In the above scheme, ppdD-C2 The nucleotide sequence of the gene is SEQ ID No.2.

[0013] The invention relates to an application of the avian Pasteurella multocida bivalent subunit vaccine in preventing avian Pasteurella multocida.

[0014] The present invention has the beneficial effect of discovering for the first time that type IV fimbriae of different serotypes of avian Pasteurella multocida fall into only two categories. A bivalent subunit vaccine prepared based on these two types of type IV fimbriae proteins can provide excellent protection against all serotypes of avian Pasteurella multocida. Compared with existing vaccines, the bivalent subunit vaccine prepared in this invention offers superior cross-protection, high safety, is endotoxin-free, and significantly reduces stress responses to the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This paper is to analyze the phylogenetic tree of the reported avian Pasteurella multocida type IV fimbriae proteins. Figure 2 is an SDS-PAGE image of two types of type IV fimbriae proteins in the present invention; Figure 3 This is a diagram showing the purification results of two types of type IV fimbriae proteins in the present invention; Figure 4 The safety and protective effect of the monovalent subunit vaccine PpdD-C1 against C48-1 and X73 strains were determined. Figure 5 The effect of the monovalent subunit vaccine PpdD-C1 on the bacterial load in tissues after challenge with C48-1 and X73 strains was determined. Figure 6 The safety and protective effect of the monovalent subunit vaccine PpdD-C2 against C48-1 and X73 strains were determined. Figure 7 The effect of the monovalent subunit vaccine PpdD-C2 on the bacterial load in tissues after challenge with C48-1 and X73 strains was determined. Figure 8 The safety and protection of the bivalent subunit vaccine PpdD-C1+PpdD-C2 against C48-1 and X73 strains were determined. Figure 9 This is a determination of the effect of the bivalent subunit vaccine PpdD-C1+PpdD-C2 of the present invention on the bacterial load in tissues after challenge with C48-1 and X73 strains. DETAILED DESCRIPTION

[0016] The present invention is further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art, and the methods used in the present invention can adopt methods commonly used in the field of vaccine preparation.

[0017] Example 1

[0018] Cloning, Expression and Purification of Type IV Fimbriae Proteins of C1 and C2 Types from Avian Pasteurella multocida 1. Materials Tryptic soy peptone agar (TSA), tryptic soy peptone broth (TSB), and LB broth were purchased from Qingdao Haibo Co., Ltd.; restriction endonucleases and Escherichia coli BL21 (DE3) competent cells were purchased from Dalian Takara Biotechnology Co., Ltd., Taq polymerase was purchased from Nanjing Novozymes, and a conventional agarose gel DNA recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0019] 2. Test methods 2.1 Avian Pasteurella multocida ppdD-C1 and ppdD-C2 Construction of gene recombination plasmid 2.1.1 ppdD-C1 and ppdD-C2 Synthesis of gene fragments Will ppdD-C1 and ppdD-C2 The gene was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and introduced at both ends. Bam HI and Sal I restriction enzyme site, the synthesized fragment was connected to the pUC57 plasmid. ppdD-C1 and ppdD-C2 The nucleotide sequences of the genes are SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0020] 2.1.2 Construction of recombinant plasmids The recovered DNA fragments were linked to the expression plasmid pET-32a using the double enzyme digestion method and transformed into the Escherichia coli BL21 (DE3) strain. The strains were selected for verification using PCR. ppdD-C1 The primer sequences for gene identification were: PpdD-C1-upstream: 5'-CGGGATCCATGAAAAAAGCCATTTTCT (Bam HI); PpdD-C1-downstream: 5'-GCGTCGACTTATGCGCAAAATCCTGCT (Sal I). ppdD-C2The primer sequences for gene identification were: PpdD-C2-upstream: 5'-CGGGATCCATGAAAAAAGCCATTTTCT (Bam HI); PpdD-C2-downstream: 5'-GCGTCGACTTATGCACAAAATCCTGCT (Sal I). The PCR reaction system consisted of 12.5 μL of high-fidelity enzyme, 1 μL of each upstream and downstream primer, 0.5 μL of genomic template, and 10 μL of ultrapure water. The PCR reaction program was: initial denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds; and extension at 72°C for 5 minutes.

[0021] The positive plasmids were picked and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared by Blast on NCBI, and the sequence homology with the target fragment was 100%, indicating that the recombinant plasmid pET-32a- ppdD-C1 and pET-32a- ppdD-C2 Build successful.

[0022] 2.2 Expression and purification of PpdD-C1 and PpdD-C2 proteins 2.2.1 Expression of PpdD-C1 and PpdD-C2 proteins The recombinant plasmids pET-32a- ppdD-C1 and pET-32a- ppdD-C2 A single colony of the strain was picked and inoculated into LB medium containing 100 μg / mL kanamycin, and cultured at 37°C, 200 rpm, and shaken overnight. The overnight culture solution was transferred to 200 mL of LB medium containing 100 μg / mL kanamycin at a ratio of 1:100, and cultured at 37°C, 200 rpm, until the OD 600 When the pH is 0.4, add IPTG solution to a final concentration of 0.1 mM and induce at 37°C for 4-6 hours. Collect the cells by centrifugation and resuspend the pellet in 20 mL of purified water. After ultrasonic lysis, centrifuge at 12,000 rpm for 30 minutes. Take 40 μL of the supernatant and pellet and add them to 10 μL of 5× SDS Loading Buffer respectively. Mix well and boil in boiling water for 10 minutes. Finally, perform SDS-PAGE to detect protein expression.

[0023] 2.2.2 Purification of PpdD-C1 and PpdD-C2 proteins Cultivate and induce the strain as described in 2.2.1, using a 500 mL culture volume. Collect the induced bacterial cells by centrifugation. Wash the pellet with PBS and resuspend in 20 mL of Buffer A. Disrupt the cells using ultrasound, remove the supernatant by centrifugation, and resuspend the supernatant and pellet in 10 mL of Buffer A. Transfer the supernatant to a pre-equilibrated Ni-NTA column and wash the column with binding buffer to remove nonspecifically bound proteins. Finally, elute the target protein with varying concentrations of imidazole, collect the eluate, and analyze it by SDS-PAGE.

[0024] 3. Results like Figure 3 As shown, the recombinant plasmid pET-32a- ppdD-C1 ( Figure 3 A) and pET-32a- ppdD-C2 ( Figure 3 B) PpdD-C1 and PpdD-C2 proteins can be successfully expressed in vitro; Figure 4 As shown, PpdD-C1 and PpdD-C2 proteins were successfully purified in vitro using Ni-NTA columns.

[0025] Example 2 Preparation of vaccine

[0026] 1. Materials BCA kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; Freund's complete adjuvant F5881 and Freund's incomplete adjuvant F5506 were purchased from Sigma.

[0027] 2. Test methods 2.1 Preparation of monovalent subunit vaccines The protein concentrations of PpdD-C1 and PpdD-C2 were determined using a BCA kit. After being diluted to 400 μg / mL using PBS solution, the proteins were mixed and emulsified with an equal volume of Freund's complete adjuvant to prepare the monovalent subunit vaccines PpdD-C1 and PpdD-C2.

[0028] 2.2 Preparation of bivalent subunit vaccines The protein concentrations of PpdD-C1 and PpdD-C2 were determined using a BCA kit. The proteins were diluted to 400 μg / mL and mixed in a 1:1 ratio to prepare a protein solution. Finally, the protein solution was mixed with an equal volume of Freund's complete adjuvant and emulsified to prepare a subunit vaccine.

[0029] Example 3 Safety and protection testing of monovalent subunit vaccines

[0030] 1. Materials 2. Test methods 28-day-old SPF chickens were randomly divided into seven groups, each consisting of 10 chickens. These groups included a blank control group, an X73 challenge group, a C48-1 challenge group, a PpdD-C1 vaccine-immunized group 1 (C48-1), a PpdD-C1 vaccine-immunized group 1 (X73), a PpdD-C2 vaccine-immunized group 2 (C48-1), and a PpdD-C2 vaccine-immunized group 2 (X73). Immunizations were administered intramuscularly in the leg using a 100 μL subunit vaccine containing 20 μg of protein. A second vaccination was administered 14 days after the first vaccination, and the challenge was performed 14 days after the second vaccination. The immunization dose for both C48-1 and X73 was 300 CFU. One week after vaccination, the protective rate and blood bacterial load were measured.

[0031] 3. Results The results are as follows Figure 4 、 Figure 6 As shown, the survival rate of the C48-1 challenge group was 40%, and the survival rate of the X73 challenge group was 70%. The monovalent vaccine PpdD-C1 containing 20 μg of PpdD-C1 had a protection rate of 70% against the X73 strain with C1 type IV pili and a protection rate of 70% against the C48-1 strain with C2 type IV pili. The monovalent vaccine PpdD-C2 had a protection rate of 100% against the C48-1 strain with C2 type IV pili and a protection rate of 60% against the X73 strain with C1 type IV pili.

[0032] The results are as follows Figure 5 、 Figure 7 As shown, after immunization with the monovalent vaccine PpdD-C1, the blood bacterial load of the X73 strain containing the same type of fimbriae can be significantly reduced on the first day, and the blood bacterial load of the C48-1 strain containing different types of fimbriae can be significantly reduced on the third day; after immunization with the monovalent vaccine PpdD-C2, the blood bacterial load of the X73 strain containing the same type of fimbriae and the C48-1 strain containing different types of fimbriae can be significantly reduced on the third day, and there is no significant effect on the blood bacterial load at other times.

[0033] The above results show that the monovalent subunit vaccine has a certain protection rate against strains with the same type of type IV fimbriae, but the protection rate against strains with different types of type IV fimbriae is lower.

[0034] Example 4 Safety and protection testing of bivalent subunit vaccines

[0035] 1. Materials 2. Test methods 28-day-old SPF chickens were randomly divided into five groups, each consisting of 10 chickens: a blank control group, an X73-challenged group, a C48-1-challenged group, an immune-challenged group-1 (X73), and an immune-challenged group-2 (C48-1). Immunizations were administered intramuscularly in the leg with a 100 μL volume. The immunizing dose of the bivalent subunit vaccine contained 10 μg each of PpdD-C1 and PpdD-C2 proteins. A second immunization was administered 14 days after the first immunization, and the challenge was conducted 14 days after the second immunization. The immunizing dose was 300 CFU for both C48-1 and X73. Immunoprotection rates and blood bacterial loads were observed one week after immunization.

[0036] 3. Results like Figure 8 As shown, the survival rate of the C48-1 challenge group was 40%, and the survival rate of the X73 challenge group was 70%. The bivalent subunit vaccine PpdD-C1+PpdD-C2, containing 10 μg of each of the PpdD-C1 and PpdD-C2 proteins, provided 80% protection against the X73 strain with C1 type IV pili and 100% protection against the C48-1 strain with C2 type IV pili.

[0037] The results of blood bacterial load determination were as follows Figure 9 As shown, after immunization with the bivalent subunit vaccine PpdD-C1+PpdD-C2, the blood bacterial load of the X73 strain can be significantly reduced on the first day; and on the third and seventh days, the blood bacterial load of the immunized group is almost zero.

[0038] The above results all indicate that the bivalent subunit vaccine PpdD-C1+PpdD-C2 provides good protection against all serotypes of avian Pasteurella multocida, and has better protection than the monovalent subunit vaccine, and can be used to prevent multiple serotypes of avian Pasteurella multocida.

Claims

1. A bivalent subunit vaccine for avian Pasteurella multocida, characterized in that: The avian Pasteurella multocida bivalent subunit vaccine is prepared by mixing and emulsifying a protein mixture consisting of PpdD-C1 protein and PpdD-C2 protein with Freund's complete adjuvant.

2. The avian Pasteurella multocida bivalent subunit vaccine according to claim 1, wherein The mass ratio of the PpdD-C1 protein to the PpdD-C2 protein is 1:0.8-1.

2.

3. The avian Pasteurella multocida bivalent subunit vaccine according to claim 1, wherein The concentrations of the PpdD-C1 protein and the PpdD-C2 protein in the subunit vaccine are both 90-110 μg / mL.

4. The method for preparing the bivalent subunit vaccine of avian Pasteurella multocida according to claim 1, wherein: The steps include: synthesis ppdD-C1 The gene was cloned into the pET-32a vector to construct the recombinant plasmid pET-32a- ppdD-C1 , the recombinant plasmid pET-32a- ppdD-C1 The protein was transformed into BL21 strain and purified to obtain PpdD-C1 protein; synthesis ppdD-C2 The gene was cloned into the pET-32a vector to construct the recombinant plasmid pET-32a- ppdD-C2 , the recombinant plasmid pET-32a- ppdD-C2 The protein was transformed into BL21 strain and purified to obtain PpdD-C2 protein; The PpdD-C1 protein and the PpdD-C2 protein are diluted with a solvent to prepare a protein mixture, which is then mixed and emulsified with Freund's complete adjuvant to obtain the avian Pasteurella multocida bivalent subunit vaccine.

5. The preparation method according to claim 4, wherein ppdD-C1 The nucleotide sequence of the gene is SEQ ID No.

1.

6. The preparation method according to claim 4, wherein ppdD-C2 The nucleotide sequence of the gene is SEQ ID No.

2.

7. Use of the bivalent subunit vaccine against avian Pasteurella multocida according to any one of claims 1 to 6 in preventing avian Pasteurella multocida.

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