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Segregative technique for purifying stimulating factor of recombined human beta lymphocyte

A technology of B lymphocytes and stimulating factors, applied in the field of separation and purification of recombinant proteins, can solve the problems of high cost, poor repeatability, and difficulty in amplification, and achieve the effect of improving the recovery rate and simplifying the process of renaturation.

Inactive Publication Date: 2005-08-31
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the shortcomings of the existing process, which are not easy to scale up, poor repeatability, and high cost, the present invention provides a renaturation process on a gel filtration column, which effectively solves the problem

Method used

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  • Segregative technique for purifying stimulating factor of recombined human beta lymphocyte
  • Segregative technique for purifying stimulating factor of recombined human beta lymphocyte
  • Segregative technique for purifying stimulating factor of recombined human beta lymphocyte

Examples

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Effect test

Embodiment 1

[0060] The expression strain BL21(DE3) was activated overnight for 12 hours, inoculated in 500ml liquid LB medium (2L shake flask) at 1:100 the next day, a total of 2 bottles, cultured until the bacterial density OD600 was 0.6, and the final concentration of IPTG was 1mM to induce expression For 5 hours, the temperature was controlled at 37°C, the shaking table speed was 220rpm / min; 5000-1000rpm / min, and the bacterial precipitate was collected by centrifugation at 4°C. For the induced expression, see figure 1 ;

[0061] Separation, purification and dissolution of inclusion bodies: 50Mm Tris-HCl, 5mM EDTA, 0.15M NaCl, pH 8.0, wash inclusion bodies, 5000rpm / min, 4°C. Centrifuge for 15min, collect the precipitate, repeat 3-4 times; 50mMTris-HCl, 3M urea, 5mM EDTA, 0.15mM NaCl, pH 8.0, wash the inclusion body for 30min, 4°C. Centrifuge for 15min, collect the precipitate, repeat 3-4 times; 0.5% Triton X-100 (v / v), 5mM EDTA, 0.15mM NaCl, pH 8.0, wash the inclusion body for 30min, ...

Embodiment 2

[0065] Determination of purity by SDS-PAGE: Prepare 15% concentration of separation gel, concentrate the final purified product 20 times, and 2 × loading buffer (125mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol blue , 10% of 2-mercaptoethanol), mixed in a boiling water bath for 5 min, and loaded for electrophoresis, stained with Coomassie Brilliant Blue R-250, such as Figure 5 .

Embodiment 3

[0067] HPLC detection of purity: Dissolve the lyophilized purified product sample and standard in PBS buffer at pH 7.4, the concentration is 100 μg / ml, the mobile phase is PBS, the temperature is 20-25 degrees, and the flow rate is controlled at 0.5 mg / ml ml, detected by OD280 UV detector, see Figure 6 , Figure 7 .

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Abstract

A process for separating and purifying recombinant human lymphocyte B stimulating factor rhsBLyS includes such steps as separating and purifying the inclusion body containing rhsBLyS, dissolving, renaturalizing by Sephacryl S-200 gel column, chromatography by DEAE sepharose, F.F. column, collecting target protein peak, and micron filtering.

Description

technical field [0001] The invention relates to a separation and purification process of recombinant protein, in particular to a separation and purification process of recombinant human B lymphocyte stimulating factor rhsBLyS. Background technique [0002] Human B lymphocyte stimulating factor (hBLyS) is a cytokine closely related to human immune regulation newly discovered by Moore PA et al. (TNF) superfamily is a type II transmembrane protein, which is mainly expressed in human peripheral blood mononuclear cells, spleen, lymph nodes, bone marrow and other tissues, and can be expressed in the extracellular soluble part (hsBLyS) under the action of certain metalloproteinases Free in peripheral blood to play a role. hsBLyS is a co-stimulatory factor of lymphocytes, which can strongly stimulate the proliferation and differentiation of activated B cells: for normal B cells, after preactivation with PMA and Anti-IgM, hsBLyS can induce a large number of proliferation And secret...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C07K14/52
Inventor 张双全曹鹏
Owner NANJING NORMAL UNIVERSITY
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