Preparation method and application of a mutant strain of Aeromonas veronii

Abstract

The present invention discloses a preparation method and application of a Aeromonas veronii mutant strain, belonging to the technical field of microbiology. The preparation method comprises the following steps: S1, extracting the whole genome of Aeromonas veronii; S2, constructing primers used for knocking out the cheZ / mshK / aerA genes; S3, using the extracted whole genome of Aeromonas veronii as a template, and using the primers to amplify the upstream and downstream homologous arms of the target gene and perform overlap extension to obtain the deleted cheZ / mshK / aerA gene fragment; S4, introducing the deleted cheZ / mshK / aerA gene fragment into the suicide plasmid pRE112 to obtain the recombinant plasmid pRE112-ΔCMA; S5, transferring the recombinant plasmid pRE112-ΔCMA into WM3064 competent cells, constructing a homologous recombinant mutant strain through conjugation transfer, and then screening by sucrose pressure to obtain the Aeromonas veronii mutant strain ΔCMA. The present invention uses molecular cloning technology, Overlap PCR technology and homologous recombination technology to perform in-frame deletion mutation on the cheZ / mshK / aerA genes of Aeromonas veronii by using a suicide vector, and obtains a stable hereditary Aeromonas veronii mutant strain ΔCM with three-gene deletion.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a preparation method and application of an Aeromonas veronii mutant strain. Background Art

[0002] Aeromonas veronii is a Gram-negative pathogenic bacterium that can infect humans, animals, and aquatic animals. It belongs to the family Aeromonadaceae and the genus Aeromonas. It is widely distributed and commonly exists in natural waters, soil, silt, and aquatic animal bodies. It has strong pathogenicity and can cause diseased fish to show symptoms such as bleeding, septicemia, ulcers, ascites, and high mortality, bringing huge economic losses to aquaculture. At the same time, it also has a great impact on humans, especially on those with low immunity, such as people suffering from AIDS, malignant tumors, cirrhosis, and diabetes. Once infected, there is a risk of death. Aeromonas veronii is usually non-pathogenic, but when the aquatic environment is harsh, the temperature changes suddenly, or when the immunity of aquatic animals declines, the pathogenic bacterium will play a pathogenic role through virulence factors such as chemotactic factors, fimbriae, flagella, and aerolysin.

[0003] In the prior art, there has been no research on developing a live attenuated vaccine by mutating the chemotactic factor, fimbriae, and aerolysin of Aeromonas veronii to reduce its pathogenicity. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method of an Aeromonas veronii mutant strain.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A preparation method of an Aeromonas veronii mutant strain includes the following steps:

[0007] S1. Extract the whole genome of Aeromonas veronii;

[0008] S2. Construct primers for knocking out the cheZ / mshK / aerA genes;

[0009] S3. Using the extracted whole genome of Aeromonas veronii as a template, use primers for amplifying the upstream and downstream homologous arms of the target gene and overlapping extension to obtain the deleted cheZ / mshK / aerA gene fragment;

[0010] S4. Introduce the deleted cheZ / mshK / aerA gene fragment into the suicide plasmid pRE112 to obtain the recombinant plasmid pRE112-ΔCMA;

[0011] S5. Transfer the recombinant plasmid pRE112-ΔCMA into the competent cells of WM3064, construct a homologous recombination mutant strain through conjugation transfer, and then obtain the Aeromonas veronii mutant strain ΔCMA through sucrose pressure screening.

[0012] Furthermore, an Aeromonas veronii mutant strain is used to prevent Aeromonas veronii infection in aquaculture.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1. Through molecular cloning technology, Overlap PCR technology and homologous recombination technology, the present invention uses a suicide vector to perform in-frame deletion mutations on the cheZ / mshK / aerA genes of Aeromonas veronii, and obtains the Aeromonas veronii mutant strain ΔCMA with three-gene deletion and stable inheritance.

[0015] 2. The Aeromonas veronii mutant strain ΔCMA provided by the present invention has significantly weakened toxicity, can provide necessary materials for the preparation of live attenuated vaccines against Aeromonas veronii, and prevent Aeromonas veronii infection in aquaculture, with important clinical value.

[0016] Other advantages, objectives and features of the present invention will be described in the subsequent specification, and to some extent will be obvious to those skilled in the art, or those skilled in the art can obtain teachings from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the objectives, technical solutions and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0018] Figure 1 It is the deletion effect diagram of the Aeromonas veronii mutant strain ΔCMA of the present invention;

[0019] Figure 2 It is the relative protection rate of different groups of Carassius auratus gibelio challenged with Aeromonas veronii of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present invention uses a suicide vector to perform in-frame deletion mutations on the cheZ / mshK / aerA genes of Aeromonas veronii, and obtains the Aeromonas veronii mutant strain ΔCMA with three-gene deletion and stable inheritance.

[0021] Example 1

[0022] S1. Extract the genomic DNA of the wild-type Aeromonas veronii virulent strain, and construct the primers used for knocking out the cheZ / mshK / aerA genes;

[0023] S2. Using the extracted whole genome of Aeromonas veronii as a template, amplify the upstream and downstream fragments of cheZ / mshK / aerA by Single PCR, and then ligate the two fragments by overlap extension PCR to obtain a fusion fragment;

[0024] S3. Ligate the fusion fragment to the suicide plasmid pRE112 and transform it into competent E. coli SM10λpir cells. After successful verification, conjugate the E. coli and the virulent strain of Aeromonas veronii. Utilizing the chloramphenicol (Cm) intolerance of Aeromonas veronii and the ampicillin (Amp) intolerance of E. coli, plate them on LB medium containing double antibiotics (Cm + Amp) for cultivation, and screen to obtain the first homologous recombinant Aeromonas veronii strain with two bands (mutant fragment ΔcheZ / mshK / aerA + non-mutant fragment cheZ / mshK / aerA);

[0025] S4. Culture in 15% sucrose medium using the sucrose intolerance of the pRE112 plasmid, screen to obtain the second homologous recombinant strain (only containing the mutant fragment ΔcheZ / mshK / aerA). After verification by molecular means and sequencing, conduct subculture to verify its genetic stability, and construct a genetically stable Aeromonas veronii ΔcheZ / mshK / aerA mutant strain, namely the Aeromonas veronii mutant strain ΔCMA.

[0026] Figure 1 This is the deletion effect diagram of the Aeromonas veronii mutant strain ΔCMA of the present invention.

[0027] Using Carassius auratus gibelio to set up a control group (wild-type Aeromonas veronii), a control group, and an experimental group (Aeromonas veronii mutant strain ΔCMA), and conduct pathogenicity and live attenuated vaccine feasibility analysis. The results are shown in Table 1. The median lethal dose (LD50) of the Aeromonas veronii mutant strain ΔCMA to Carassius auratus gibelio increased significantly.

[0028] Table 1

[0029]

[0030] After immunizing Carassius auratus gibelio for 4 weeks and then challenging, the results are as Figure 2 shown. It was found that the final relative protection rate RPS of the inactivated Aeromonas veronii vaccine group was 53.73 ± 10.87%. And there was no new death number of Carassius auratus gibelio after immunization in the Aeromonas veronii ΔCMA group. The final RPS of the ΔCMA-AV group was 73.97 ± 3.57%, which was significantly higher than that of the inactivated vaccine group (p < 0.05).

[0031] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in terms of form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a mutant strain of Aeromonas veronii, characterized in that: It includes the following steps: S1. Extract the whole genome of Aeromonas veronii, and construct primers used for knocking out the cheZ / mshK / aerA genes. S2. Using the extracted whole genome of Aeromonas veronii as a template, use primers to amplify the upstream and downstream homologous arms of the target gene and perform overlap extension to obtain the deleted cheZ / mshK / aerA gene fragment. S3. Introduce the deleted cheZ / mshK / aerA gene fragment into the suicide plasmid pRE112 to obtain the recombinant plasmid pRE112-ΔCMA. S4. Transfer the recombinant plasmid pRE112-ΔCMA into the WM3064 competent cells, construct a homologous recombination mutant through conjugation transfer, and then screen through sucrose stress to obtain the Aeromonas veronii mutant strain ΔCMA in which the three genes cheZ / mshK / aerA are knocked out.

2. Application of the mutant strain obtained by the preparation method of an Aeromonas veronii mutant strain according to claim 1 in the preparation of a vaccine for preventing Aeromonas veronii infection in aquaculture.

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