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Aeromonas hydrophila heat shock protein subunit vaccine and preparation method thereof

A technology of Aeromonas hydrophila and subunit vaccine, applied in the fields of genetic engineering and vaccinology, can solve the problems of toxic and side effects, short validity period, high storage and transportation conditions of live attenuated vaccines, and achieve high protection rate, operation easy effect

Inactive Publication Date: 2014-08-27
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation methods of the above two vaccines are simple, but the use of inactivated vaccines in aquaculture animals requires large doses and multiple immunizations, while the storage and transportation conditions of the attenuated live vaccines are extremely high, the validity period is short, and there is a risk of reversion , so they may cause greater toxic side effects
The development of vaccines for diseases related to turtles and turtles is far behind that of fish. There is no practically available long-term storage and high-efficiency vaccines on the market to prevent and control Vibrio sinensis diseases

Method used

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  • Aeromonas hydrophila heat shock protein subunit vaccine and preparation method thereof

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Effect test

Embodiment 1

[0020] Preparation of recombinant subunit vaccine AH DnaK

[0021] 1.1 Cloning of the heat shock protein gene: using the genomic DNA of Aeromonas hydrophila as a template, using the primer AH DnaK F1 (5'-CCCGGGCATATGGGTAAAATCATCGGTATTGA-3', the sequence is shown in SEQ ID No.2, and the underlined bases are SmaⅠ restriction site) and AH DnaK R1 (5'- CCCGGGCTCGAGCTTCTTGTCGTCTTTCACTTC-3', the sequence is shown in SEQ ID No.3, the underlined base is the SmaⅠ restriction site) as primers for PCR amplification, PCR conditions It is: 94°C for 5 minutes to pre-denature template DNA, then 94°C for 30s, 55°C for 1min, 72°C for 1min, after 5 cycles, adjust to 94°C for 30s, 68°C for 1min, 72°C for 1min, after 25 cycles, extend at 72°C for 10min; After the PCR product was purified, it was ligated with the vector pEASY-Simple-T1 at room temperature for 10 minutes, and the mixture was transformed into E. coli DH5α in a medium containing 100 μg / ml ampicillin, 40 μg / ml X-Gal and 24 μg / ml isopr...

Embodiment 2

[0026] Immune Application of Recombinant Subunit Vaccine DnaK

[0027] 2.1 Vaccination preparation

[0028] Get the AH DnaK protein and Freund's complete adjuvant and mix it to obtain the AH DnaK protein vaccine injection with a final concentration of 100 μg / ml. The PBS composition is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na 2 HPO 4 12H 2 O, 0.024% NaH 2 PO 4 , and the rest is water.

[0029] 2.2 Immunization application of recombinant subunit vaccine AH DnaK. Sixty soft-shelled turtles (each weighing about 10 g) were randomly divided into two groups, 30 in each group, and named as group A and group B respectively. Each soft-shelled turtle in group A was injected intraperitoneally with 100 μl of the above-mentioned 2.1 vaccine injection, and each soft-shelled turtle in group B was injected intraperitoneally with 100 μl of the above-mentioned PBS buffer solution of 2.1. After 21 days, the second immunization injection was performed. Each soft-shelled turtle in ...

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Abstract

Belonging to the technical field of gene engineering and vaccinology, the invention relates to an aeromonas hydrophila heat shock protein subunit vaccine and preparation method thereof. The vaccine antigen contains the amino acid sequence shown as SEQIDNo.1. The preparation method comprises: cloning of heat shock protein gene; construction of plasmid pETDnaK; and inducible expression of plasmid pETDnaK. The aeromonas hydrophila heat shock protein subunit vaccine provided by the invention has the advantages of: 1. safety and no toxicity, thus avoiding atavism of attenuated vaccine and possible water pollution caused by repeated immunization by a lot of inactivated vaccines; 2. high protection rate, with the immunoprotection efficiency of the recombinant subunit vaccine AHDnaK on aeromonas hydrophila up to 81.8%; and 3. simple operation, i.e. being injectable after being mixed with a cheap commercial adjuvant Freund's adjuvant.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and vaccinology, and in particular relates to an Aeromonas hydrophila heat shock protein subunit vaccine and a preparation method thereof. Background technique [0002] Aeromonas hydrophila belongs to Vibriaceae, Aeromonas genus, and is the primary pathogenic bacteria of various aquatic animals. Aeromonas hydrophila strains with high adhesion can produce highly toxic exotoxins . After Aeromonas hydrophila invades aquatic animals, it proliferates in the intestinal tract, and then enters the liver, kidney and other tissues, causing these organs and blood lesions, leading to the outbreak of severe epidemic diseases such as sepsis in aquatic animals. [0003] At present, its prevention and treatment still mainly rely on traditional medicines. The development of new anti-Aeromonas hydrophila vaccines in farmed fish has been a research hotspot in recent years, but it mainly focuses on who...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02C12N15/70C12N15/66C12N15/31C12N15/10A61P31/04
Inventor 党伟陆洪良高原杜卫国
Owner HANGZHOU NORMAL UNIVERSITY
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