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Swine cholera attenuated salmonella recombinant strain for expressing haemophilus parasuis Omp 26 gene

A technology of Haemophilus suis and cholerasuis, which is applied in the field of recombinant strains of attenuated Salmonella choleraesuis, which can solve the problems of large differences in virulence of strains and inability to type

Pending Publication Date: 2019-04-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, the vaccine for preventing HPS disease is an inactivated vaccine, but the serotypes of Haemophilus parasuis are diverse, and a large proportion cannot be typed, and the virulence of the strains varies greatly.

Method used

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  • Swine cholera attenuated salmonella recombinant strain for expressing haemophilus parasuis Omp 26 gene
  • Swine cholera attenuated salmonella recombinant strain for expressing haemophilus parasuis Omp 26 gene
  • Swine cholera attenuated salmonella recombinant strain for expressing haemophilus parasuis Omp 26 gene

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Embodiment 1

[0040] Embodiment 1 (design primer pair)

[0041] This embodiment provides a primer pair for amplifying the Omp 26 gene of the HPS standard type 5 SH0165 strain, which is a primer designed according to the published sequence Gene ID of the NCBI database: 219691637, including primer 1 and primer 2;

[0042] The nucleotide sequence of primer 1 is shown in sequence 1 in the sequence listing, that is, the upstream primer (Omp 26-up): 5'-GCGTCGACAAAAAATTTATTTAAACTTGC-3';

[0043] The nucleotide sequence of primer 2 is as shown in sequence 2 in the sequence listing, namely the downstream primer (Omp 26-down): 5'-CCAAGCTTTTTTTCACTTCTTCTGG-3'.

Embodiment 2

[0044] Embodiment 2 (construction of recombinant vector)

[0045] This embodiment constructs a recombinant vector, the steps include: extracting the DNA of Haemophilus parasuis type 5 SH0165 strain, and then using the primer pair in Example 1 to amplify the Omp 26 gene, wherein the PCR amplification condition is: 94°C Pre-denaturation for 5 min; denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, a total of 32 cycles; final extension at 72°C for 10 min; gel recovery of the amplified PCR product (gel electrophoresis as shown in Figure 7 shown) and purified, then ligated with the pMD18-T vector to obtain the ligated product; the ligated product was transferred into competent cells DH-5α, identified by enzyme digestion, screened positive clones and sequenced to obtain pMD18-T-Omp 26; Simultaneously digest the non-resistant plasmid vector pYA3493 with EcoR I and Hind III (the restriction site of pYA3493 is as follows Figure 8 As shown, the ...

Embodiment 3

[0046] Embodiment 3 (construct a strain Haemophilus parasuis outer membrane protein gene Omp 26 recombinant cholerasuis attenuated Salmonella strain)

[0047] This embodiment constructs a strain of Haemophilus parasuis outer membrane protein gene Omp 26 recombinant choleraesuis attenuated Salmonella strain, the steps include: constructing the asd gene deletion strain C500Δasd of Salmonella choleraesuis; and then introducing the recombinant vector pYA3493(+) -Omp 26 was electrotransformed into the asd gene deletion strain C500Δasd of Salmonella choleraesuis, specifically, 10 μl of the desalted recombinant plasmid pYA3493(+)-Omp 26 was added to 100 μl of competent cells C500Δasd, mixed gently and immediately transferred to the pre- In a cold 0.2cm electric cup, blot up the water stains on the outside of the electric cup, and then put it into the sample tank of the electric cup for electrotransformation according to the following conditions: voltage 2.0KV, capacitance 5μF; pulse r...

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Abstract

The invention relates to a swine cholera attenuated salmonella recombinant strain for expressing a haemophilus parasuis Omp 26 gene. Omp 26 is selected as an exogenous gene, a non-resistant plasmid pYA3493 is selected as a vector, and a swine cholera attenuated salmonella C500 asd gene deletion strain is adopted as a receptor strain to form an Asd+ balanced expression system. The asd gene can be stably inherited under the condition of no exogenous selection pressure, the problem of previous unstable inheritance of exogenous genes can be solved, the system has no marker of any antibiotic gene,a biological safety problem cannot be generated, and foundation is laid for developing the system into a safe, efficient and live vaccine vector; meanwhile, the outer membrane protein Omp 26 of haemophilus parasuis (HPS) can be well expressed and can also serve as special protein for researching clinical detection, epidemiological investigation and immune detection of the HPS. A basic idea is provided for research on bivalent vaccines of swine cholera and haemophilus parasuis.

Description

technical field [0001] The invention relates to the technical field of genetic engineering vaccines for animal bacterial diseases, in particular to constructing a recombinant strain of cholerasuis attenuated Salmonella choleraesuis expressing the Omp26 gene of Haemophilus parasuis. Background technique [0002] Salmonella is an intracellular parasite, and the vaccine strain can effectively present antigens, and induce specific humoral and cellular immune responses against exogenous antigens while stimulating an anti-Salmonella immune response. The Salmonella carrier itself has an immune adjuvant effect, and its lipopolysaccharide ( LPS) as an internal adjuvant can stimulate host cells to release various cytokines. In view of this, using the auxotrophic (Asd gene) attenuated Salmonella as the expression vector of the genetically engineered live vaccine for mucosal immunization has incomparable advantages. Based on the fact that the attenuated intracellular bacteria can be di...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/74C12N15/66C12N1/21C07K14/285C12R1/42
CPCC07K14/285C12N15/66C12N15/74
Inventor 张建民陈政权
Owner SOUTH CHINA AGRI UNIV
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