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Method for preparing recombinant duck interleukin-2 protein and its application

A technology of interleukin and protein, applied in the field of duck interleukin 2 protein preparation, can solve the problems of unfavorable protein recovery and purification, low expression efficiency, etc., achieve strong antibody secretion ability, good stability, and improve the effect of vaccine immunity

Inactive Publication Date: 2005-12-14
ZHEJIANG UNIV
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  • Abstract
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Problems solved by technology

In 1999, Stepaniak et al. carried out in-depth research on the expression of chicken IL-2 in vitro, and expressed the chicken IL-2 gene with the leader peptide removed in the E. coli Pgex-2t system, and the obtained protein had the characteristics of chicken IL-2 protein Biological activity, but the expression efficiency is only 63 μg / L, and most of them are insoluble proteins, which is not conducive to protein recovery and purification

Method used

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  • Method for preparing recombinant duck interleukin-2 protein and its application
  • Method for preparing recombinant duck interleukin-2 protein and its application

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preparation example Construction

[0018] The steps of the preparation method of recombinant duck interleukin 2 protein are as follows:

[0019] 1) Design PCR primers to clone the cDNA fragments SEQ ID No.1 and SEQ ID No.3 of duck interleukin-2 from the spleen lymphocytes of Shaoxing shelduck and American duck;

[0020] 2) The above-mentioned duck interleukin-2 cDNA and containing P BAD The prokaryotic expression vector pBAD / His B of the promoter is constructed into the expression vector pBAD / His B / dkIL-2; or the above-mentioned duck interleukin-2 cDNA is combined with P AUG1 The eukaryotic expression vector pMETαA of the promoter was constructed into the secretory expression vector pMETαA / dkIL-2;

[0021] 3) Using RM as the basal medium to amplify Escherichia coli engineering bacteria LMG194 by fermentation;

[0022] 4) Using BMDY as the basal medium, amplify yeast engineered bacteria PMAD11 and PMAD16 by fermentation, and add nutrients such as nitrogen source and carbon source at different expression times;...

Embodiment 1

[0035] Embodiment 1. Design and synthesis of oligonucleotide primers

[0036] A pair of primers at the 5' end and the 3' end were designed according to the chicken IL-2 nucleotide sequence reported by Sundick et al. The 5' end primer and the 3' end primer introduce the Hind III restriction site. Synthesize the following two primers:

[0037] 5' end primer: 5'-GC GGATCC AACACTGACAAGATGTGC-3'

[0038] 3' end primer: 5'-GC GGATCC GTAGGTTACTGAAATTTA-3'

Embodiment 2

[0039] Example 2. Isolation of spleen lymphocytes

[0040] The spleens of ducklings of two different strains bred to 12 days old were aseptically collected by carotid artery bleeding, cut into pieces and placed in a Ca-free 2+ , Mg 2+ Ions in PBS (717mmol / L K 2 HPO 4 , 283mmol / L KH 2 PO 4 , pH7.2), centrifuged at 300×g for 10min at 4°C, transferred the supernatant to a centrifuge tube containing an equal volume of lymphocyte separation medium, and centrifuged at 500×g for 30min at 4°C, the capillary extended into the mononuclear cell layer, along the Gently aspirate all cells from the tube wall. Then wash twice with PBS, then wash once with RPMI1640 culture medium (without calf serum), stain with trypan blue (0.1%) and count viable cells, then use RPMI1640 growth culture medium (with 10% calf serum, 100IU / ml penicillin and 100μg / ml streptomycin) the cells were made into 2×10 6 / ml of cell suspension. Add 10 μg / ml final concentration of ConA to the cell suspension, divi...

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Abstract

The invention discloses a preparation method and application of recombinant duck interleukin 2 protein. Duck interleukin-2 cDNA fragments amplified by RT-PCR, prokaryotic expression vector pBAD / his B vector and eukaryotic expression vector pMETαA were used to construct high-efficiency expression plasmids, and the recombinant expression vectors were transformed into Escherichia coli LMG194 and yeast strains PMAD11 and PMAD16 respectively induced expression. After induction of Escherichia coli, the crude product of duck interleukin-2 can be obtained by ultrasonic cracking and centrifugation to remove precipitation, and the pure product of duck interleukin-2 can be obtained by protein purification system. The crude or pure duck interleukin-2 can be used as immune adjuvant, disease resistance additive and disease treatment drug after adding protective agent. The preparation process of duck interleukin-2 monoclonal antibody and polyclonal antibody is simple and economical, and can be used as a good immunosuppressant. The recombinant duck interleukin-2 protein prepared by applying the genetic engineering technology in the invention has the characteristics of simple technological process, low production cost, good stability, high biological activity and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method and application of duck interleukin-2 protein. Background technique [0002] In 1997, Sundick et al. used the method of constructing cDNA library to obtain chicken IL-2 gene from chicken spleen for the first time. In 1998, Choi et al. cloned chicken IL-2 gene with a signal sequence length of 430 nucleotides into PUC18 vector and eukaryotic expression The vector pcDNA3 is expressed in Escherichia coli and CHO-K1 cells respectively, and the two proteins obtained have the biological activity of chicken T lymphocyte proliferation. In 1999, Stepaniak et al. carried out in-depth research on the expression of chicken IL-2 in vitro, and expressed the chicken IL-2 gene with the leader peptide removed in the E. coli Pgex-2t system, and the obtained protein had the characteristics of chicken IL-2 protein Biological activity, but the expression eff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/39A61K39/395A61P37/06C07K14/55C12N15/26
Inventor 周继勇王金勇吴建祥陈吉刚
Owner ZHEJIANG UNIV
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