Natural immune receptor TLR5S gene of Saddletail grouper and novel application of protein encoded with natural immune receptor TLR5S gene
An oblique grouper and natural immunity technology, applied in the field of molecular biology, can solve the problems of great harm, economic loss of fishery and breeding, and high lethality
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Problems solved by technology
 Cloning of Example 1 TLR5S Gene of Plaque-banded Grouper
 (1) Extraction of total RNA from the head and kidney of the grouper
 Healthy grouper was taken, anesthetized on ice for 3 minutes, head kidney tissue was separated, and total RNA was extracted with Trizol reagent.
 (2) Synthesis of the first strand of cDNA
 1 μg of total RNA sample from the head and kidney of the grouper was taken and treated with DNase to remove the contamination of genomic DNA, mixed with RNAOligodT, and subjected to reverse transcription, and the obtained product was stored at -20°C for future use.
 (3) Cloning of the complete cDNA sequence of the TLR5S gene of the grouper
 Design specific primers, the upstream primer is TLR5S-ORF-F: 5`-ATGTGGACGCTGGGTCTTCA-3`; the downstream primer is TLR5S-ORF-R: 5`-CTGCTGTGTGATGTCATCAG-3`, the size of the amplified ORF fragment is 1935bp, electrophoresis The result is as figure 1 As shown, the target band ...
 Example 2 Construction and verification of eukaryotic expression vector pcDNA4.0-TLR5S
According to the full-length cDNA sequence of TLR5S gene, BamHI and KpnI double restriction sites were selected to design primers, in which the upstream primer was EcTLR5S-BamHI-F: 5`-CGCGGATCCGCCACCATGTGGACGCTGGGTCTTCA-3`; the downstream primer was EcTLR5S-KpnI-R: 5`- CCGCTCGAGCTGCTGTGTGATGTCATCAG-3`. Using the pGEM-T plasmid containing the TLR5S coding gene as a template, carry out PCR amplification, and the size of the specific amplification product is about 2000bp, and connect the TLR5S with restriction sites to the DNA that has been double-enzymatically digested and purified with BamHI and KpnI In the final pcDNA4.0 eukaryotic expression vector, the recombinant expression vector was obtained, and the gel electrophoresis results of the recombinant expression vector verified by PCR are shown in figure 2 , the correctness of the sequence was verified by sequencing, and the...
 Example 3 Overexpression Effect of Recombinant Plasmid pcDNA4.0-TLR5S
 Inoculate an appropriate amount of 293T cells into a 12-well plate, transfect when the cells grow to 70%-80%, and replace with serum-free Opti-MEM culture medium before transfection. Each well was transfected with 1500ng recombinant plasmid pcDNA4.0-TLR5S, or pcDNA4.0 blank vector control. Six hours after transfection, the medium was replaced with DMEM medium containing 5% fetal bovine serum. The cells were collected 36 hours after transfection, the total protein was extracted, and the expression of EcTLR5S fusion protein was detected by western blot. The experimental results were as follows: image 3 As shown, β-actin was used as an internal reference protein. The results showed that the recombinant plasmid was constructed successfully.
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