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Method for preparing recombinant human interleukin-11

A technology of interleukin and fusion protein, which is applied in the preparation method of peptides, chemical instruments and methods, organic chemistry, etc., to achieve the effects of avoiding the renaturation step of inclusion bodies, improving recovery rate and efficient purification

Inactive Publication Date: 2011-08-03
DONGGUAN TAILI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] But so far, no one has prepared rhIL-11 from Trx-rhIL-11 fusion protein simply by using two chromatographic methods: nickel ion chelation affinity chromatography and CM cation chromatography.

Method used

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  • Method for preparing recombinant human interleukin-11
  • Method for preparing recombinant human interleukin-11
  • Method for preparing recombinant human interleukin-11

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Design of DNA Sequence for Expressing rhIL-11

[0041] Compared with natural human interleukin 11, the amino acid sequence of human interleukin 11 expressed in the present invention lacks the first amino acid (proline) at the N-terminus, and its amino acid sequence is shown in SEQ ID NO:1. The nucleotide sequence encoding the rhIL-11 is shown in SEQ ID NO:2.

[0042] According to the IL-11 coding sequence listed in SEQ ID NO: 2, the stop codon TAA was introduced at the 3' end, and the DNA coding sequence GACGACGACGACAAG of the enterokinase digestion recognition site was introduced at the 5' end. Such a DNA sequence was named sequence 3 (see SEQID NO: 3), and Shanghai Sangon Bioengineering Technology Service Co., Ltd. was entrusted to artificially synthesize the sequence by means of whole gene synthesis. To facilitate subsequent subcloning, the synthetic gene was cloned into pUC18 (Fermentas Life Science Company) for preservation and further cloning of the synthetic DNA...

Embodiment 2

[0108] Embodiment 2 rhIL-11 freeze-dried powder injection

[0109] The rhIL-11 prepared above was taken, and freeze-dried powder was prepared according to the following formula.

[0110] Table 4 freeze-dried powder formula

[0111]

[0112] Ingredients Content

[0113]

[0114] rhIL-11 1.00g

[0115] NaCl 5.85g

[0116] Na 2 HPO 4 12H 2 O 2.187g

[0117] NaH 2 PO 4 2H 2 O 0.608g

[0118] Glycine 30g

[0119] Add water for injection to 1000ml

[0120]

[0121] The pH value was adjusted to 7.0, and after aseptic filtration, it was divided into 3ml / vials. Then freeze-dry to make a freeze-dried powder injection and store at 4°C.

Embodiment 3

[0122] Example 3 Determination of rhIL-11

[0123] 1. SDS-PAGE purity analysis

[0124] Adopt SDS-PAGE system (Guo Yaojun, "Protein Electrophoresis Experimental Technology", Science Press, 1999) to carry out non-reducing electrophoresis, scan and measure with Bio-Rad Gel Doc 2000 gel imaging system, rhIL-11 purity is 98.90% (see image 3 in track 5).

[0125] 2. RP-HPLC purity analysis

[0126] The chromatographic column is Delta-Pak C18 5 μ m 3.9 × 150 (Waters Co.), from the eluent (at 95% by volume dH 2 O and 0.1 vol% trifluoroacetic acid (TFA) in 5 vol% acetonitrile) was changed to the eluent (in 95 vol% acetonitrile and 5 vol% dHO 2 0.1 vol% TFA in O) for linear gradient elution for 40 min, flow rate 1 ml / min, and UV detection at 220 nm.

[0127] Analysis results showed that the purity of rhIL-11 prepared by the above method was 99.28%. RP-HPLC analysis results see Figure 4 and Table 5.

[0128] table 5

[0129] serial number

Retention time (minutes) ...

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Abstract

The invention provides a method for preparing recombinant human interleukin-11 (rhIL). The method comprises the following steps: 1) providing a fusion protein, wherein from the N-end to the C-end, the fusion protein is thioredoxin-(His)6-proteolytic enzyme recognition site-rhIL-11 or (His)6-thioredoxin-proteolytic enzyme recognition site-rhIL-11; 2) using the proteolytic enzyme to perform enzyme cutting to the fusion protein and obtain an enzyme cutting product, wherein the enzyme cutting product contains thioredoxin and rhIL-11; and 3) using the nickel ion chelate affinity column chromatography to purify the enzyme cutting product, and performing gradient elution to the column to obtain rhIL-11, wherein the thioredoxin and the rhIL-11 are both adsorbed on the column. By adopting the method provided by the invention, the rhIL-11 can be purified rapidly, conveniently and efficiently and the recovery rate can be greatly increased.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a method for preparing recombinant human interleukin-11. Background technique [0002] Interleukin-11 (Interleukin-11, IL-11) was first discovered in 1986 by D.A.Williams et al. of Harvard Medical School. IL-11 is a multifunctional cytokine, and its most basic function lies in the regulation of hematopoiesis. It stimulates the differentiation of human hematopoietic erythrocytes and megakaryocyte precursors and induces the generation and maturation of megakaryocytes, ultimately leading to an increase in the number of platelets. In addition, it also has various biological activities such as promoting the recovery of digestive tract epithelial damage and regulating adipocyte differentiation (Du X.X., Williams D.A., Blood, 1997, 89(11): 3897-3908). In 1990, Paul S.R. et al. (Proc.Natl.Acad.Sci.USA, 1990, 87:7512-7516) cloned the cDNA of this cytokine, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/22C07K1/18
Inventor 韩为跃叶学君杨立明阳勇张仁怀何凯汪猜张风豪
Owner DONGGUAN TAILI BIOTECH
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