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Pseudomonas plecoglossicida DNA vaccine and preparing method and application thereof

A technology of Pseudomonas ayumi and DNA vaccines, applied in the fields of molecular biology and immunology, can solve the problems of not being fully developed and DNA vaccine attempts have not been reported, and achieve a high protection rate effect

Inactive Publication Date: 2018-09-07
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the research on the Pseudomonas ayucidal vaccine has not been fully launched, and there is no report on the DNA vaccine.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of DNA vaccine plasmid pCI-pcrV:

[0032] Using the genomic DNA of Pseudomonas ayucididae NB2011 as a template, primers P1 / P2 were used for PCR amplification. The PCR conditions were: 94°C for 60s to pre-denature the template DNA, then 94°C for 45s, 59°C for 45s, 72°C for 45s, and 30°C. After one cycle, the terminal extension was carried out at 72°C for 8 min. PCR products were purified with Sangon DNA purification kit. Digest the purified PCR product and plasmid pCI-neo with EcoRI enzyme and SalⅠ enzyme, recover the corresponding fragment, add T 4 DNA ligase, establish the ligation reaction, transform E. coli DH5α after ligation at 16°C for 2 hours, culture on LB solid culture containing ampicillin for 18-24 hours, screen the transformant to extract the plasmid, and identify it as recombinant after EcoRI and SalⅠ double enzyme digestion Plasmid pCI-pcrV.

[0033] PCR primers are:

[0034] P1:5'-GGAATTCATGGCAAGGATCGAAGAGG-3' (the sequence shown in SEQ I...

Embodiment 2

[0039] Application of vaccine pCI-pcrV

[0040] Step 1) Preparation of vaccine and original plasmid preparation: Dilute the above-mentioned pCI-pcrV plasmid (DNA vaccine) in PBS to a final concentration of 100 μg / ml, which is the vaccine preparation solution; Dilute the original plasmid pCI-neo in PBS to The final concentration is 100 μg / ml, which is the original plasmid preparation solution.

[0041] The composition of the PBS by weight percentage is: 0.8% NaCl, 0.02% KCl, 0.358% Na 2 HPO 4 12H 2 O, 0.024% NaH 2 PO 4 , and the balance is water.

[0042] Step 2) vaccination of the vaccine: 150 large yellow croakers (each weighing about 100 g) were randomly divided into 3 groups, 50 in each group, and these 3 groups were named groups A, B and C respectively. Groups A and B were injected intraperitoneally with the vaccine preparation solution and original plasmid preparation solution of the above step (1) respectively, and each fish in group C (control group) was injected ...

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Abstract

The invention discloses a pseudomonas plecoglossicida DNA vaccine and a preparing method and application thereof. The pseudomonas plecoglossicida DNA vaccine is characterized in that the pseudomonas plecoglossicida DNA vaccine is pCI-pcrV; pseudomonas plecoglossicida NB2011 genome DNA serves as a template, and is subjected to PCR amplification through a primer P1 / P2, the amplified products are purified to be connected with plasmid pCI-neo, and recombinant plasmid pCI-pcrV is obtained, and is the pseudomonas plecoglossicida DNA vaccine, wherein the sequence of a target gene pcrV is shown in SEQID NO:1. According to the pseudomonas plecoglossicida DNA vaccine, the immune protective rate of pseudomonas plecoglossicida infection is 70% or above, and therefore the high protective rate is achieved. The immune protective rate of the pseudomonas plecoglossicida DNA vaccine in 8 weeks after immunization is not obviously reduced, and therefore the pseudomonas plecoglossicida DNA vaccine has thelong-acting advantage.

Description

technical field [0001] The invention relates to the fields of molecular biology and immunology, in particular to the construction and application of a Pseudomonas ayucidia DNA vaccine, that is, the Pseudomonas ayucidia DNA vaccine, its preparation method and its application. Background technique [0002] Pseudomonas plecoglossicida is the pathogenic bacteria of large yellow croaker visceral white spot disease, which has caused serious losses in the large yellow croaker aquaculture industry in the past ten years. Visceral white spot disease caused by this bacteria has no obvious symptoms, but white nodules of different sizes appear in internal organs such as spleen and kidney in the late stage of disease development, which usually occurs in winter and spring when the water temperature is low, and fish basically do not eat. The disease cannot be effectively controlled by conventional methods such as feeding oral drugs, and immunoprophylaxis may be an important way to control t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/104A61P31/04C12N15/85C12N15/66C12N15/31
CPCA61K39/104A61K2039/53A61P31/04C07K14/21C12N15/66C12N15/85C12N2800/106
Inventor 毛芝娟张恒泽高丽婷万里
Owner ZHEJIANG WANLI UNIV
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