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Pro-urokinase gene mutant and preparation method thereof

A pro-urokinase, gene technology, applied in the field of bioengineering, can solve the problems of gene sequence optimization, location dispersion, etc., to achieve the effect of optimizing genetic codons

Inactive Publication Date: 2013-06-12
NANCHANG WANHUA BIOCHEM PHARMA
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The positions of these codons in the gene sequence are scattered, and it is difficult to completely optimize the gene sequence by simple operations such as gene site-directed mutation

Method used

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  • Pro-urokinase gene mutant and preparation method thereof
  • Pro-urokinase gene mutant and preparation method thereof
  • Pro-urokinase gene mutant and preparation method thereof

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Embodiment Construction

[0014] In order to clearly show the innovation of the present invention, the nucleotide sequence of the natural prourokinase cDNA gene is listed in Sequence 2, and the nucleotide sequence of the newly designed gene mutant of the present invention is listed in Sequence 1. The amino acid sequence of the natural pro-urokinase is given in sequence 4, and the amino acid sequence encoded by the mutation of the pro-urokinase gene is shown in sequence 5.

[0015]The invention not only optimizes all the genetic codes, but also properly modifies the two ends of the gene, making it easy to carry out further genetic operations such as sequencing analysis and construction of expression vectors. The object of the present invention is to develop a novel pro-urokinase gene suitable for expression in Escherichia coli, so as to produce pro-urokinase through E. coli fermentation technology. The nucleotide sequence of the mutant gene synthesized by the present invention has many differences with ...

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Abstract

The invention relates to a pro-urokinase gene mutant and a preparation method thereof. On the premise of keeping the amino acid composition and sequence of a protein unchanged, two nucleotides G and T are added on the 5' end of the gene sequence, and a recognition sequence GTCGAC of endonuclease Sal1 is added on the 3' end of the sequence. The nucleotide sequence of the gene mutant is shown in a sequence table 1. The preparation method comprises the following steps: (1) synthesizing a single-chain nucleotide segment; (2) performing PCR (polymerase chain reaction) to generate a double-chain DNA (deoxyribonucleic acid) segment; (3) splicing the whole sequence of the pro-urokinase gene mutant; and (4) determining and analyzing the sequence of the pro-urokinase gene mutant. Through a method of artificially synthesizing the whole gene, the invention optimizes the whole genetic codon, appropriately modifies both ends of the gene and ensures that sequencing analysis, expression vector construction and other further genetic manipulations can be easily performed. Thus, the invention develops a novel pro-urokinase gene suitable for being expressed in Escherichia coli, so that pro-urokinase can be conveniently produced through an Escherichia coli fermentation technology.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a prourokinase gene mutant and a preparation method thereof. Background technique [0002] Pro-urokinase (pro-UK, single-chain urokinase plasminogen activator, scu-PA) is a single-chain serine protease. The mature protein molecule consists of 411 amino acid residues and has a molecular weight of 54KD. Under the action of plasmin, trypsin and kallikrein, prourokinase breaks between lysine 158 and leucine 159 (Ile), and two peptide chains A and B are formed after the break , the two chains are connected to each other through the disulfide bond formed between the 148th and 279th cysteine ​​(Cys148-Cys279), forming a high molecular weight urokinase (HMW-UK). Polymer urokinase has the same molecular weight as prourokinase. High molecular weight urokinase can be further hydrolyzed under the action of plasmin, resulting in a break between Lys135-Lys136, losing the first 135 amino acids ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/58C12N15/10
Inventor 陈永福熊晓云
Owner NANCHANG WANHUA BIOCHEM PHARMA
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