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A double gene deletion Salmonella enteritidis, its construction method and a vaccine containing the double gene deletion Salmonella enteritidis

A Salmonella Enteritidis, dual-gene technology, applied in the field of gene knockout, can solve the problems of systemic transmission, easy induction of wild strains, etc., and achieve the effects of reducing fluid secretion, enhancing mucosal immunity, and reducing the number of net proliferations

Active Publication Date: 2019-10-08
CHONGQING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the above-mentioned deficiencies in the prior art, the technical problem solved by the present invention is: it is difficult for the Salmonella enteritidis live vaccine in the prior art to produce a strong colonization inhibitory effect on wild strains, and it is easy to induce systemic transmission of wild strains, and It also cannot effectively induce the technical problem of cross-protection against other non-host adaptive serotypes of Salmonella, and provides a double-gene deletion Salmonella enteritidis with good clearance effect in the host and excellent immune effect, its construction method and containing the double gene Vaccine missing Salmonella Enteritidis

Method used

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  • A double gene deletion Salmonella enteritidis, its construction method and a vaccine containing the double gene deletion Salmonella enteritidis
  • A double gene deletion Salmonella enteritidis, its construction method and a vaccine containing the double gene deletion Salmonella enteritidis
  • A double gene deletion Salmonella enteritidis, its construction method and a vaccine containing the double gene deletion Salmonella enteritidis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Proliferation of Salmonella enteritidis and extraction of genome

[0038] Inoculate Salmonella enteritidis D1203 in liquid LB medium, culture overnight at 37°C, take 1.5mL of cultured bacteria, place in an Eppendorf centrifuge tube, centrifuge at 10,000g for 2min, discard the supernatant, add 1.5mL of PBS buffer to resuspend the precipitate, Centrifuge at 10,000 g for 2 minutes, discard the supernatant, add 240 μL of lysozyme to the pellet, and add 15 μL of lysozyme, ice-bath at 0°C for 30 minutes, add 15 μL of 10% SDS, mix well, add proteinase K, and bathe in water at 65°C for 30 minutes , add 200 μL of 5M NaCl solution, shake, add 500 μL of phenol / chloroform / isoamyl alcohol (volume ratio 1:24:25) solution, mix well, centrifuge at 12000 g for 10 min, take the supernatant into a centrifuge tube, record its volume, add etc. The volume of frozen absolute ethanol was placed in a -80°C refrigerator for 10 minutes, centrifuged at 12000g for 10 minutes, the supernat...

Embodiment 2

[0039] Construction of embodiment 2 rfaH gene knockout mutant strain

[0040] 2.1 Overlap PCR

[0041] According to the Salmonella enteritidis genome sequence published on GeneBank, the upstream primer rfaH-1F and the downstream primer rfaH-1R of the upstream homology arm sequence rU of the rfaH gene were designed, and the upstream primer rfaH- of the downstream homology arm sequence rD of the rfaH gene was designed. 2F and downstream rfaH-2R, the nucleotide sequence of the rfaH-1F is shown in SEQ ID NO.3, the nucleotide sequence of the rfaH-1R is shown in SEQ ID NO.4, the rfaH-2F The nucleotide sequence of rfaH-2F is shown in SEQ ID NO.5, and the nucleotide sequence of rfaH-2F is shown in SEQ ID NO.6. Using the above-mentioned extracted Salmonella enteritidis D1203 genomic DNA as a template, the upper and lower homology arm sequences rU and rD of rfaH were amplified by PCR method. The 50 μL PCR amplification system of rU and rD is shown in Table 1:

[0042] Table 1 PCR amp...

Embodiment 3

[0060] Example 3 Construction of Salmonella Enteritidis ΔrfaHΔsopB Double Deletion Strain

[0061] 3.1 Overlap PCR

[0062] According to the Salmonella enteritidis genome sequence published on GeneBank, the upstream primer sopB-1F and the downstream primer sopB-1R of the upstream homology arm sequence sU of the sopB gene were designed, and the upstream primer sopB- of the downstream homology arm sequence sD of the sopB gene was designed. 2F and downstream primer sopB-2R, the nucleotide sequence of the sopB-1F is shown in SEQ ID NO.9, the nucleotide sequence of the sopB-1R is shown in SEQ ID NO.10, the sopB- The nucleotide sequence of 2F is shown in SEQ ID NO.11, and the nucleotide sequence of sopB-2R is shown in SEQ ID NO.12. Using Salmonella enteritidis D1203 genomic DNA as a template, the upper and lower homology arm sequences sU and sD of sopB were amplified by PCR. The 50 μL PCR amplification system and PCR reaction conditions of sU and sD are the same as those of rU and...

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Abstract

The invention discloses a double-gene-deficient Salmonella enteritidis strain and an establishment method thereof, and a vaccine containing the double-gene-deficient Salmonella enteritidis strain. The double-gene-deficient Salmonella enteritidis strain is a Salmonella enteritidis strain of which rfaH gene and sopB gene are knocked out. According to the establishment method, a homologous recombination process and a sucrose-sensitive gene negative screening process are adopted to knock out the rfaH gene and sopB gene in the Salmonella enteritidis strain. The vaccine comprises the double-gene-deficient Salmonella enteritidis strain prepared by the method. The cellular immunity and mucosa immunity of the double-gene-deficient Salmonella enteritidis strain are enhanced; and the double-gene-deficient Salmonella enteritidis strain has favorable protective effects and favorable immune effects, has favorable removing effects in the host, can be colonized and removed in the body within 3-4 weeks, and thus, has excellent safety performance.

Description

technical field [0001] The invention belongs to the technical field of gene knockout, and in particular relates to a double gene deletion Salmonella enteritidis, a construction method thereof and a vaccine containing the double gene deletion Salmonella enteritidis. Background technique [0002] Salmonella Enteritidis is an important zoonotic pathogen. Poultry infected with Salmonella is the most common reservoir of Salmonella. Humans are usually infected with Salmonella by ingesting contaminated eggs or undercooked poultry meat. Worldwide, food poisoning caused by ingestion of poultry and egg products contaminated by Salmonella Enteritidis has occurred in all countries and the number is increasing. Therefore, Salmonella enteritidis not only seriously hinders the development of poultry industry, but also threatens the health of humans and animals, and has become an important issue in the fields of medicine, veterinary medicine and public health. At present, antibiotics are m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/66A61K39/112A61P31/04
Inventor 胡勇林治华舒茂王远强邱荣蓉廖羚茜
Owner CHONGQING UNIV OF TECH
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