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

A technology of interleukin and protein, which is applied in the field of preparation of recombinant goose interleukin-2 protein, can solve the problems of unfavorable protein recovery and purification, low expression efficiency, etc., and achieve simple operation, remarkable effect, high stability and biological The effect of academic activity

Inactive Publication Date: 2004-11-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stepaniak et al. (1999) carried out more 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 Escherichia coli Pgex-2t system, and the protein obtained had natural chicken IL-2 The biological activity of the protein, but the expression efficiency is only 63μg / L, and most of them are insoluble proteins, which is not conducive to the recovery and purification of proteins

Method used

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preparation example Construction

[0018] The preparation and purification process of the established crude product is simple, has good stability and high activity. This process has the following characteristics: (1) The gsIL-2 protein expressed by E. coli is mainly stored in the engineered bacteria in the form of fusion protein, and the target protein can be released by ultrasound, and the crude gsIL-2 can be obtained without other treatments. (2) The secreted yeast expression vector pMETαB is selected, and most of the expressed gsIL-2 protein is secreted into the medium, and the gsIL-2 protein can be obtained without any treatment Crude product, and the target protein has good biological activity; (3) The non-secretory yeast expression vector PMET B is selected, and the expressed gsIL-2 protein is mainly stored in the engineered bacteria in the form of fusion protein, and gsIL can be obtained by ultrasonic treatment -2 Crude protein, and the crude protein has relatively high biological activity; (4) The Ni-NTA Ag...

Embodiment 1

[0022] Example 1. Design and synthesis of oligonucleotide primers

[0023] According to the IL-2 nucleotide sequences of duck (AF294323), chicken (AF017645) and turkey (AJ007463) registered in GenBank, a pair of RT-PCR primers were designed:

[0024] Upstream primer, 5’-AATACTAGCACAGAGACAACCAG-3’

[0025] Downstream primer, 5’-TTACTGAAATTTATTAAATATCATCTA-3’

Embodiment 2

[0026] Example 2. Isolation of splenic mononuclear lymphocytes

[0027] The spleen of 1 month old Zhejiang East White Goose was collected aseptically, cut into pieces and placed without Ca 2+ , Mg2+ Ion in PBS (717mmol / L K 2 HPO 4 , 283mmol / L KH 2 PO 4 , PH7.2), centrifuge at 300×g 4℃ for 10 min, take the supernatant at 500×g 4℃ for 30 min to collect lymphocytes, then wash twice with PBS, and then wash once with RPMI1640 culture medium (without calf serum) After counting the viable cells by staining with trypan blue (0.1%), use RPMI1640 growth medium (containing 10% calf serum, 100IU / ml penicillin and 100μg / ml streptomycin) to prepare the cells into 1×10 7 / ml of cell suspension. The cell suspension was added with a final concentration of 10mg / L ConA, divided into cell culture plates, cultured in a 5% carbon dioxide incubator at 40°C for 16 hours to collect lymphocyte cultures and extracted with Trizol reagent one-step method to extract total cell RNA.

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Abstract

The invention discloses a recombinant goose interleukin-2(gsIL-2) protein preparing method and its use. The DNA fraction of RT-PCR amplified Zhedong white goose interleukin-2, prokaryotic expression carrier pBAD / HisB, and eukaryotic expression carriers pMET alpha B and pMETB compose expression particles pBAD / HisB / gsIL-2, pMET alpha B / gsIL-2 and pMETA / gsIL-2, which are converted to Escherichia coli LMG194, microzyme PMAD11 and PMAD16, respectively, then inducing expression. After inducing, making ultrasonic wave cracking and deposit elimination by centrifugation on Escherichia coli and microzyme PMAD16 to obtain crude products of gsIL-2 protein, PMAD11 expressed gsIL-2 is mainly separated in the culture medium, centrifugating and taking supernatant to obtain gsIL-2 protein crude products. Using Ni-NTAArgarrose protein purifying system, thus able to faster obtain gsIL-2 pure product, and the crude and pure products can act as immune assistant and disease-curing drugs. 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 relates to the field of biotechnology, in particular to a method for preparing recombinant goose interleukin-2 protein and its use. Background technique [0002] 1. Schauenstein et al. (1982) first discovered that chicken spleen lymphocytes secrete a cytokine with similar activity to mammalian interleukin-2. Subsequently, Schnetlzler et al. (1983) used ammonium sulfate precipitation and ammonium bicarbonate resuspension , Sephadex G-100 gel filtration obtains two types of active isolates, the molecular weights of which are between 9~11.5KDa and 19.5~21.5Kda, respectively. Vainio et al. later used the same method to obtain a molecule with a molecular weight of 13KDa, and they used isoelectric focusing to estimate the PI of chicken IL-2 to be 5.9. Frederickson et al. (1987) used Phenyl-sepharose, anion exchange and superose-12 column to separate two types of active substances. One molecular weight is 14KDa, and the secon...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61P31/00A61P31/12A61P33/00C07K14/55C12N15/26C12P21/00
Inventor 周继勇陈吉刚王金勇腾巧泱吴建祥
Owner ZHEJIANG UNIV
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