Interfusion protein of human interleukin 15 and Fe

A technology of interleukin and fusion protein, applied in the field of genetically engineered human interleukin-15 and Fc fusion protein, which can solve the problems of small molecular weight, limited biological activity, and short half-life of natural IL-15

Active Publication Date: 2007-08-29
上海海欣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to the small molecular weight of natural IL-15 and its short half-life after in vivo application, its biological activity in vivo is limited.

Method used

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  • Interfusion protein of human interleukin 15 and Fe
  • Interfusion protein of human interleukin 15 and Fe
  • Interfusion protein of human interleukin 15 and Fe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Cloning of human IL-15 functional gene and Fc fragment gene of human IgG4

[0049] 1. Cloning of IL-15 functional gene:

[0050] Adherent mononuclear cells were isolated from the peripheral blood of normal people, and after being stimulated by adding LPS for 4 hours, the total RNA was extracted by one-step method of guanidine isothiocyanate, the first strand of cDNA was synthesized by AMV reverse transcriptase, and PCR was carried out separately using it as a template To amplify the wild-type human mature IL-15 cDNA sequence, the primers used are:

[0051] Upstream primer: P1 5'AACTGGGTGAATGTAATAAG3'

[0052] Downstream primer: P2 5'AGAAGTGTTGATGAACA TTTG3'

[0053] The estimated size of the PCR product of human IL-15 is 790bp, the PCR reaction volume is 50μl, the primer concentration is 0.4μM, and the amplification parameters are 94°C for 30 seconds, 51°C for 45 seconds, and 72°C for 50 seconds. After 30 cycles, the product line 2% agarose gel electrophor...

Embodiment 2

[0058] Example 2: Construction of Yeast Secretion Vector Encoding IL15-Fc Fusion Protein

[0059] Using pMD18T-IL15 and pMD18T-Fc as templates, primers were designed to amplify the IL15 and Fc fragments, and then IL15 was connected to the modified α signal peptide on the pPICK vector, while the Fc fragment was connected to the downstream region of IL15 to form a gene encoding IL15 - The carrier of the Fc fusion protein, named pPIC9K-IL15-Fc.

[0060] (a) Amplification of IL-15 fragments

[0061] For the IL15 fragment, the primer sequences used for amplification are:

[0062] Upstream primer IL15-F: -CGAGAAAAGAAACTGGGTTAACGTTATCTC-

[0063] Downstream primer IL15-RE: -CGGAATTCGGAAGTGTTGATGAACAT-

[0064] Using pMD18T-IL15 as a template, the IL15 fragment was amplified by conventional PCR method. The amplification conditions were 97°C, 2min; 94°C, 30s, 58°C, 30s, 72°C, 45s, a total of 25 cycles; then 72°C, 10min; and finally stored at 4°C. As a result, a 360bp target fragme...

Embodiment 3

[0086] Example 3: Establishment and Identification of Yeast Engineering Strains Expressing IL15-Fc Fusion Protein

[0087] Take 5-10 μg of the recombinant plasmid pPIC9K-IL15-Fc and the control plasmid pPIC9K large pumping plasmid, and after linearization by SacI digestion, transform the yeast strain GS115 by electric shock method, and use MD (glucose minimal medium) plate after transformation to screen for occurrence homologous recombination His + clone. Mix all the transformed clones with water, and take 10 5 Each yeast was spread on the G418 gradient resistance YPD plate (G418 concentrations were 0, 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0 and 4.0 mg / ml). Incubate at 30°C for 2-5 days. Pick a number of recombinant colony inoculum on each gradient resistance plate in 20ml of BMGY medium, culture in a shake flask at 30°C, grow to the bacterial cell concentration A600nm=2~6, collect the bacteria by centrifugation, and replace it with 5-10 times The volume (100ml) of BMMY ...

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Abstract

A human interleukin 15-Fc fusion protein composed of interleukin 15 and the Fc fragment of human immunoglobulin, which are linked via joining peptide, the nucleic acid ofr coding it, the expression carrier containing said nucleic acid, its composite medicine for preventing and treating microbial infection and its preparing process are disclosed.

Description

technical field [0001] The present invention relates to the technical field of utilizing recombinant DNA, more specifically, the present invention relates to genetically engineered human interleukin-15 (Interleukin-15, IL-15) and Fc fusion protein, its application and preparation method. Background of the invention [0002] Interleukin 15 (IL15) is a newly discovered cytokine of interleukins, which has similar biological activities to interleukin 2 (IL-2), can promote the differentiation and proliferation of B cells, induce the proliferation of NK cells, Cytotoxic activity, cytokine production, it cooperates with IL12 to promote NK cells to produce IFN-γ, which plays an important role in regulating the function of NK cells and the body's immunity [1-7]. [0003] First, IL-15, as part of the early immune response to anti-microbial infection in the body, can continue to function in an environment that is not conducive to the expression of pro-inflammatory cytokines (such as IL...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/54C12N15/62C12N15/63A61K39/395A61K38/20A61P31/00
Inventor 王建莉
Owner 上海海欣生物技术有限公司
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