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Method for preparing human interleukin 29 and recombinant IL-29 engineering strain

A technology for interleukin and production method, which is applied to the production method of human interleukin and the field of recombinant engineering bacteria, can solve the problems of high polypeptide activity, large expression amount, small expression amount, etc. Effects of Expression Efficiency

Inactive Publication Date: 2005-09-21
SHANTOU UNIV MEDICAL COLLEGE
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Problems solved by technology

[0003] The purpose of the present invention is to overcome the common problems of low biological activity and small expression of the product in the existing IL-29 production method, and provide a production method of human IL-29, which can express IL-29 efficiently, and the obtained product IL-29 -29 polypeptide has high activity and large expression

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  • Method for preparing human interleukin 29 and recombinant IL-29 engineering strain
  • Method for preparing human interleukin 29 and recombinant IL-29 engineering strain
  • Method for preparing human interleukin 29 and recombinant IL-29 engineering strain

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Embodiment

[0034] (1) Synthetic PCR primers: According to the sequence of the human interleukin IL-29 cDNA gene sequence in GenBank and the LIC site sequence on the pET-44 Ek / LIC vector (see the instructions of Novagen, Cat 711433), the Omiga 2.0 software was used to design as follows Primers:

[0035] Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAAGCCCAC-3

[0036] Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;

[0037] (2) PCR amplification: On the MJ Research PTC-200 gradient PCR amplification instrument, hotstart PCR amplification was adopted. The pcDNA3.1 / V5-His-TOPO-IL-29 plasmid was used as a template. Set the annealing temperature gradient, and the cycle conditions are: ①95°C denaturation for 15 minutes; ②94°C for 45s, 72°C for 30s, the annealing temperature decreases by 1.0°C per cycle, 72°C for 45s, 5 cycles; ③94°C for 45s, set the annealing temperature gradient from 68°C to 70°C for 30s, 72°C for 45s, 30 cycles; ④ 72°C extension for 10min. The PCR products w...

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Abstract

The invention discloses a method for producing a body interleukins29 and restructuring the engineering bacteria IL-29, which comprises artificial synthetic primers, PCR augmenting mature peptides of IL-29 coding areas, the construction of IL-29 pronucleus expression carrier pET44-IL-29, transforming into colibacillus to restructure engineering bacteria IL-29, culturing the engineering bacteria to highly effective express IL-29, and purifying IL-29. In the invention, adopting a less dependent interconnection reaction clone process (LIC) to precise and quick-speed get the mature peptides coding areas of IL-29, adopting the terminator codon of colibacillus preference to increase the expression efficiency, one-step purifying using a S protein affinity chromatography column to get the very purified production.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a production method of human interleukin and recombinant engineering bacteria thereof. Background technique [0002] Human interleukin 29 (IL-29) is produced by some viruses or double-stranded RNA (dsRNA) activated by various human cells such as peripheral blood mononuclear cells (PMBC), dendritic cells (DC) and HeLa cells Cytokines (Sheppard, P et al, 2003, Nature Immunology, 4(1):63-68). IL-29 has a low level of homology with interferon (IFN) and IL-10, and IL-29 has 81% homology with IL-28A; the chromosome location is 19q13.13. Similar to interferon, IL-29 induces cells to produce a variety of intracellular proteins that mediate its biological activity and can selectively act on different types of target cells. Therefore, IL-29 can be used as a substitute for IFN for the treatment of tumors, viral diseases, etc., and has potential application prospects in clinical applicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54C12N1/21C12N15/09C12N15/24
Inventor 何韶衡李明才
Owner SHANTOU UNIV MEDICAL COLLEGE
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