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A Subunit Vaccine of Actinobacillus Pleuropneumoniae

A technology of subunit vaccines and pleuropneumonia, applied in vaccines, veterinary vaccines, bacterial peptides, etc., can solve the problems of multiple components and can not achieve rApxIIIAN, and achieve the effect of low overall cost and easy production

Active Publication Date: 2022-02-25
HUAZHONG AGRI UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The applicant found through previous research (Master's thesis "Preparation of Porcine Infectious Pleuropneumonia Subunit Vaccine and Evaluation of Immune Effect" (Author Liu Ting, Huazhong Agricultural University, 2019) that the recombinant proteins rApxIARH, rApxIIAM, rApxIIIAN, The immunoprotective effect of the subunit vaccine combined with rOmlA and rTbpB was comparable to that of the commercialized subunit inactivated vaccine (the subunit inactivated vaccine of Actinobacillus pleuropneumoniae from Merck & Co. Epco APP)), its disadvantage is that there are many components, and rApxIIIAN cannot be expressed in a soluble form

Method used

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  • A Subunit Vaccine of Actinobacillus Pleuropneumoniae
  • A Subunit Vaccine of Actinobacillus Pleuropneumoniae
  • A Subunit Vaccine of Actinobacillus Pleuropneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] (1) Construction of recombinant expression plasmids

[0016] Using the genomic DNA of Actinobacillus pleuropneumoniae as a template, the target fragment was amplified by PCR using the primers shown in Table 1.

[0017] Table 1 Primer information

[0018]

[0019]

[0020] The amplified fragments of interest and the plasmid pET-20b(+) were digested with NdeI and XhoI respectively, and then ligated and transformed into Escherichia coli DH5α to finally construct the recombinant expression plasmids pET20b-apxIIAM, pET20b-omlA and pET20b -tbpB. These recombinant expression plasmids were identified by enzyme digestion and sequencing, all of which were in line with expectations, without any mutation in the sequence, and successfully inserted into the pET20b vector.

[0021] (2) Expression and purification of recombinant protein

[0022] The constructed recombinant expression plasmids pET20b-apxIIAM, pET20b-omlA and pET20b-tbpB were transformed into Escherichia coli BL...

Embodiment 2

[0025] The concentrations of the purified recombinant proteins in Example 1 were measured by the BCA method, and the recombinant proteins were combined in a certain proportion and then mixed with aluminum hydroxide adjuvant in equal volumes to obtain three components with a content of each recombinant protein of 200 μg / mL Recombinant subunit vaccines are used in the following immune challenge experiments.

[0026] Vaccine sterility test: Aseptically draw 200 μL of the prepared vaccine into a plate coated with TSA+NAD (nicotinamide adenine dinucleoside, 5 μg / mL), culture at 37° C. overnight, no colonies grow on the plate.

Embodiment 3

[0028] The safety and effectiveness of the three-component recombinant subunit vaccine obtained in Example 2 were verified through piglet clinical trials, and the piglets were protected by the Actinobacillus pleuropneumoniae subunit inactivated vaccine of Merck & Co. Epco APP) for comparison. Before the pigs were immunized, the blood was collected and the serum was tested with the Actinobacillus pleuropneumoniae differential diagnosis kit to ensure that the test pigs were all negative pigs.

[0029] The 8-week-old piglets were divided into 3 groups for immunization, with 5 piglets in each group, and the experimental groups were as follows:

[0030] Vaccine group I: rApxIIAM, rOmlA, rTbpB three-component recombinant subunit vaccine;

[0031] Vaccine group II: MSD Actinobacillus pleuropneumoniae subunit inactivated vaccine (containing 1 outer membrane protein and 3 toxins ApxI, ApxII, ApxIII);

[0032] Blank control group: PBS + aluminum hydroxide adjuvant.

[0033] Immune...

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Abstract

The invention discloses a porcine infectious pleuropneumoniae actinobacillus subunit vaccine, which belongs to the field of veterinary vaccines. The Actinobacillus pleuropneumoniae subunit vaccine of the present invention comprises protein 1-3 combinations whose amino acid sequences are respectively shown in SEQ ID NO.1-3. The truncated proteins selected in the combination of antigenic proteins of the present invention are highly homologous to all serotypes of APP, and their preparation into a subunit vaccine can provide cross-protection to different serotypes of Actinobacillus pleuropneumoniae; these truncated proteins are all Expressed in a soluble form, it is easy to produce and low in cost.

Description

technical field [0001] The invention relates to the field of veterinary vaccines, in particular to a porcine infectious pleuropneumoniae Actinobacillus subunit vaccine. Background technique [0002] Porcine contagious pleuropneumoniae (PCP) is a respiratory infectious disease caused by porcine infectious pleuropneumoniae (Actinobacillus pleuropneumoniae, APP), usually characterized by sudden onset, short course, high morbidity and mortality . The disease often occurs from April to May and September to November, and has obvious seasonality. In addition, various factors can induce the disease such as poor ventilation, crowding, group transfer, humidity, and long-distance transportation. This disease is especially common in fattening pigs, and it is common for death (the most acute mortality rate is as high as 100%) and mixed infection (with PRRS, PRV, HPS, etc.) caused by acute outbreaks to cause slow growth or even death; Pigs that survive or are subclinically infected may ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/05A61P31/04C07K14/195
CPCA61K39/05A61P31/04C07K14/195A61K2039/552A61K2039/55505
Inventor 贝为成刘迦慧刘婷张年章宝陈焕春
Owner HUAZHONG AGRI UNIV
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