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Production method of recombined human urinary trypsin inhibitor

A technology of trypsin inhibition, human albumin, applied in the field of biology

Inactive Publication Date: 2013-04-03
CHENGDU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to overcome the defect of existing production recombinant human urinary trypsin inhibitor, provide a kind of yield height, the method that quality guarantees the production recombinant human urinary trypsin inhibitor

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  • Production method of recombined human urinary trypsin inhibitor
  • Production method of recombined human urinary trypsin inhibitor
  • Production method of recombined human urinary trypsin inhibitor

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Embodiment Construction

[0015] A method of producing recombinant human urinary trypsin inhibitor (rh-UTI), comprising:

[0016] After the human h-UTI gene was fused with the first domain gene sequence of human serum albumin, it was inserted into the special expression vector PlCZa for expression. Fermentation is carried out in a fermenter, and the key parameters of the fermentation process are pH6.0, 30°C, dissolved oxygen between 20% and 30%, and the induction time is 50 hours; after the induction, the fermentation supernatant is collected by centrifugation, and NaCl is added , adjust the pH of the fermentation supernatant to 7.4 with NaOH, add phosphate to a final concentration of 50mM, and membrane filter to obtain a fermentation broth that can be used for Ni2+-chelation chromatography loading; 50mM imidazole elutes the DoI-UTI fusion protein , desalting and converting the DoI-UTI into the enzymatic digestion buffer solution of enterokinase, and after the enzyme digestion is completed, the rh-UTI ...

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Abstract

The invention discloses a method for producing a recombined human urinary trypsin inhibitor. The method comprises the steps of: fusing an h-UTI gene of a human body respectively with a first structural domain gene sequence in human serum albumin, inserting a fused body into a special expression carrier PICZa to be expressed, carrying out fermentation culture on a constructed engineering strain in a fermentation tank, and after the induction is completed, collecting fermenting supernatant in a centrifugal manner, adding NaC1, adjusting the pH of the fermenting supernatant to be at 7.4 by using NaOH, adding phosphate until reaching the final concentration of 50nM, and carrying out membrane filtration to obtain fermentation broth which can be used for Ni2 plus or minus chelating chromatography sampling. DoI-UTI fusion protein is eluted with 50mM imidazole, desalination is carried out and DoI-UTI is converted into a digestion buffer solution of enterokinase, and rh-UTI is obtained by a chelating chromatography medium and an ion exchange medium after digestion is completed. The method can solve the difficulty of nonuniformity of h-UTI expression in yeast, low expression output and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a production method of recombinant human urinary trypsin inhibitor. Background technique [0002] As early as 1985, Japan used human urinary trypsin inhibitor (h-UTI) extracted from human urine for clinical practice, and in 1994, Guangzhou Techpool Biochemical Pharmaceutical Co., Ltd. also extracted h-UTI. - UTI is used clinically. The main indications of h-UTI are acute pancreatitis, acute exacerbation of chronic pancreatitis, acute circulatory failure, adjuvant drugs in tumor, shock and surgery, prevention and treatment of renal function damage during cisplatin chemotherapy, adjuvant treatment and precursor of AIDS Prevention and treatment of miscarriage, etc. The extracted ingredients have clear clinical efficacy, few side effects, and low production cost. However, as a class of drugs extracted from human urine, due to its low content, difficulty in collecting human urine, and hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12R1/84
Inventor 苟兴华邬晓用邹亮刘达玉王卫唐仁勇邹凯
Owner CHENGDU UNIV
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