Recombinant human kallikrein

A technology of kalalidin and recombinant protein, which is applied in the field of recombinant human kalalidin, and can solve the problems that there are no high-molecular-weight recombinant human kalalidin research reports.

Active Publication Date: 2015-08-12
UNICOHEALTH CO LTD
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  • 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 Ka

Method used

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  • Recombinant human kallikrein
  • Recombinant human kallikrein
  • Recombinant human kallikrein

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...

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+ , adding 2mM ManNAc (N-acetyl-D-aminomannose) to the feeding medium can increase the level of glycosylation. 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 o...

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Abstract

The invention relates to a high molecular weight recombinant human kallikrein and its preparation method and use. The high molecular weight recombinant human kallikrein is obtained by a mammalian cell culture production method and a specific purification method. A SDS-PAGE electrophoresis determination result shows that the high molecular weight recombinant human kallikrein is glycoprotein with molecular weight of 43-49KDa and contains 3 N-Link glycosylation binding sites respectively located at Asn78, Asn84 and Asn141. Compared with the existing human urine-derived human kallikrein, the high molecular weight recombinant human kallikrein has a complex and complete glycosylation level and obviously reduces a decarboxyamidation level. The high molecular weight recombinant human kallikrein can substantially prolong a half life in vivo and reduce side effects. The invention also relates to a research on application of the high molecular weight recombinant human kallikrein in cerebral infarction treatment.

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 vivo and reduce side effects. The invention also relates to the research on the application of the recombinant human kalalidin in treating cerebral infarction. 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 f...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/85A61K38/48A61P7/02
CPCA61K38/00C12N9/64C12N15/85
Inventor 张玉杰黄虎李强
Owner UNICOHEALTH CO LTD
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