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Recombinant Salmonella pullorum, preparation method and application

A technology of Salmonella pullorum and recombinant plasmids, applied in the field of genetic engineering of animal bacteria, can solve the problems of residual virulence, high production cost, stimulation of the body's cellular immunity and mucosal immunity, etc., achieve broad market application prospects and reduce production costs , the effect of good biosecurity

Active Publication Date: 2020-02-11
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, traditional vaccines are mainly used for immunization prevention of ND in my country, which has problems such as high production cost, residual virulence, and inability to stimulate the body to produce cellular immunity and mucosal immunity.

Method used

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  • Recombinant Salmonella pullorum, preparation method and application
  • Recombinant Salmonella pullorum, preparation method and application
  • Recombinant Salmonella pullorum, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0059] The name of the recombinant Salmonella pullorum in this example is Salmonella pullorum C79-13ΔcrpΔasd (pYA3493-HN) (Salmonella enteria serovar Pullorum C79-13ΔcrpΔasd (pYA3493-HN)), preservation number: CCTCC NO: M 2015305, preservation date : May 15, 2015, depository unit: China Center for Type Culture Collection (CCTCC), deposit address: China.Wuhan.Wuhan University.

[0060] The preparation method of recombinant Salmonella pullorum comprises the following steps:

[0061] (1) Prepare the gene fragment of the chicken Newcastle disease protective antigen HN protein capable of prokaryotic expression

[0062] a. Design of primers required for preparing the prokaryotic expression plasmid pET-32a-HN, the sequences of which are shown in Table 1 below.

[0063] Table 1 prepares the primer sequences required for the prokaryotic expression plasmid pET-32a-HN

[0064]

[0065] The underlined parts of the primers in Table 1 are restriction sites.

[0066] Wherein, the prime...

Embodiment 2

[0080] In this example, the application of recombinant Salmonella pullorum (CCTCC NO: M 2015305) in the preparation of genetic engineering vaccines includes: streaking culture of recombinant bacteria C79-13ΔcrpΔasd (pYA3493-HN) on LB solid medium, picking A single colony was placed in LB liquid medium, and cultured until the concentration of viable bacteria reached 2.5×10 10 CFU / mL. The bacteria liquid was centrifuged at a low speed to collect the bacteria, and mixed according to the ratio of Salmonella C79-13ΔcrpΔasd (pYA3493-HN):50% skimmed milk powder (volume:volume) at a ratio of 2:1. Divide into 1.5mL / bottle, freeze-dry in a vacuum freeze dryer, seal the cap after freeze-drying, and store at -20°C for later use.

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Abstract

The invention discloses recombinant S. Pullorum, as well as a preparation method and an application thereof, belonging to the technical field of animal bacterial gene engineering. The name of the recombinant S. Pullorum is S. Pullorum C79-13deltacrpdeltaasd(pYA3493-HN), the preservation number is CCTCC NO:M 2015305, the newcastle diseases virus protective antigen gene-HN gene expressed by the recombinant S. Pullorum is the important immunogenic gene segment for the newcastle diseases virus, and has good immune protection. The recombination strain derives from the S. Pullorum standard strain C79-13, completely reserves the immune efficacy of C79-13 for S. Pullorum, has weaker toxicity than that of C79-13, has good bio-safety, and can simultaneously provide protection aiming at S. Pullorum and newcastle disease pathogenes. Meanwhile, the recombinant strain contains no resistance markers, and completely accords with the requirements on the bio-safety of vaccines.

Description

technical field [0001] The invention relates to a recombinant Salmonella pullorum, in particular to an attenuated Salmonella pullorum expressing the main protective antigen of Newcastle disease virus without a resistance marker, and a preparation method and application of the recombinant Salmonella pullorum, belonging to Technical field of genetic engineering of animal bacteria. Background technique [0002] Salmonella (Salmonella) is an intracellular parasitic Gram-negative enterobacteriac with invasiveness. The most common Salmonella species are Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis and Salmonella pullorum. Salmonella has strong pathogenicity to both humans and animals, and can cause gastrointestinal diseases in humans and animals. Salmonella has a strong viability in the external environment. After being weakened by genetic engineering, it still has Strong aggressiveness. [0003] Initially, the vaccine for preventing Salmonella was an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/295A61K39/17A61K39/112A61P31/04A61P31/14C12R1/42
Inventor 郁川程相朝余祖华翟崇凯何雷贾艳艳廖成水李静汪洋张春杰李银聚吴庭才
Owner HENAN UNIV OF SCI & TECH
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