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Haemophilus parasuis with double-gene deletion and construction method thereof

A technology for gene deletion of Haemophilus parasuis, applied in the field of Haemophilus parasuis and its construction, can solve the problem of unclear pathogenic mechanism of Hps

Active Publication Date: 2014-06-18
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the pathogenic mechanism of Hps is still unclear. The aroA gene and omp2 gene are considered to be the potential virulence genes of Hps, and there are deep research reports in other bacteria

Method used

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  • Haemophilus parasuis with double-gene deletion and construction method thereof
  • Haemophilus parasuis with double-gene deletion and construction method thereof
  • Haemophilus parasuis with double-gene deletion and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] A Haemophilus parasuis with double gene deletion is constructed by the following method:

[0059]1) PCR identification of strain H10: referring to the method of PCR identification of Hps established by Oliveira, S. et al. in 2001, a pair of primers SEQ ID NO: 11 and SEQ ID NO: 12 were synthesized, and 16S was amplified using the overnight cultured bacterial solution as a template For rRNA fragments, the PCR reaction system is 25 μL: 10xTaqbuffer 2.5 μL, 2 mmol / L dNTPs 0.5 μL, 16S rRNA-1 and 16S rRNA-2 (10 μmol / L) 1 μL each, Taqase 0.5 μL, template 1 μL, ddH2O to 25 μL. The PCR reaction program is: 94°C for 4min, then enter the PCR cycle: 94°C for 30s, 58°C for 30s, 72°C for 1min, 30 cycles; finally, extend at 72°C for 10min; the amplified product is identified by 0.8% agarose gel electrophoresis, and the UV Observed under the lamp; a single band was amplified with a size of about 800bp, which was consistent with the expected 821bp fragment, proving that the strain H10 w...

Embodiment 2

[0077] The steps are the same as in the example, except that the identification of the clones in step 7) adopts the conventional method. Pick the single colonies grown after transformation, inoculate them in 3ml of liquid medium containing the corresponding antibiotics, and culture them at 37°C and 250r / min for 6h Left and right until the medium becomes turbid; take 100 μL of the bacterial solution, centrifuge at 5000r / min for 10min, discard the supernatant, resuspend the bacterial cells with 30μL of sterilized double distilled water, boil the cell suspension for 10min, then ice-bath for 10min, 12000r / min Centrifuge for 1 min, and take the supernatant as a PCR template for PCR identification.

[0078] Result identification

[0079] 1. Amplification of the front and rear arm fragments of the aroA gene: such as Figure 6 As shown, the electrophoresis results showed a band of about 700bp, consistent with the expected 710bp fragment. The rear arm ΔaroA-DN of the aroA gene was am...

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Abstract

The invention discloses haemophilus parasuis with double-gene deletion. The haemophilus parasuis is a double-gene deletion strain, wherein an aroA gene of the strain deletes 394131-394631 nucleotides, an omp2 gene deletes 181258-181643 nucleotides, and the accession number of the strain is CCTCC NO: M2013460. The haemophilus parasuis disclosed by the invention mainly utilizes a modern genetic engineering principle and a molecular biological means to delete the Hps potential virulent factors, namely the aroA gene and the omp2 gene, constructs the Hps resistance marker-free double-gene deletion strain, investigates the biological features, the virulence and the like, and further lays a foundation for further development of novel efficient genetic engineering live vaccines capable of providing high cross-protection efficacy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double-gene-deleted Haemophilus parasuis and a construction method thereof. Background technique [0002] Haemophilus parasuis disease is a new bacterial infectious disease that seriously harms the pig industry in recent years, causing multiple serositis, arthritis and meningoencephalitis in pigs. The incidence rate is generally 10-15%, and the mortality rate is It can reach more than 50%, which has aroused people's increasing attention. The serotypes of Haemophilus parasuis (Hps), the pathogenic bacterium of the disease, are complex and diverse. According to the Kieleetin. The serotype of more than % of the isolates could not be determined. Existing data show that Hps has obvious endemic characteristics, and the inactivated vaccine prepared based on the whole bacteria of one or some specific serotype strains has a very low cross-protection rate between different serotypes, which...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/74A61K39/102A61P31/04C12R1/21
Inventor 贾爱卿王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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