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Application of phage depolymerase ORF38 protein in pasteurella multocida capsule typing identification

A Pasteurella, typing and identification technology, applied in the biological field, can solve problems such as Pasteurella that have not yet been found

Pending Publication Date: 2022-02-08
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Capsule typing of Pasteurella multocida using depolymerase has not been found so far

Method used

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  • Application of phage depolymerase ORF38 protein in pasteurella multocida capsule typing identification
  • Application of phage depolymerase ORF38 protein in pasteurella multocida capsule typing identification
  • Application of phage depolymerase ORF38 protein in pasteurella multocida capsule typing identification

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The construction of embodiment 1 recombinant protein

[0029] 1. Gene Amplification

[0030] The target fragment gene ORF38 (Gene ID: 54980989) was amplified using phage PHB01 as a template.

[0031] Forward primer: 5'-CCG GAATTC ATGTCATCAAGATATCAGTA-3'EcoRI

[0032] Reverse primer: 5'-GC GTC GAC TCACAATTGACACCATTTATTCA-3'SalI

[0033] The amplification system and amplification conditions are as follows:

[0034]

[0035] PCR reaction program: pre-denaturation at 98°C for 5 min; pre-denaturation at 98°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 10 min, and storage at 4°C.

[0036] 2. Construction of recombinant plasmids

[0037] (1) 200ng of the recovered target fragment and 100ng of the recovered pET-28a vector were digested with restriction endonucleases EcoRI and SalI at 37°C for 2h;

[0038] (2) Ligate the digested product overnight at 4°C under the action of T4 ligase;

[0039] (3) After...

Embodiment 2

[0059] Activity and stability of embodiment 2 protein

[0060] 1. Dot-spot assay for protein activity

[0061] Take 300μL PmD bacterial solution and mix it with 6mL TSB medium containing 0.75% agar, pour the lower plate, and after the plate is naturally dried, dilute the purified depolymerase ORF38 protein by two times, and take 5 μL (0.15 μg) of ORF38 protein was spotted onto the prepared double-layer plate, and incubated at 37°C for 12 hours to observe the results.

[0062] The result is as figure 2 As shown, the cleavage activity can still be seen when the protein is diluted to the 8th power.

[0063] 2. Protein pH stability

[0064] PmD exopolysaccharide (EPS) was extracted by hot phenol method. The lyophilized EPS was dissolved by adding appropriate amount of PBS buffer solution with different pH to reach the required concentration. Take 900 μL EPS diluent (EPS is 5 mg / mL) and mix with 100 μL ORF38 protein (30 μg / mL), and incubate at 37° C. for 1 h. The degradation...

Embodiment 3

[0069] Capsule typing of embodiment 3 Pasteurella multocida

[0070] Of the 55 strains of PmD preserved in our laboratory, 10 strains of Pm with capsular serology A, 5 strains of Pm with capsular serology B, 1 strain of Pm with capsular serology E, and 1 strain of Pm with capsular serology For Pm of F, 10 strains of Bordetella bronchis, 10 strains of Salmonella, 10 strains of Escherichia coli, 10 strains of Staphylococcus aureus, 10 strains of Haemophilus parasuis and 10 strains of Streptococcus were cultured to the logarithmic phase, and 300 μL of the above After the bacterial solution was mixed with 6 mL of TSB medium containing 0.75% agar, the lower plate was poured. After the plate was dried, the ORF38 protein was diluted with PBS (pH=6.0) buffer solution, 5 μL (0.15 μg) of ORF38 protein was spotted onto the double-layer plate, and incubated upside down in a 34°C-38°C incubator for 12 hours.

[0071] Table 1 is the ORF38 protein cleavage profile test. Using 5 μL (0.15 μg...

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Abstract

The invention discloses application of phage depolymerase ORF38 protein in pasteurella multocida capsule typing identification, wherein the amino acid sequence of the protein is shown as SEQ ID No.2, and the nucleotide sequence for coding the protein is shown as SEQ ID No.1. According to the invention, it is found for the first time that the phage depolymerase ORF38 protein only has a splitting effect on PmD and does not have a splitting effect on pasteurella multocida (including A, B, E and F) of other capsule serotypes and other bacteria such as escherichia coli, salmonella, haemophilus parasuis, bordetella bronchii, streptococcus, staphylococcus aureus and the like, so that the phage depolymerase ORF38 protein can be used for PmD capsule typing identification; and the phage depolymerase ORF38 protein has the advantages of simplicity, rapidness, accuracy and the like.

Description

technical field [0001] The invention belongs to the field of biology, and relates to the application of phage depolymerase ORF38 protein in the identification of Pasteurella multocida capsule typing, and also relates to a method for identification of Pasteurella multocida capsule typing. Background technique [0002] Pasteurella multocida (Pm) is a pathogen that can cause a variety of pasteurellosis in livestock and poultry, and is a serious hazard to animal husbandry. The pathogen has a wide host range and can be transmitted among a variety of animals, causing hemorrhagic sepsis or infectious pneumonia in animals. The important thing is that Pm can also infect humans. Individuals with unsound immune systems who are scratched or bitten by animals carrying pathogenic Pm can lead to death due to sepsis. In addition, wine infectious atrophic rhinitis (ARS) in pigs is usually associated with Pasteurella multocida type D (PmD). This disease is widespread and prevalent in pig far...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/34C12Q1/04C12R1/01
CPCC12Q1/34C12Q1/04Y02A50/30
Inventor 陈义宝刘玉庆刘正洁胡明赵效南张庆
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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