Recombinant expression method of human lysozyme

A technology of human lysozyme and expression cassette, applied in the field of biology

Inactive Publication Date: 2015-01-14
上海万特医药科技(集团)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this strategy has limitations, increasing the copy number is not necessarily accompanied by an increase in the expression of the target protein

Method used

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  • Recombinant expression method of human lysozyme
  • Recombinant expression method of human lysozyme
  • Recombinant expression method of human lysozyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the vector construction that contains human lysozyme coding sequence

[0057] The nucleotide sequence of the natural human lysozyme cDNA is as SEQ ID NO: 1 (the 1-54th position at the 5' end encodes a signal peptide), which encodes the amino acid sequence shown in SEQ ID NO: 16.

[0058] In order to utilize the expression of human lysozyme, the sequence was designed and modified, and the 1-54th signal peptide coding sequence was removed at the 5' end of human lysozyme cDNA, and the coding sequence CTC GAG AAG AGA with part of the 3' end of the yeast α signal peptide was added (SEQ ID NO:8), a synthetic sequence such as SEQ ID NO:2, at the 3' end of the DNA is the translation termination signal TAA, the DNA is inserted into the PUC18 vector (purchased from New England Biolabs), named as PUC18- HLY.

Embodiment 2

[0059] Embodiment 2, human lysozyme cDNA sequence optimization

[0060] Considering the codons preferred by Pichia pastoris, the ratio design of AT:GC, the optimization of mRNA secondary structure, the size and distribution of the base AT-rich region, the GC cluster (GC cluster) or the G cluster (G cluster) On the basis of a series of factors such as distribution, the natural lysozyme cDNA sequence (SEQ ID NO: 1) is optimized, and the sequence GAATTC of the EcoRI restriction site is added to the 3' end, and the optimized lysozyme matures The peptide cDNA sequence is SEQ ID NO:3.

Embodiment 3

[0061] Example 3. Construction of phase bean lectin signal peptide-optimized lysozyme cDNA sequence

[0062] Phaseolin signal peptide (PHA) sequence has a nucleotide sequence such as SEQ ID NO:4, and an amino acid sequence such as SEQ ID NO:15. Artificially synthesize the PHA-HLY (optimized) DNA sequence, add the PHA signal peptide coding sequence and the EcoRI restriction site at the 5' end to obtain the PHA-HLY sequence, and its sequence is the EcoRI restriction site sequence followed by the PHA signal peptide The sequence (SEQ ID NO:4) is followed by the sequence-optimized mature peptide sequence of lysozyme, followed by the terminator sequence, and finally the EcoRI restriction site sequence. The artificially synthesized full sequence is SEQ ID NO:5. The synthetic sequence was inserted into the PUC18 vector to construct a PUC18-PHA-HLY (optimized) vector.

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Abstract

The invention relates to an efficient expression method of a human lysozyme. By comprehensively considering various factors affecting expression and then optimizing the cDNA sequence of the human lysozyme, a lysozyme expression box is established by use of the signal peptide of phaseolus vulgaris agglutinin and the mature peptide of the human lysozyme and used for expressing the human lysozyme; and as a result, the expression efficiency of the lysozyme is greatly improved. The lysozyme expressed by the efficient expression method of the human lysozyme is close to the natural lysozyme and is extremely high in enzymatic activity.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a method for highly expressing human lysozyme. Background technique [0002] Human lysozyme contains 130 amino acids and has a molecular weight of 14.7kD. Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in bacteria. Its hydrolysis site is the β-1.4 glycosidic bond between the 1-carbon atom of N-acetylmuramic acid (NAM) and the 4-carbon atom of N-acetylglucosamine (NAG). Decompose the insoluble mucopolysaccharides of the cell wall into soluble glycopeptides, resulting in the release of the contents of the cell wall rupture and the lysis of the bacteria. The cell wall of G+ bacteria is almost entirely composed of peptidoglycan, while only the inner layer of G- bacteria is peptidoglycan. Therefore, lysozyme is stronger in destroying the cell wall of Gram-positive bacteria G+...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N15/56C12N15/63C12N1/19
CPCC12N9/2462C12Y302/01017
Inventor 孙九如
Owner 上海万特医药科技(集团)有限公司
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