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Bordetella bronchiseptica gene deletion strain, vaccine prepared from Bordetella bronchiseptica gene deletion strain and application

A technology of gene deletion strain, Bordetella sanguinis, applied in the field of animal bacterial genetic engineering, can solve problems such as deletion, and achieve the effects of stable traits, clear genetic background and good biological safety

Active Publication Date: 2012-09-19
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because Bordetella bronchiseptica contains multiple virulence factors, there are obvious differences in virulence and pathogenicity among different strains, and it is not clear which one or which type of virulence-related genes play a role in pathogenicity and immunity. Related functions play a decisive role, therefore, there is a certain degree of blindness in choosing to delete one or several target genes

Method used

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  • Bordetella bronchiseptica gene deletion strain, vaccine prepared from Bordetella bronchiseptica gene deletion strain and application
  • Bordetella bronchiseptica gene deletion strain, vaccine prepared from Bordetella bronchiseptica gene deletion strain and application
  • Bordetella bronchiseptica gene deletion strain, vaccine prepared from Bordetella bronchiseptica gene deletion strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction and identification of Bordetella bronchiseptica LY1aroA gene deletion strain

[0026] 1. Primer design (for gene cloning and molecular detection)

[0027] According to the aroA gene sequence (GenBank No: AF315119) of the reported Bordetella bronchiseptica strain RB50, two pairs of primers (ar1 / ar2 and ar3 / ar4, see Table 1) were designed to obtain the virulent strain of Bordetella bronchiseptica. LY1 (LY1 is a virulent strain of wild-type Bordetella bronchiseptica isolated from pigs, which was deposited in the China Center for Type Culture Collection (CCTCC) on December 23, 2011, and the preservation number: CCTCC NO: M2011478.) in the genome Respectively amplify the upstream and downstream fragments aroA1 (upper arm) and aroA2 (lower arm) of the aroA gene. The sizes of the amplified fragments are 927bp and 887bp respectively. The Kpn I and BamH I restriction sites are introduced into the two ends of the upper arm and the two ends of the lower arm ...

Embodiment 2

[0039] Example 2 Bordetella bronchiseptica gene deletion strain aroA - Analysis of growth characteristics of LY1

[0040] gene deletion strain aroA - LY1 was cultured at 37°C for 36 hours on a TSA plate containing 10% calf serum, and the diameter of the colony was about 0.7mm, which was significantly smaller than that of the parent strain LY1 (1.5mm) (see Figure 5 ). Parent strain LY1 and gene deletion strain aroA - LY1 from 10 6 CFU / mL to start culturing, sampling every 1h, and counting viable bacteria. The results showed that the gene deletion strain aroA - LY1 grew significantly slower than the parental strain LY1 (see Image 6 ), the mean generation interval (Mean generation time) was 38.7min, which was 12.6min longer than that of the parental strain (26.1min), indicating that the growth rate of the gene deletion strain LY1 was significantly slower than that of the parental strain LY1.

Embodiment 3

[0041] Example 3 Gene deletion strain aroA - Genetic stability of LY1

[0042] The gene deletion strain aroA prepared by the present invention - Streak culture of LY1 on a TSA plate containing 10% calf serum, pick a single colony on the TSA plate containing 10% calf serum, culture in a 37°C incubator for 24 hours, and then pick a single colony on the cultured plate Transfer to a TSA plate containing 10% calf serum for 50 consecutive transfers, and observe the colony size. The results showed that the gene deletion strain aroA - The colony diameter of LY1 is still about 0.7mm, which is significantly smaller than that of the parent strain LY1 (about 1.5mm), and still conforms to aroA - The growth rate of the LY1 gene deletion strain was obviously slow. At the same time, use primers ar1 / ar4 (see Table 1) to carry out PCR identification, gene deletion strain aroA - Inheritance of the aroA deletion gene in LY1 see Figure 7 . Figure 7 It shows that the 1, 10, 20, 30, 40 and ...

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Abstract

The invention relates to a Bordetella bronchiseptica gene deletion strain, vaccine prepared from the Bordetella bronchiseptica gene deletion strain and application of the strain. The aroA gene deletion strain, namely aroA-LY1 (Bordetella bronchiseptica), is preserved in the China Center for Type Culture Collection (CCTCC) on 23 December 2011, and the preservation number is CCTCCNO: M 2011479. The Bordetella bronchiseptica gene deletion strain disclosed by the invention has excellent immune protection efficacy and weak toxicity without any adverse side effect on immunized pigs and is safer compared with the traditional vaccine. Therefore, the vaccine which is prepared from the gene deletion strain built by a parent strain has wide market application prospect. In addition, the Bordetella bronchiseptica gene deletion strain disclosed by the invention retains the immune protective efficacy aiming at Bordetella bronchiseptica infection, and has clear genetic background and stable characteristics, so that the Bordetella bronchiseptica gene deletion strain has better biosecurity. The Bordetella bronchiseptica gene deletion strain disclosed by the invention does not contain a resistance tag and totally accords with the biological security requirement of vaccine.

Description

technical field [0001] The invention relates to a gene-deleted strain of Bordetella bronchiseptica without resistance marker, a vaccine constructed by it and the application of the vaccine, which belong to the technical field of animal bacterial genetic engineering. Background technique [0002] Bordetella bronchiseptica (Bb) is a small Gram-negative aerobic bacillus that is closely related to B. pertussis (Bp) and B. parapertussis (B. .parapertussis, Bpp) belong to the Bordetella genus. Bb can widely infect a variety of mammals, especially pigs, rabbits, and dogs. It manifests as acute and chronic inflammation of the respiratory tract, collectively known as Bordetella. In pigs, Bordetella bronchiseptica can cause pneumonia and atrophic rhinitis (AR) in pigs, and is also an important pathogenic factor of porcine respiratory disease complex (PRDC). Porcine Bordetella disease, represented by atrophic rhinitis, has spread all over the developed countries of the pig industry. ...

Claims

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Application Information

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IPC IPC(8): C12N1/20A61K39/10A61P31/04C12R1/01
Inventor 赵战勤薛云冯书营
Owner HENAN UNIV OF SCI & TECH
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