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Recombinant expression, separation and purification method of human ribonuclease 4 protein

A ribonuclease and protein technology, which is applied in the fields of biotechnology and biopharmaceuticals, can solve the problems of lack of application prospects, high cost of polypeptide synthesis, and no method for prokaryotic expression and purification of human RNASE4 protein reported in literature, and achieves high yield and reduced Production cost, simple operation effect

Inactive Publication Date: 2015-02-04
ZHEJIANG UNIV
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Problems solved by technology

However, the cost of peptide synthesis is too high, and it lacks application prospects as a drug production
So far, no literature has reported the prokaryotic expression and purification method of human RNASE4 protein

Method used

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  • Recombinant expression, separation and purification method of human ribonuclease 4 protein
  • Recombinant expression, separation and purification method of human ribonuclease 4 protein
  • Recombinant expression, separation and purification method of human ribonuclease 4 protein

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Embodiment 1

[0042] Embodiment 1, the method for recombinant expression and separation and purification based on human ribonuclease 4 protein, the following steps are carried out successively:

[0043]First, optimize the codons of the RNASE4-encoding gene:

[0044] According to the "Escherichia coli synonymous codon preference table" and so on, the gene encoding RNASE4 was transformed, and the codon-optimized sequence obtained was shown in SEQ ID NO.2. In order to facilitate the construction of subsequent prokaryotic expression vectors, restriction endonuclease BamH I and Nhe I sites (such as SEQ ID NO .2 underlined). The synthetic gene is constructed on the pUC57 vector, which is convenient for the construction of the prokaryotic expression plasmid in the next step.

[0045] Second, construct the RNASE4 protein prokaryotic expression plasmid:

[0046] The synthetic RNASE4 gene was inserted into the prokaryotic expression vector pET-11a. The specific experimental process was as follows:...

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Abstract

The invention discloses a gene for coding a human ribonuclease 4-based recombinant protein. The nucleotide sequence of the gene is represented by SEQ ID NO:2, and the amino acid sequence of the human ribonuclease 4-based recombinant protein is represented by SEQ ID NO:1. The invention also discloses a recombinant expression, separation and purification method of the human ribonuclease 4 protein. The method comprises the following steps: optimizing the codon of the RNASE 4 encoding gene to make the optimized codon suitable for prokaryotic expression of Escherichia coli; constructing an RNASE 4 protein prokaryotic expression plasmid pET-11a-RNASE 4; inducing Escherichia coli BL21(DE3 / pET-11a-RNASE4) to express the recombinant RNASE4 protein; and separating the recombinant RNASE4 protein, and purifying the recombinant RNASE4 protein. The protein is used for preparing auxiliary medicines for treating neurodegenerative diseases.

Description

technical field [0001] The invention relates to the recombinant expression of human ribonuclease 4 protein and its separation and purification method, as well as its application in the treatment of neurodegenerative diseases, belonging to the fields of biotechnology and biopharmaceuticals. Background technique [0002] Ribonuclease-4 (Ribonuclease-4, RNASE4) protein was initially purified and identified from tumor cell culture fluid, and was called "tumor ribonuclease". The mature RNASE4 protein is a secreted single-chain basic protein consisting of 119 amino acid residues, with a relative molecular weight of about 13.8kDa. As a member of the ribonuclease A superfamily, RNASE4 protein mainly participates in various biological processes by hydrolyzing substrate RNA. Like other members of the family, the RNASE4 protein specifically hydrolyzes the 5'-phosphoester bond at the 3' end of the pyrimidine nucleotide on the RNA chain to produce oligonucleotides or nucleotides. The R...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/22C12N15/70A61K38/46A61P25/28A61P25/16A61P25/02
Inventor 盛静浩许正平李斯琪陈光弟
Owner ZHEJIANG UNIV
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