High-level secretory expression method for human interleukin 8, and yeast transformant
An interleukin, secretory expression technology, applied in the field of high-level secretory expression of human interleukin 8 and yeast transformants, can solve the problems of reducing active protein yield, low protein yield, inactivation of target products, etc. Efficiently stabilize protein in large quantities, prevent degradation, and ensure the effect of biological activity
Inactive Publication Date: 2011-11-09
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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At present, Escherichia coli is mainly used in the market for expression, and the expression product usually exists in the form of inclusion bodies, and the active form of interleukin-8 can only be obtained through complex operations of denaturation and renaturation, which has always reduced the yield of active protein; in addition, prokaryotic expression The interleukin-8 usually exists in the form of fusion protein, and the removal of the fusion non-interleukin-8 fragment requires complex and high-cost enzyme digestion steps to obtain a protein with the same primary structure as the natural interleukin-8; third, the prokaryotic host expresses especially Expression in Escherichia coli may cause toxicity due to the presence of LPS, so it is often necessary to analyze and determine the toxicity of the expressed purified product; finally, to obtain a high-purity protein often requires multi-step purification operations, the more purification steps More, the lower the protein yield, and more likely to lead to the inactivation of the target product
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[0032] The present invention uses the Pichia X-33 strain and the integrated expression plasmid pPICZαA. The vectors were all purchased from Invritrogen, USA.
[0033] 1. Clone the whole rhIL-8 gene:
[0034] Design a pair of primers, take total mRNA from human bone marrow stromal cells as template, first amplify total cDNA by RT-PCR, and then use this cDNA as template to amplify a complete human recombinant IL-8 gene fragment with IL-8 specific primers , PCR conditions are: 95°C pre-denaturation, 4min, one thermal cycle; 95°C thermal denaturation for 30s, 55°C tempering for 30s, 72°C extension for 40s, 28 thermal cycles; 72°C renaturation for 10min. The obtained amplified fragment was connected to the pMD20-T vector (purchased from Guangzhou TAKARA Company), and identified by PCR, restriction digestion and nucleic acid sequencing, and the determined sequence was consistent with the IL-7 sequence published by Genebank.
[0035] The PCR amplification primers are:
[0036] 5’-GC CTC GA...
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Abstract
The invention discloses a high-level secretory expression method for human interleukin 8, and yeast transformant. The method mainly comprises the following steps: cloning a human IL-8 gene; constructing a eukaryotic expression vector; transforming a recombinant vector into a eukaryotic yeast host; obtaining the yeast transformant in high-level secretory expression through screening; expressing recombinant human IL-8 protein in the yeast host; and quickly obtaining the recombinant human IL-8 protein with the purity up to more than 95 percent by a two-step method of performing ammonium sulfate precipitation and CM-cation exchange chromatography. The invention changes the prior method of using prokaryotic host Escherichia coli to express IL-8, explores the method of using eukaryotic host Pichia pastoris to express the IL-8, screens out the yeast transformant for high-level secretory expression, and quickly obtains the high-purity recombinant human IL-8 protein through ammonium sulfate precipitation and CM-cation exchange chromatography. The method has the advantages of not only ensuring the biological activity of the IL-8 but also quickly obtaining a large amount of stable protein.
Description
Technical field [0001] The present invention relates to the application of recombinant DNA technology to produce genetically engineered protein drugs, in particular to a method for high-level secretion and expression of human interleukin 8 and yeast transformants. Background technique [0002] Interleukin 8 belongs to the CXC subfamily. Monocytes and endothelial cells can express synthetic interleukin 8. It is a multi-source and multifunctional cytokine, which can initiate and promote inflammation. Sex granulocytes have chemotaxis and activation effects, chemotactic effects on T cells, can activate basophils, and promote the division and chemotaxis of keratinocytes, stimulate blood vessel formation and release precursor substances. Recently, it has been discovered that interleukin 8 can be used as a diagnostic molecular target for many diseases. For example, pregnant women with high serum interleukin 8 levels are more likely to have schizophrenia in their offspring; the level of ...
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IPC IPC(8): C12N15/81C12P21/02C12N1/19C12R1/84
Inventor 吴东海李洪波李侍武金守光徐爱民
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI