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AHFGDS (aeromonas hydrophila five-gene deletion strain) attenuated bacteria without antibiotic markers and application

A Aeromonas hydrophila, antibiotic-free technology, applied in the direction of antibacterial drugs, microbial-based methods, medical preparations containing active ingredients, etc., can solve the problem of inability to stimulate correct immune response, low immune response, loss of protection Sexual antigens and other issues to avoid safety issues, improve disease resistance phenotype, and enhance immunity

Active Publication Date: 2016-08-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] my country's vaccines for the prevention and treatment of diseases caused by Aeromonas hydrophila are mainly inactivated vaccines, but inactivated vaccines usually have the following defects: the inactivation process leads to partial or complete loss of protective antigens, which can only cause unstable or low-level Immune response, even unable to stimulate the correct immune response and cause side effects, etc.

Method used

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  • AHFGDS (aeromonas hydrophila five-gene deletion strain) attenuated bacteria without antibiotic markers and application
  • AHFGDS (aeromonas hydrophila five-gene deletion strain) attenuated bacteria without antibiotic markers and application
  • AHFGDS (aeromonas hydrophila five-gene deletion strain) attenuated bacteria without antibiotic markers and application

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Experimental program
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Effect test

Embodiment 1

[0031] Acquisition of attenuated Aeromonas hydrophila AHFGDS without antibiotic markers:

[0032] Through genetic engineering, the five genes of aerA, alt, ahp, ast and hly of the highly virulent Aeromonas hydrophila ZYAH72 were knocked out, and after screening, a strain of Aeromonas hydrophila with five genes was finally obtained Deletion strain AHFGDS (hereinafter referred to as AHFGDS), the obtained mutant strain does not have the five genes described ( figure 1 ), so it is an attenuated strain. The strain was sent to the China Center for Type Culture Collection on December 29, 2015 for preservation, named after classification: Aeromonas hydrophila five-gene deletion strain AHFGDS, address: Wuhan University, Wuhan, China. Deposit number: CCTCC NO: M2015798.

[0033] The highly virulent Aeromonas hydrophila ZYAH72 of the present invention (hereinafter referred to as ZYAH72) is a highly virulent wild strain isolated and screened by the applicant from the diseased crucian ca...

Embodiment 2

[0037] Qualitative and quantitative hemolysis assays showed reduced cytotoxicity of AHFGDS:

[0038] In the qualitative test, single colonies of ZYAH72 and AHFGDS were picked respectively, and spotted on the sheep blood plate, and it was found that the hemolytic circle formed by AHFGDS on the blood plate was smaller than that of ZYAH72 ( image 3 ).

[0039] Fresh sheep red blood cells were diluted to 1% with saline.

[0040] Select a single clone on the plate, culture it in TSB medium for 24 hours, centrifuge at 12000rpm for 15min, and take the supernatant. Fresh sheep red blood cells were diluted to 1% with physiological saline, 100 μL each was mixed with 100 μL sheep red blood cells (1% content), incubated at 37° C. for 2 hours, and then the OD540nm absorbance value was measured. It can be observed that AHFGDS significantly reduces the toxicity to cells compared with the wild-type strain ZYAH72 ( Figure 4 ). This is due to the deletion of the hemolysin gene in AHFGDS. ...

Embodiment 3

[0042] 4-week-old female Balb / c mice were used as experimental subjects to prove that the toxicity of AHFGDS was weakened:

[0043] The mice used in the experiment were purchased from the Center for Disease Control and Prevention of Wuhan City, Hubei Province, and those that had no abnormal changes in the laboratory for 3 days were used as experimental mice.

[0044] Mice infection experiment: adjust the bacterial concentration of ZYAH72 and AHFGDS to 1*10 6 CFU / mL, each mouse was intraperitoneally injected with 200 μL bacterial solution, observed every 3 hours, and recorded the number of dead mice. The observation time lasted for one week. After injecting the wild strain ZYAH72, 8 mice died within 24 hours, while those injected with AHFGDS Mice did not die, indicating that the toxicity of AH FGDS to mice was significantly weakened ( Figure 5 ).

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Abstract

The invention discloses AHFGDS (aeromonas hydrophila five-gene deletion strain) attenuated bacteria without antibiotic markers and an application. Based on high-virulent strain ZYAH72, five virulence factors including aerA, alt, ahp, ast and hly are deleted with a genetic engineering method and a marker-free screening strategy, an AHFGDS with five genes deleted is obtained, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) No:M2015798. Compared with wild bacteria, the strain can still be subjected to a strong reaction with positive serum even if the pathogenicity is remarkably weakened. Experiments prove that the strain serving as an attenuated live vaccine can effectively protect susceptible aquatic animals against infection of wild AHFGDSs.

Description

technical field [0001] The invention relates to a live attenuated vaccine for freshwater aquaculture fish, in particular to an attenuated Aeromonas hydrophila without antibiotic markers and its application. Background technique [0002] Bacterial diseases outbreaks in fish have a high frequency of occurrence and a wide range of prevalence, which has become an important bottleneck restricting my country's freshwater aquaculture industry. [0003] Aeromonas hydrophila is an important fish pathogen. The bacterial sepsis caused by it seriously endangers the aquaculture industry in my country and even the world. Among them, the freshwater fish aquaculture industry is very serious, causing huge economic losses to the freshwater aquaculture industry in my country. A major acute infectious disease. [0004] Aeromonas hydrophila infection of fish causes extremely fast pathological changes, and studies have shown that the bacteria has a variety of pathogenic factors involved in the pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61K39/02A61P31/04C12R1/01
CPCA61K39/0208A61K2039/522A61K2039/552C12N1/205C12R2001/01
Inventor 周洋李锦铨林蠡王小红魏顺李智喻蔚
Owner HUAZHONG AGRI UNIV
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