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Swine cholera attenuated salmonella recombinant strain for expressing haemophilus parasuis GAPDH gene

A technology for Haemophilus suis and cholera suis, which is applied in the field of recombinant strains of Salmonella suis attenuated, can solve the problems of serotype complex infection persistence, difficulty in infection, etc.

Inactive 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

[0003] Due to the complex serotype of Haemophilus parasuis and the persistence of infection caused by it, it is becoming more and more difficult to prevent and control the infection of Haemophilus parasuis in pigs.

Method used

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

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Embodiment 1

[0026] Embodiment 1 (design primer pair)

[0027] This embodiment provides a pair of primers for amplifying the GAPDH gene of the HPS standard type 5 SH0165 strain, which is based on the gene sequence of Haemophilus parasuis, using Oligo6.0 software analysis, to design a pair of GAPDH primers, and add Restriction sites EcoR I, Hind III and in-box and protective bases. Analyzed by computer BLAST software, it has good specificity; includes primer 1 and primer 2; the nucleotide sequence of primer 1 is shown in sequence 1 in the sequence table, that is, the upstream primer (GAPDH-up): 5'-GTAAAAGATGCTGAAGAATTCATGGCAATTAAAATTG- 3';

[0028] The nucleotide sequence of primer 2 is shown as sequence 2 in the sequence listing, ie the downstream primer (GAPDH-down): 5'-GCAAGTTTAAATAAATTTTTTTAGCCTTTGTAGTTG-3'.

[0029] The GAPDH gene-specific primers used in the present invention are self-designed according to the gene sequence and synthesized by Guangzhou Tianyi Huiyuan Biotechnology S...

Embodiment 2

[0030] Embodiment 2 (construction of recombinant vector)

[0031]In this embodiment, a recombinant vector is constructed, and the steps specifically include the following steps.

[0032] Extract the DNA of Haemophilus parasuis type 5 serum strain SH0165 (preserved by the Department of Poultry Diseases, South China Agricultural University), and then use the primer pair in Example 1 to amplify the GAPDH gene, wherein the PCR amplification conditions are: 94 ° C pre-denaturation 5min; denaturation at 94°C for 45s, annealing at 55°C for 30s, extension at 72°C for 90s, 34 cycles, and finally extension at 72°C for 10min; the amplified PCR product was recovered using a product purification kit to recover the target fragment.

[0033] After the amplified product was electrophoresed on an agarose gel, the target band was cut under ultraviolet light, recovered and purified with a gel recovery kit, and the specific steps were carried out according to the instructions.

[0034] The resul...

Embodiment 3

[0038] Embodiment 3 (construction of a strain Haemophilus parasuis outer membrane protein gene GAPDH recombinant cholerasuis attenuated Salmonella strain)

[0039] In this example, an attenuated Salmonella strain of cholerasuis recombinant with the outer membrane protein gene GAPDH of Haemophilus parasuis was constructed, and the specific steps were as follows.

[0040] Construct the asd gene-deleted strain C500Δasd of Salmonella choleraesuis; then electrotransfer the recombinant vector pYA3493(+)-GAPDH of Example 2 into the asd gene-deleted strain C500Δasd of Salmonella choleraesuis, specifically take 10 ul of the desalted recombinant plasmid pYA3493-GAPDH and add Put it into 100ul competent cells C500Δasd, mix gently and immediately transfer to a pre-cooled 0.2cm electric transfer cup, blot up the water stains on the outside of the electric transfer cup, and then put it into the sample chamber of the electrotransfer instrument according to the following conditions for electro...

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Abstract

The invention relates to a swine cholera attenuated salmonella recombinant strain for expressing a haemophilus parasuis GAPDH gene. A balanced lethal vector system of a salmonella choleraesuis C500 delta asd deletion strain is utilized, an important immunogenicity gene GAPDH of haemophilus parasuis is connected with pYA3493 vectors in host bacteria, and correspondingly an HPS-Sal recombinant vaccine strain is constructed, wherein the recombinant strain lacks an asd gene on a salmonella choleraesuis genome and contains a recombinant plasmid pYA-GAPDH capable of expressing the asd gene and the GAPDH gene of haemophilus parasuis in the strain. It is shown by detection that the recombinant strain has a good expression effect. It is indicated that outer membrane protein GAPDH can be used as basic protein for researching clinical detection, epidemiological investigation and immunodetection of haemophilus parasuis in the later period, and because of the particularity of vectors and receptor bacteria, a basic idea is provided for creation of 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 an attenuated Salmonella choleraesuis recombinant strain expressing the GAPDH gene of Haemophilus parasuis. Background technique [0002] Haemophilus parasuis, also known as Glaser's disease, is a commensal bacterium that exists in the upper respiratory tract of healthy pigs, but it is also the pathogen that causes Glaser's disease. The main features are polyserositis, meningitis, and arthritis. Haemophilus parasuis can affect young pigs from 2 weeks to 4 months. It mainly occurs after weaning and nursery. It is usually seen in pigs aged 5 to 8 weeks. The incidence rate is generally 10% to 15%. The mortality rate can reach 50%. With the emergence of production management problems in recent years, Haemophilus parasuis has become one of the main pathogens causing morbidity and mortality in pigs, causing huge economic...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/74C12N15/66C12N9/02C12N1/21C12R1/42
CPCC12N9/0008C12N15/66C12N15/74
Inventor 张建民陈政权
Owner SOUTH CHINA AGRI UNIV
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