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Production method of recombination human interleukin-4

An interleukin and cell technology, applied in the field of genetic engineering, can solve the problems of low expression, low yield of IL-4, and large loss of target protein

Inactive Publication Date: 2007-12-19
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been reports on the expression and purification of IL-4 at home and abroad, but the expression level is not high, generally accounting for 5-10% of the total protein of the bacteria, and it is expressed in the form of inclusion bodies. After the inclusion bodies are refolded, the target protein is lost. Very large, so that the final yield of IL-4 is very low

Method used

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  • Production method of recombination human interleukin-4
  • Production method of recombination human interleukin-4
  • Production method of recombination human interleukin-4

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Experimental program
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Embodiment 1

[0060] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0061] According to the natural amino acid sequence of human interleukin-4, according to the codon preference, under the condition of not changing the amino acid sequence, the target gene sequence (SEQ ID NO: 1) of the recombinant human interleukin-4 protein is synthesized completely. The homology between the optimized IL-4 gene sequence and the natural IL-4 gene sequence is 93% (see FIG. 1 ). The gene was cloned into pET-30a(+) and verified by sequencing.

[0062] The expression vector construction method is shown in Figure 2. The target human interleukin-4 gene was amplified by PCR, and the PCR recovery product of the gene IL-4 and the plasmid pET30a(+) were respectively treated with Nde I and EcoRI double digestion, and the corresponding fragments were recovered after digestion and ligated with T4 DNA Enzymes were ligated overnight at 16°C. The ligation ...

Embodiment 2

[0064] Example 2 Expression of Recombinant Human Interleukin-4 and Determination of Expression Form

[0065] Select a well-confirmed expression engineering strain BL21(DE3) / pET30a(+) / IL-4, culture it with LB medium and induce expression with IPTG, detect whether there is any target band by SDS-PAGE electrophoresis, and analyze its expression. A part of the fermented bacterial liquid was centrifuged, resuspended in pH 8.0, 20 mM Tris buffer, ultrasonically disrupted in an ice bath, centrifuged to collect ultrasonic supernatant and ultrasonic precipitation, detected by SDS-PAGE electrophoresis, and determined its expression form. Analysis of the ultrasonic supernatant and ultrasonic precipitation revealed that the target protein was mostly in the precipitate, indicating that the target protein was expressed in the form of inclusion bodies (see Figure 3).

Embodiment 3

[0066] Example 3 Large-scale fermentation of recombinant human interleukin-4

[0067] Get the expression engineered bacteria BL21(DE3) / pET30a(+) / rhIL-4 identified as positive clones, culture it in LB containing 30ug / ml kanamycin for 14-16 hours; to OD 600 1 to 3, as the secondary seed solution. Prepare 2.96L of improved M9 fermentation medium (Glucose 1%, Yeast extract 0.5%, K 2 HPO 1 0.5%, KH 2 PO 4 0.35%, (NH 4 ) 2 HPO 4 0.35%, MgSO 4 0.025%, CaCl 2 0.1%), pour it into a cleaned fermenter, after autoclaving the tank, cool to 30-40°C, put the cultured secondary seed liquid into the fermenter at 1.5% inoculum, cultivate at 37°C, and Constantly adjust the ventilation flow rate and stirring speed according to the dissolved oxygen value, and control the dissolved oxygen above 40%. Sampling every hour to measure OD 600 value. Grow to OD 600 When it reaches about 5.0, the induction starts. Add IPTG to the final concentration of 1mM in the fermenter for induction....

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Abstract

This invention provides the coding sequence of recombination human interleukin-4, and method for producing said interleukin-4, and also the expression vactor and host cells used for said method. In this invention, pET system for expression of IL-4 is firstly used for obtaining high leven expression strain; and by controlling fermentation condition, the higher level of protein expression is obtained. Based on this and after large scale of research of purification of recombination human interleukin-4 of inclusion body expression, we obtain the method of purification of IL-4. By using this invention method, recombination human interleukin-4 protein can be obtained with high yield and high purity, to meet the clinical needs. This invention method is of high efficiency, simple and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a method for efficiently producing recombinant human interleukin-4 (IL-4), including the construction of related engineering bacteria, the expression and purification process of recombinant human interleukin-4. Background technique [0002] In 1982, Howard discovered that there was a growth factor in the culture supernatant of T cells that promoted the proliferation of B cells, which was initially named B cell growth factor-1 (BCSF-1). Some laboratories call it B cell stimulating factor-1 (BSF-1), T cell growth factor-2 (T cell growth-2, TCGF-2). In 1986, the gene was successfully cloned, and it was named interleukin-4 (interleukin-4, IL-4) internationally. [0003] IL-4 is a cytokine produced by helper T cells (Th cells), which mainly acts on B cells. It can activate B cells in a dormant state, and then promote the proliferation and different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/24C07K14/54C12N15/63C12P21/02C12N1/21C07K1/14
Inventor 黄阳滨孙九如张翊任军陈子珺徐晓燕刘勇张甘良
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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