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Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof

A gene deletion vaccine, Salmonella technology, applied in the direction of antibacterial drugs, medical preparations containing active ingredients, bacteria, etc., can solve the problems that do not meet the biological safety requirements, can not be used as vaccine strains, etc., to achieve broad market application prospects, Good immune protection, weak toxicity effect

Inactive Publication Date: 2011-02-02
HENAN UNIV OF SCI & TECH
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  • Abstract
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  • Claims
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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; Although the method of source recombination leading to target gene mutation is accurate, the obtained mutant strains often contain resistance markers in the end, and cannot be used as vaccine strains for vaccine production because they do not meet the biosafety requirements

Method used

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  • Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof
  • Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof
  • Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Salmonella choleraesuis C78-2 crp Construction of gene deletion strains

[0032] 1. Primer design (for gene cloning and molecular detection)

[0033] Refer to literature (Xu Yindi, et al. Construction and identification of a balanced lethal vector system for △crp △asd deletion strain of Salmonella choleraesuis C500 strain. Acta Biological Engineering, 2006, 22(3):366–372) and the reported Salmonella typhi strain Ty2 of crp Gene sequence (GenBank No: AE016847) designed 2 pairs of primers (pr1 / pr2 and pr3 / pr4, see Table 1), from the virulent strain of Salmonella choleraesuis C78-2 (purchased from the National Veterinary Microorganism Collection Center of the China Veterinary Drug Administration). strains) were amplified separately in the genome crp Gene Upstream and Downstream Fragments crp1 (upper arm) and crp2 (lower arm), the sizes of the amplified fragments are 1048 bp and 1743 bp respectively, and the two ends of the upper arm are respectively ...

Embodiment 2

[0044] Example 2 Identification and biological characteristics of Salmonella choleraesuis C78-2 crp gene deletion mutant strain C7821

[0045] 1. Phenotype identification of gene deletion strain C7821

[0046] The gene deletion strain C7821 (experiment number: △crpC78-2) and the parental strain C78-2 were streaked and inoculated on solid LB plates, and then transferred to glucose, maltose, lactose, sucrose, rhamnose, mannose, arabinose, xylose , dulcitol, urea and other carbon sources and H 2 S and other biochemical identification tubes (purchased from Hangzhou Tianhe Company) were used for biochemical reactions. O and H antigens were identified according to the instructions of serum factors (purchased from China Veterinary Drug Administration). The results showed that the biochemical characteristics of the gene deletion strain C7821 were not completely consistent with the parent strain C78-2. Parental strain C78-2 can utilize maltose, and red colonies appear on MacConkey a...

Embodiment 3

[0051] Example 3 Preparation of Salmonella choleraesuis Gene Deletion Vaccine

[0052] The obtained Salmonella choleraesuis gene deletion mutant strain C7821 (experiment number: △ crp C78-2) for identification, each generation was inoculated on 1% maltose MacConkey agar medium to observe the color of the colony, and the use of Salmonella choleraesuis crp Genetic PCR detection was performed to identify the genetic stability of the missing bacteria. After 50 passages, it was found that the colonies of the deletion mutant strain C7821 could not turn red on 1% maltose MacConkey agar medium, and still met crp Phenotypic characterization of gene deletion; and PCR identification crp The gene is still missing 320 bp, and heredity is stable. The Salmonella choleraesuis gene deletion mutant strain C7821 was cultured on LB solid medium, and a single colony was picked and cultured in LB liquid medium until the concentration of viable bacteria reached 1×10 10 CFU / mL. Add gelati...

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Abstract

The invention relates to a salmonella choleraesuis gene deletion mutant C7821 without a resistant marker, with collection number being CCTCCNO: M2010102. The invention also relates to a salmonella choleraesuis gene deletion vaccine prepared by the mutant. The gene engineering strain of the mutant is derived from the salmonella choleraesuis C78-2 strain (which is a commercial strain purchased from the China Veterinary Culture Collection Center of China Institute of Veterinary Drugs Control). The main virulence adjusting gene crp of the mutant deletes 320bp, so that the bacteria can not ingest cAMP from the mammals and grow more slowly, and the virulence of the bacteria is greatly reduced, thus the mutant has high safety. The animal tests prove that the gene deletion mutant strain has good protective immunity. The gene deletion vaccine prepared by the invention can stimulate the swine to generate protective immune response resistant to salmonella choleraesuis homologous wild strains and effectively prevent infection with salmonella choleraesuis.

Description

technical field [0001] The invention relates to a gene deletion mutant strain of Salmonella choleraesuis without resistance marker, and also relates to a gene deletion vaccine of Salmonella choleraesuis prepared by the strain, which belongs to the technical field of genetic engineering of animal bacteria. Background technique [0002] Salmonella choleraesuis ( Salmonella cholerae suis ) is the main pathogen that causes paratyphoid fever in piglets aged 2 to 4 months. After infection, pigs can easily cause typical symptoms such as acute sepsis, chronic necrotic enteritis, and intractable diarrhea. Infection with other diseases or untimely treatment will result in a higher mortality rate and cause heavy losses. Animals infected with Salmonella or their products are contaminated, which can easily cause food poisoning after human consumption. Therefore, this pathogen is also of great significance in public health. [0003] Salmonella choleraesuis belongs to the genus Salmonell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/112C12N1/20A61P31/04C12R1/42
Inventor 程相朝赵战勤郁川张春杰李银聚吴庭才王臣余祖华廖成水
Owner HENAN UNIV OF SCI & TECH
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