Trachinotus ovatus-derived streptococcus agalactiae DNA vaccine as well as preparation method and application thereof

Technology of a DNA vaccine, Streptococcus lactis, applied in the field of molecular biology

Active Publication Date: 2021-05-11
BEIBU GULF UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the construction of pcDNA-internalin recombinant eukaryotic p

Method used

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  • Trachinotus ovatus-derived streptococcus agalactiae DNA vaccine as well as preparation method and application thereof
  • Trachinotus ovatus-derived streptococcus agalactiae DNA vaccine as well as preparation method and application thereof
  • Trachinotus ovatus-derived streptococcus agalactiae DNA vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: the preparation of oval pomfret source Streptococcus agalactiae DNA vaccine (hereinafter also referred to as DNA vaccine pcDNA-internalin)

[0022] 1) Internalin gene sequence cloning

[0023] According to the nucleotide sequence of the internalin gene on NCBI (GenBank accession number: MW321594, SEQ ID NO: 2), design and synthesize a pair of specific primers, wherein the forward primer is: InF: 5'-CG GAATTC ATGAAAGGTCAAAAAAATTATTGCT-3' (the underline is the EcoR I restriction site, SEQ ID NO: 3), the reverse primer is InR:5'-CCG CTCGAG TTAATGGTGATGATGACCTACATGTG-3' (the underline is the Xho I restriction site, SEQ ID NO: 4). The reaction system for PCR amplification is: 0.5 μL of Streptococcus agalactiae genomic DNA template, 0.5 μL of each primer (InF and InR), 5 μL of 10×Ex Taq Buffer, 25 μL of DNA polymerase Mix, ddH 2 O to make up to 50 μL. PCR amplification conditions were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 s, a...

Embodiment 2

[0030] Embodiment 2: DNA vaccine pcDNA-internalin immunizes pomfret ovata

[0031] DNA vaccine preparation solution and original plasmid preparation solution (also known as no-load preparation solution): Dilute the DNA vaccine pcDNA-internalin in sterile saline to a final concentration of 100 μg / mL, which is the vaccine preparation solution. Dilute the pcDNA plasmid in sterile saline to a final concentration of 100 μg / mL, which is the blank preparation solution.

[0032] 360 healthy pomfrets (each weighing about 120g) were randomly divided into 3 groups, 40 in each group, with 3 replicates in each group. They were DNA vaccine group, empty group and control group (normal saline group). The large oval pomfret was immunized by back intramuscular injection. DNA vaccine group: each fish was injected with 100 μL of pcDNA-internalin recombinant eukaryotic plasmid at a concentration of 100 μg / mL; empty group: each fish was injected with 100 μL of a concentration of 100 μg / mL. mL emp...

Embodiment 3

[0033] Example 3: Expression of DNA vaccine pcDNA-internalin in pompano pomfret tissue and detection of antiserum titer

[0034] On the 28th day after immunization, the muscle, head kidney, gill, brain, liver and spleen tissues of pomfret were collected, and the expression of the target gene was detected at the DNA level and RNA level. Using the genomic DNA of the above-mentioned tissues and the reverse-transcribed cDNA extracted from RNA as templates, PCR amplification was carried out using internalin-specific primers InF and InR, and the amplified products were identified by 1% agarose gel electrophoresis. The results showed that internalin could be expressed in the above-mentioned tissues at the DNA and RNA levels during 28 days of immunization (such as Figure 5 shown).

[0035] On the 7th day, 14th day, and 21st day after immunization, 3 pomfrets were randomly selected from each group, blood was collected from the tail vein, left at room temperature for 1 hour, at 4°C fo...

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Abstract

The invention discloses a trachinotus ovatus-derived streptococcus agalactiae DNA vaccine as well as a preparation method and application thereof. The nucleotide sequence of the vaccine is shown as SEQ ID NO: 1, and the preparation method of the vaccine comprises the following steps: designing primers according to an internalin gene sequence, carrying out PCR (Polymerase Chain Reaction) amplification, connecting the obtained product with pMD-18T, transforming DH5alpha, selecting positive bacterium clones, carrying out amplification culture, extracting plasmids, and verifying to obtain pMD-internalin recombinant plasmids; performing enzyme digestion with restriction enzymes EcoR I and Xho I on the obtained plasmids and pcDNA3.1 (+) respectively, ligation of enzyme digestion products with T4DNA ligase and transformation of DH5alpha cells, selecting positive bacterium clones, extracting plasmids and obtaining the trachinotus ovatus-derived streptococcus agalactiae DNA vaccine through verification. The vaccine disclosed by the invention is relatively high in immune protection rate and relatively long in immune protection period.

Description

technical field [0001] The invention relates to a DNA vaccine for fish to resist bacterial infection, in particular to a DNA vaccine of Streptococcus agalactiae derived from oval pomfret and its preparation method and application, belonging to the field of molecular biology. Background technique [0002] Trachinotus ovatus, commonly known as golden pomfret, is a warm-water pelagic fish. Due to its fast growth rate and attractive market prospects for export processing, the scale of cage culture has been increasing year by year, and it has gradually become the One of the main species of marine culture in the south. However, due to many factors such as the rapid expansion of the breeding scale, the decline of germplasm, the poor management of the breeding, the deterioration of the environment in the coastal sea area, and the improper use of drugs, the occurrence of bacterial diseases and streptococcal diseases in the pomfret is becoming more and more frequent. huge losses to t...

Claims

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Application Information

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IPC IPC(8): A61K39/09A61P31/04C12N15/31C12N15/79
CPCA61K39/092A61P31/04C07K14/315C12N15/79A61K2039/53A61K2039/552
Inventor 蔡小辉彭银辉杨绍宇陈泓霖龙群能王龙文胥鹏
Owner BEIBU GULF UNIV
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