Mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, vaccine and application thereof

A gene deletion vaccine, Salmonella technology, applied in the field of animal bacterial genetic engineering, can solve the problems of not being able to be used as a vaccine strain, not meeting the biosafety requirements, etc., and meeting the vaccine biosafety requirements, good immune protection, and good safety. Effect

Inactive Publication Date: 2011-08-03
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, there is randomness in the mutation method mediated by transposons, which is only suitable for the screening of mutants with obvious changes in phenotype, and mutant strains that do not cause obvious phenotypic changes cannot be obtained through these methods; So

Method used

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  • Mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, vaccine and application thereof
  • Mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, vaccine and application thereof
  • Mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Salmonella typhimurium SL1344 cya Construction of gene deletion strain SL13441

[0038] 1, cya Related primer design

[0039] Refer to http: / / www.ncbi.nlm.nih.gov / registered Salmonella typhimurium LT2 strain cya Gene sequence (GenBank No: AE006468.1) design 7 pieces cya Related primers (see figure 1 , Table 1), for Salmonella typhimurium parent strain SL1344 (gifted by Nanjing Agricultural University) and gene deletion strain ?cya Construction and characterization of SL1344. All primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0040] Table 1 PCR primers

[0041]

[0042] 2. Salmonella typhimurium SL1344 cya Gene Upstream and Downstream Fragments cya 1 (upper arm) with cya Cloning of 2 (lower arm)

[0043] Put Salmonella typhimurium SL1344 on LB solid medium (10g tryptone, 5g yeast extract, 10g sodium chloride, 12g agar powder, ultrapure water to 1000mL, autoclave at 121°C for 20min) in a zigzag shape Line, cultured...

Embodiment 2

[0049] Example 2 Salmonella typhimurium cya Biological characteristics of gene deletion strain SL13441

[0050] 1. Phenotype identification of gene deletion strain SL13441

[0051] The serotype of the deletion strain SL13441 was identified according to the instructions of Salmonella single-factor serum (purchased from Zhejiang Ningbo Tianrun Bio-Pharmaceutical Co., Ltd.). The gene deletion strain SL13441 (experiment number: deletion strain ?cya SL1344) and the parental strain SL1344 were inoculated on MacConkey agar plates with and without 1% maltose, and then transferred to glucose, maltose, lactose, sucrose, arabinose, xylose, mannitol and other carbon sources and H 2 S and other biochemical identification tubes (purchased from Hangzhou Tianhe Microbial Reagent Co., Ltd.) were used to study their biochemical characteristics. The results showed that the serotype of the gene deletion strain SL13441 was consistent with that of the parental strain SL1344, which was still 1,...

Embodiment 3

[0059] Example 3 Preparation of Salmonella typhimurium cya gene deletion strain SL13441 vaccine

[0060] The gene deletion strain SL13441 (experiment number: deletion strain?cyaSL1344) was streaked and cultured on LB solid medium for 24 hours, the culture was scraped and washed with sterile saline, centrifuged at 10000r / min for 5min, the supernatant was discarded, and an appropriate amount of Sterilize the suspension in saline. Centrifuge at 10000r / min for 5min, wash 1-2 times. After suspending the bacteria with sterilized normal saline, serially 10-fold dilution was used to determine the number of viable bacteria by plate counting method, and finally the appropriate bacterial concentration was adjusted with sterilized normal saline according to the needs of the test to obtain a spare vaccine for the test.

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Abstract

The invention relates to a mouse-typhus salmonella gene-deletion mutant strain without containing resistance marks, a vaccine and application thereof, belonging to the technical field of animal bacterium genetic engineering. The invention utilizes a mediated allelic exchange technique of recombinant suicidal plasmid for obtaining the mouse-typhus salmonella gene-deletion mutant strain (Salmonellatyphimurium SL13441) (the preservation number is CCTCC NO: M2010301) without containing the resistance marks. The mutant strain is deficient in an important toxicity regulating gene-cya gene in mouse-typhus salmonella, so that the toxicity of the mouse-typhus salmonella is obviously weakened after the deficiency of the cya gene, but the mouse-typhus salmonella still has immunogenicity. The invention also discloses the Salmonella gullinarum gene-deletion vaccine prepared by utilizing the gene-deletion mutant strain and an application of the gene-deletion mutant strain in preparing the vaccine, and the prepared vaccine meets the requirement on biological safety. The gene-deletion vaccine prepared in the invention can effectively prevent infection of Salmonella gullinarum and has the advantages of good stability and safety and low cost, is convenient to use and has obvious economic and social benefits.

Description

technical field [0001] The present invention relates to a Salmonella typhimurium gene deletion mutant strain without resistance marker, and also relates to a Salmonella typhimurium gene deletion vaccine without resistance marker and the application of the strain in vaccine preparation, which belongs to animal bacterial gene field of engineering technology. Background technique [0002] Salmonella typhimurium ( Salmonella typhimurium , St) belongs to Salmonella group B, is a group of host non-specific enteropathogenic bacteria, has a wide host spectrum, exists in many poultry (chicken, duck, pigeon, etc.), livestock (pig, cattle, sheep, horse, dog , cats, etc.), rodents, birds and human intestines. According to experiments on sugar fermentation and tartrate utilization, the bacteria can be divided into 38 biotypes, and phages can be used to divide them into 207 phage types (Lu Chengping. 2001. Veterinary Microbiology (Third Edition). Beijing: China Agricultural Publishing ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09A61K39/112A61P31/04C12R1/42
Inventor 程相朝廖成水赵战勤张春杰李银聚吴庭才李小康汪洋
Owner HENAN UNIV OF SCI & TECH
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