Edwardsiella tarda subunit vaccine, and preparation and application thereof

A delayed Edwards, subunit vaccine technology, applied in the field of immunology, can solve the problem of no commercial vaccine, and achieve the effect of simple application method and high immune protection rate

Inactive Publication Date: 2014-04-02
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the prevention and treatment of Edwardsiella tarda mainly relies on antibiotics. Due to the adverse effects of antibiotics on ecology, environment and food safety, their application in aquaculture is subject to many restrictions at home and abroad.
Immunization is a safe and effective way to prevent

Method used

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  • Edwardsiella tarda subunit vaccine, and preparation and application thereof
  • Edwardsiella tarda subunit vaccine, and preparation and application thereof
  • Edwardsiella tarda subunit vaccine, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The construction method of Edwardsiella tarda vaccine expression vector:

[0020] Step 1) Construction of plasmid pGMTD: Edwardsiella tarda LSE40 (preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, preservation number: CGMCC No.7199, taxonomically named Edwardsiella tarda, preservation date: On January 23, 2013, the address of the preservation unit is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) Genomic DNA was used as a template, and the primer EDF (5'-GCGC GGATCC GACGACTATCGACAGCGGCAG-3', the underlined base is the BamHI restriction endonuclease point) and EDR (5'-GCGC AAGCTT GGACATGCGTACGCTGCT-3', the underlined base is the HindIII restriction endonuclease as the point) for PCR amplification (the PCR instrument was purchased from Takara Company, model TP600). The volume of the PCR reaction system is 25 μL: 2.5 μL of 10×PCR buffer solution, 0.5 μL of 0.2 mM dNTP, 1.5 mM MgCl 2 2.0 μL, 0.5 μL ea...

Embodiment 2

[0024] The preparation method of Edwardsiella tarda vaccine protein:

[0025] The resulting plasmid pETD was transformed into competent Escherichia coli BL21(DE3) (Tiangen Biochemical Technology Co., Ltd., Beijing), cultured on LB solid medium containing 50 μg / ml kanamycin, screened positive colonies, and named it BL21 / pETD. Culture BL21 / pETD in LB liquid medium containing 50μg / ml kanamycin, culture at 37°C for 12-16 hours, and then transfer to 100ml containing 50μg / ml In the fresh LB liquid medium of kanamycin, culture at 37°C at 120rpm to OD 600 0.6-0.8, add IPTG to a final concentration of 1mmol / L, continue to cultivate for 4-6 hours, then centrifuge at 5000g, 4°C for 10min, collect the bacteria and suspend them in 5-10ml PBS, and the obtained bacteria suspension is lysed by ultrasonic Bacteria until the bacterial suspension is clear (200w, ultrasonic 5s, interval 15s, 20-30min). The obtained clarified bacterial liquid was centrifuged at 10,000 g at 4° C. for 30 min, an...

Embodiment 3

[0028] Use of Edwardsiella tarda subunit vaccine:

[0029] Step 1) Oil emulsion vaccine preparation. Mix 92% (volume ratio) No. 10 white oil, 6% (volume ratio) Siben-80 and 2% (volume ratio) aluminum stearate (heated and melted) to form an oil-emulsion adjuvant. Dilute the vaccine protein purified in Example 2 above to 200 μg / ml in PBS; mix the diluted vaccine protein with oil-emulsion adjuvant in equal volumes to form an oil-emulsion vaccine.

[0030] Step 2) Immunization application of subunit vaccine. Experimental turbot with a body weight of 10-12g was kept temporarily in a 1000L aquarium equipped with a flowing circulating water treatment and purification system, and the culture water temperature was maintained at 16-18°C. After 1 week of temporary breeding, they were randomly divided into 3 groups into a 500L test aquarium, with 50 fish in each group, and continued to be temporarily raised for 1 week. In the test aquarium, the water was changed once a day in the morni...

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Abstract

The invention relates to the field of immunology, and specifically relates to an Edwardsiella tarda subunit vaccine and a preparation method thereof. The protein gene of the vaccine has a base sequence shown in SEQ ID No.1 in a sequence table, and the vaccine protein coded by the protein gene has an amino acid sequence as shown in SEQ ID No.2 in the sequence table. The subunit vaccine provided by the invention is mixed with an oil emulsion adjuvant, and then capable of effectively exciting the generation of specific antibodies after immunizing the fish through intraperitoneal injection; the Edwardsiella tarda subunit vaccine is capable of efficiently improving the ability of resisting the infection of the Edwardsiella tarda of the immune fish and thus can be applied to controlling the Edwardsiella tarda of the fish.

Description

technical field [0001] The invention relates to the field of immunology, in particular to an Edwardsiella tarda subunit vaccine and its preparation and application. Background technique [0002] Edwardsiella tarda is a Gram-negative bacillus that infects fish, amphibians, reptiles, and mammals. The bacterium is one of the important pathogenic bacteria of freshwater and marine cultured fish and wild fish, causing hemorrhagic sepsis and causing significant economic losses in fish farming worldwide. In my country, Edwardsiella tarda not only infects soft-shelled turtle, eel, tilapia and other freshwater cultured species, but also infects important species such as bass, flounder, turbot, tongue sole, American redfish, large yellow croaker, grouper and red sea bream. Mariculture species are very harmful to my country's aquaculture. At present, the prevention and treatment of Edwardsiella tarda mainly relies on antibiotics. Due to the adverse effects of antibiotics on ecology, en...

Claims

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Application Information

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IPC IPC(8): A61K39/02C12N15/70C12P21/02A61P31/04C12R1/01C12R1/19
Inventor 莫照兰李贵阳黄倢李杰
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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