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Synthetic method of enterokinase light chain gene and preparation method of expression product of enterokinase light chain gene

A technology of light chain gene and bovine enterokinase, applied in the field of bovine enterokinase light chain protein, can solve problems such as limited source of enterokinase, difficulty in practical application, and enterokinase contamination

Inactive Publication Date: 2011-05-18
YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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  • Abstract
  • Description
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Problems solved by technology

However, the source of natural enterokinase is limited, and the enterokinase extracted from animal tissue is contaminated with other proteases, which brings difficulties to practical application

Method used

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  • Synthetic method of enterokinase light chain gene and preparation method of expression product of enterokinase light chain gene
  • Synthetic method of enterokinase light chain gene and preparation method of expression product of enterokinase light chain gene
  • Synthetic method of enterokinase light chain gene and preparation method of expression product of enterokinase light chain gene

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Embodiment Construction

[0018] 1. Recombinant enterokinase expression vector pPICZαA-EK L build

[0019] 1. Artificial synthesis of recombinant enterokinase gene

[0020] According to the bovine enterokinase light chain EK disclosed in Genebank L Nucleic acid sequence (AY682203) and amino acid sequence, selected codons preferred by Pichia pastoris, artificially synthesized bovine enterokinase light chain EK L The full-length gene sequence (the nucleotide sequence of SEQ-3 in the sequence listing).

[0021] The total synthesis of the gene sequence adopts the method of overlapping PCR. Use DNAMAN to analyze the restriction site of sequence SEQ-3, find 2 natural restriction sites NdeI (CA / TATG), PstI (CTGCA / G) and divide it into 3 segments, namely BS1 (183bp), BS2 ( 240bp), BS3 (282bp). Each segment is then designed as several 60-68bp oligonucleotide fragments for synthesis, and each oligonucleotide fragment has 18-20bp overlapping bases. A pair of linker primers were designed for BS1, BS2, and BS3 ...

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Abstract

The invention relates to a synthetic method of an enterokinase light chain gene and a preparation method of an expression product of the enterokinase light chain gene, in particular to cow enterokinase light chain protein produced by adopting a DNA (Deoxyribonucleic Acid) recombination technology. The encoding gene of the protein is shown in a sequence (3) of a sequence list. The invention further discloses a fermentation and purification process of the cow enterokinase light chain protein. The optimized fermentation and purification method of the invention is utilized to successfully express the cow enterokinase light chain protein with high biological activity in a secretory expression mode. The cow enterokinase light chain protein is used as a tool protease for the specific cutting of fusion protein and is particularly suitable for researching the biological engineering pharmaceutical industry and gene engineering, biochemistry, molecular biology, and the like.

Description

Technical field: [0001] The invention relates to the bovine enterokinase light chain protein produced by DNA recombination technology, and the key is the cloning of the enterokinase light chain gene and the fermentation expression and separation and purification of the product. Recombinantly expressed enterokinase is used as a tool protease for specific cleavage of fusion proteins, and is especially suitable for bioengineering pharmaceutical industry and genetic engineering, biochemistry, molecular biology and other research. Background technique: [0002] Enterokinase (Enterokinase) is a serine protease present in the mammalian duodenum, which was first discovered by Schepowalnickow in Pavlov's laboratory in 1899 (The Journal of Biological Chemistry, Vo1.246, August 25, 1971, PP. 5031-5039.). In 1939, Kunitz confirmed that enterokinase is a trypsinogen activator (J Gen Physiol, March 30, 1939). [0003] So far, natural enterokinase has been purified from humans, mice, pig...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/63C12N15/81C12N9/12C12R1/84
Inventor 张同姜松杨子义李光伟岳妙殊李晓雯
Owner YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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