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

Inactive Publication Date: 2006-10-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Compared with humans and mammals, due to the species specificity of poultry, the research on IL-2R is still relatively lagging behind
In 2001, Lillehoj et al. registered the chicken CD25 mRNA sequence on NCBI, but there are no reports on chicken IL-2 receptor protein

Method used

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  • Chicken interleukin-2-receptor alpha chain extracellular region protein monoclonal antibody, and its preparing method
  • Chicken interleukin-2-receptor alpha chain extracellular region protein monoclonal antibody, and its preparing method

Examples

Experimental program
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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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Abstract

The invention relates to chicken interleukin-2 acceptor alpha chain chCD25 cyst outside albumen monoclonal antibody and the manufacture method. It expanding the nucleotide section of coding cyst outside albumen and connecting to carrier constructing recombination expression plasmid, transforming the plasmid into coliform BL21(DE3), the IPTG inducing transforming bacilli expressing chCD25 cell outside albumen, using Ni-NTA Agarose albumen purification system to separate purified recombined chCD25 cell outside albumen, fusing the purified cell outside albumen with splenocyte and myeloma SP2/0 cell, taking HAT selective cultivating, filtering the indirect ELISA cultivating supernatant, taking limiting dilution to gain stable excreting anti chCD25 cell outside albumen monoclonal antibody hybridoma strain; expanding cultivation and injecting BALB/c, the anti-chCD25 monoclonal antibody ascetic fluid would be gained.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the technology of genetic engineering and antibody engineering, in particular to chicken interleukin-2 receptor alpha chain chCD25 extracellular region protein monoclonal antibody and a preparation method thereof. Background technique [0002] Interleukin-2 (IL-2) is mainly a lymphokine secreted by T lymphocytes, which affects the growth, development, differentiation and maturation, and biological functions of T cells, B cells, and NK cells. important regulators. In recent years, IL-2 has also been widely used as an immune enhancer in the treatment of AIDS, autoimmune diseases and tumors, and has achieved remarkable results. Enter the S phase and activate the body's immune cells. It can be seen that the IL-2 receptor IL-2R plays an important role in the biological function of IL-2. In-depth study of the biological characteristics and functions of IL-2R is helpful for understanding the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70C12P21/08
Inventor 周继勇滕巧泱郭军庆
Owner ZHEJIANG UNIV
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