Recombinant human interleukin1 receptor antagon (rhIL-1ra) with low pyrogen and its high efficiency preparation process

A technology of interleukin and receptor antagonist, applied in the field of recombinant human interleukin 1 receptor antagonist and its high-efficiency preparation, achieving high purity, strong activity, and good market prospects

Active Publication Date: 2010-06-23
SICHUAN HENGXING BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current low pyrogen recombinant human interleukin-1 receptor antagonist and its preparation method have not been reported yet

Method used

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  • Recombinant human interleukin1 receptor antagon (rhIL-1ra) with low pyrogen and its high efficiency preparation process
  • Recombinant human interleukin1 receptor antagon (rhIL-1ra) with low pyrogen and its high efficiency preparation process
  • Recombinant human interleukin1 receptor antagon (rhIL-1ra) with low pyrogen and its high efficiency preparation process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0033] Example 1 Separation and amplification of target gene hIL-1ra cDNA

[0034] 1. Human U937 bone marrow mononuclear cells (myelomonocytic cells, purchased from ATCC, USA) were cultured. The total RNA of cultured human U937 bone marrow mononuclear cells was extracted with a total RNA kit, and used for RT-PCR reaction after electrophoresis inspection.

[0035] 2: According to the human IL-1ra cDNA sequence published by Cater D.B. et al., design and synthesize upstream primer P1 and downstream primer P2 for amplifying human IL-1ra cDNA (84-539):

[0036] Upstream primer P1: 5'-TTT CAT ATG CGA CCC TCT AGA GGG AAA AAATCC-3' (SEQ NO.1) has an additional translation initiation codon (ATG) and an NdeI restriction site at the 5' end.

[0037] Downstream primer P2: 5'-AAA GGA TCC TTA TTA GTC CTC CTC CTG GAA GTAGAA-3' (SEQ NO.2). A BamHI restriction site and a stop codon TAA are added at the 3' end.

[0038] 3. Amplification of hIL-1ra cDNA

[0039] Take 4-5 μg of total RNA as a...

Embodiment 2

[0040] Example 2 Construction of expression vector and transformation

[0041] 1. Source of expression vector

[0042] The expression vector pBV220 was purchased from Beijing Yuanping Biotechnology Company.

[0043] 2. The source and biological characteristics of the host bacteria

[0044] The host strain DH5α was purchased from Promega, USA.

[0045] Genotype: SupE44, ΔLacV169 (φ80LacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1.

[0046] 3 Expression plasmid construction, transformation, enzyme digestion identification

[0047] 3.1. Preparation of Competent Cells E.coli DH5α (J. Sambrook D.W. Russell Science Press, author of "Molecular Cloning Experiment Guide", August 2002)

[0048] Pick the freeze-dried bacteria DH5α, put it into 20mL LB culture medium, cultivate overnight at 30°C and 250rpm, transfer 4mL culture solution into another 20mL LB culture medium, and cultivate it at 30°C and 250rpm until the OD600 is about 0.4. Transfer to another new, sterile 50mL ce...

Embodiment 3

[0076] Example 3 Screening and large-scale expression of pBV220-IL-1ra high-efficiency expression engineering strains

[0077] 1. Main instruments and equipment

[0078] PCR instrument Mastercycler Personal, purchased from Eppendof;

[0079] Laser scanning densitometer GS-700, purchased from Bio-Rad;

[0080] SCF-24 shaker (purchased from Shanghai Institute of Centrifugal Machinery)

[0081] Fermentation tank KSB6231 MFR K3T, purchased from KoBio Tech Co.LTD, the working volume of the working tank is 300L, and the seed tank is 30L.

[0082] 2. The expression plasmid pBV220-IL-1ra was transformed into Escherichia coli DH5α by conventional methods to obtain engineering bacteria.

[0083] 3. Randomly pick 6 single-colony transformants (numbered 1, 2, 3, 4, 5, 6) with uniform size and shape from the LB plate with a sterilized toothpick, and inoculate them in 5 mL of LB medium ( Amp+100μg / mL) test tube, 30°C, 220rpm, shake the flask overnight. The seed solution of 6 single col...

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Abstract

The invention discloses a low pyrogen recombination human leucocyte mesonium 1 acceptor antagonist (rhIL-1ra) and preparing method, wherein the endotoxin content of rhIL-1ra is smaller than 1Eu / mg rhIL-1ra. The invention comprises the following steps: a, increasing cDNA sequence of rhIL-1ra; constructing recombination plasmid of cDNA sequence with encode rhIL-1ra; b, converting recombination plasmid into bacillus coli to get inversion rotor; c, expressing rhIL-1ra from inversion rotor; purifying and removing heat; getting product yield. The invention simplifies the step, which reduces the cost and improves the product.

Description

technical field [0001] The invention relates to the field of bioengineering and pharmacy, in particular to a recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and its efficient preparation method and application. Background technique [0002] Interleukin-1 receptor antagonist (IL-1Ra) is a protein found in the culture supernatant of human monocytes stimulated by IgG, which can specifically bind IL-1 Receptor binding, competitively prevents the binding of IL-1 to its receptor, and blocks the activation of IL-1 target organ receptor signaling pathway, so that IL-1 cannot exert biological effects, and can be used for rheumatoid joints inflammation treatment. In 1990, scientists Eisenberg, S.P. et al. first cloned its gene (cDNA) and expressed it in Escherichia coli ([1] Cater D.B., et al. Nature Vol 344.12 April 1990, 633-637). Complete human IL-1ra is composed of 152 amino acids with a molecular weight of 22KD. The sugar group is not necessary for its biologica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/54C12N15/09C12N15/10C12N15/24C12N15/63C12N15/70C12N1/21C12P21/00A61K38/20A61K9/00A61P19/02
Inventor 彭红卫赵斌杨伟张宝华周裕诚
Owner SICHUAN HENGXING BIOMEDICAL
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