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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com