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Recombinant human kininogenase and application thereof

A technology of kalalidin and recombinant protein, which is applied in the field of recombinant human kalalidin, and can solve problems such as the lack of research reports on high molecular weight recombinant human kalalidin

Active Publication Date: 2015-08-26
UNICOHEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It has been reported that the molecular weight of recombinant human Kalaleylin is similar to that of human urinary Kalaleylin. So far, there has been no research report on high molecular weight recombinant human Kalaleylin at home and abroad.

Method used

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  • Recombinant human kininogenase and application thereof
  • Recombinant human kininogenase and application thereof
  • Recombinant human kininogenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Constructing the Gene Expression Vector Encoding the Human Kawailin Recombinant Protein

[0073] The leader peptide and mature protein gene encoding human kalalidin were obtained by artificial synthesis, with a full length of 771bp (sequence 1 and figure 1 ), this DNA fragment was inserted between the restriction enzyme site KpnI in the vector such as pUC57, and the plasmid pUC-human angioretin was obtained. The accuracy of the synthesized fragments was verified by DNA sequencing.

[0074] The mouse DHFR gene, promoter and terminator fragments containing restriction sites EcoRI (5' end) and HindIII (3' end) were obtained by artificial synthesis, and inserted into the mammalian cell expression vector pCMV / ZEO (Invitrogen ) in the corresponding restriction site, the vector pCMV-DHFR was obtained, and the sequences of DHFR, promoter and terminator contained in it could be verified by DNA sequencing.

[0075] The obtained complete gene sequence encoding human k...

Embodiment 2

[0076] Example 2. Stable Expression of Recombinant Human Kalatin in Mammalian Host Cells

[0077] In order to express the recombinant human kalalidin protein, the constructed gene expression vector pBudCE4.1-DHFR-human kalalidin recombinant protein was transfected into a mammalian host cell line. The preferred host cell line is the DHFR enzyme-deficient CHO-cell (DHFR--CHO), and for the host cell to be more suitable for expressing the secreted protein, and the cell can reach a higher density, we will improve the DHFR--CHO cell, improve Methods include transfecting proteases and altering cellular metabolic pathways. The cell transfection method is preferably electroporation, but other methods including calcium phosphate co-sedimentation, lipofection, and protoplast fusion can also be used. In electroporation, add 10 μg of plasmid linearized with PvuI to 2–5×107 cells in a cuvette using a Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) set at 250 V electric fiel...

Embodiment 3

[0079] Example 3. Cell production of recombinant human kalalin protein

[0080] The cell lines obtained in Example 2 were first adapted to suspension culture in shake flasks. Human kalalin is a glycosylated protein, and the glycosylation level of the protein is directly related to its activity and side effects after being made into a drug. For protein, we optimized the culture medium, and added some substances to increase the glycosylation level of recombinant human kalalidin through a comparative experiment in small shake flask culture. Add 100 μM Cu to basal medium T300 2+ , the feeding medium was supplemented with 2 mM ManNAc (N-acetyl-D-mannose amino). When the shake flask culture of the cells was stable, in order to obtain more human kalalin recombinant protein, the cells were expanded to a sufficient amount, and batch culture was carried out in a 7L bioreactor. The set parameters of the bioreactor are: temperature 37° C., pH 6.90, dissolved oxygen 50%, and rotation sp...

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Abstract

The invention relates to high-molecular-weight recombinant human kininogenase, and a preparation method and application thereof. Specifically, the invention relates to the high-molecular-weight recombinant human kininogenase prepared by using a mammal cell culture production method and specific purifying technology for the first time. According to results of SDS-PAGE electrophoresis determination, the molecular weight of the high-molecular-weight recombinant human kininogenase is 43 to 49 KDa; the high-molecular-weight recombinant human kininogenase is glycoprotein and contains three N-Link glycosylation binding sites which are respectively located at Asn78, Asn84 and Asn141; and compared with kininogenase originated from human urine, the high-molecular-weight recombinant human kininogenase has a more complicated and more complete glycosylation level and obviously reduces a deamidation level. The high-molecular-weight recombinant human kininogenase obtained in the invention can substantially prolong in-vivo half-life and reduce side effects. The invention also relates to application of the high-molecular-weight recombinant human kininogenase in treatment of kidney failure.

Description

technical field [0001] The invention relates to a recombinant human vasocalelin, its preparation method and application. More specifically, the present invention relates to the preparation of recombinant protein by means of molecular biology and host cell production methods, and obtaining high-molecular-weight recombinant human kalalidin for the first time through a specific purification process. The high-molecular-weight recombinant human kalalidin obtained by the invention can significantly prolong the half-life in the body and reduce side effects, and the invention also relates to the application research of the recombinant human kalalidin in treating renal failure. Background technique [0002] Human kininogenase is a glycoprotein containing 238 amino acids, which can degrade kininogen into kinin, and the combination of kinin with specific receptors on the target organ can produce a series of biological effects, such as dilating blood vessels and increasing Blood flow, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/22C07K1/18A61K38/17A61P13/12
CPCA61K38/17C07K14/47
Inventor 李强张玉杰
Owner UNICOHEALTH CO LTD
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