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Process for preparing medicine grade recombined human urine trypase inhibitor

A technology of fusion protein and preparation process, which is applied in the direction of protease inhibitors, peptide preparation methods, botany equipment and methods, etc. It can solve the difficulties in determining the clinical value of rh-UTI, uneven N-terminal, and no large-scale production, etc. question

Inactive Publication Date: 2006-01-04
上海万兴生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the U.S. patent (US5407915, Human bikunin variants as proteinase inhibitors, and medications containing these) applied for by Bayer Aktiengesellschaft of Germany in 1995 proved well that glycosylation is not necessary for its activity. The N-terminus of the rh-UTI molecule expressed by Saccharomyces cerevisiae has a serious heterogeneity problem: 60% rh-UTI has a natural N-terminus, while 40% of the N-terminus lacks an alanine (Ala)
However, the yield of rh-UTI produced by existing yeast or Escherichia coli is too low, and the homogeneity of the protein cannot be guaranteed. Although there are some research reports, there have been no research reports on large-scale production and animal model experiments. Therefore, relevant The clinical value of rh-UTI remains highly elusive

Method used

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  • Process for preparing medicine grade recombined human urine trypase inhibitor
  • Process for preparing medicine grade recombined human urine trypase inhibitor
  • Process for preparing medicine grade recombined human urine trypase inhibitor

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the structure of recombinant human urine trypsin inhibitor (rh-UTI) engineering strain

[0035] 1. The acquisition of rh-UTI gene and the construction of expression vector PICZa-DoI-UTI

[0036] Referring to the human Bikunin gene sequence in Genbank, two primers for hBIK5 and hBIK3 were synthesized:

[0037] hBIK5:5`-cat ggtacc gat gat gat gac aaa gct gtg cta ccc caa gaa gag-3`

[0038] hBIK3:5`-cat gcggccgc tta cag cag ctc ctc atc acc-3`

[0039] Using human normal fetal liver tissue cDNA (Lot: A604235, Biochain Institute, Inc) as a template, the human Bikunin gene fragment (see SEQ-1) was PCR-produced according to the following system and conditions.

[0040] 50ul PCR system:

[0041] cDNA (template) -------------------------- 2.0ul

[0042] hBIK5 (forward primer) ---------------------5.0ul

[0043] hBIK3 (reverse primer) ---------------------5.0ul

[0044] dNTP-------------------------------2.5ul

[0045] 10XPfu buffer --------------------...

Embodiment 2

[0056] Example 2: Fermentation and purification of recombinant human urinary trypsin inhibitor

[0057] The pilot fermentation and purification process selected GS115-UTI bacterial classification, and carried out in a 30L fermenter according to the description in the Invitrogen fermentation operation guide, the whole process of fermentation and purification (see Figure 4 with Figure 5 ) can be briefly described as:

[0058] 1. Preparation of Seed Solution

[0059]Streak the strain on the YPD plate, place it upside down in a 30°C incubator for 48 hours, pick a single clone and transfer it to a 250ml shaker flask filled with 25ml of YPD culture medium, and culture it at 30°C and 280rpm for 18 hours to OD 600 At 4 to 6 o'clock, after microscopic examination of the yeast with normal morphology and no bacterial contamination, transfer it to a 1L shaker flask containing 500ml of YPD culture medium at a ratio of 1:500, culture at 30°C and 280rpm for 18 hours, OD 600 After reachi...

Embodiment 3

[0074] Example 3: Confirmation of the properties of recombinant human urinary trypsin inhibitor

[0075] The rh-UTI prepared according to the method in Example 2 has been studied in terms of protease inhibitory activity, specific activity, protein purity, molecular weight determination, isoelectric point, N-terminal-C-terminal sequencing, etc. according to the requirements of SFDA quality inspection standards. Tested to determine its basic properties.

[0076] 1. Protease inhibitory activity of h-UTI-specific activity determination

[0077] 1.1. Analysis of the inhibitory effect on trypsin

[0078] Use benzoyl-L-arg-p-NA as substrate, according to the method (Geige&Fritz, Methods of Enzymatic Analysis, Vol V, 3 rd ed., Bergmeyer (ed.), Verlag Chemie, Weinheim, p.121, 1984), and the released p-NA was detected with a spectrophotometer at a wavelength of 405 nm. Enzymes and inhibitors were pre-warmed for 15 minutes prior to adding substrate. The results showed that rh-UTI cou...

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Abstract

The extraction human urinary trypsase inhibitor (hUTI) from urea has been widely applied clinically. The present invention describes how to constitute fusion protein engineering strain capable of secreting and expressing recombinant human urinary trypsase inhibitor (rhUTI) in Pichia pastoris. Through fermentation, rough purification, restriction, fine purification and serial property comparison tests, engineering strain with yield, purity and pharmacological characteristic meeting the requirement of large scale production and clinical application and rhUTI producing process are finally determined.

Description

technical field [0001] The present invention describes the production and preparation of a kind of glycoprotein drug extracted from human urine in methanol yeast (Pichia pastoris) by recombinant DNA technology---Human urinary trypsin inhibitor (Human urinary trypsin inhibitor, h-UTI), especially the construction and fermentation of the methanol yeast engineering strain used for production, the purification of the expression product, the confirmation of its properties, and the drug efficacy test. Background technique [0002] In 1909, Bauer and Reich reported the presence of a trypsin inhibitor in human urine, the so-called human urinary trypsin inhibitor (Human urinary trypsin inhibitor, h-UTI), but it was not until 1977 that Sumi et al. This glycoprotein molecule is purified from the urine. h-UTI is the degradation product of Inter-α-trypsin inhibitor (I-α-TI) in human plasma. Studies have confirmed that the I-α-TI protein in human plasma is composed of three heavy chains...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C07K1/14C07K14/81A61K38/57
Inventor 黄秀东
Owner 上海万兴生物制药有限公司
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