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Secretory expression method for recombinant human IL-15

An IL-15, high-expression technology, applied in the field of genetic engineering, can solve the problems of low expression, low yield, difficult purification, etc.

Inactive Publication Date: 2008-03-05
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Abstract
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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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  • Secretory expression method for recombinant human IL-15
  • Secretory expression method for recombinant human IL-15

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Embodiment 1

[0028] The construction of embodiment 1 expression plasmid and the acquisition of high expression engineering strain

[0029] The expression vector construction method is shown in Figure 2. Human IL-15 target gene was amplified by PCR, and double-digested with BamHI+EcoRI (MBI, 2*Tango TM , 37°C) PCR product and pTHiohisA plasmid (purchased from Invitrogen). The two fragments were ligated with T4 DNA ligase, and the ligated product was transformed into E. coli DH5

[0030] α (purchased from Promega Company), clones were selected on LB plates containing ampicillin, plasmids were prepared in small quantities, and positive clones were screened out by double enzyme digestion / PCR identification. A large number of confirmed pTHiohisA / IL-15 plasmids were amplified and identified by double digestion with BamHI+EcoRI; after the pTHiohisA / IL-15 plasmid was verified by sequencing, it was transformed into Escherichia coli BL21 (DE3) (purchased from Promega Company), and the Positive cl...

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Abstract

The present invention provides the coding sequence of recombinant human interleukin-15 (IL-15), the method of constituting engineering cell for high expressing recombinant human IL-15 protein, and the constitution of corresponding expression vector. Through optimizing human IL-15 gene and constituting to proper expression vector, the IL-15 is suitable for secreting expression in colibacillus. Through screening, high expressing recombinant human IL-15 engineering cell is obtained.

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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IPC IPC(8): C12N15/09C12N15/24C12N1/21
Inventor 黄阳滨孙九如张翊任军梁光军
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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