Optimized king cobra venom protease inhibitor gene OH-TCI as well as expression method and application thereof

A protease inhibitor, OH-TCI technology, applied in the field of molecular biology, can solve the problems of unsatisfactory processing and low yield, and achieve the effects of low production cost, rapid growth and simple operation

Inactive Publication Date: 2014-04-23
WEIFANG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention aims at the disadvantages of using the Escherichia coli expression system to produce the king cobra venom protease inhibitor OH-TCI in the prior art. The expression of the exogenous protein exists in the periplasmic space, the post-translational processing is not ideal, and the yield is low. A kind of optimized king cobra venom protease inhibitor gene OH-TCI and its expression method and application

Method used

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  • Optimized king cobra venom protease inhibitor gene OH-TCI as well as expression method and application thereof
  • Optimized king cobra venom protease inhibitor gene OH-TCI as well as expression method and application thereof
  • Optimized king cobra venom protease inhibitor gene OH-TCI as well as expression method and application thereof

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Embodiment 1

[0025] 1. Selection, optimization and synthesis of OH-TCI gene

[0026] 1. Selection of OH-TCI gene

[0027] The novel protease inhibitor OH-TCI isolated from king cobra venom belongs to the Kunitz-family, and its mature molecule consists of 58 amino acids. It is the only protein molecule reported to have excellent inhibitory activity on both trypsin and chymotrypsin.

[0028] 2. Optimization of OH-TCI gene sequence

[0029] The inherent characteristics of exogenous genes such as codon bias are important factors affecting the effective expression of exogenous proteins. According to the codon preference of Escherichia coli, the present invention optimizes the low-frequency codons of the Escherichia coli gene of the OH-TCI gene to high-frequency codons, so as to increase the translation speed and expression amount of the target protein. The nucleotide sequence of the OH-TCI gene is compared with the sequence of the OH-TCI gene in Genbank figure 1 shown.

[0030] exist figur...

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Abstract

The invention provides an optimized king cobra venom protease inhibitor gene (i)OH-TCI (/i) as well as expression carrier and an application thereof. The optimized king cobra venom protease inhibitor gene (i)OH-TCI (/i) is synthesized, recombinant plasmids pUC18-T-OH-TCI and pET43.1-OH-TCi are constructed and are transferred into escherichia coli, positive converters are screened, and the experiment results show that the recombinant escherichia coli secretion expresses fusion protein NusA-OH-TCI, and after enzyme digestion of thrombin, recombinant rOH-TCI which can remarkably inhibit activity of trypsin and chymotrypsin. A king cobra venom protease inhibitor OH-TCI with high expression amount and high activity can be obtained through an escherichia coli expression system, and the defects that the cost is high and the expression amount is small when OH-TCI is obtained in other modes are overcome. The inhibitor has wide application prospects in industry such as medicine and chemical engineering.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an optimized king cobra venom protease inhibitor gene OH-TCI and its expression method and application. Background technique [0002] At present, the snake venom Kunitz-type protease inhibitor OH-TCI has been isolated for the first time from the domestic Cobra family King Cobra. family members. OH-TCI and Aprotinin belong to Knitz-type protease inhibitors, and the mature peptides of both are composed of 58 amino acid residues. However, the inhibitory activity of trypsin and chymotrypsin between the two was significantly different. Aprotinin is a broad-spectrum serine protease inhibitor that can inhibit plasmin, kallikrein and protect platelets. Clinically, it is mainly used to treat acute pancreatitis and reduce intraoperative bleeding. [0003] OH-TCI is obtained from a small amount of king cobra venom. Due to its difficult source, it cannot be produce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/15C12N15/70C07K14/81A61K38/57A61P1/18A61P7/04
Inventor 孙同毅
Owner WEIFANG MEDICAL UNIV
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