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

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

Inactive Publication Date: 2004-11-10
ZHEJIANG UNIV
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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

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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 and clone the cDNA fragments SEQ No.1 and SEQ No.2 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 at 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 at 4°C for 30 minutes, 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 suspen...

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Abstract

The invention discloses a recombinant duck interleukin-2 protein preparing method and its use, the DNA fraction of RT-PCR amplified duck interleukin-2c of different ducks and prokaryotic expression carrier pBAD / HisB, as well as eukaryotic expression carrier pMET alpha A to compose high-efficacy expression particles, recombining and converting these carriers to Escherichia coli LMG194, and microzyme strains PMAD11 and PMAD16, respectively and then inducing expression. After inducing, making ultrasonic wave cracking and deposit elimination by centrifugation on Escherichia coli to obtain crude products of duck interleukin-2 and using protein purifying system to obtain duck interleukin-2 pure products. The pure or crude of duck interleukin-2 can act as immune assistant, anti-disease additive and disease-curing drug. The duck interleukin-2 monoclonal and polyclonal antibodies have simple preparing procedure, and can act as good immune inhibitor. It applied genetic engineering technique to prepare recombinant protein gsIL-2, and has simple technical flow, low production cost, good stability, high bioactivity and other characters.

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 ef...

Claims

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Application Information

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Patent Type & Authority Applications(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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