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Haemophilus parasuis engineering strain with hhdA gene deletion and without resistance maker and construction method thereof

A technology of Haemophilus suis without resistance markers, which is applied in the field of construction of Haemophilus parasuis strains, and can solve the problems of unclear biological functions and the like

Active Publication Date: 2014-01-01
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is speculated that this gene may be related to the virulence of HPS, but the biological function of this gene in Haemophilus parasuis is still unclear

Method used

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  • Haemophilus parasuis engineering strain with hhdA gene deletion and without resistance maker and construction method thereof
  • Haemophilus parasuis engineering strain with hhdA gene deletion and without resistance maker and construction method thereof
  • Haemophilus parasuis engineering strain with hhdA gene deletion and without resistance maker and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The embodiment uses the Haemophilus parasuis serotype 5 wild-type H11 bacterial strain ( Haemophilus parasuis HPS-H11) (hhdA +) (preserved in China Center for Type Culture Collection (CCTCC) on July 6, 2013, address: China, Wuhan, Wuhan University, deposit number is CCTCC NO: M 2013316) as the parent strain , respectively design specific primers for the upstream and downstream sequences of the hhdA gene, amplify the upstream and downstream homology arms, use the method of overlapping PCR to connect the upstream and downstream sequences, construct the suicide plasmid vector PEMOC2ΔhhdA, and transfer PEMOC2ΔhhdA into the parental strain by combining the transfer method Among them, the strain without resistance marker deletion of hhdA gene (HPSΔhhdA) was finally obtained after screening and identification by corresponding methods. Specific steps are as follows:

[0052] 1. Amplification of the upstream and downstream sequences of the HPS-hhdA gene

[0053] According ...

Embodiment 2

[0075] Identification of Genetic Stability of Gene Deletion in HPSΔhhdA Strain

[0076] Streak culture the hhdA gene deletion strain HPSΔhhdA prepared in Example 1 on a TSA (10 μg / mL NAD, 10% fetal bovine serum) plate, pick a single colony and inoculate it on TSB (10 μg / mL NAD, 10% fetal bovine serum ) medium, cultured at 72°C and 200 r / min for 16 h, and the proliferated bacterial liquid was one generation. Then the first-generation bacterial solution was streaked on the TSA plate, and a single colony was picked and inoculated into the TSB medium for overnight shaking at 72°C. The proliferated bacterial solution was the second generation, which was repeatedly passaged in TSA and TSB medium. Continuously cultivated for 21 generations, every 3 generations, the proliferated bacterial liquid was amplified and identified by PCR with hhdA-up-F and hhdA-down-R primers, the amplification system was 25 μL: bacterial liquid template 1 μL, 10×PCR buffer 2.5 μL, 25 mmol / L MgCl 2 1 μL...

Embodiment 3

[0079] Identification of Growth Characteristics of HPSΔhhdA Strain

[0080] The hhdA gene-deleted strain HPSΔhhdA and the wild-type strain prepared in Example 1 were rejuvenated, respectively inoculated in TSB medium, cultured with shaking at 37 °C for about 12 h, and plate colonies were counted as seed liquid. According to the counting results, the deletion strain and the wild type strain were divided into 1×10 7 The colonies were inoculated into 5 mL of TSB liquid medium, and cultured with shaking at 37 °C 200 r / min, during which samples were taken every 2 h for plate colony counting, and the experimental results were drawn into a curve to analyze and identify the growth characteristics of the gene-deleted strains. Such as Figure 8 The indicated HPSΔhhdA strains had similar growth characteristics to the wild-type strain.

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Abstract

The invention discloses a haemophilus parasuis (HPS) strain with hhdA gene deletion and without a resistance maker and a construction method thereof. The construction method comprises the following steps: connecting upstream and downstream sequences of an hhdA gene of an HPS wild type strain by an overlapping PCR (Polymerase Chain Reaction) technology; constructing to a suicide plasmid vector (PEMOC2 delta hhdA) and transferring into an E.beta 2155 strain; transferring the PEMOC2 delta hhdA into the HPS wild type strain by a conjugal transfer method to screen out a single-exchanged positive combined son strain; carrying out limiting dilution continuous cultivation; and screening to obtain HPS delta hhdA of the HPS strain with hhdA gene deletion and without the resistance maker. The HPS delta hhdA strain constructed by the method has the growth characteristics and protective immunogenicity similar to those of a parent strain; an animal subjected to HPS delta hhdA strain immunizing does not produce an antibody for resisting hhdA protein, so that the HPS delta hhdA strain can be used as a vaccine for distinguishing the wild type strain in the processes of preventing, controlling and detecting haemophilus parasuis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of animal bacteria, and relates to a method for constructing a HhdA gene-deleted Haemophilus parasuis strain without a resistance marker. Background technique [0002] Haemophilus parasuis (HPS) is a polymorphic, nicotinamide adenine dinucleotide (NAD)-dependent, non-motile, Gram-negative fine bacterium belonging to the family Pasteurellaceae. belongs to. The pathogen can cause polyserositis, arthritis and meningitis in pigs, mainly characterized by Glösser's Disease. At present, the disease is widespread and prevalent in many countries and regions, and it is the primary cause of morbidity and death in weaned piglets aged 2 weeks to 4 months; clinically, the pathogen is often associated with porcine reproductive and respiratory syndrome virus, porcine round The mixed infection of pathogenic microorganisms with immunosuppressive effects such as ring virus and swine fever virus has cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/74A61K39/102A61P31/04C12R1/21
Inventor 宋帅李春玲杨冬霞李淼岳磊
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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