Duck interleukin 2 acceptor alpha strand lyoregion protein monoclone antibody and its preparation method
A technology of monoclonal antibody and extracellular region, which is applied in the fields of genetic engineering and antibody engineering, can solve problems such as duck IL-2 receptor and IL-2R research lag, and achieve simple preparation method, good immunogenicity, highly specific effect
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Embodiment 1
[0026] Embodiment 1. Design and synthesis of oligonucleotide primers
[0027] The upstream primers were designed and synthesized according to the chCD25 nucleotide sequence registered in GenBank (No.AF143806). Upstream primer [23]: 5'-TCCTTGCTAGTCCTTGGTAGTTCTAGGGCTAACCATGCAGTAGACGGCT-'3, downstream universal primer CDS: 5'-GTCACGGGTGACAGCCACTC-3', used to amplify the cDNA fragment encoding duCD25. Primer R-32a-up: 5'-GGGTACCGACGACGACGACAAGGGCAAATGCCCACCTGTT-'3 (contains KpnI restriction site) and R-outside: 5'-CAAGCTTTCAATGATGATGATGATGGTGGGATGGATGAGCTGAACC-3' (contains HindIII restriction site) for amplifying duCD25 extracellular region of DNA fragments.
Embodiment 2
[0028] Example 2. Isolation and cultivation of duck spleen mononuclear cells
[0029] The duck spleen was aseptically collected, placed in a sterile plate, and washed twice with PBS. Add RPMI 1640 washing solution, roll the spleen with a curved glass rod under aseptic conditions, and gradually squeeze the spleen cells into the liquid phase. After repeatedly blowing and blowing the Pasteur tube to disperse the cells, transfer to a 15mL sterile centrifuge tube and centrifuge at 500×g 20°C for 5min, discard the supernatant, suspend the lymphocytes with 5mL PBS, and gently add to the tube wall containing an equal volume of lymphocyte separation In the centrifuge tube of the separation liquid, avoid damaging the liquid level of the separation liquid. Centrifuge at 500×g for 30 minutes at 20°C. The capillary extends into the white misty cell layer (intermediate phase), gently aspirate the white intermediate phase, transfer it to a centrifuge tube containing 40mL PBS, repeat the op...
Embodiment 3
[0030] Example 3. RT-PCR amplification of duCD25 full-length cDNA fragment
[0031] The total RNA of duck spleen mononuclear cells was extracted with the Trizol RNA extraction kit, and the first strand of duCD25 cDNA was synthesized with the Reverse Transcription Systemkit; the reverse transcription product was amplified by PCR with primers [23] and CDS, and the reaction conditions were: : Denaturation at 94°C for 45s, annealing at 52°C for 45s, extension at 72°C for 1min, a total of 30 cycles, and finally extension at 72°C for 10min, check PCR amplification products by 1% agarose gel electrophoresis.
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