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Expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist

A technology of human serum albumin and receptor antagonist, applied in the field of protein expression system, can solve the problem of low expression level of fusion protein, and achieve the effects of low immunogenicity, improved expression level and favorable clinical application.

Active Publication Date: 2013-07-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention provides an expression system for the fusion protein of human serum albumin and interleukin-1 receptor antagonist to solve the problem of low expression level of the fusion protein in the existing expression system

Method used

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  • Expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist
  • Expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist
  • Expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Constructing plasmid pPIC9-IGH, and obtaining the DNA sequence that can express IGH protein

[0036] Using the plasmids PGEM-T-HSA and PGEM-T-IL1ra disclosed in Patent Application No. 200810060568.7 as templates, primers IL1ra up (SEQ ID NO.16), IL1ra dn (SEQ ID NO.17) were designed according to the sequences of IL1ra gene and HSA gene ), HSAup (SEQ ID NO.18) and HSA dn (SEQ ID NO.19) were amplified by PCR. In view of the fact that the base sequence of the BamH I restriction site just encodes -GS-, the codon encoding the connecting peptide -GGGGS- is specially designed at the 5' end of IL1ra dn, so that the BamH I restriction site is also designed. And a BamHI restriction site is also added at the 5' end of HSA up, so that the IL1ra gene and the HSA gene can be fused by BamHI restriction restriction and connection. The IL1ra gene amplification product was digested with Xho I and BamH I and recovered, the HSA gene amplification product was digested with BamH I...

Embodiment 2

[0038] Example 2 Construction of recombinant plasmid pPICZαB-IGH

[0039] The empty pPICZαB vector was digested with Xho I and Not I, and the vector fragment was recovered by electrophoresis. The digested pPICZαB vector and the IGH expression sequence obtained in Example 1 were mixed in an appropriate ratio, and ligated with T4 ligase in a water bath at 16°C for 12 hours to form pPICZαB-IGH (see figure 1 ). After pPICZαB-IGH was transformed into DH5α, spread on LB agar plates containing 25 μg / mL Zeocin and culture overnight at 37°C. Select several clones on the plate and inoculate them into 5 mL LB liquid medium containing 25 μg / mL Zeocin, and culture overnight at 37°C. The obtained positive clones were sent to Shanghai Sangon for sequencing verification.

Embodiment 3

[0040] Example 3 Transformation of recombinant plasmid pPICZαB-IGH into GS115

[0041] The correctly verified pPICZαB-IGH plasmid obtained in Example 2 was linearized with Pme I and then transformed into Pichia pastoris GS115 by electroporation. The transformed cells were incubated in YPD liquid medium at 30°C for 2 hours, and then spread to YPDS agar plates (1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, 2% agarose, 1M sorbitol) containing 100, 700 or 1500 μg / mL Zeocin. After culturing at 30°C for 3 days, about 200, 4, and 0 single colonies grew on YPDS agar plates with 100, 700, or 1500 μg / mL Zeocin, respectively. Pick 5 (named L1~5) and 4 (named H1~4) single colonies on YPDS agar plates with 100 μg / mL Zeocin and 700 μg / mL Zeocin, respectively.

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Abstract

The invention discloses an expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist. The expression system comprises a host cell and an expression vector transferred into the same. The expression vector comprises a first expression vector with an inserted fusion protein gene and a second expression vector with an inserted protein disulfide isomerase gene.The fusion protein gene comprises the human serum albumin and the interleukin-1 receptor antagonist. The host cell is Pichia pastoris GS115. The co-expression host of PDI (protein disulfide isomerase) and IGH (immunoglobulin heavy) is established, and expression index of the IGH is evidently increased. The PDI is expressed intracellularly, the IGH secretory expression is subjected to extracellular secretory expression, and accordingly no newly generated other proteins occur when concentration of the IGH in collected medium supernate increases.

Description

technical field [0001] The invention relates to a protein expression system, in particular to an expression system of a fusion protein of human serum albumin and an interleukin-1 receptor antagonist. Background technique [0002] The Pichia pastoris expression system is a relatively successful eukaryotic expression system for exogenous proteins in recent years. Compared with the E. coli expression system, it can better express complex proteins and can complete Post-translational modifications such as disulfide bond formation. Compared with mammalian cells and insect cell expression systems, it has the advantages of simple operation, high expression, and easy industrialization and amplification. [0003] Interleukin 1 receptor antagonist (IL1ra) is a protein that can bind to IL1 receptor, and when it binds to IL1 receptor, it can block the biological activity of IL1. Therefore, a certain concentration of IL1ra can neutralize the biological activities of these two cytokines ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/84
Inventor 陈枢青沈其
Owner ZHEJIANG UNIV
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