Method for yeast cell to express human interleukin 10

An interleukin, yeast expression technology, applied in the direction of fermentation, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of protein can not form disulfide bond space folding, low recovery rate, increase production cost and other problems

Inactive Publication Date: 2004-06-23
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Escherichia coli is a commonly used genetic engineering strain. In E. coli, hIL-10 is expressed in the form of inclusion body, which is an inactive denatured protein. After purification and renaturation, some proteins still cannot form accurate disulfide bonds and proper spaces. Folding affects the biological activity of hIL-10, and the process is complicated, the recovery rate is low, and the production cost is increased

Method used

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  • Method for yeast cell to express human interleukin 10
  • Method for yeast cell to express human interleukin 10
  • Method for yeast cell to express human interleukin 10

Examples

Experimental program
Comparison scheme
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Embodiment Construction

[0054] 1. Artificially synthesized hIL-10 whole gene, the nucleotide sequence is as follows:

[0055] 10 20 30 40 50 60

[0056] ATGCATTCTT CTGCTTTGTT GTGTTGTTTG GTTTTGTTGA CTGGTGTTAG AGCTTCTCCA

[0057] TACGTAAGAA GACGAAACAA CACAACAAAC CAAAACAACT GACCACAATC TCGAAGAGGT

[0058] 70 80 90 100 110 120

[0059] GGTCAAGGTA CTCAATCTGA AAATTCTTGT ACTCATTTTC CAGGTAATTT GCCAAATATG

[0060] CCAGTTCCAT GAGTTAGACT TTTAAGAACA TGAGTAAAAG GTCCATTAAA CGGTTTATAC

[0061] 130 140 150 160 170 180

[0062] TTGAGAGATT TGAGAGATGC TTTTTCTAGA GTTAAGACTT TTTTTCAAAT GAAGGATCAA

[0063] AACTCTCTAA ACTCTCTACG AAAAAGATCT CAATTCTGAA AAAAAGTTTA CTTCCTAGTT

[0064] 190 200 210 220 230 240

[0065] TTGGATAATT TGTTGTTGAA GGAATCTTTG TTGGAAGATT TTAAGGGTTA CTTGGGTTGT

[0066] AACCTATTAA ACAACAACTT CCTTAGAAAC AACCTTCTAA AATTCCCAAT GAACCCAACA

[0067] 250 260 270 280 290 300

[0068] CAAGCTTTGT CTGAAATGAT TCAATTTTAC TTGGAAGAAG TTATGCCACA AGCTGAAAAT

[0069] GTTCGAAACA...

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Abstract

The method for yeast cell to express human interleukin 10 (hIL-10) has the technological scheme including artificially synthesizing all-gene sequence of hIL-10 including signal-carrying hIL-10 sequence, named shIL-10, and no-signal hIL-10 sequence, named nshIL-10; adding to the ends with EcorR I enzyme incising sequence and Not I enzyme incising sequence or with EcorR I enzyme incising sequences; double incising with EcorR I and Not I the integral expression vector pPIC3.5K and Ppic9K of destination gene shIL-10, nshIL-10 and Pichia yeast; constituting recombinant plasmid pPIC3.5K / shIL-10 and Ppic9K / nshIL-10; converting Pichia yeast cell via electrical perforation and plasmodic granule process; integrating recombinant plasmid and destination gene to yeast chromosome; and applying methanol to induce Pichia yeast for intracellular soluble expression and extracellular secretion type expression. The present invention is superior to expression in colibacillus.

Description

technical field [0001] The invention relates to a manufacturing method of human interleukin 10, more specifically relates to the application of yeast cells to express human interleukin 10, and belongs to the high-tech research field of biotechnology. Background technique [0002] In 1989, Fiorentino et al. found that mouse CD4+ Th2 cells secreted a substance that could inhibit Th1 cells from producing cytokines (such as IFN), which was called cytokine secretion inhibitory factor (CSIF), and later collectively called interleukin 10 (IL-10). 10). [0003] IL-10 is an important cytokine in normal human body. The mature IL-10 has 160 amino acid residues, the monomer molecular weight is 18.7KD, and contains disulfide bonds formed by 4 cysteines (12-108, 62 -114). The active form is a homologous oligodimer with a molecular weight of 39KD linked by non-covalent bonds. [0004] Human IL-10 is secreted by a variety of cells, mainly produced by Th2 cells, and exerts immunosuppressi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/81C12P21/02
Inventor 邹全明井申荣
Owner ARMY MEDICAL UNIV
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