Chicken interleukin-2-receptor alpha chain extracellular region protein monoclonal antibody, and its preparing method
A technology of monoclonal antibody and extracellular region, which is applied in the field of genetic engineering and antibody engineering, can solve the problems of chicken IL-2 receptor protein and IL-2R research lag, and achieve simple preparation method and good immunogenicity , the effect of high antibody titer
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Embodiment 1
[0026] Embodiment 1. Design and synthesis of oligonucleotide primers
[0027] Two pairs of primers were designed and synthesized according to the chCD25 nucleotide sequence registered in GenBank (No.AF143806). Primers chCD25F: 5’-ACCATTTGCTGAC CACAGAG-3’ and chCD25R: 5’-ATTATGCACACTTTTTTTG-3’ are used to amplify the full-length cDNA fragment of chCD25; primers chCD25ECF: 5’-AT GAATTCGATAAATGCCCACGTCTTTTC-3' (EcoRI restriction site) and chCD25ECR: 5'-CG GTC GAC TCAGGATAGCTGCTTGTTTATAG-3' (containing SalI I restriction site) was used to amplify the DNA fragment encoding the extracellular region of chCD25.
Embodiment 2
[0028] Example 2. Isolation of Chicken Spleen Mononuclear Cells
[0029] Aseptically collect chicken spleen, cut it into pieces and place it in a Ca-free 2+ , Mg 2+ ions in PBS solution (717mmol / L K 2 HPO 4 , 283mmol / L KH 2 PO 4 , pH 7.2), centrifuged at 300×g 4°C for 10 minutes, transferred the supernatant to a centrifuge tube containing an equal volume of lymphocyte separation medium, and centrifuged at 500×g 4°C for 30 minutes, the capillary extended into the mononuclear cell layer, along the tube wall Gently suck out all the cells, wash twice with PBS solution, wash once with serum-free RPMI 1640 culture solution, and count live cells after staining with 0.1% trypan blue solution; use 10% calf serum, 100IU / ml penicillin and 100±g / ml streptomycin in RPMI1640 growth medium to make the cells into 5×10 6 cells / ml of cell suspension; add ConA at a final concentration of 10±g / ml to the cell suspension, dispense into cell culture plates, place in a 5% carbon dioxide incuba...
Embodiment 3
[0030] Example 3.RT-PCR amplification of chCD25 full-length cDNA fragment
[0031] The total RNA of chicken spleen mononuclear cells was extracted with the Trizol RNA extraction kit, and the first strand of chCD25 cDNA was synthesized with the Reverse Transcription Systemkit reverse transcription kit; the reverse transcription product was amplified by PCR with primers chCD25F and chCD25R, and the reaction conditions were as follows: 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, and PCR amplification products were checked by 1% agarose gel electrophoresis.
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