Subunit vaccine, and preparation method and application thereof

A vaccine composition, Pasteurella porcine technology, applied in the direction of bacterial antigen components, antibacterial drugs, etc., can solve the problems of the immune effect of the attenuated live vaccine being susceptible to the environment, limited protective effect, and affecting virulence, so as to improve immune protection effects, avoiding adverse reactions, and reducing immunization costs

Active Publication Date: 2016-04-20
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Toxin-producing Pasteurella multocida mainly causes progressive atrophic rhinitis in pigs, which can lead to atrophy of pig turbinates, deformation of nose and snout, and affected bone development of the whole body, so that growth of pigs is slow, and it is easy to develop secondary complex pneumonia. The pig industry causes huge economic losses
[0004] At present, the vaccines used clinically to prevent Pasteurella multocida are mainly inactivated vaccines and attenuated live vaccines. The inactivated vaccines have poor cross-protection ability and short protection period against different serotypes of Pasteurella; The effect is easily affected by factors such as the environment and there is a risk of strong toxicity
Whether it is an inactivated vaccine or a live attenuated vaccine, repeated immunizations are required, and the protective effect is very limited in the face of attacks by alien strains

Method used

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  • Subunit vaccine, and preparation method and application thereof
  • Subunit vaccine, and preparation method and application thereof
  • Subunit vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1P.multocidaOmpA, OmpH, PlpE gene PCR amplification and construction of prokaryotic expression plasmid

[0058] 1.1 Design of primers

[0059] According to the OmpA gene sequence of the P.multocidapm-113 strain (accession number: JX435111.1) published in GenBank, the OmpH and PlpE gene sequences in the full-length gene sequence of the pm-70 strain (accession number: AE004439.1), use Primer5.0 software designed 3 pairs of primers respectively. The 5' ends of the upstream and downstream primers were respectively introduced with BamHI, XhoI restriction sites and protective bases to amplify the full-length sequences of OmpA, OmpH, and PlpE genes. The sequences of the 3 pairs of primers See Table 1, where the underlined part is the introduced restriction site, and the primers were synthesized by Invitrogen.

[0060] Table 1 Primer Sequence

[0061]

[0062]

[0063] 1.2 Extraction of Genomic DNA from P.multocida A Type HN5 Strain

[0064] Streak the Paste...

Embodiment 2

[0079] Example 2 Construction of recombinant expression strains BL21-OmpA, BL21-OmpH and BL21-PlpE

[0080] 2.1 Transformation of E.coliBL-21 Competent Cells with Recombinant Plasmid and Expression of Recombinant Protein

[0081] In Example 1, the recombinant plasmids sequenced correctly were transformed into E. coliBL-21 competent cells, and the recombinant expression strains BL21-OmpA, BL21-OmpH, and BL21-PlpE were respectively obtained. At the same time, the empty plasmid pET-32a was transformed into E. coliBL-21 in the same way. .coliBL-21 competent cells, coated with ampicillin-containing LB solid medium, cultured at 37°C for 16 hours, picked a single colony that grew well and inoculated them in 10ml of ampicillin-containing LB liquid medium, cultured with shaking at 37°C and 200rpm for 2h . OD of bacteria solution 600 When the value reaches 0.6, take 1 mL of uninduced expression bacteria solution and empty plasmid bacteria solution as samples before induction, add IPTG...

Embodiment 3

[0089] The preparation of embodiment 3Pm vaccine

[0090] 3.1 Preparation of recombinant Pm subunit vaccine

[0091] Combine r-OmpA, r-OmpH, and r-PlpE recombinant proteins according to a certain ratio, add a certain amount of Gel adjuvant (Seppic, France) and mix to make a Pm subunit vaccine containing three recombinant protein components with different concentrations , the concentrations of the three recombinant protein components in each vaccine are shown in Table 2.

[0092] Table 2 Protein content of different vaccines

[0093]

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Abstract

The invention provides a vaccine composition. The vaccine composition comprises an immunizing dose of porcine Pasteurella multocida antigens, i.e., OmpA protein, OmpH protein and PlpE protein. A plurality of protein antigens in the vaccine composition are used in combination and cooperatively stimulate immunoreactions of an organism; and the vaccine composition can exert good protection effect only through one immunization and exerts good protection effect on mixed infection with porcine Pasteurella multocida of different serotypes. The invention also provides a preparation method for the vaccine composition. The protein antigens are expressed and generated in large quantities through gene engineering means, so large-scale production is benefited. Testing results show that the vaccine composition can effectively protect pigs from mixed infection with porcine Pasteurella multocida of different serotypes. Application of the vaccine composition is of active practical significance to providing of a perfect approach for preventing and / or treating porcine Pasteurella multocida infection and to purifying of porcine Pasteurella multocida.

Description

technical field [0001] The invention relates to a subunit vaccine. The preparation method and application of the vaccine belong to the field of veterinary biological products. Background technique [0002] Pasteurella multocida (Pasteurella multocida, Pm) belongs to the Pasteurella family Pasteurella genus Pasteurella multocida species, is an encapsulated Gram-negative short bacillus, facultative anaerobic. According to the difference of its capsule and lipopolysaccharide, Pm can be divided into 5 serogroups A, B, D, E, and F and 16 serotypes. Type A mainly causes fowl cholera, and types B and E mainly cause hemorrhagic sepsis. The toxin-producing type D mainly causes atrophic rhinitis in pigs. The pathogen is prevalent all over the world and has a wide range of hosts. It can infect a variety of animals and cause disease. Different animals have different symptoms and pathological changes after infection. [0003] Pasteurella multocida in pigs is mostly caused by PmA, B, an...

Claims

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Application Information

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IPC IPC(8): A61K39/116A61P31/04
Inventor 张许科孙进忠金云云田克恭
Owner PU LIKE BIO ENG
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