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Avian Pasteurella multocida gene knockout strain mediated by Ngpiwi protein and construction method and application thereof

A Pasteurella and gene knockout technology, applied in the field of avian Pasteurella multocida gene knockout strain and its construction, can solve the problem that the weakening mechanism is unclear, the gene is difficult to knock out, and good cross immunity cannot be produced. protection, etc.

Active Publication Date: 2019-01-25
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1) Conventional inactivated vaccines generally use the popular virulent strains of Pasteurella avian to prepare whole-bacteria inactivated vaccines, as well as pasteurella multocida of serum A:1, A:3 and A:4 types Trivalent oil-emulsion inactivated vaccines are made, and propolis dual inactivated vaccines are made from Pasteurella poultry and E. There is no good cross-immune protection, and it only has the effect of immune protection against the infection of strains of the same serotype
[0006] 2) Traditional attenuated live Pasteurella avian vaccines mainly include clinically isolated attenuated strains and attenuated strains weakened by physical and chemical mutagenesis. Wide range and low production cost, but because the weakening mechanism is not clear, there is a risk of strong virulence
[0011] Due to the difficulty of knocking out the genes in the genome of Pasteurella multocida, the study of genetically engineered attenuated vaccines against Pasteurella multocida lags behind

Method used

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  • Avian Pasteurella multocida gene knockout strain mediated by Ngpiwi protein and construction method and application thereof
  • Avian Pasteurella multocida gene knockout strain mediated by Ngpiwi protein and construction method and application thereof
  • Avian Pasteurella multocida gene knockout strain mediated by Ngpiwi protein and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0141] The specific method for constructing the temperature-sensitive suicide basic plasmid pSHK5Ts-NgPiwi for knocking out the target gene of Pasteurella multocida comprising the following steps:

[0142] 1) Using SEQ ID No.6 and SEQ ID No.7 as templates to design NgPiwi fragments and RBS fragment amplification fusion primers and identification primers for NgPiwi fragments; respectively:

[0143] NgPiwi forward and reverse amplification primers:

[0144] NgPiwi-L: ATGACAGTGATTGACCTCGATTCG,

[0145] NgPiwi-RH-R: GCAAGAAAAAATATCAATTAGAGGAATCCGACAT;

[0146] Forward and reverse amplification primers for RBS:

[0147] RBS-RH-L: TGTCGGATTCCTCTAATTGATATTTTTTTCTTGC,

[0148] RBS-R: TATGCACTCCTATTTATTTAACTAAGTTGAC;

[0149] Primers to identify NgPiwi fusions to the plasmid:

[0150] NgPiwi-JD-F: CAACCCGGTAAGACACGACTTATC,

[0151] NgPiwi-JD-R: ATCTGTAACATCATTGGCAACGC;

[0152] 2) Fusion primers were designed using the temperature-sensitive suicide plasmid pSHK5Ts and NgPiwi-RBS...

Embodiment 2

[0195] The acquisition of the P. multocida hyae gene ORF partial sequence deletion bacterial strain △hyae-GX-PM comprises the following steps:

[0196] 1) Using the ORF sequence of the capsular synthesis protein gene + 300bp sequences upstream and downstream (referred to as the ORF sequence of the hyae gene and the 300bp sequences upstream and downstream) as shown in SEQ ID No.8 as a template, design primers for knocking out the partial sequence of the hyae gene ORF , respectively:

[0197] Left homology arm forward and reverse primers:

[0198] hyaE-KpnI-L-1: CATGGTACCGGTTATTATCATTGGAC,

[0199] hyaE-L-2: AACAGATGCAGTGAGATCTTGGTTTACTTCAATAATTTCC;

[0200] Right homology arm forward and reverse primers:

[0201] hyaE-R-3: TGAAGTAAACCAAGATCTCACTGCATCTGTTCAATC,

[0202] hyaE-SacI-R-4: AAAGAGCTCGAGTAAGCCACTTAAACGG;

[0203] Primers on both sides of the left and right homology arms of the ORF partial sequence of the hyae gene to be deleted on the genome:

[0204] hyaE-ID-F: ...

Embodiment 3

[0257] The acquisition of the Pasteurella multocida lyi gene ORF sequence deletion strain Δlyi-GX-PM, the main steps are the same as in Example 2, and the differences include the following steps:

[0258] 1) Using the lyi ORF sequence and the upstream and downstream 1000bp sequences, as shown in SEQ ID No.9, as a template, design primers for knocking out the lyi gene ORF sequence, respectively:

[0259] Left homology arm forward and reverse primers:

[0260] lyi-SacI-L1:AAGAGCTCAGATGTTATTGAGTCTGCG,

[0261] lyi-RH-L2: TTTAGGAGTTTTTTATGTAAGTCAATACTGATC;

[0262] Right homology arm forward and reverse primers:

[0263] lyi-RH-R3: TGATCAGTATTGACTTACATAAAAACTCCTAAATTC,

[0264] lyi-BamHI-R4: TTGGATCCTGACTTTGTCTTTAACACTGC;

[0265] Primers on both sides of the left and right homology arms of the lyi gene ORF sequence to be deleted on the genome:

[0266] lyi-ID1F: TGGTGGCGTTGACTCTTCTGTCAC,

[0267] lyi-ID1R: AAATTACGAGCGATGGCCTCG;

[0268] Primers for internal identification...

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Abstract

The invention discloses an avian Pasteurella multocida gene knockout strain mediated by Ngpiwi protein and a construction method and application thereof. The avian Pasteurella multocida gene knockoutstrain is a deleted strain which deletes an ORF sequence or a partial sequence of a latent virulence-related gene on the avian Pasteurella multocida genome. Firstly, that recombinant plasmid with Ngpiwi and the left and right homologous arm of the target gene sequence to be deleted is successfully construct; secondly, the recombinant plasmid was electroporated into Pasteurella multocida and induced to double-exchange recombination at 28 DEG C, the strains deleted the target gene sequence were screened by PCR, and then the plasmid was induced to lose at 42 DEG C, thus the strains deleted the potential virulence gene sequence were obtained. The genetic operating system has the advantages of small plasmid, easy operation, wide application, no potential miss-targeting effect, high knockout efficiency and no resistance gene screening marker, which provides an excellent tool for the development of genetic engineering vaccine of avian Pasteurella multocida.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, in particular to a Ngpiwi protein-mediated knockout strain of Pasteurella multocida gene in poultry and its construction method and application. Background technique [0002] Fowl cholera is a hemorrhagic and septic infectious disease caused by Pasteurella multocida infecting poultry. The morbidity and mortality are very high, and death occurs within 1-3 days. Fowl cholera is a severe disease in chicken farms in some areas of my country, often causing serious economic losses, and is one of the most serious infectious diseases to the poultry industry. In October 2011, Yu Chengjie et al. isolated a strain of Pasteurella multocida with capsular genotype A type from laying hens in a chicken farm in Guangxi, and named it GX-PM. Animal regression experiments showed that the strain It is highly pathogenic to chickens, and 100 CFU can cause all 10-week-old chickens to die within 3...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/66A61K39/102A61P31/04C12R1/01
CPCA61K39/102A61K2039/522A61P31/04C07K14/285C12N15/66C12N15/74
Inventor 张安定付磊韩丽项耀祖靳泽华
Owner HUAZHONG AGRI UNIV
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