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Human interleukin-10 gene sequenc and E coli containing the said gene sequence

An interleukin and gene sequence technology is applied in the field of Escherichia coli engineering bacteria to achieve the effects of ensuring activity, mild reaction conditions and high cutting efficiency

Inactive Publication Date: 2007-03-21
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the huge number of patients with such diseases, such as rheumatoid arthritis (RA) is the most common systemic immune disease, which is distributed worldwide. In the United States, the incidence rate of RA is 0.3-0.5%, and the incidence rate of RA in my country is 0.4-0.7%, the number of patients is more than 5 million, and there is no specific drug to treat these diseases. Moreover, human interleukin-10 has not yet formed a drug that can be industrialized in the field of biotechnology, so the development of this drug will bring Bring huge economic and social benefits

Method used

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  • Human interleukin-10 gene sequenc and E coli containing the said gene sequence
  • Human interleukin-10 gene sequenc and E coli containing the said gene sequence
  • Human interleukin-10 gene sequenc and E coli containing the said gene sequence

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Experimental program
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Effect test

Embodiment Construction

[0058] 1. Cloning and sequencing of hIL-10 gene

[0059] According to the human interleukin-10 gene sequence reported by Genbank, some rare codons in the sequence were replaced, and the IL-10 gene was synthesized by Shanghai Sangon Bioengineering Co., Ltd. containing codons favored by E. coli and frequently used, 5 Add the EcoR I recognition site (AATTC) at the 'end and the Hind III recognition site (AGCTT) at the 3' end, and clone it into the pUC18 plasmid. The sequencing results were consistent with expectations (see Figure 3).

[0060] 2. Construction and identification of pTRX-hIL-10 recombinant plasmid

[0061] It was cloned into the prokaryotic fusion expression vector pTRX constructed in our laboratory according to conventional methods to construct the expression plasmid pTRX-hIL-10 (see Figure 6).

[0062] Upstream primer 5'CGG GGT ACC GAT GAC GAT GAC AAG TCT CCG GGC CAG GGC 3’

[0063] Kpn I Enterokinase site

[0064] Downstream primer 5'TG...

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Abstract

The present invention relates to the antificial synthesis of human interleukin-10 gene sequence and E. Coli containing the sequence. The present invention features that 35 sites of human interleukin-10 are codon substituted into E. Coli preference codon; pTRX-hIL-10 expression plasmid is constituted on the basis to obtain stable and efficient expression in E. Coli; and throgh separation and purification, hIL-10 recombinant protein with high purity and high activity is prepared. The present invention realizes in vitro expression of human interleukin-10 and this sets the foundation of producing the medicine with the matter.

Description

Technical field: [0001] The invention relates to a human interleukin-10 gene sequence and Escherichia coli engineering bacteria containing the gene and capable of producing immunosuppressive and anti-inflammatory active substances. Background technique: [0002] Human interleukin-10 (hIL-10) is directly derived from the expression product of human genes, which cannot be prepared by traditional biopharmaceutical technology. It is one of modern biotechnology to obtain recombinant human interleukin-10 (rhIL-10) with natural biological activity and curative effect by using various advanced modern molecular biology techniques such as artificially synthesized genes, molecular cloning, expression and purification of target proteins . High-efficiency expression and isolation and purification of eukaryotic short peptide genes are key issues in genetic engineering. According to the structural characteristics of different polypeptides, different expression methods are required. For ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/24C12N1/21C12P21/02
Inventor 徐安龙吴文言杨红彭立胜钟肖芬梁东卫剑文杨文利
Owner SUN YAT SEN UNIV
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