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Human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and preparation method thereof

An interleukin, site-directed mutagenesis technology, applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as non-specificity of cell responses

Inactive Publication Date: 2013-07-31
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] IL-29 must bind to the corresponding receptor on the surface of the target cell to exert its effect. IL-29 and IL-28 share a heterodimeric receptor complex composed of IL-28R1 and IL-10R2, of which IL-28R1 is the binding receptor. Subunit, specific for cellular response to IL-29, IL-10R2 is an auxiliary subunit, non-specific for cellular response to IL-29

Method used

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  • Human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and preparation method thereof
  • Human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and preparation method thereof
  • Human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Cloning of human interleukin 29 variant encoding gene

[0022] 1. Design and Synthesis of PCR Primers

[0023] According to pPIC9K in GenBank M Carrier Factor signal peptide sequence and human IL-29 gene sequence, use Oligo7 software to design a pair of specific primers as follows:

[0024] Upstream primer: 5'- CTCGAGAAAAAGA GGCCCTGTCCCCCACTTCC-3'

[0025] Downstream primer: 5'- GCGGCCGC TCAGGTGGACTCAGGGTGG-3'

[0026] Mutation primer: 5'-TCCAATGCGTCC T TGGCC C TCTTGAAGCTCGCTA-3'

[0027] Added restriction endonuclease Xho I recognition site (CTCGAG) and protease Kex2 recognition site (GAGAAAAGA, encoding product is Glu-Lys-Arg) in the upstream primer, added Not I site (GCGGCCGC ), in order to obtain the mutation product, the 13th base in the mutation primer is designed as T, and the triplet code composed of this base is mutated from AGG to AAG, and the 35th amino acid encoded by the triplet code is composed of arginine (Arg ) is mutated to lysin...

Embodiment 2

[0030] Example 2: Construction of recombinant eukaryotic expression vectors expressing human IL-29 variants

[0031] Extraction of pUCm-29Z and vector pPIC9K M (from Invitrogen Company, modified by our laboratory and applied for a patent, application number: 201110410391.0), each take 20 μL of the plasmid and perform double digestion with restriction endonuclease Xho I and NotI respectively, and recover about 560 bp of the target Gene fragment and vector pPIC9K M For large fragments, use T 4 DNA ligase (BBI Company) was connected in a water bath at 16°C for 20 hours, and the ligation product was transformed into competent Escherichia coli JM109, then inoculated on LB culture plates containing kanamycin, cultured overnight at 37°C, and positive colonies were picked to inoculate the liquid Amplify in LB medium, extract the recombinant eukaryotic expression plasmid and name it pPIC9K M -hIL-29Z; identification of recombinant eukaryotic expression plasmid pPIC9K by Xho I and No...

Embodiment 3

[0032] Example 3: Transformation of Yeast GS115 with Recombinant Eukaryotic Expression Plasmid and Screening of High Copy Recombinant Engineering Bacteria

[0033] Extract pPIC9K identified by sequencing M - hIL-29Z plasmid, take 20 μL and digest it with restriction endonuclease Sal I to linearize it, separate and purify it by 0.7% agarose gel electrophoresis, and recover the linearized pPIC9K M - hIL-29Z; mixed linearized pPIC9K M - hIL-29Z and yeast GS115 competent cells were electrotransformed according to the method of the Pichia pastoris expression manual (Invitrogen).

[0034] Spread the transformation product on the MD culture plate without His, culture at 30°C for 2-3 days, select the dominant colony that grows vigorously on the MD plate and inoculate it on the YPD plate containing 0.5mg / mL G418, and culture at 30°C for 2-3 days , select the vigorously growing dominant colony and inoculate it on a YPD plate containing 1mg / mL G418, culture it at 30°C for 2 to 3 days, ...

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Abstract

The invention provides a human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and a preparation method thereof. The preparation method comprises the following steps of 1, carrying out artificial design synthesis of a site-directed mutagenesis primer, 2, carrying out site-directed mutagenesis of 98th and 104th basic groups of a hIL-29 mature peptide encoding gene by the site-directed mutagenesis primer, 3, constructing a recombinant eukaryotic expression vector containing a hIL-29 mutant encoding gene, and carrying out pichia pastoris transformation to obtain recombinant engineered yeast, 4, carrying out culture of the recombinant engineered yeast, and adding 1.5% (v / v) of methanol into the culture solution so that hIL-29 mutant expression is induced, and 5, through SP-Sepharose Fast Flow cation-exchange chromatography, carrying out purification of the hIL-29 mutant in the supernatant of the culture solution. The engineered yeast obtained by the invention can be subjected to high-density culture, can secrete and express the hIL-29Z with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine, and is suitable for industrial preparation of hIL-29 mature peptide mutants.

Description

technical field [0001] The present invention provides a human interleukin 29 mature peptide 33rd lysine, 35th arginine site-directed mutation into arginine, lysine variant (hIL-29Z) and its preparation method, belonging to biological engineering field. Background technique [0002] Human interleukin 29 (Interleukin29, IL-29) is a new cytokine discovered in recent years, also known as interferon λ (Interferon29, IFNλ1), which belongs to the IFNλ family (IFNλs) together with IL-28A and IL-28B. ). [0003] Studies have found that IL-29 initiates signal transduction and exerts biological effects by binding to a special heterodimer receptor complex. It shares the same Jak-STAT signaling pathway with type I interferon and promotes a set of common Therefore, IL-29 exhibits some of the same properties as type I interferon, such as anti-virus, anti-proliferation, anti-tumor in vivo and immune regulation and other biological activities. [0004] IL-29 must bind to the corresponding...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/20C12N1/19C07K14/555C12P21/02C12R1/84
Inventor 陈伟朱荣葛春蕾陆源郑海军邬敏辰吴静
Owner JIANGNAN UNIV
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