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Recombinant human pancreatic kininogenase-containing medicine for treating and/or preventing cerebral infarction

A kallikrein and drug technology, applied in the field of recombinant human kallikrein drugs, can solve the problems of unknown influence of enzyme activity, limited human urine protein resources, difficulty in collecting human urine, etc. The effect of excellent performance

Inactive Publication Date: 2010-12-01
GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its amino acid composition is the same as the amino acid sequence of human pancreatic kininogenase, but there are Glu / Lys two amino acids at the 162 position of the kininogenase protein extracted from human urine. Effect of activity unknown
The clinical use of human urinary kininogenase found that too fast intravenous infusion would lead to adverse side effects such as a sharp drop in blood pressure, which brought certain risks to patients
In addition, it is difficult to collect human urine, and human urine protein resources are very limited

Method used

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  • Recombinant human pancreatic kininogenase-containing medicine for treating and/or preventing cerebral infarction
  • Recombinant human pancreatic kininogenase-containing medicine for treating and/or preventing cerebral infarction
  • Recombinant human pancreatic kininogenase-containing medicine for treating and/or preventing cerebral infarction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Cloning of human pancreatic kininogenase gene (KLK1 gene)

[0047] Materials and methods: Using human kidney total RNA as a template, the full-length cDNA of KLK was firstly obtained through a reverse transcription kit (Invitrogen, USA). Then using the cDNA as a template, first amplify the two fragments of 1-496bp (taking ATG as 1) and 476-789bp of KLK respectively, and then use the 5' and 3' end primers to splice the two fragments into Complete KLK gene. PCR reaction conditions for amplifying the KLK fragment: denaturation at 94°C for 3 minutes; 30s at 94°C; 30s at 62°C; 30s at 72°C; 34 cycles of amplification;

[0048] The primers used to amplify KLK 1-496 are:

[0049] Upstream primer: 5'GCCTCGCCCTGTCCCTGGGGGGGACTGGTGCTGCGCCCCCGATTCAGTCCCGGATTGTGG3'

[0050] Downstream primer: 5'AATTCTCTGGTTCGATGCTGC 3'

[0051] The primers used for PCR amplification of 476-789bp fragments are:

[0052] Upstream primer: 5'GCAGCATCGAACCAGAGAATTTCTCATTTCCAGATGATCTC3'

...

Embodiment 2

[0060] Embodiment 2: Contain the construction of recombinant KLK1 gene expression plasmid

[0061] The full-length KLK1 gene was inserted into the Xho I and EcoR I sites of the vector pcDNA3.1 / myc-His(-)A containing a strong CMV promoter, and the pcDNA3.1-KLK1 eukaryotic expression plasmid was constructed.

[0062] Primers used to construct pcDNA3.1-KLK1:

[0063] Upstream primer: 5'GTGA CTCGAG ACCATGGGGTTCCTGGTTCTGTGC 3' (Xho I restriction site is underlined)

[0064] Downstream primer: 5'ATCT GAATTC TCAGGAGTTCTCCGCTATGGTGTC 3' (the underline is the EcoR I restriction site).

[0065] In addition, in order to construct a fusion protein containing human pancreatic kininogenase and human IgG1 Fc fragment to express more stable in vivo, the human IgG1 Fc fragment was inserted at the EcoR I and BamH I sites of pcDNA3.1-KLK1, and the pcDNA3.1-KLK1-Fc eukaryotic expression plasmid. The Fc fragment is located at the C-terminus of the KLK1 gene, connected by an EcoR I restricti...

Embodiment 3

[0066] Example 3: Expression and preparation of recombinant human pancreatic kininogenase in eukaryotic cells

[0067] method:

[0068] 1. Expression of KLK1 gene in CHO cells:

[0069] CHO cells were cultured in DMEM medium containing 10% fetal bovine serum in 5% CO 2, Incubated at 37°C. Cationic liposomes (LipofectAMINE 2000, Invitrogen Corporation) were used to transfect CHO cells with the pcDNA3.1-KLK eukaryotic expression plasmid prepared in Example 2, and to screen transfected cells with G418; and then to identify G418 by enzyme activity assay and Western Blot Screened monoclonal stable cell lines.

[0070] 2. Large-scale culture of host cells expressing recombinant human pancreatic kininogenase:

[0071] a) The cell line containing the expressed recombinant protein is cultured with DMEM medium (pH7.20) containing 5% fetal bovine serum, and subcultured in a serum-free medium using a conventional method, and then cultured in small-scale suspension batches , placed i...

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Abstract

The invention relates to the use of recombinant human pancreatic kininogenase composite in the preparation of medicine for treating and / or preventing the cerebral infarction. Recombinant human pancreatic kininogenase is prepared by being combined with a host cell expressing the recombinant protein by means of molecular biology technology, thus having obvious treatment and / or prevention function(s) for the cerebral infarction. The recombinant human pancreatic kininogenase is generally used in a form of pharmaceutical composite such as freeze-dried powder injection or liquid injection.

Description

(1) Technical field [0001] The invention relates to a recombinant human pancreatic kininogenase drug, its preparation method and its application in pharmacy. More specifically, the present invention relates to the preparation of recombinant human pancreatic kininogenase by combining molecular biology techniques with large-scale cell culture methods, and also relates to pharmaceutical compositions containing recombinant human pancreatic kininogenase. The present invention further relates to the use of recombinant human pancreatic kininogenase in the preparation of medicines for treating and preventing cerebral infarction. (2) Background technology [0002] Human pancreatic kininogenase can act on the kininogen substrate to release kinin with vasoactive activity. The combination of kinin with specific receptors on the target organ can produce a series of biological effects, such as dilating blood vessels to increase blood flow, Improve blood circulation, and also increase the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48C12N15/57C12N9/76A61P9/10
Inventor 傅和亮侯永敏吴蓉蓉王晓岩
Owner GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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