Full-length complementary deoxyribonucleic acid (cDNA) sequence of Paralichthys olivaceus pattern recognition Toll-like receptor TLR21 and applications thereof
A technology of pattern recognition receptors and flounder, which is applied in the fields of application, receptor/cell surface antigen/cell surface determinant, and microbial determination/inspection, etc., and can solve the problem that there is no immune prevention technology and treatment method.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0066] Isolation of head and kidney tissue from flounder:
[0067] Vibrio anguillarum (provided by Tianjin Aquatic Disease Control Center) was inoculated in Luriai-Bertani liquid medium with a pH value of 7.5 and supplemented with 2% NaCl, and cultured at 28°C for 24 h with constant temperature shaking (150 r / min). The OD value was measured at 550 nm, and the bacterial concentration and the total number of cells were calculated. The bacteria were collected by centrifugation, washed twice with PBS, the cell pellet was suspended in 0.5% formaldehyde solution and inactivated for 36 h at 28°C, and the bacteria were washed three times with PBS to prepare 10 6 Individual / mL concentration of inactivated Vibrio anguillarum bacteria liquid.
[0068] Healthy flounder (about 0.5 kg) purchased in the market was kept in an aquarium at 25°C for three days, and 1 mL of the above-mentioned Vibrio anguillarum inactivated by formaldehyde (or non-inactivated Vibrio anguillaria) was injected i...
Embodiment 2
[0070] Extraction and purification of total RNA:
[0071] (1) Take 100 mg of head kidney tissue, cut it into pieces with scissors, add 1 mL of Trizol reagent (Invitrogen, USA) and 10 μL of heparin, grind the homogenate thoroughly in a homogenizer, then transfer to a 1.5 mL RNase-free centrifuge tube and shake to mix. Mix well, and place at room temperature for 5 minutes to fully lyse.
[0072] (2) Centrifuge at 4°C and 12,000 rpm for 5 minutes, and transfer the supernatant to a new RNase-free centrifuge tube.
[0073] (3) Add 0.1 mL of 5 mol / L NaCl to each tube and mix well, then add 0.3 mL of chloroform to each tube, shake vigorously for 15 s, place at room temperature for 3 min, and centrifuge at 12,000 r / min at 4°C After 15 min, the supernatant was aspirated and transferred to another clean 1.5 mL centrifuge tube without aspirating the middle layer as much as possible.
[0074] (4) Add isopropanol of equal volume to the obtained supernatant to each tube, mix gently, pla...
Embodiment 3
[0078] Cloning and sequencing of intermediate fragments:
[0079] (1) Primer design:
[0080] Firstly, download the full-length cDNA sequence and amino acid sequence of the model organism zebrafish TLR21 gene from GeneBank, and use the downloaded sequence to blast the fish TLR21 gene with high homology to zebrafish on NCBI. The taxonomic status of flounder is the closest to that of grouper. The primers were designed using Primer 5 primer design software, using the grouper TLR21 sequence (GU198366) as a template, combined with the sequence comparison results of grouper, redfin puffer and zebrafish as follows: For correction, because the gene is relatively long, two pairs of degenerate primers were designed for TLR21 mid-sequence amplification.
[0081] TLR21-F1: 5'-TATCGTTACAAACMGHATYCT-3'
[0082] TLR21-R1: 5'-AAGATTYCCRAAAACMTC-3'
[0083] TLR21-F2: 5'-AATBTGTCCTBTAACWACAT-3'
[0084] TLR21-R2: 5'-GTTCCTCAAAGCCTTTCCA-3'
[0085] (2) cDNA first-strand synthesis:
[00...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More