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Method for producing recombined human interleukin-15

A technology of human interleukin and interleukin, applied in the field of genetic engineering, can solve the problems of low expression, low yield, and difficult purification

Inactive Publication Date: 2008-04-02
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

② For normal B cells, IL-15 can induce their proliferation after preactivation with myristate phorbol acetate (PMA) and anti-IgM antibody, but not for resting B cells
[0008] There have been reports on the expression and purification of IL-15 at home and abroad, but the expression level is not high, generally accounting for 5-10% of the total bacterial protein, or the purification is difficult and the yield is low

Method used

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Examples

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Embodiment 1

[0027] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0028] Expression vector construction method: obtain the target human IL-15 gene by PCR amplification, and use Nco I+EcoRI double digestion (MBI, 2*Tango TM , 37℃) PCR product and BL21(DE3)STAR TM / plysS plasmid (purchased from Invitrogen). The two fragments were ligated with T4 DNA ligase, and the ligated product was transformed into Escherichia coli DH5α (purchased from Promega), clones were selected on an LB plate containing ampicillin, and plasmids were prepared in small quantities, and screened out by double enzyme digestion / PCR identification Positive clone. A large number of confirmed pBV220 / IL-15 plasmids were amplified and identified by Nco I+EcoRI double enzyme digestion; after the pBV220 / IL-15 plasmid was verified by sequencing, it was transformed into Escherichia coli BL21(DE3)STAR TM / plysS (purchased from Promega Company), positive clones were...

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Abstract

The present invention provides an encoding sequence of recombinant human interleukin-15 as well as a construction method of high expression recombinant human IL-15 engineered cells and construction of related expression vectors. The present invention is very suitable for expressing in escherichia coli by optimizing the human IL-15 gene and constructing in appropriate expressing vectors, and at the same time high expression recombinant human IL-15 engineered cells are obtained by screening.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention provides a coding sequence of recombinant human interleukin-15, a method for constructing engineered cells with high expression of recombinant human interleukin-15, and construction of corresponding expression vectors. Background technique [0002] In May 1994, the American Seattle Immunex Biotechnology Research and Development Company and the US FDA's Biological Products and Research Center obtained the culture supernatants of the African green monkey kidney epithelial cell line CV-1 / EBNA and the adult leukemia T cell line HuT-102 respectively. A soluble cytokine was found in It is capable of sustaining T cell proliferation and has many of the same biological functions as interleukin 2 (IL-2). The amino acid sequence of the mature protein of this factor is different from any protein in the GeneBank and EMBL databases, so this new cytokine was named in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/24C12N15/63
Inventor 黄阳滨郭学彦任军朱飞云
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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